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Nima Momeni, Accused in Murder of Tech Exec Bob Lee, Seeks To Move Trial Out of San Francisco  

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The murder trial of Nima Momeni , the alleged killer of tech executive Bob Lee, has been set for March 15 in San Francisco, but defense attorneys on Thursday asked a judge to move the trial out of the city, citing the "prejudicial" publication of photos of Momeni in jail.

Momeni appeared in court beside his legal team, which includes San Francisco lawyer Tony Brass and two Florida attorneys, Saam Zangeneh and Bradford Cohen. Momeni’s mother was also in court for the hearing, where she raised her hands in the shape of a heart after her handcuffed son came into the room. 

Zangeneh filed a motion to change venue because he said his client could not receive a fair trial due to unfair media coverage, including the recent publication by The Standard of photos of Momeni in his jail cell . 

"There was a photographer that went into the jail, and that photographer was allegedly there to take pictures of solitary confinements,” Cohen said. “In my 27 years, I’ve never seen a photographer go into a jail with a camera and start taking pictures of a defendant before trial. It is highly prejudicial.” 

Superior Court Judge Loretta Giorgi set a hearing regarding the motion for a change of venue for Jan. 25.

RELATED: Nima Momeni’s New Lawyer Won Lil Wayne, Kodak Black Pardons From Trump

Prosecutors have alleged that Momeni drove Lee, the Cash App founder, to a secluded street under the Bay Bridge in the early morning hours of April 4 and stabbed him twice with a kitchen knife, leading to his death hours later in a hospital.  

Momeni has been charged with murder and has been held at San Francisco’s jail in San Bruno since mid-April. Photos of Momeni in his cell published by The Standard this week revealed that he has been reading books on psychology and Napoleon Bonaparte as he awaits trial.

Momeni’s attorneys were aware of the photos prior to publication, and refused to comment to The Standard about them. They did not request that they not be published.

Outside of court on Thursday morning, Zangeneh, the attorney, said Lee was a "local luminary" and noted that hundreds of thousands of people in the Bay Area work in the tech sector.

"It's kind of like, if a Los Angeles Laker is the victim of a homicide, do you think [his accused killer] could get a fair trial in L.A.?"

"A freelance photographer had permission to take photos inside the jail and Nima Momeni gave the photographer permission to have his photo taken. We contacted Momeni's legal team prior to publication, and they did not raise objections or concerns about the publication of the images," said The Standard's editor-in-chief, Julie Makinen. "The Standard believes the images were entirely newsworthy."

Who Will Testify at Trial?

Prosecutors have said Momeni was upset because Lee encouraged Momeni’s sister, Khazar Momeni, to do “inappropriate” things the day before at the home of Lee’s alleged drug dealer, Jeremy Boivin, according to court documents.

But Momeni’s attorneys have argued in filings that the facts of the case do not amount to a cold-blooded killing. 

The coming trial could include many of the people who were with Lee in his last hours, including Khazar Momeni and her husband , as well as expert witnesses, Brass said in October. 

“It’s essential for us to give the complete story,” Brass said. “We are not trying to hide anything. If anything, we are trying to tell a complete story about how this incident occurred.” 

Monday’s arrest of Khazar Momeni in San Francisco on suspicion of driving under the influence and hit-and-run brought more media attention to a family already under the spotlight. 

The other expected witnesses, Brass said, include everyone who spent any time with Lee in the hours before his death. That would include Boivin , who witnesses said gave Khazar Momeni drugs the day before Lee was killed during a party at Boivin's apartment where Lee was present. The woman who called Khazar Momeni’s husband and brother to come and get her from the party could also testify.

“We are trying to establish the relationship between Bob and Boivin,” Brass said previously. “He’s the designer drug dealer to the stars.”

Brass said that once a date has been set, each side has to share their witnesses with each other.

Correction : This story has been updated to note the change-of-venue motion will be considered at a hearing Jan. 25.

Jonah Owen Lamb can be reached at [email protected]

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Nima Momeni’s attorneys ask to move trial, claiming SF has pro-tech bias

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Attorneys for Nima Momeni, the accused killer of tech executive Bob Lee, have requested to have the murder case moved from San Francisco, where they suggested Momeni would not get a fair trial because the city’s large tech population saw Lee as a “celebrity.” 

They also suggested today, after a hearing to set a trial date, that a San Francisco jury could be biased against Momeni after new photos apparently purchased by the San Francisco Standard and published this week showed Momeni dressed in an orange jumpsuit in his jail cell. 

“This was a hit job, and there’s going to be ramifications,” said Saam Zangeneh, Momeni’s Florida-based attorney. 

Zangeneh added: “What they did, purchasing pictures that they knew were not supposed to be taken, were not supposed to be [legal], … I think there’s civil liability. I think there’s partial criminal liability.”

The Standard ‘s editor-in-chief, Julie Makinen, said in a statement that a freelance photographer with permission to take photos inside the jail got clearance from Momeni to have his photo taken.

“We contacted Momeni’s legal team prior to publication, and they did not raise objections or concerns about the publication of the images,” Makinen added. “The Standard believes the images were entirely newsworthy.”

In the photos, Momeni is smiling through the glass window of his cell, and appears to be posing for the photos — but Zangeneh insisted this was not the case, that the photos were “happenstance,” and Momeni did not agree to be photographed. Momeni’s walls are lined with art, and he has books, like the biography of Napoleon and a psychology book, on his desk. 

“The article, coupled with those pictures, is one of the most disgusting things that I’ve seen in a case where a defendant is innocent until proven guilty,” said Bradley Cohen, another member of Momeni’s legal team. 

Cohen said he had never seen an instance where a photographer was permitted into a jail pod to take photos of a defendant before trial — and said that the cameraman had been told not to photograph individuals. 

San Francisco Superior Court Judge Loretta Giorgi, who is hearing the case, seemed to agree with Momeni’s attorneys. 

“I saw that, and the moment I saw it, I notified the sheriff. And they are looking into all of it,” Giorgi said in court. Giorgi said that internal and external investigations would follow. 

The sheriff’s spokesperson, Tara Moriarty, said a reporter and photographer from the LA Times had been permitted into the jail for a story on administrative separation cells.

“Regrettably, the LA Times photographer violated our established media rules, thereby unethically violating the conditions imposed, which were put in place to protect the security and privacy of those incarcerated,” Moriarty said. She added that the Sheriff’s Office “condemns such actions unequivocally, as they compromise the mutual trust between law enforcement agencies and the media … the well-being of incarcerated individuals, and the integrity of information shared with the public.”

The photographer, for his part, told the San Francisco Chronicle that he had cleared his photographs with a jail representative.

Momeni allegedly stabbed Lee in the street in April, and has been in jail since his arrest on April 13 . The killing initially sparked outrage in the city over out-of-control crime, as Lee was found alone and bleeding out in the street the night of the stabbing.

But it later became clear that Momeni and Lee knew each other, and prosecutors have suggested that the two men had argued about Lee’s relationship with Momeni’s sister, Khazar Momeni. 

Momeni’s defense attorneys filed two motions today in court: One to change venue, and another demanding a hearing for prosecutors to explain a violation of a court order. They said that prosecutors have not yet released Momeni’s vehicle to his family, despite a court ruling demanding they do so . 

The two motions will be heard at a status conference on Jan. 25. A trial date was tentatively set for March 15. 

Zangeneh said that the motion to change venue was only sped up by the release of Momeni’s photos, suggesting that the team had already been planning to ask that the trial be moved out of San Francisco due to the massive tech influence here, and Lee’s position as a well-known founder of CashApp and executive of other companies. 

“I think that Bob Lee is akin to a celebrity in the tech sector, and a vast majority of San Franciscans are part of that industry,” Zangeneh said. “If a Los Angeles Laker is the victim of a homicide, you think that the defendant would be able to get a fair trial in LA? I don’t think so.” 

Only about 14 percent of San Francisco residents work in tech.

Momeni’s attorneys were also furious about the release of the photos, and while taking questions from the press, refused to take any from the San Francisco Standard reporter who was in court today. 

Zangeneh even went so far as to suggest a connection between Lee and Michael Moritz, the owner of the Standard . 

“I’m not sure if there’s a nexus between Bob Lee’s family and Big Tech and the San Francisco Standard,” Zangeneh said. “And if there’s some sort of, like, marriage that’s going on here to be able to influence the jury — there may be, but I’ll tell you what, we’re going to look into it.” 

Bob Lee killing: Murder trial pushed back as attorneys process case

Bob Lee killing: Nima Momeni denied bail

Nima Momeni pleads ‘not guilty’ again, seized BMW to be released

Eleni balakrishnan.

REPORTER. Eleni reports on policing in San Francisco. She first moved to the city on a whim more than 10 years ago, and the Mission has become her home. Follow her on Twitter @miss_elenius.

Join the Conversation

This is truly preposterous. All communities have more and less well-known members. We do not ship cases involving them out to Turlock. More important, I’ve served on SF juries. I can tell you there are not many techies on them. More retired municipal workers than anything.

The deteriorating ethics of The San Francisco Standard is abhorrent–a blend of yellow journalism and billionaire megaphone propaganda politics. Storyteller Michael Moritz is using his wealth to manipulate readers and voters while obscuring his financed networks’ activities a la Rothschild. Industry tycoons and financiers cultivating their vision of the future and controlling reality for the rest of us is an old story and very undemocratic. Readers beware of The San Francisco Standard! For another recent example, just look at their article, “Y Combinator CEO Garry Tan Is Organizing San Francisco’s Moderate Political Agenda.” There they collude with a political group founded by their own CEO, Griffin Gaffney, and funded by Michael Moritz, owner of The San Francisco Standard, called TogetherSF, without anywhere in the article disclosing their association with the organizers at Tan’s political organizing event, in clear violation of their own Standards & Ethics Policy. When they were challenged on this ethical lapse, their editor-in-chief, Julie Makinen, doubled-down and refused to inform readers of their hidden hand in creating the “news” that they then themselves “report on” as The San Francisco Standard embarks on its mission to transform San Francisco in the more conservative image of its creator and owner. That is not news. That’s manipulative political perspectival propaganda posing as news which abuses the trust of its readers and discredits itself as a news organization worthy of the respect of readers.

Where will there not be some form of bias? Guantanamo?

I was surprised myself to see those pics. Maybe not illegal, but a series of ethical lapses and questionable portrayals of notifications and permissions.

Momeni would have been far better served if he had a public defender representing him. I just hope, for his sake, that these guys agreed to represent him for free.

I thought “tech bias” would work the other way, that Momeni would garner sympathy from the jury pool for putting a drug addled tech warlord out of our collective misery.

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https://www.wsj.com/articles/nima-momeni-bob-lee-suspect-arraignment-san-francisco-3ef6fdb

Suspect in Bob Lee Killing Pleads Not Guilty

Nima momeni’s attorney says defense is a combination of an accident and self defense.

Updated May 18, 2023 4:46 pm ET

Paula Canny, an attorney for Nima Momeni, also argued that her client shouldn’t be detained ahead of a trial, but a San Francisco superior court judge ordered Momeni to remain behind bars. Momeni didn’t speak during the hearing.

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• Published: 02 October 2018

The effect of NIMA matching in adult unrelated mismatched hematopoietic stem cell transplantation – a joint study of the Acute Leukemia Working Party of the EBMT and the CIBMTR

• Julia Pingel 1 ,
• Tao Wang 2 , 3 ,
• Yvonne Hagenlocher 1 ,
• Camila J. Hernández-Frederick 1 ,
• Arnon Nagler 4 ,
• Michael D. Haagenson 5 ,
• Katharina Fleischhauer 6 ,
• Katharine C. Hsu   ORCID: orcid.org/0000-0003-2827-5324 7 ,
• Michael R. Verneris 8 ,
• Stephanie J. Lee 2 , 9 ,
• Mohamad Mohty 10 ,
• Emmanuelle Polge 11 ,
• Stephen R. Spellman 5 ,
• Alexander H. Schmidt 1 &
• Jon J. van Rood 12 , 13  

Bone Marrow Transplantation volume  54 ,  pages 849–857 ( 2019 ) Cite this article

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1 Citations

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• Haematological diseases
• Preclinical research

Hematological malignancies can be cured by unrelated donor allogeneic HSCT and outcomes are optimized by high-resolution HLA matching at HLA-A, -B, -C, -DRB1 and -DQB1 (10/10 match). If a 10/10 match is unavailable, 9/10 matches may be suitable. Fetal exposure to non-inherited maternal antigens (NIMA) may impart lifelong NIMA tolerance modulating the immune response, as shown in adult haploidentical transplantation. In cord blood transplantation, NIMA matching lowered rates of aGvHD and TRM; in haploidentical transplantation, sibling donors with non-shared maternal antigens showed less grade II-IV aGvHD. This retrospective analysis examined if 9/10 matched unrelated donor HSCT benefits from NIMA matching. DKMS contacted 1,735 donors and obtained 733 (42%) maternal samples. NIMA-matched and -mismatched cases with a minimum follow-up of 1 year were compared by univariate and multivariate analyses adjusted for co-variates for OS, DFS, relapse, TRM and a/cGvHD. The study population ( N  = 445) comprised 31 NIMA-matched and 414 NIMA-mismatched cases. No significant differences between NIMA-matched and NIMA-mismatched groups were found for any outcomes with similar OS and TRM rates within both groups. This study provides the proof of principle that NIMA matching is possible in the unrelated donor HSCT setting; larger studies may be able to provide significant results.

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Acknowledgements

We thank Carlheinz Müller from the German National Bone Marrow Donor Registry ZKRD for providing additional HLA information for patients, Jon van Rood for his persistence in moving HSCT forward with his dedication and research for NIMA matching and related topics. Without his initiative, scientific directions and valuable advice, this study would not have been possible. We thank Christina Peters from the pediatric working group of EBMT for granting access to data from the pediatrics working group of the EBMT for this study and Cladd Stevens for revisions of our NIMA match assignments, as well as the donors and their mothers for their participation and cooperation in this study. The CIBMTR is supported primarily by Public Health Service Grant/Cooperative Agreement 5U24CA076518 from the National Cancer Institute (NCI), the National Heart, Lung and Blood Institute (NHLBI) and the National Institute of Allergy and Infectious Diseases (NIAID); a Grant/Cooperative Agreement 4U10HL069294 from NHLBI and NCI; a contract HHSH250201200016C with Health Resources and Services Administration (HRSA/DHHS); two Grants N00014-17-1-2388 and N0014-17-1-2850 from the Office of Naval Research; and grants from *Actinium Pharmaceuticals, Inc.; *Amgen, Inc.; *Amneal Biosciences; *Angiocrine Bioscience, Inc.; Anonymous donation to the Medical College of Wisconsin; Astellas Pharma US; Atara Biotherapeutics, Inc.; Be the Match Foundation; *bluebird bio, Inc.; *Bristol Myers Squibb Oncology; *Celgene Corporation; Cerus Corporation; *Chimerix, Inc.; Fred Hutchinson Cancer Research Center; Gamida Cell Ltd.; Gilead Sciences, Inc.; HistoGenetics, Inc.; Immucor; *Incyte Corporation; Janssen Scientific Affairs, LLC; *Jazz Pharmaceuticals, Inc.; Juno Therapeutics; Karyopharm Therapeutics, Inc.; Kite Pharma, Inc.; Medac, GmbH; MedImmune; The Medical College of Wisconsin; *Mediware; *Merck & Co, Inc.; *Mesoblast; MesoScale Diagnostics, Inc.; Millennium, the Takeda Oncology Co.; *Miltenyi Biotec, Inc.; National Marrow Donor Program; *Neovii Biotech NA, Inc.; Novartis Pharmaceuticals Corporation; Otsuka Pharmaceutical Co, Ltd. – Japan; PCORI; *Pfizer, Inc; *Pharmacyclics, LLC; PIRCHE AG; *Sanofi Genzyme; *Seattle Genetics; Shire; Spectrum Pharmaceuticals, Inc.; St. Baldrick’s Foundation; *Sunesis Pharmaceuticals, Inc.; Swedish Orphan Biovitrum, Inc.; Takeda Oncology; Telomere Diagnostics, Inc.; and University of Minnesota. The views expressed in this article do not reflect the official policy or position of the National Institute of Health, the Department of the Navy, the Department of Defense, Health Resources and Services Administration (HRSA) or any other agency of the U.S. Government. *Corporate Members

Authorship contributions

JvR: Study design and interpretation of the results. JP: Study design, data collection, interpretation of results, manuscript preparation, revision and approval. TW: Statistical analysis, interpretation of results, manuscript preparation, manuscript revision and approval. MDH: Data collection, statistical analysis, manuscript preparation, manuscript revision and approval. SRS: Study design, data collection, interpretation of results, manuscript writing, manuscript revision and approval. YH: HLA allele frequency data and comparisons, manuscript preparation, manuscript revision and approval. CJH-F: Data collection, results interpretation, manuscript revision and approval. AN: Study design, data contributions, results interpretation, manuscript revision and approval. MM: Study design, data contributions, results interpretation, manuscript revision and approval. KF: Study design, manuscript revision and approval. KCH: Study design, manuscript revision and approval. MRV: Study design, manuscript revision and approval. SJL: Study design, results interpretation, manuscript revision and approval. EP: Study design, data management, data collection, manuscript revision and approval. AHS: Study design, data collection, results interpretation, manuscript revision and approval

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Julia Pingel, Yvonne Hagenlocher, Camila J. Hernández-Frederick & Alexander H. Schmidt

Center for International Blood and Marrow Transplant Research, Medical College of Wisconsin, Milwaukee, WI, USA

Tao Wang & Stephanie J. Lee

Division of Biostatistics, Institute for Health and Society, Medical College of Wisconsin, Milwaukee, WI, USA

Sheba Medical Center, Sheba Cord Blood Bank, Tel-Hashomer, Israel

Arnon Nagler

Center for International Blood and Marrow Transplant Research, Minneapolis, MN, USA

Michael D. Haagenson & Stephen R. Spellman

Institute for Experimental Cellular Therapy, University Hospital Essen, Essen, Germany

Katharina Fleischhauer

Memorial Sloan Kettering Cancer Center, New York, NY, USA

Katharine C. Hsu

University of Colorado - Children’s Hospital, Aurora, CO, USA

Michael R. Verneris

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

Stephanie J. Lee

Department of Haemotology, Saint Antoine Hospital, Paris, France

Mohamad Mohty

EBMT (European Society for Blood and Marrow Transplantation), Department of Haematology, Saint Antoine Hospital, Université Pierre et Marie Curie, INSERM UMR 938, Paris, France

Emmanuelle Polge

Matchis Foundation, Leiden, The Netherlands

Jon J. van Rood

Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands

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Pingel, J., Wang, T., Hagenlocher, Y. et al. The effect of NIMA matching in adult unrelated mismatched hematopoietic stem cell transplantation – a joint study of the Acute Leukemia Working Party of the EBMT and the CIBMTR. Bone Marrow Transplant 54 , 849–857 (2019). https://doi.org/10.1038/s41409-018-0345-8

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Received : 12 June 2018

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Accepted : 30 August 2018

Published : 02 October 2018

Issue Date : June 2019

DOI : https://doi.org/10.1038/s41409-018-0345-8

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Sister of Nima Momeni, man accused of killing Bob Lee, arrested in San Francisco

Posted: November 29, 2023 | Last updated: November 29, 2023

The sister of Nima Momeni, the man accused of killing tech executive Bob Lee , was arrested Monday in San Francisco on suspicion of driving under the influence and hit-and-run, according to police.

Khazar Momeni, 38, was arrested following a car crash in the area of Geary and Larkin streets, police said.

A man who works in the neighborhood said a white Mercedes coupe crashed into two parked cars and then stopped. Police said they arrived to find Khazar at the wheel.

According to a police report, two members of the fire department's Street Crisis Response Team told officers they saw Khazar inhaling from a whip-it cartridge.

Whip-it cannisters contain nitrous oxide, sometimes called laughing gas. The report said officers found two of them in Khazar's car and started a DUI investigation.

Khazar was booked into jail on the following charges: driving under the influence, hit-and-run, fail to drive within the lane, and failure to provide insurance at the scene of an accident, police said.

The district attorney's office didn't have her case as of Tuesday and had no comment. The office of prominent cosmetic surgeon Dr. Dino Ellyassnia, Khazar's husband, also wouldn’t comment.

Khazar has been seen regularly in court during her brother Nima's appearances. He is facing murder charges in the death of Lee, founder of Cash App.

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Bone-chilling photos show cash app founder bob lee’s alleged killer nima momeni smiling in jail.

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New photos have emerged showing Cash App founder Bob Lee’s alleged murderer Nima Momeni smiling behind bars as he awaits trial.

Momeni was ready for his photo-op, with the accused killer nonchalantly posing behind the glass and metal reinforced door of his cell at San Bruno jail in photos obtained by The Post show.

The accused killer, who faces life in prison if convicted of killing the tech mogul, seemed to smooth down his closely cropped hair before striking a crossed-armed pose while smirking and smiling at the camera in his bright orange jumpsuit.

Among his things, a psychology book can be seen in the pics as well as a 2015 biography of Napoleon Bonaparte.

He also seemed to have a few photos and a piece of paper taped to his wall, alongside another stacks of books in the photos, which were taken in August.

Momeni has been accused of stabbing Lee to death with a kitchen knife under the Bay Bridge in San Francisco in early April after arguing with him about his sister, Khazar Elyassnia.

Lee, 43, was transported to a hospital where he later died after being found unresponsive and bleeding badly.

Momeni was arrested 11 days later and has since been denied bail, as his family members have called him a “lunatic and psychotic” and prosecutors warned the court that he was dangerous to the public.

Momeni has been housed in a solitary cell for the past seven months as he awaits trial. Outside of his lawyers, his mother has been the only one to visit him, according to the San Francisco Standard .

Prosecutors have alleged Momeni was upset because the tech executive had encouraged Elyassnia – arrested for a DUI earlier this week – to do “inappropriate” things.

Lee was found with cocaine and ketamine in his system and it was subsequently revealed he had frequented drug and sex parties with Momeni’s sister, according to the Wall Street Journal.

Elyassnia had texted Lee around the time he was being murdered, allegedly saying: “Just wanted to make sure your doing ok Cause I know nima came wayyyyyy down hard on you.”

Police do not suspect she was involved in the killing.

The men were spotted leaving Millennium Tower — where his sister lives with her plastic surgeon husband — in surveillance footage shortly before Lee was killed.

Momeni is expected back in court on Thursday. His trial date has yet to be set.

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NIMA (National Imagery and Mapping Agency)

The National Imagery and Mapping Agency (NIMA) was formed in October, 1996, to provide the United States military and intelligence agencies with up-to-date and accurate imaging and geospatial information. NIMA is a Department of Defense agency and is a member of the United States intelligence community. NIMA uses satellite and aerial imaging equipment to produce maps that can be used by both military planners and soldiers in the field.

NIMA assumed the duties of the Defense Mapping Agency, the Central Imagery Office, the Defense Dissemination Program Office, the National Photographic Interpretation Center, and parts of the Defense Intelligence Agency, the National Reconnaissance Office (NRO), Defense Airborne Reconnaissance Office, and the Central Intelligence Agency . NIMA now serves as the sole source for mapping and imaging needs of the U.S. military and intelligence agencies.

NIMA uses satellite photographic, radar, and infrared imaging information to create and analyze a database of cartographic and geodetic images. NIMA can then customize these images to suit the needs of its customers. NIMA's database allows the creation of two-dimensional and three-dimensional (elevation) models of any part of the world. NIMA also catalogs man-made and natural features, which can be used for navigational or intelligence purposes.

For information gathering, NIMA uses Department of Defense, NRO, and other government owned imaging satellites. NIMA also contracts out for the use of privately owned imaging satellites in a cost-saving effort. NIMA declassifies many of the images obtained from these commercial satellites for use by American allies.

The National Imagery and Mapping Agency contributes to achieving United States foreign policy and national security objectives by providing intelligence agencies and policymakers with current imagery information. Military and civilian intelligence agencies use NIMA's cartographic and geospatial intelligence to monitor the proliferation of nuclear, chemical, and biological weapons, track arms shipments, and ensure that global treaties are being upheld.

NIMA's primary function is to provide accurate geospatial information for combat planning and support. NIMA tailors its products to fit the needs of its target audience. When the United States began military operations in Afghanistan in 2001, American military forces had little information on Afghan geography and topography. NIMA assisted the various U.S. forces involved in this conflict by quickly producing high quality maps for strategists and soldiers. NIMA used its resources to produce different maps for different operations. Maps for Naval aviators included detailed information about targets for the U.S. bombing campaign. Maps for special operations forces noted possible food and water locations, as well as the locations of enemies and non-combatants.

NIMA provided similar logistical support for Operation Iraqi Freedom in 2003. During the planning stages of the war, NIMA provided policymakers and military coordinators with maps that included the locations of enemy forces, suspected chemical and biological weapons depots, and potential government and military targets. Maps also noted strategic locations, including oil wells. During combat, NIMA's technologically advanced imaging systems supplied U.S. forces with near real-time maps that allowed American forces to engage enemy combatants before visually confirming the enemies' presence.

NIMA is currently working on the Shuttle Radar Topography Mission (SRTM), a mission that recorded elevation data for most of the Earth 's surface. By accumulating elevation data from a single source, NIMA will be able to produce a uniform elevation map of the Earth.

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Finding Time

Researchers say time is an illusion. so why are we all obsessed with it.

Geoff Brumfiel

Scientists at the National Institute for Standards and Technology are developing ever-smaller and more accurate clocks. M. Hummon/NIST hide caption

Scientists at the National Institute for Standards and Technology are developing ever-smaller and more accurate clocks.

America's official time is kept at a government laboratory in Boulder, Colo., and according to the clock at the entrance, I was seven minutes behind schedule.

I rush across the campus of the National Institute of Standards and Technology (NIST) and arrive at the end of a long hallway where physicist Jeff Sherman was waiting patiently.

"Sorry I'm running late," I tell him.

"It's OK, we only measure the nanoseconds," Sherman jokes.

It's never been easier to know what time it is. NIST broadcasts the time to points across the country. It's fed through computer networks and cellphone towers to our personal gadgets, which tick in perfect synchrony. Humanity's ever-improving agreement on the time smooths communication and transportation, and it lubricates our economy.

But time has another side to it, one that the clocks don't show.

"A lot of us grow up being fed this idea of time as absolute," says Chanda Prescod-Weinstein , a theoretical physicist at the University of New Hampshire. But Prescod-Weinstein says the time we're experiencing is a social construct. Real time is actually something quite different. In some of the odder corners of the Universe, space and time can stretch and slow — and sometimes even break down completely.

For many people, this unruly version of time is "radical," she says. But as technology to better count the time grows ever more sophisticated, our everyday understanding of time itself may need to start changing.

A Sisyphean task

To get a sense of where the rigid time that governs most of our lives comes from, Sherman takes me into a beige-colored laboratory crammed with experimental equipment and computers. There are three big boxes looming above lab benches, holding three high-precision atomic clocks. Each box is labeled with a name: One's called George, another Fiona, and the third is Elvis.

"They all have quirks and personalities," Sherman explains. "When they fail at 2 a.m. you want to have a little bit of compassion for them, so you give them names."

George, Fiona and Elvis are just part of the 21-clock ensemble NIST uses to generate the official time. These three clocks tick using hydrogen atoms. The atoms are excited using radiofrequency energy and then sent into a chamber. Once inside, they decay, emitting a specific frequency of light.

Think of it as striking an atomic tuning fork, Sherman says.

Shots - Health News

Finding time: how did covid warp our sense of time it's a matter of perception.

The excited hydrogen emits "a tone of light," he says. The rest of the clock "is an instrument that tries to sample — tries to listen — to a little bit of that light and count the cycles of oscillation in that light."

Those light cycles are the "tick" of the clock. By averaging a subset of the 21 clocks together, NIST has created a system that can count the time to within one quadrillionth of a second. That means the government's clock can keep time to within a second over the course of about 30 million years.

In another room, that timing signal is sent out across the United States, and via satellite to other government laboratories in other parts of the world with clocks of their own.

It's an impressive system, but there's a catch. You've got to keep counting. If you stop, if you blink, you don't know the time anymore.

"In exchange for this wonderful idea," Sherman says, "you're now beholden to count forever and not lose track."

The MAN in "time management"

The ever-advancing NIST clock is one way to understand time. But theoretical physicist Prescod-Weinstein bristles at that definition. She says this version of time is just the time the government wants you to think about.

"The management of what counts as correct time and what time it is in any given place is deeply related to authority," she says.

The time from this lab is used to run our lives. It says when planes take off and land, when markets open and close, when schoolchildren arrive at class. It controls computer networks, navigation tools and much, much more.

Governments around the world aren't just providing the time as an altruistic service to citizens, Prescod-Weinstein argues. It's about keeping society organized and efficient. It's about increasing economic productivity.

And this is why people feel so tense about the time — it's actually a technology being thrust upon them. "Capitalism sucks, and I think a lot of people's relationship to why time is not cool, is structured by the resource pressures that we feel," she says.

Wibbly wobbly timey wimey

True time is actually much more flexible than most people realize, Prescod-Weinstein says. According to Einstein's general theory of relativity, space and time are tied together, and space-time can bend and curve.

"The way to think about it is that that curvature is stretching out time," she says.

As time stretches, it slows.

New Clock May End Time As We Know It

The best-known force that stretches time is gravity. The more gravity somebody experiences, the slower time passes for them when compared to someone in a lower gravitational field.

The effect is minuscule compared to a human lifespan, but it is real and measurable. Boulder, Colo. is a mile above sea level. That means the gravitational field is slightly weaker, and time ticks by a little faster.

But modern technology can't deal with flowy time like this. As a result, the timekeepers at Boulder and elsewhere make corrections to ensure these different flows of time look like they're ticking in lockstep. The same goes for satellites farther from Earth, like those that make up the Global Positioning System. The system works by measuring the time difference between several satellites carrying clocks in space to the time that people measure on the ground.

Without correctly calibrating for the difference of the ticking clocks inside the GPS satellites, the system wouldn't be as accurate, Prescod-Weinstein says. "Any kind of system that uses GPS requires general relativity," she says. "You have to understand what time flow of the satellite is like and how that is different from time flow from Earth."

Far out time

Even farther from Earth, time gets really freaky.

In places where gravity is very strong, time as we understand it can break down completely. At the edge of black holes, for example, the powerful gravitational pull slows time dramatically, says Prescod-Weinstein. And upon crossing the black hole's point of no return, known as its event-horizon, she says space and time flip.

Far from the Earth, time gets extremely weird. Black holes can cause it to stretch and even break down entirely. NASA/JPL-Caltech hide caption

Far from the Earth, time gets extremely weird. Black holes can cause it to stretch and even break down entirely.

"You end up in a region where space now has an arrow, and it's one direction ... and time doesn't have an arrow like it did before," she says. "There's really no sense of time."

At the edge of the observable Universe, there's something else happening, according to Katie Mack , an astrophysicist at the Perimeter Institute for Theoretical Physics in Canada. The Universe is expanding from the Big Bang, and that expansion is stretching time too.

"When you see things in the really distant Universe, because of the expansion of the Universe, it takes longer for things to happen," she says. The effect is known as cosmological time dilation and it's far more powerful than the tiny time changes seen near Earth.

Compare for example two identical stars that explode: One nearby, and one far away. "If we see a star exploding, and that star takes about 10 days to go from the brightest part of the explosion to dim again; if we look at it in the very distant Universe it might take 20-30 days," Mack says.

The far away star isn't exploding more slowly, time is ticking more slowly, at least from our perspective.

Mack, who's written a book called The End of Everything (Astrophysically Speaking) , says that many billions of years into the future, time could get stranger still. The Universe is expanding, and because of entropy, energy and matter are becoming more and more evenly spread out across the ever-growing void. In its final state, the Universe may end up as an inert cloud of energy and matter, where everything is evenly distributed.

In this gray nothingness, "there is no future, there's no arrow of time anymore," she says. At that point time has no real meaning, she says. "It has no direction."

Subatomic madness

So time, as we understand it, has some really big problems, but it also has some really tiny ones, too. In fact, some scientists who study the microscopic interactions of fundamental particles are questioning the idea of time itself.

"Space-time is doomed," says Nima Arkani-Hamed , a physicist at the Institute for Advanced Study in Princeton, N.J.

Arkani-Hamed has reached this conclusion over the past decade, as he's tried to gain a better understanding of what's happening inside the world's most powerful atom-smashers. These giant devices use huge amounts of energy to collide together subatomic particles. Calculating each collision as it happens should, according to quantum mechanics, require hundreds of pages of complex mathematics. Yet inexplicably, a much simpler equation can work just as well.

Some theorists believe that collisions within the world's most powerful particle accelerators can only be explained by something other than space-time. Max Brice/CERN hide caption

Some theorists believe that collisions within the world's most powerful particle accelerators can only be explained by something other than space-time.

Arkani-Hamed believes the concept of time itself may be behind the discrepancy. In physics equations, time is used to keep track of the sequence of things as events unfold. But he has come to believe that organizing particle collisions according to "when" unnecessarily complicates the mathematics. Instead, he's experimenting with abstract geometric shapes that can describe events without using time.

Arkani-Hamed says that these shapes can't yet replace the idea of time, but he believes that at some point time itself will be supplanted by some other theory of what makes the Universe tick.

"It's unlikely to survive in the fundamental principles of an even deeper understanding of physics," he says.

Ticktock, ticktock

The scientists back at NIST are well aware of all the problems with time.

"We are really focused on measuring time, but ... it's pretty hard to really define it in a satisfactory way," says John Kitching , who heads the atomic devices and instrumentation group at NIST.

Kitching takes me to a lab where his team is developing their latest marvel — an atomic clock that can be mass-produced and put on a microchip.

"The vision for this type of clock is to essentially enable atomic timing to be used everywhere," he says. "Imagine — an atomic clock in every computer and in every cellphone."

Just as humans keep improving their maps, they must improve their clocks, Kitching says. Better clocks can make GPS more accurate, computer networks faster, and enable new technologies in everything from stock trading to astronomy.

And that's important because in the end, Kitching says, he actually agrees with Chanda Prescod-Weinstein, the theoretical physicist who believes that time as we experience it is a technology rather than a fundamental part of the Universe. He too sees time is a human affair — a result of neurons firing and memories formed and books written.

"It's my belief that the forward passage of time is mostly a human construct," he says. "From a physics sense, it doesn't have that much value."

And yet, he adds, the NIST lab's relentless counting of seconds, minutes, hours and days is being used for everything from navigation to power production. For humans, there is enormous social value in trying to tame nature's unruly sense of time.

"We need to arrange to meet at a certain place at a certain time, " he says. "If we don't have those two pieces of information, then we're going to miss each other."

This story is part of our periodic science series "Finding Time — a journey through the fourth dimension to learn what makes us tick."

• standard time
• general relativity
• theory of relativity
• theoretical physics
• alarm clock
• quantum physics
• Standard Model of Particle Physics
• Arrow Of Time

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Open Access

Peer-reviewed

Research Article

Conserved NIMA kinases regulate multiple steps of endocytic trafficking

Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing – original draft

Affiliation Department of Molecular Biology, College of Agriculture Life Sciences, and Natural Resources, University of Wyoming, Laramie, Wyoming, United States of America

Roles Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization

Affiliations Department of Biochemistry & Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, United States of America, The Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, United States of America

Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

Roles Data curation, Formal analysis, Methodology, Validation, Visualization

Roles Investigation, Methodology, Validation

Affiliation Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey, United States of America

Roles Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing

Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing

Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

* E-mail: [email protected]

• Braveen B. Joseph, 
• Naava Naslavsky, 
• Shaonil Binti, 
• Sylvia Conquest, 
• Lexi Robison, 
• Ge Bai, 
• Rafael O. Homer, 
• Barth D. Grant, 
• Steve Caplan, 
• David S. Fay

• Published: April 26, 2023
• https://doi.org/10.1371/journal.pgen.1010741
• Peer Review
• Reader Comments

Human NIMA-related kinases have primarily been studied for their roles in cell cycle progression (NEK1/2/6/7/9), checkpoint–DNA-damage control (NEK1/2/4/5/10/11), and ciliogenesis (NEK1/4/8). We previously showed that Caenorhabditis elegans NEKL-2 (NEK8/9 homolog) and NEKL-3 (NEK6/7 homolog) regulate apical clathrin-mediated endocytosis (CME) in the worm epidermis and are essential for molting. Here we show that NEKL-2 and NEKL-3 also have distinct roles in controlling endosome function and morphology. Specifically, loss of NEKL-2 led to enlarged early endosomes with long tubular extensions but showed minimal effects on other compartments. In contrast, NEKL-3 depletion caused pronounced defects in early, late, and recycling endosomes. Consistently, NEKL-2 was strongly localized to early endosomes, whereas NEKL-3 was localized to multiple endosomal compartments. Loss of NEKLs also led to variable defects in the recycling of two resident cargoes of the trans-Golgi network (TGN), MIG-14/Wntless and TGN-38/TGN38, which were missorted to lysosomes after NEKL depletion. In addition, defects were observed in the uptake of clathrin-dependent (SMA-6/Type I BMP receptor) and independent cargoes (DAF-4/Type II BMP receptor) from the basolateral surface of epidermal cells after NEKL-2 or NEKL-3 depletion. Complementary studies in human cell lines further showed that siRNA knockdown of the NEKL-3 orthologs NEK6 and NEK7 led to missorting of the mannose 6-phosphate receptor from endosomes. Moreover, in multiple human cell types, depletion of NEK6 or NEK7 disrupted both early and recycling endosomal compartments, including the presence of excess tubulation within recycling endosomes, a defect also observed after NEKL-3 depletion in worms. Thus, NIMA family kinases carry out multiple functions during endocytosis in both worms and humans, consistent with our previous observation that human NEKL-3 orthologs can rescue molting and trafficking defects in C . elegans nekl-3 mutants. Our findings suggest that trafficking defects could underlie some of the proposed roles for NEK kinases in human disease.

Author summary

Intracellular trafficking is an evolutionary conserved process whereby cargoes, which include proteins, lipids, and other macromolecules, are internalized by cells, packaged into vesicles, and distributed to their proper places within the cell. This study demonstrated that two conserved NIMA-related kinases, NEKL-2 and NEKL-3, are required for the transport of multiple cargoes in the epidermis of C . elegans . NEKL-2 and NEKL-3 function at organelles, called endosomes, to regulate their morphology and control the sorting of cargoes between different intracellular compartments. In the absence of NEKL activities, various cargoes, including components of the BMP and Wnt signaling pathways, were misregulated. Our studies are further supported by results showing that the human counterparts of NEKL-3, NEK6 and NEK7, were also required for maintaining endosome morphologies and for the proper sorting of cargo in human cells. Notably, NIMA-kinases are well studied for their roles in cell cycle regulation, and overexpression of these kinases is linked to cancer formation and poor prognosis. Our study suggests their role in cancer progression could be partly due to the abnormal intracellular trafficking of conserved signaling components with known roles in cancer formation.

Citation: Joseph BB, Naslavsky N, Binti S, Conquest S, Robison L, Bai G, et al. (2023) Conserved NIMA kinases regulate multiple steps of endocytic trafficking. PLoS Genet 19(4): e1010741. https://doi.org/10.1371/journal.pgen.1010741

Editor: Andrew D. Chisholm, University of California San Diego, UNITED STATES

Received: December 22, 2022; Accepted: April 11, 2023; Published: April 26, 2023

Copyright: © 2023 Joseph et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Raw data contained in S1 File .

Funding: This project was supported by NIH R35 GM136236 to DSF, BBJ, SB, SC, LR and ROH (University of Wyoming), NIH R01 GM135326 to BDG, GB (Rutgers, State University of New Jersey), NIH R35 GM144102 to SC and NN (University of Nebraska Medical Center), and by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (P20GM103432) to DF, BBJ and SB (University of Wyoming). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The NIMA ( n ever i n m itosis gene a ) family of serine/threonine kinases was first identified in a genetic screen for mutants that fail to enter mitosis in the filamentous fungus Aspergillus nidulans [ 1 – 4 ]. The human genome encodes 11 NIMA-related kinases or NEKs (NEK1–NEK11). Consistent with the pro-mitotic function of NIMA in A . nidulans , several mammalian NEKs regulate cell division processes including spindle assembly and centrosome separation (NEK1, NEK2, NEK5–NEK7, and NEK9) and cytokinesis (NEK1, NEK2, NEK6, and NEK7) [ 5 – 16 ]. Correspondingly, misregulation of NEKs can cause aberrant cell proliferation, and overexpression of several NEKs, including NEK6, NEK7, and NEK9, is associated with multiple cancers and cardiac hypertrophy [ 8 , 17 – 20 ]. More recently, attention on NEK7 has focused on its role as an activator of NLRP3-inflammasomes, which are critical for the immune response against microbial pathogens [ 21 , 22 ]. Other NEK family members (e.g., NEK1, NEK4, and NEK8) were identified as causal factors in mouse models of polycystic kidney disease or have been linked to human ciliopathies [ 23 – 30 ]. Several NEK family members, including NEK8 and NEK9, span multiple functional categories, as NEK8 has been linked to cancer and cell cycle control [ 18 , 26 , 31 , 32 ] and NEK9 has been implicated in ciliogenesis and ciliopathies [ 25 , 33 ]. These findings suggest that individual NEKs have tissue- or cell type–specific functions, which may affect different developmental and disease-associated processes.

C . elegans has four NIMA-related kinases, NEKL-1, NEKL-2, NEKL-3, and NEKL-4. NEKL-2, a homolog of human NEK8 and NEK9, contains a highly conserved kinase domain and a short disordered region at the C terminus [ 34 ]. NEKL-2 is expressed in a punctate pattern in the large epidermal syncytium, hyp7, where it colocalizes with the conserved ankyrin repeat proteins MLT-2 (ANKS6 homolog) and MLT-4 (INVS homolog). MLT-2 and MLT-4 are required for the proper localization of NEKL-2, and their physical association is conserved in vertebrates [ 34 – 36 ]. NEKL-3, a close homolog of human NEK6 and NEK7, contains a kinase domain only and is also expressed in a punctate pattern within hyp7. NEKL-3 forms a complex with a third conserved ankyrin repeat protein, MLT-3/ANKS3, which is required for the proper localization of NEKL-3 in C . elegans and NEK7 in vertebrates [ 37 ]. We have shown that NEKL-2 and NEKL-3, along with their MLT binding partners, are required for the completion of molting in C . elegans , as a strong loss of function in any of these proteins leads to molting defects in early larval stages [ 34 , 38 – 40 ]. Moreover, mutation of the kinase domain of nekl-2 or nekl-3 leads to uniform molting defects indicating that the catalytic activity of the NEKLs is essential for the promotion of molting [ 34 , 39 ].

Molting is an essential feature for C . elegans to develop through its four larval stages and to reach adulthood. During molting, the old cuticle, an apical extracellular matrix (aECM) made up of proteins and lipids, detaches from the underlying epidermis and is broken down as a new cuticle is synthesized underneath. This process of aECM remodeling requires the trafficking of macromolecules required for the synthesis, degradation, and recycling of cuticle components [ 41 , 42 ]. We previously reported that NEKL-2 and NEKL-3 regulate clathrin-mediated endocytosis (CME) at the apical membrane of hyp7. Our findings indicated that loss of NEKL-2 or NEKL-3 leads to a defect in the uncoating of apical clathrin-coated vesicles, thereby preventing the flow of apical cargo through the endocytic pathway. Consistent with this, an apically expressed low-density lipoprotein–like receptor, LRP-1/Megalin, was observed to be trapped at or near the apical membrane in NEKL-depleted animals [ 34 , 43 ]. LRP-1 internalization by hyp7 is essential for molting, as it is required for the uptake of cholesterol, a substrate required for hormonal signaling pathways that coordinate the molting cycle [ 41 , 44 , 45 ].

The involvement of NEKLs in CME is also strongly supported by our genetic screens, which identified subunits of the clathrin adapter protein complex, AP2, as strong suppressors of both molting and trafficking defects in nekl-2 and nekl-3 mutants. Likewise, loss of FCHO-1, an allosteric activator of AP2, alleviates molting and trafficking defects in nekl mutants [ 43 ]. Our collective data indicate that NEKL-2 and NEKL-3 directly or indirectly promote clathrin uncoating and suggest that AP2 activity can also affect the uncoating process. However, a possible role for NEKLs in other aspects of intracellular trafficking was not explored.

In this study, we further investigated the role of NEKLs at different membranes and in different compartments within the endocytic network. Importantly, we found that NEKL-2 and NEKL-3 have distinct functions within different endosome types and are required for basolateral trafficking and cargo recycling. Importantly, we extended our studies to human cell lines and demonstrated that the human NEKL-3 orthologs, NEK6 and NEK7, also appear to function at multiple points within the endocytic pathway in mammals. These studies provide new insights into the biological roles of NEK/NEKL family members, which may have relevance to their roles in human development and disease.

NEKL-2 and NEKL-3 colocalize with endosomal markers

Previously, we reported that NEKL-2 and NEKL-3 are specifically expressed and required within the large epidermal syncytium of C . elegans , hyp7 [ 34 , 38 ]. Moreover, both NEKL-2 and NEKL-3 are cytoplasmic and are localized to variably sized actin-rich puncta or aggregates, which are reminiscent of endosomal compartments [ 34 , 38 ]. To determine the subcellular compartments in which NEKLs may function, we carried out colocalization experiments using endogenously tagged NEKL strains, together with reporters for early (P rab-5 ::GFP::RAB-5) and late (P hyp7 ::GFP::RAB-7) endosomes [ 46 – 50 ]. In the case of NEKL-3, we observed robust colocalization of NEKL-3::mKate with GFP::RAB-5 and GFP::RAB-7 (~50% Manders’ overlap with each marker), such that a majority of detectable NEKL-3 may be associated with early-to-late endosomes ( Fig 1A–1F, 1A’–1F’ and 1M ). In contrast, whereas ~40% of NEKL-2::mKate colocalized with RAB-5::GFP, only ~10% colocalized with RAB-7::GFP, suggesting that NEKL-2 may play a greater role in early steps of endocytic trafficking ( Fig 1G–1L, 1G’–1L’ and 1M ). We also note that we previously reported the association of NEKL-2–MLT-2–MLT-4 with actin-rich puncta that form along the boundary of hyp7 and the seam cells, which do not appear to be endosomal and may represent adhesion junctions [ 38 , 39 ]. In addition, we have observed some diffuse reticulated signal from fluorescently tagged NEKL-3 and NEKL-2 in the cytoplasm of hyp7 ( Fig 1 ), which could correspond in part to the endoplasmic reticulum [ 39 ].

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Colocalization assays were carried out in adult worms expressing either NEKL-2::mKate or NEKL-3::mKate with endosomal markers P rab-5 ::GFP::RAB-5 or P hyp7 ::GFP::RAB-7. (A–C, A’–C’, G–I, and G’–I’) Representative confocal images of adult worms expressing either NEKL-3::mKate (A–C and A’–C’) or NEKL-2::mKate (G–I and G’–I’), along with the early endosomal marker P rab-5 ::GFP::RAB-5. (D–F, D’–F’, J–L, and J’–L’) Representative confocal images of adult worms expressing either NEKL-3::mKate (D–F and D’–F’) or NEKL-2::mKate (J–L and J’–L’) and late endosomal marker P hyp7 ::GFP::RAB-7. Yellow arrowheads indicate overlap. Scale bar in A = 10 μm for A–L. Scale bar in A’ = 1 μm for A’–L’. (M) Manders’ coefficient was calculated for all the worms with the indicated backgrounds and plotted to determine the fraction of overlap between NEKL-2– or NEKL-3–positive pixels and the indicated markers. Error bars represent the 95% confidence intervals. p-Values were obtained by comparing means using an unpaired t-test: ****p < 0.0001, ***p < 0.001, *p < 0.05; ns, not significant (p > 0.05). Raw data are available in S1 File .

https://doi.org/10.1371/journal.pgen.1010741.g001

The differences observed between NEKL-2 and NEKL-3 localization are consistent with our previous report that NEKL-2 and NEKL-3, along with their MLT co-partners, exhibit distinct localization patterns in hyp7 [ 39 ]. Consistent with this, we find that the Manders’ overlap between fluorescently tagged NEKL-2 and NEKL-3 is <20% ( S1 Fig ), suggesting that the cellular functions of these kinases are distinct, despite having similar terminal molting-defective phenotypes. Notably, although a loss of NEKL-2 and NEKL-3 lead to defects in clathrin uncoating [ 43 ], neither showed strong colocalization with a marker for the AP2 clathrin-adapter protein complex, APA-2::mScarlet ( S2 Fig ). This suggests that the previously described impact of NEKLs on clathrin uncoating may be indirect or that levels of NEKLs at clathrin-coated vesicles are below detection. Notably, both NEKLs colocalize with RAB-5, which plays a role in the uncoating of clathrin-coated vesicles [ 51 ].

NEKLs are required for normal endosome morphologies

Given the observed colocalization of NEKLs with endosomes, we next wanted to determine if the loss of NEKLs led to an observable defect in the morphologies of endosomal compartments. During the process of endocytosis, endosomes undergo maturation, which is accompanied by changes in shape, molecular composition, and function. In the case of early/sorting and recycling endosomes, tubular extensions extend and pinch off from larger vesicles, allowing proteins to be recycled to specific destinations such as the plasma membrane and trans-Golgi [ 52 – 55 ]. Disruptions to endosomal functions can lead to a wide variety of defects including alterations in cargo localization and abundance.

To avoid indirect consequences caused by molting defects, we assessed the roles of NEKLs on endosomal morphology using the auxin-inducible degron (AID) system, in which endogenously tagged NEKL-2::AID and NEKL-3::AID were depleted in day-1 adults after the addition of auxin [ 43 , 56 , 57 ]. Depletion of NEKL-2::AID led to an increase in the overall size of the early endosome compartment (based on total intensity and area measurements; P rab-5 ::GFP::RAB-5) and, most strikingly, to an increase in the extent of tubulation ( Fig 2A, 2B and 2D–2F ). This phenotype suggests a requirement for NEKL-2 in the process of vesicle fission at early endosomes. In contrast, loss of NEKL-2 had more modest effects on the late endosome compartment (P hyp7 ::GFP::RAB-7), consistent with the observed strong colocalization of NEKL-2 to early, but not late, endosomes ( Fig 2G, 2H and 2J–2L ). Specifically, although the mean number of vesicles was decreased in NEKL-2–depleted worms as compared with wild type, the morphology of late endosomes was not strongly affected, and the observed changes could be due in part to gross alterations in the early endosome caused by NEKL-2 depletion.

(A–C, G–I, M–O) Confocal imaging was used to examine the effects of NEKL-2 (B, H, N) and NEKL-3 (C, I, O) loss relative to wild type (WT; A, G, M) in hyp7 of day-2 adult worms after auxin treatment. Representative images are shown. (A–C) Imaged worms expressed P rab-5 ::GFP::RAB-5 in the indicated backgrounds. (D–F) The mean intensity (D), vesicle area (E), and the roundness of the puncta (F) were plotted for worms expressing P rab-5 ::GFP::RAB-5. (G–I) Imaged worms expressed P hyp7 ::GFP::RAB-7 in the indicated backgrounds. (J–L) The mean intensity (J), vesicle area (K), and the number of vesicles (L) were plotted for worms expressing P hyp7 ::GFP::RAB-7. (M–O) Imaged worms expressed mScarlet::RAB-11, a marker for recycling endosomes, in the indicated backgrounds. (P–R) The mean intensity (P), vesicle area (Q), and perimeter of the vesicles (R) were plotted for individual worms expressing mScarlet::RAB-11. Area is in square pixels; perimeter is the number of pixels in the boundary of the object. Error bars represent the 95% confidence intervals. p-Values were obtained by comparing means using an unpaired t-test: ****p < 0.0001, ***p < 0.001, *p < 0.05; ns, not significant (p > 0.05). Raw data are available in S1 File .

https://doi.org/10.1371/journal.pgen.1010741.g002

In the case of NEKL-3::AID, depletion led to strong defects at both early and late endosomes, consistent with the localization of NEKL-3 to both compartments. In the case of early endosomes, however, the effects of NEKL-3 loss differed markedly from those after loss of NEKL-2 ( Fig 2B–2F ). Rather than increased tubulation, loss of NEKL-3 led to increased variability in the size and shape of early endosomes accompanied by an increase in total signal intensity. Thus, while NEKL-2 and NEKL-3 both act at early endosomes, their roles in controlling early endosome morphology appear to be distinct. Depletion of NEKL-3 also led to a strong overall decrease in the abundance of late endosomes, which was associated with a decrease in total intensity, vesicle size, and changes in vesicle morphology ( Fig 2G and 2I–2L ). These defects may be due to a combination of the roles for NEKL-3 in directly regulating late endosomes and the potential secondary effects associated with its effects on early endosomes. We also note that even in the absence of auxin, NEKL-3::AID worms are smaller than wild-type worms and show some arrest due to molting defects, which may be attributable to a partial LOF caused by the AID tag [ 43 ].

We also examined the effects of NEKL loss on recycling endosomes, which were marked with endogenously (CRISPR) tagged RAB-11. Whereas NEKL-2::AID depletion had little or no effect on mScarlet::RAB-11 localization, we observed a strong increase in tubulation after loss of NEKL-3:AID, which was accompanied by an increase in total intensity and vesicle size ( Fig 2M–2R ). For technical reasons, we were unable to carry out colocalization studies between the NEKLs and either mScarlet::RAB-11 or GFP::RAB-11. Our observations, however, suggest that NEKL-3 may also function at recycling endosomes, possibly to control vesicle fission. In contrast to endosomal compartments, depletion of NEKL-2::AID and NEKL-3::AID had little or no effect on the morphology of the Golgi compartment, based on a P hyp7 ::AMAN-2::mNeonGreen marker ( S3 Fig ). Nevertheless, the total number of AMAN-2–positive vesicles was variably reduced in these backgrounds, with strongest effects shown for NEKL-3::AID ( S3A–S3C Fig ), which could be caused by reduced endosome-to-Golgi recycling. Consistent with an indirect effect, a multi-copy NEKL-3::mCherry reporter [ 34 ] showed very low levels of colocalization with AMAN-2:: mNeonGreen ( S3D Fig ). Collectively, our studies demonstrate a role for NEKL-2 and NEKL-3 in regulating endosomal morphologies and further suggest direct functions within endosomal compartments.

NEKLs are required for basolateral cargo uptake by hyp7

We previously showed that NEKLs are required for CME at the apical membrane of hyp7 ( Fig 1A and 1B ) [ 44 , 45 , 58 ]. Specifically, depletion of NEKL-2 or NEKL-3 led to the accumulation of both clathrin and LRP-1, a low-density lipoprotein–like receptor, at the apical hyp7 surface [ 34 , 43 , 59 , 60 ]. Consistent with this, C . elegans LRP-1 endocytosis is dependent on clathrin and the clathrin adapter protein complex, AP2, analogous to findings in mammals [ 34 , 43 , 60 – 64 ]. To determine whether NEKL kinases are also required for trafficking at basolateral membranes of hyp7, we examined the internalization of two basolaterally localized BMP-family receptors. SMA-6 (Type I BMP receptor) and DAF-4 (Type II BMP receptor) form a heteromeric complex that binds to DBL-1 (BMP ligand), leading to the regulation of genes controlling C . elegans body size and male mating structures [ 65 ]. The internalization of SMA-6 depends on CME, as loss of AP2 function leads to the accumulation of SMA-6 at the basolateral membrane of intestinal cells [ 66 ]. In contrast, DAF-4 internalization does not require AP2, suggesting that it is endocytosed through a clathrin-independent mechanism [ 66 ].

To investigate possible roles for NEKLs in basolateral endocytosis, we expressed SMA-6::GFP and DAF-4::GFP using a hyp7-specific promoter and examined expression at the membrane of hyp7 adjacent to the seam cell (referred to hereafter as the lateral membrane) and at the membrane that forms the interior boundary of hyp7 (referred to hereafter as the basal membrane; Fig 3A and 3B ). Notably, the mean intensity of P hyp7 ::SMA-6::GFP was increased by ~1.3-fold or ~2.3-fold when NEKL-2 or NEKL-3 were depleted, respectively ( Fig 3C–3E and 3H ). Moreover, a strong increase (~4.8-fold) in the total level of P hyp7 ::DAF-4::GFP was observed after NEKL-3::AID depletion ( Fig 3F, 3G and 3I ), suggesting that NEKL-3 may also be required for non-clathrin–dependent endocytosis or at the downstream junction of these pathways. In contrast, P hyp7 ::DAF-4::GFP was not detectably affected by depletion of NEKL-2 ( Fig 3H and 3J ), suggesting that NEKL-2 may not be required for non-CME or may have a more limited role in endocytosis at the basolateral membrane relative to NEKL-3. Accumulation of these markers after NEKL-3 depletion was most evident at or near the basal hyp7 membrane, indicating a defect in plasma membrane uptake and/or processing through the early steps of endocytosis. In addition, some accumulation at or near the basolateral membrane with seam cells was also observed ( S4 Fig ). Together these results show that NEKL-2 and NEKL-3 play variable roles in the endocytosis of basolateral BMP receptors, although much stronger effects were observed for NEKL-3 in this process.

(A) Cross-sectional view of an adult worm depicting the position of the hyp7 syncytium (blue grey) and seam cell syncytium (red). Specific membrane domains of hyp7 are indicated: apical, facing externally directly underneath the cuticle (brown); basal, facing internally (purple); lateral, facing the sides and bottom of the seam cell (green). Dashed lines indicate the medial imaging plane used to acquire the images. (B) Top-down view of the long axis of the worm’s body indicating the positions of the hyp7 and seam cell syncytium along with indicated membranes as in A. (C–E) Representative confocal images of P hyp-7 ::SMA-6::GFP expression in auxin-treated wild-type (C), nekl-2 :: aid (D), and nekl-3 :: aid (E) day-2 adults. (F, G) Representative confocal images of P hyp-7 ::DAF-4::GFP expression in auxin-treated wild-type (G), nekl-2 :: aid (H), and nekl-3 :: aid (I) day-2 adults. Green and purple arrowheads (D,E,G) indicate seam and basal membranes (where detectable), respectively. Note that it is often not possible to identify the precise lateral/basolateral boundaries of hyp7 in the SMA-6::GFP and DAF-4::GFP lines in wild type because of the curved nature of the lateral membrane and because of low levels of these cargos marking the membrane. Scale bar in C = 10 μm for C–E, F, and G. (F, J) Mean intensity values for P hyp-7 ::SMA-6::GFP (F) and P hyp-7 ::DAF-4::GFP (J) expression were plotted for individual adults. The two highest datapoints in nekl-3 :: aid ; DAF-4::GFP (3J; 615 and 1233) were omitted for clarity of presentation. Error bars represent the 95% confidence intervals. Statistical significance was determined using a two-tailed, unpaired t-test: **p < 0.01, ****p < 0.0001. Raw data are available in S1 File .

https://doi.org/10.1371/journal.pgen.1010741.g003

NEKLs are required for cargo sorting

Membrane cargoes that are internalized by endocytosis are first delivered to sorting/early endosomes after which they may be recycled back to their original compartment(s) or routed to the lysosome for degradation. Previous studies have characterized the recycling routes for two conserved cargo proteins, MIG-14/Wntless and TGN-38/TGN38, both of which originate from the trans-Golgi [ 67 – 71 ]. MIG-14 is a transmembrane protein that binds to Wnt ligands in the Golgi and is responsible for delivering Wnts to the plasma membrane for secretion. Once at the plasma membrane, MIG-14 is endocytosed and recycled back to the Golgi through a retrograde pathway that requires the retromer complex [ 72 ]. In the absence of retromer function, MIG-14 is missorted to the lysosome and is degraded [ 67 , 68 ]. Likewise, TGN-38 is a trans-Golgi resident that is also recycled from the plasma membrane to the trans-Golgi by a retromer-associated retrograde pathway [ 67 , 73 , 74 ].

In wild-type adults, both P hyp7 ::MIG-14::GFP and P hyp7 ::TGN-38::GFP markers were observed in punctate structures of varying size throughout the cytoplasm, with some accumulation occurring at the lateral surface ( Fig 4A and 4E ). After depletion of NEKL-3::AID, P hyp7 ::MIG-14::GFP expression was decreased ~2.7-fold, whereas depletion of NEKL-2::AID resulted in at most a subtle decrease in total P hyp7 ::MIG-14::GFP levels (~1.2-fold; p = 0.09) ( Fig 4A–4D ). In the case of P hyp7 ::TGN-38::GFP, marker expression was decreased by ~2.8-fold after depletion of either NEKL-2::AID or NEKL-3::AID ( Fig 4E–4H ). Such findings further highlight differences between NEKL-2 and NEKL-3 with respect to their effects on specific cargoes and suggest that MIG-14::GFP and TGN-38::GFP may be missorted to the lysosomal degradative pathway after loss of NEKLs.

(A–C, E–G, and I–J) Representative confocal images of P hyp-7 ::MIG-14::GFP (A–C) and P hyp-7 ::TGN-38::GFP (E–G and I–J) expression within hyp7 in auxin-treated wild-type (A, E), NEKL-2::AID (B, F), NEKL-3::AID (C, G), NEKL-2::AID; cup-5 (I), and NEKL-3::AID; cup-5 (J) day-2 adults. Scale bar in A = 10 μm for A–C, E–G, I, and J. (D, H) Mean pixel intensity values of P hyp-7 ::MIG-14::GFP and P hyp-7 ::TGN-38::GFP expression for individual worms. Error bars represent the 95% confidence intervals. Statistical significance was determined using a two-tailed, unpaired t-test: ****p < 0.0001; ns, not significant (p > 0.05). Raw data are available in S1 File .

https://doi.org/10.1371/journal.pgen.1010741.g004

To test if NEKL depletion leads to the aberrant degradation of cargoes, we used CRISPR methods to introduce a partial loss-of-function mutation into cup-5/trpml1 , which encodes a conserved channel protein required for the maturation of lysosomes and whose loss leads to reduced lysosomal function [ 75 – 77 ]. Notably, reduction of CUP-5 activity led to an ~1.5-fold increase in the total expression of P hyp7 ::TGN-38::GFP in both NEKL-2::AID and NEKL-3::AID depleted strains ( Fig 4I–4K ). Moreover, accumulation of P hyp7 ::TGN-38::GFP was detected in moderate-to-large internal structures in these strains, which may correspond to defective lysosomes. Together these results indicate that, in addition to functioning in membrane cargo uptake, NEKL-2 and NEKL-3 are also required for the correct sorting and recycling of internalized cargo. We note that loss of cup-5 in an otherwise wild-type background also led to the accumulation of P hyp7 ::TGN-38::GFP, suggesting that some portion of TGN-38 is normally delivered to the lysosome in wild-type animals and that it’s degradation depends on lysosomal function ( S5 Fig ). TGN-38 accumulations, however, appeared larger in cup-5 single mutants relative to NEKL::AID; cup-5 strains (Figs 4 and S5 ). One possible explanation for this difference is that NEKL depletion may directly or indirectly affect lysosomal morphology or lysosome abundance.

Depletion of NEKL-3::AID but not NEKL-2::AID also led to a reduction in the levels of P hyp7 ::MIG-14::GFP at the hyp7 membrane surrounding the ALM and PLM neurons, which run longitudinally through hyp7 along the anterior-posterior axis ( S6 Fig ). Altogether our results indicate that loss of NEKLs leads to a range of defects affecting endosomal compartments as well as cargo internalization and sorting. Moreover, although nekl-2 and nekl-3 mutants appear superficially identical with respect to molting defects, they act in largely distinct subcellular compartments and lead to different subcellular phenotypes when depleted.

The mammalian homologs of NEKL-3, NEK6 and NEK7, are required for normal endocytic trafficking in human cells

Human NEK6 and NEK7 are both ~70% identical and ~85% similar to NEKL-3 and can rescue molting and trafficking-associated defects when expressed in C . elegans nekl-3 mutants [ 43 ]. Nevertheless, a systematic examination of potential roles for NEK6 and NEK7 in intracellular trafficking has not been undertaken. Using siRNA approaches, we were able to substantially reduce protein levels of NEK6 and NEK7 within 48 h of oligonucleotide transfection into cell lines ( Fig 5A ). As a first measure, we examined the effects of NEK6 and NEK7 knockdown on the cation-independent mannose 6-phosphate receptor (M6PR) in HeLa cells grown at steady state. M6PR is responsible for the delivery of mannose 6-phosphate–tagged lysosomal hydrolases from the trans-Golgi network (TGN), and thus cycles rapidly between the TGN and late endocytic organelles. Accordingly, M6PR is highly sensitive to disruptions in endocytic trafficking, which lead to alterations in its distribution.

(A) siRNA knockdown of NEK6 and NEK7 was validated by western blotting of HeLa cells that were mock-transfected or transfected with oligonucleotides specific for NEK6 (left panel) or NEK7 (right panel). GAPDH was used as the loading control (lower panels). (B–G) Mock-transfected cells (B and enlarged area in C), NEK6 siRNA–transfected cells (D and enlarged area in E), or NEK7 siRNA–transfected cells (F and enlarged area in G) were plated on coverslips and immunostained with antibodies against mannose 6-phosphate receptor (M6PR). (K, L) The mean intensity of M6PR immunostaining (K) and the mean area of M6PR distribution (L) of individual cells in mock-transfected, NEK6 siRNA–transfected, and NEK7 siRNA–transfected cells. (H–J) Saturated and zoomed micrographs demonstrating the distribution and intensity of mannose 6-phosphate receptor immunostaining in mock-transfected (H), NEK6 siRNA–transfected (I), and NEK7 siRNA–transfected cells (J). The dashed lines indicate individual cells. Scale bar in B = 10 μm for B, D, and F; scale bar in C = 10 μm for C, E, and G; scale bar in J = 10 μm for H–J. Statistical significance was determined using Student’s unpaired t-test. Raw data are available in S1 File .

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Relative to control cells, the mean fluorescence intensity of M6PR-containing endosomes showed a significant decrease (~3-fold) in both NEK6 and NEK7 knockdown cells ( Fig 5B–5G and 5K ). These findings suggest that, analogous to observations for MIG-14 and TGN-38 in C . elegans , M6PR may be aberrantly targeted for lysosomal degradation after loss of the NEKL-3 orthologs. In addition, the distribution of M6PR in NEK6/7-depleted cells was altered, with M6PR showing a 2- to 3-fold increase in the area of dispersal ( Fig 5H–5J and 5L ). Although this increased M6PR dispersal might reflect slightly larger cell sizes in the NEK-depleted cells, M6PR appears to be more homogeneously distributed in the knockdown cells, an effect that has been associated with defects in endosome-to-Golgi transport via the retromer complex [ 78 ]. These data imply that both NEK6 and NEK7 are involved in the regulation of endosome-to-Golgi retrograde trafficking.

Given that loss of NEKLs led to a major impact on endosomes, we next assessed the impact of NEK6 and NEK7 depletion on EEA1, a well-characterized early endosomal protein that binds to endosomal PI3P via its FYVE domain [ 79 – 81 ]. Upon siRNA-mediated depletion of NEK6 or NEK7 ( Fig 6A ) and immunostaining with antibodies against EEA1, endosomal size was increased in cells depleted of NEK6 or NEK7, with those lacking NEK7 displaying especially large ring-like structures decorated by EEA1 ( Fig 6B–6G and 6H ). In contrast, the number of EEA1 endosomes was not significantly altered upon depletion of either NEK6 or NEK7 ( Fig 6I ). As a further test, we performed NEK6/7 siRNA knockdown in glioblastoma cells and observed an increase in the size of EEA1-marked vesicles after depletion of NEK7 but not NEK6 ( S7 Fig ), suggesting cell type–specific requirements for NEK functions. Together, these data suggest a role for NEK6 and NEK7 at endosomes, potentially in the regulation of fusion and/or fission events.

(A) siRNA knockdown of NEK6 (left panel) and NEK7 (right panel) in HeLa cells was confirmed by western blotting (B–G) Mock-transfected cells (B and enlarged area in C), NEK6 siRNA–transfected cells (D and enlarged area in E), or NEK7 siRNA–transfected cells (F and enlarged area in G) were plated on coverslips and immunostained with antibodies against the early/sorting endosome marker protein EEA1. Yellow boxes in B, D, and F indicate the area of higher magnification shown in C, E, and G, respectively. (H) The mean size of EEA1-containing endosomes in mock-transfected, NEK6 siRNA–transfected, and NEK7 siRNA–transfected cells. (I) The number of EEA1-containing endosomes in mock-transfected, NEK6 siRNA–transfected, and NEK7 siRNA–transfected cells. Scale bar in B = 10 μm for B, D, and F; Scale bar in C = 5 μm for C, E, and G. Statistical significance was determined using Student’s unpaired t-test. Raw data are available in S1 File .

https://doi.org/10.1371/journal.pgen.1010741.g006

To further examine the role of NEK6 and NEK7 at endosomes, we analyzed the effect of their depletion on tubular recycling endosomes marked by the protein MICAL-L1 [ 82 – 85 ]. After depletion of NEK6 or NEK7 with siRNA oligonucleotides ( Fig 7A ), HeLa cells were fixed and immunostained with antibodies against endogenous MICAL-L1. Inhibition of NEK6 or NEK7 led to an increase in the MICAL-L1 tubular endosomal surface area relative to controls ( Fig 7B–7E ). Similar observations were observed for glioblastoma cells, with depletion of either NEK6 or NEK7 leading to increased tubulation ( S8 Fig ). Together, these data indicate roles for NEK6 and NEK7 in endosomal morphology and function in mammalian cells and support the possibility of their involvement in fusion and/or fission activities.

(A) siRNA knockdown of NEK6 (left panel) and NEK7 (right panel) was validated by western blotting of HeLa cells. (B–D) Mock-transfected cells (B), NEK6 siRNA–transfected cells (C), or NEK7 siRNA–transfected cells (D) were plated on coverslips and immunostained with antibodies against the tubular recycling endosome marker protein MICAL-L1. (E) The surface area of MICAL-L1–containing endosomes in mock-transfected, NEK6 siRNA–transfected, and NEK7 siRNA–transfected cells. Scale bar in D = 10 μm for B–D. Statistical significance was determined using Student’s unpaired t-test. Raw data are available in S1 File .

https://doi.org/10.1371/journal.pgen.1010741.g007

NIMA kinases are required for proper endosome morphology

In this study we have demonstrated that C . elegans NEKLs and human NEKs are important for maintaining the proper morphology of endosomal compartments. Loss of NEKL-2 led to enlarged early endosomal compartments with noticeably longer tubular extensions. These changes could indicate a failure of early endosomes to undergo fission, an expansion of early endosomes caused by abnormal cargo retention, or both. Notably, the effect of NEKL-3 loss on early endosomes was distinct from that of NEKL-2, as no increase in tubular extensions was observed. The shape of early endosomes, however, became irregular, and the mean intensity of the early endosomal marker GFP::RAB-5 was increased. This suggests that NEKL-2 and NEKL-3 have distinct roles in regulating early endosome morphology. Notably, siRNA-mediated knockdown of human NEK6 or NEK7 also led to an increase in the size of EEA-1–positive early endosomes in HeLa cells. However, whereas knockdown of NEK7 led to an increase in the size of early endosomes in glioblastoma cells, inhibition of NEK6 did not show this effect, suggesting that NEK6 and NEK7 may regulate early endosomal morphology in a cell type–specific manner.

Consistent with their distinct localization patterns, depletion of NEKL-3 had much stronger effects on late endosomal compartments than did loss of NEKL-2. We note, however, that effects on late endosomes could be due in part to perturbations of early endosomes caused by loss of NEKL-2 and NEKL-3. For technical reasons, we could not determine the expression of NEKLs in recycling endosomes. However, an increase in membrane tubulation was observed with recycling endosomes in hyp7 after loss of NEKL-3 but not NEKL-2. Collectively, these results indicate that NEKL-2 and NEKL-3 have important but distinct roles in regulating endosomal morphology.

NIMA kinases may broadly affect cargo uptake and recycling

We examined two Golgi residents, MIG-14 and TGN-38, to better understand how NEKLs may influence the endocytic recycling process. We observed a dramatic reduction in the MIG-14 and TGN-38 signal after depletion of NEKL-3. In contrast, loss of NEKL-2 led to a strong reduction in TGN-38, but only minimal effects on MIG-14. These findings suggest that specific cargoes are variably missorted to the lysosomal degradative pathway when NEKL-2 or NEKL-3 is depleted, as was previously reported for mutants affecting actin assembly and the sorting nexin, SNX3 [ 67 , 68 ]. Consistent with this, expression of TGN-38 was rescued when lysosomal function was inhibited in NEKL::AID strains. Interestingly, defects consistent with abnormal recycling were also observed for M6PR when NEK6 and NEK7 were depleted in HeLa cells, consistent with M6PR being incorrectly targeted to lysosomes. Together these findings suggest that NIMA family kinases may have conserved functions in the sorting of cargoes from early endosomes.

Previously, we reported that NEKL-2 and NEKL-3 regulate endocytosis at the apical membrane of hyp7 [ 43 ]. In this study, we found that the internalization of a basolateral membrane cargo, SMA-6, was also affected in worms with NEKL-2 or NEKL-3 depletion. Our data also suggest that NEKL-3 may have a role in clathrin-independent endocytosis, as loss of NEKL-3 led to an accumulation of a putative clathrin-independent cargo, DAF-4, near the basolateral surface. One potential caveat to this interpretation is that in the presence of ligand, SMA-6 and DAF-4 would be predicted to form a complex [ 86 ], and thus SMA-6 or DAF-4 could potentially affect each other’s uptake or retention. For example, membrane retention of endogenous SMA-6 could impact the localization and uptake of DAF-4::GFP. However, SMA-6 is expressed at very low levels [ 87 , 88 ], and thus it seems unlikely that endogenous plasma-membrane associated SMA-6 could indirectly lead to the strong membrane/juxta-membrane retention of the more highly expressed DAF-4::GFP. Altogether, these findings point to roles for NEKL-3 in both CME and non-CME.

We note that neither SMA-6 nor DAF-4 appeared to be incorrectly targeted to lysosomes after NEKL depletion, which differs from what we observed for MIG-14 and TGN-38. We speculate that SMA-6 and DAF-4 may be trapped at or near the basolateral surface in NEKL-3::AID depleted worms, as our previous study failed to detect clathrin accumulation at basolateral membranes of hyp7 [ 43 ]. Moreover, we failed to detect GFP::RAB-5 accumulation at or near basolateral surface in NEKL-3::AID depleted worms, suggesting that the basolateral accumulation of SMA-6 and DAF-4 is not within a RAB-5–marked early endosomal compartment. This buildup SMA-6 and DAF-4 could be due plasma membrane accumulation, internalized juxta-membrane vesicles, or a combination of the two.

What are the core functions of NEKLs in intracellular trafficking?

In a previous study, we reported that NEKL-2 and NEKL-3 regulate CME at the apical membrane and that they promote the uncoating of clathrin from internalized vesicles [ 43 ]. Our current study, however, suggests that the primary sites for trafficking regulation by NEKL-2 and NEKL-3 may be endosomes, and we failed to observe strong colocalization of NEKL-2 or NEKL-3 to AP2–decorated clathrin coated pits and vesicles. Thus, the clathrin-uncoating defects we previously observed could in part be an indirect consequence of endosomal defects. In this context, it is worth noting that RAB-5, which colocalized with both NEKL-2 and NEKL-3, partially localizes to clathrin-coated pits and promotes clathrin and AP2 uncoating [ 51 , 89 ]. Moreover, human RAB5 has been detected at both early endosomes and clathrin-coated vesicles [ 46 , 90 ]. Thus, it remains possible that low levels of NEKL-2 and NEKL-3 may exert direct effects on clathrin uncoating, possibly in conjunction with RAB-5.

The effects we observed on multiple cargoes and compartments indicate that NEKL-2 and NEKL-3 may have widespread functions in intracellular trafficking. Likewise, effects on multiple compartments were observed after human NEK6 and NEK7 knockdowns. This could be due to NIMA kinases regulating distinct targets at different locations within the endocytic network. Alternatively, NIMAs may act on a smaller number of targets, which in turn have widespread functions in trafficking.

In this latter category, one compelling model is that NIMAs could regulate endosomal actin. Actin polymerization is deployed throughout endocytosis to provide mechanical forces needed to bend membranes, facilitate vesicle fission, and promote short-range vesicle transport [ 91 – 95 ]. For example, actin filaments are enriched at sorting endosomes and promote the fission of tubular extensions [ 52 , 96 ]. Actin assembly also plays a role in the sorting of macromolecules from early endosomes and their transport to the trans-Golgi [ 97 , 98 ]. Consistent with this idea, our previous studies showed that actin organization was strongly disrupted in nekl mutants and that nekl molting defects can be suppressed by loss of function in the conserved actin regulator CDC-42, along with one of its effectors, SID-3/ACK1/2 ( a ctivated C DC42 k inase) [ 38 ]. Moreover, activated (GTP-bound) CDC-42 was strongly upregulated in nekl mutants [ 38 ]. Interestingly, based on protein-interaction studies, CDC42 was reported to interact with both NEK6 and NEK7 and could therefore connect the NEKL–MLT network to actin polymerization and to their roles throughout the endocytic network. Future studies will seek to characterize the functional link between NIMA kinases and the actin cytoskeleton, along with their specific roles in regulating endocytosis.

Human NEKs in endocytosis and cancer

Although human homologs of NEKL-3 (NEK6, NEK7) and NEKL-2 (NEK9) have largely been studied for their roles in cell division, several studies have hinted at potential functions in endocytosis [ 99 – 104 ]. A high-throughput siRNA-based screen of mammalian protein kinases indicated that siRNA-mediated knockdown of NEK6 and NEK7 strongly disrupts CME in HeLa cells [ 99 ]. Moreover, genome-wide siRNA studies showed that knockdown of multiple NEK kinases, including NEK6, resulted in the abnormal uptake of endocytic cargoes [ 101 ]. A more recent study identified NEK6 as a potential downstream target of NRP-1, which is required for transferrin endocytosis by Trypanosoma brucei [ 105 ]. Moreover, proteomic studies of NEK6 and NEK7 identified interactions with several trafficking components including alpha and beta subunits of the AP2 adaptor complex (NEK7) [ 102 , 103 ]. Such reports are consistent with our current findings that NEK6 and NEK7 play roles in endocytic trafficking in human cells. Lastly, our ability to rescue both molting and trafficking defects through the expression of human NEKs in C . elegans nekl mutants suggests that these functions are conserved [ 43 ].

Finally, our studies provide evidence that NEKLs may affect two highly conserved signaling pathways, BMP and Wnt, which are misregulated in many human cancers [ 106 – 108 ]. Previous studies have described the role of endocytosis in cancers including how endocytosis adapts to the needs of cancer cells, a phenomenon known as “adaptive CME” [ 109 – 111 ]. For example, upregulation of clathrin light-chain (CLCb) leads to an increased rate of CME; this in turn alters the trafficking of oncogenic epidermal growth factor receptor (EGFR) and promotes cancer cell migration and metastasis [ 112 ]. Thus, it is possible that NIMA kinases, which are overexpressed in many tumors, could be driving cancer formation in part through their roles in trafficking as well as by their previously described functions in mitosis [ 19 , 113 – 117 ]. Future studies will elucidate how NIMA kinases control trafficking, which will aid in understanding their involvement in human disease.

Materials and methods

Strains and maintenance.

C . elegans strains were maintained according to standard protocols [ 118 ] and were propagated at 22°C unless stated otherwise. Strains used in this study are listed in S1 Table . Loss-of-function alleles of cup-5 ( fd395 and fd397 ) were generated by the CRISPR-Cas9 protocol described by Ghanta et al. [ 119 ]. Sequences for the sgRNA and repair template are given below. Capital letters represent the altered or inserted nucleotides.

sgRNA: 5′-aacgatgcgcttattatcat-3′

Repair Template: 5′-aacttaaatttttataaaaattacctgaaactcgatggatatttttgcaacGGTCTCaatgataataagcgcatcgttgaccacaatcatcacataccacaaattcag-3′

Reporter strain construction

Plasmids for C . elegans hyp7-specific expression used the promoter from semo-1/Y37A1B . 5 (P hyp-7 ) [ 120 ]. Vector details are available upon request. Cloning was performed using the Gateway system (Invitrogen) and modified versions of hygromycin-resistant and miniMos-enabled vector pCFJ1662 (gift from Erik Jorgensen, University of Utah; Addgene #51482). pDONR221 entry vectors containing coding regions for rab-5 , rab-7 , mig-14 , tgn-38 , aman-2 , daf-4 , and sma-6 were transferred into hyp7 destination vectors with the Gateway LR clonase II reaction to generate C-/N-terminal fusions. Single-copy integrations were obtained using miniMos technology [ 121 ].

Image acquisition

Confocal fluorescence images in Figs 1 – 4 and S1 and S3 and S5 were acquired using an Olympus IX83 inverted microscope with a Yokogawa spinning-disc confocal head (CSU-W1). z-Stack images were acquired using a 100×, 1.35 N.A. silicone oil objective. cellSense 3.1 software (Olympus Corporation) was used for image acquisition. Fluorescence images in S2 Fig were acquired using an Olympus IX81 inverted microscope with a Yokogawa spinning-disc confocal head (CSU-X1). MetaMorph 7.7 software was used for image acquisition. z-Stack images were acquired using a 100×, 1.40 N.A. oil objective.

Confocal images for Figs 5 – 7 and S6 and S7 were acquired using an Olympus IX83 inverted microscope with a Yokogawa spinning-disc confocal head (CSU-W1) or with a Zeiss LSM 800 microscope with a 63×, 1.40 N.A. objective with appropriate filters.

Image analysis

Mean intensity (measured in arbitrary units, a.u.), quantification of various shape parameters, and the colocalization analysis were performed using Fiji software [ 122 ]. To quantify mean intensity (Figs 2D, 2J, 2P , 3H, 3I , 4D, 4H, 4K and S3B ) for a z-plane of interest, rolling ball background subtraction was performed (radius = 50 pixels), and the polygon selection tool was used to choose the region of hyp7 in which the mean intensity was quantified.

To quantify shape parameters, a z-plane of interest was selected, and the minimum filter (radius = 10.0 pixels) was applied to the raw image. The filter-applied image was then subtracted from the raw image using the image calculator function. Images were then thresholded, and the Despeckle function was used to remove noise corresponding to 1 pixel in size. Finally, various shape parameters were quantified using the Shape Descriptor plugin.

For colocalization studies, the raw z-stack images were deconvoluted using the Wiener deconvolution algorithm (cellSense 3.1 software). The desired z-plane was extracted from both deconvoluted and raw z-stack images. Then, the Gaussian filter (radius = 10 pixels) was applied to the deconvoluted image, which was subtracted from the original deconvoluted image using the image calculator function. Next, these subtracted images were thresholded to obtain binary images to be used as masks. These binary masks were combined using the “AND” Boolean operation to the background-subtracted (rolling ball algorithm; radius = 50 pixels) raw images. Using the polygon tool, the region of hyp7 was selected in the combined images, and coloc2 plugin was used to calculate Manders’ coefficient.

The mean area and intensity of fluorescence in HeLa cells Fig 5K and 5L was obtained with Zeiss LSM Zen software, after outlining a region of interest. EEA1-positive endosome size ( Fig 6H ) was measured using Imaris software, and MICAL-L1 tubular endosome surface area ( Fig 7E ) was quantified using ImageJ software., or the number of MICAL-L1 tubular recycling endosomes were counted manually ( S8 Fig ), since number and area are generally correlated. Endosome size in the human cell studies was quantified using NIH ImageJ. Size parameters were set for 0 –infinity. Brightness parameters were selected to eliminate recognition of background by ImageJ’s particle counter while optimizing selection of true positive fluorescent pixels. Statistical analyses was performed in Prism by first testing the assumption of normal distribution with the D’Agostino and Pearson normality test. Statistical significance was then calculated with an unpaired two-tailed t-test.

Auxin treatment

Auxin (indole-3-acetic acid) was purchased from Alfa Aesar. A 100× stock auxin solution (0.4 M) was made by dissolving 0.7 g of auxin in 10 ml of 100% ethanol. A mixture of 25 μl of stock auxin solution and 225 μl of autoclaved deionized water was added to each plate containing day-1 adult worms.

Rabbit antibodies against NEK6 were obtained from Mybiosource (Cat# MBS94186), rabbit antibodies against NEK7 were obtained from Novus (Cat# 31110), mouse horseradish peroxidase (HRP)-conjugated antibodies against GAPDH were obtained from Proteintech (Cat# HRP-60004), rabbit antibodies against EEA1 were obtained from Cell Signaling (Cat# 3288), and mouse polyclonal antibodies against MICAL-L1 were obtained from Novus (Cat# H00085777-B01P).

Immunoblotting

HeLa cells were lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, and 0.5% deoxycholate. Proteins from the lysates were separated by SDS-PAGE, transferred to nitrocellulose filters, and immunoblotted with antibodies using standard methods.

siRNA knockdown

Cells were transfected with siRNA oligonucleotides from Sigma (600 nM NEK6 600 or 100 nM NEK7) using Dharmafect transfection reagent (Dharmacon) for 48 h, prior to validation of knockdown by immunoblotting using GAPDH as the loading control as described above.

All statistical tests were performed using software from Prism GraphPad.

Supporting information

S1 fig. colocalization between nekl-2 and nekl-3..

Colocalization assays were carried out in adult worms expressing both NEKL-2::mNeonGreen and NEKL-3::mKate. (A–C, A’–C’, D–F, and D’–F’) Representative images of the apical region of hyp7 in adult worms expressing NEKL-2::mNeonGreen and NEKL-3::mKate (A–C and A’–C’) as well as the medial plane of hyp7 (D–F and D’–F’). Here the medial plane is considered the plane ~1 μm below the apical surface. Scale bar in A = 10 μm for A–F. Scale bar in A’ = 1 μm for A’–F’. (G, H) Manders’ coefficient was calculated and plotted for individual worms in nekl-2 :: mNeonGreen ; nekl-3 :: mKate strains. The fraction of NEKL-2::mNeonGreen puncta overlapping with NEKL-3::mKate puncta in the apical and medial plane (G) and vice versa (H) are shown. Raw data are available in S1 File .

https://doi.org/10.1371/journal.pgen.1010741.s001

S2 Fig. Colocalization of NEKLs with clathrin-coated pits.

(A–F) Colocalization assays were performed on strains expressing either NEKL-2::mNeonGreen (A–C) or NEKL-3::mNeonGreen (D–F) with APA-2::mScarlet, the alpha subunit of the AP2 adaptor complex, which is present in clathrin-coated pits. Note that these images were collected on a different confocal microscope and thus appear somewhat different than images in the paper (see Materials and Methods ). Scale bar in A = 10 μm in A–F.

https://doi.org/10.1371/journal.pgen.1010741.s002

S3 Fig. Effects of NEKL depletion on Golgi compartments.

(A) Representative confocal images of P hyp7 ::AMAN-2::mNeonGreen expression in the indicated backgrounds. (B, C) Mean intensity of P hyp7 ::AMAN-2::mNeonGreen expression (B) and the mean number of P hyp7 ::AMAN-2::mNeonGreen-positive vesicles (C) for individual worms were plotted in the graphs. (D) Representative confocal images of P hyp7 ::AMAN-2::mNeonGreen co-expressed with a functional multi-copy NEKL-3::mCherry reporter. Error bars represent the 95% confidence intervals. p-Values were obtained by comparing means using an unpaired t-test: ****p < 0.0001, ***p < 0.001; ns, not significant (p > 0.05). Raw data are available in S1 File .

https://doi.org/10.1371/journal.pgen.1010741.s003

S4 Fig. Three-dimensional rendering of effects of NEKL deletion on basolateral cargoes.

(A–C) Three-dimensional plots showing the individual pixel intensity along the x and y planes for the fluorescent images presented in Fig 3C–3E . (D,E) Three-dimensional plots showing the pixel intensity value along the x and y planes for the fluorescent images presented in Fig 3F and 3G . We note that the basal boundary occurs in the x axes at approximately 40 μm and 10 μm whereas the seam cell boundary occurs between 20–30 μm.

https://doi.org/10.1371/journal.pgen.1010741.s004

S5 Fig. Effects of cup-5 loss of function on a membrane cargo, TGN-38.

Representative images of P hyp-7 ::TGN-38::GFP expression in auxin-treated day-2 adults in wildtype and cup-5 mutant background. Red arrows indicate accumulation of cargoes in vesicle-like structures. Mean intensity values of the P hyp-7 ::TGN-38::GFP expression for individual worms are plotted in the graph. Error bars represent the 95% confidence intervals. p-Values were obtained by comparing means using an unpaired t-test: ****p < 0.0001, ns, not significant (p > 0.05). Raw data are available in S1 File .

https://doi.org/10.1371/journal.pgen.1010741.s005

S6 Fig. Effects of NEKL depletion on cargo trafficking near the neural membrane.

Representative images of P hyp-7 ::MIG-14::GFP expression in auxin-treated wild-type, nekl-2 :: aid , and nekl-3 :: aid day-2 adults in the apical/medial plane. Red arrows show the presence of MIG-14::GFP expression in wild-type and nekl-2 :: aid adults at the hyp7 membrane surrounding the ALM neuron. The red arrowhead indicates the absence of MIG-14::GFP expression in nekl-3 :: aid worms. Scale bar = 10 μm in the three lower-magnification images. The yellow bracket along the ALM neuron in wild type indicates the region show in the higher magnification image (yellow outline). The bar graph shows the percentage of worms exhibiting P hyp-7 ::MIG-14::GFP expression surrounding the ALM/PLM neurons in the indicated backgrounds. Numbers of worms are indicated for each bar. Error bars represent the 95% confidence interval. p-Values were obtained using Fisher’s exact test: ****p < 0.0001. Raw data are available in S1 File .

https://doi.org/10.1371/journal.pgen.1010741.s006

S7 Fig. Effect of NEK6 or NEK7 depletion in early endosomal compartments in glioblastoma cells.

Mock-transfected cells, NEK6 siRNA-transfected cells, or NEK7 siRNA-transfected cells were plated on coverslips and immunostained with antibodies against the sorting endosome marker protein EEA1. Average sizes of early/sorting endosomes are shown for the three conditions, with significant differences observed between the mock-transfected cells and both siRNA treatments as well as between NEK6 siRNA and NEK7 siRNA-transfected cells. Yellow box in panels indicates region of enlarged inset. Scale bars = 10 μm.

https://doi.org/10.1371/journal.pgen.1010741.s007

S8 Fig. Effects of NEK6 or NEK7 depletion on recycling endosomes in glioblastoma cells.

Glioblastoma cells were mock transfected or were transfected with NEK6 siRNA or NEK7 siRNA. Transfected cells were then plated on coverslips and immunostained with antibodies against the tubular recycling endosome marker protein MICAL-L1. Average numbers of MICAL-L1 tubular endosomes per field were determined for the three conditions, with significant differences observed between the mock-transfected cells and both siRNA treatments. Yellow box in panels indicates region of enlarged inset. Scale bars = 10 μm.

https://doi.org/10.1371/journal.pgen.1010741.s008

S1 Table. List of all the strains used in this study.

https://doi.org/10.1371/journal.pgen.1010741.s009

S1 File. Compilation of raw data used in this study.

https://doi.org/10.1371/journal.pgen.1010741.s010

Acknowledgments

We thank Amy Fluet for editing this manuscript.

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• PMC10166553

Conserved NIMA kinases regulate multiple steps of endocytic trafficking

Braveen B. Joseph

1 Department of Molecular Biology, College of Agriculture Life Sciences, and Natural Resources, University of Wyoming, Laramie, Wyoming, United States of America

Naava Naslavsky

2 Department of Biochemistry & Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, United States of America

3 The Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, United States of America

Shaonil Binti

Sylvia conquest, lexi robison.

4 Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey, United States of America

Rafael O. Homer

Barth d. grant, steve caplan, david s. fay, associated data.

The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Raw data contained in S1 File .

Human NIMA-related kinases have primarily been studied for their roles in cell cycle progression (NEK1/2/6/7/9), checkpoint–DNA-damage control (NEK1/2/4/5/10/11), and ciliogenesis (NEK1/4/8). We previously showed that Caenorhabditis elegans NEKL-2 (NEK8/9 homolog) and NEKL-3 (NEK6/7 homolog) regulate apical clathrin-mediated endocytosis (CME) in the worm epidermis and are essential for molting. Here we show that NEKL-2 and NEKL-3 also have distinct roles in controlling endosome function and morphology. Specifically, loss of NEKL-2 led to enlarged early endosomes with long tubular extensions but showed minimal effects on other compartments. In contrast, NEKL-3 depletion caused pronounced defects in early, late, and recycling endosomes. Consistently, NEKL-2 was strongly localized to early endosomes, whereas NEKL-3 was localized to multiple endosomal compartments. Loss of NEKLs also led to variable defects in the recycling of two resident cargoes of the trans-Golgi network (TGN), MIG-14/Wntless and TGN-38/TGN38, which were missorted to lysosomes after NEKL depletion. In addition, defects were observed in the uptake of clathrin-dependent (SMA-6/Type I BMP receptor) and independent cargoes (DAF-4/Type II BMP receptor) from the basolateral surface of epidermal cells after NEKL-2 or NEKL-3 depletion. Complementary studies in human cell lines further showed that siRNA knockdown of the NEKL-3 orthologs NEK6 and NEK7 led to missorting of the mannose 6-phosphate receptor from endosomes. Moreover, in multiple human cell types, depletion of NEK6 or NEK7 disrupted both early and recycling endosomal compartments, including the presence of excess tubulation within recycling endosomes, a defect also observed after NEKL-3 depletion in worms. Thus, NIMA family kinases carry out multiple functions during endocytosis in both worms and humans, consistent with our previous observation that human NEKL-3 orthologs can rescue molting and trafficking defects in C . elegans nekl-3 mutants. Our findings suggest that trafficking defects could underlie some of the proposed roles for NEK kinases in human disease.

Author summary

Intracellular trafficking is an evolutionary conserved process whereby cargoes, which include proteins, lipids, and other macromolecules, are internalized by cells, packaged into vesicles, and distributed to their proper places within the cell. This study demonstrated that two conserved NIMA-related kinases, NEKL-2 and NEKL-3, are required for the transport of multiple cargoes in the epidermis of C . elegans . NEKL-2 and NEKL-3 function at organelles, called endosomes, to regulate their morphology and control the sorting of cargoes between different intracellular compartments. In the absence of NEKL activities, various cargoes, including components of the BMP and Wnt signaling pathways, were misregulated. Our studies are further supported by results showing that the human counterparts of NEKL-3, NEK6 and NEK7, were also required for maintaining endosome morphologies and for the proper sorting of cargo in human cells. Notably, NIMA-kinases are well studied for their roles in cell cycle regulation, and overexpression of these kinases is linked to cancer formation and poor prognosis. Our study suggests their role in cancer progression could be partly due to the abnormal intracellular trafficking of conserved signaling components with known roles in cancer formation.

Introduction

The NIMA ( n ever i n m itosis gene a ) family of serine/threonine kinases was first identified in a genetic screen for mutants that fail to enter mitosis in the filamentous fungus Aspergillus nidulans [ 1 – 4 ]. The human genome encodes 11 NIMA-related kinases or NEKs (NEK1–NEK11). Consistent with the pro-mitotic function of NIMA in A . nidulans , several mammalian NEKs regulate cell division processes including spindle assembly and centrosome separation (NEK1, NEK2, NEK5–NEK7, and NEK9) and cytokinesis (NEK1, NEK2, NEK6, and NEK7) [ 5 – 16 ]. Correspondingly, misregulation of NEKs can cause aberrant cell proliferation, and overexpression of several NEKs, including NEK6, NEK7, and NEK9, is associated with multiple cancers and cardiac hypertrophy [ 8 , 17 – 20 ]. More recently, attention on NEK7 has focused on its role as an activator of NLRP3-inflammasomes, which are critical for the immune response against microbial pathogens [ 21 , 22 ]. Other NEK family members (e.g., NEK1, NEK4, and NEK8) were identified as causal factors in mouse models of polycystic kidney disease or have been linked to human ciliopathies [ 23 – 30 ]. Several NEK family members, including NEK8 and NEK9, span multiple functional categories, as NEK8 has been linked to cancer and cell cycle control [ 18 , 26 , 31 , 32 ] and NEK9 has been implicated in ciliogenesis and ciliopathies [ 25 , 33 ]. These findings suggest that individual NEKs have tissue- or cell type–specific functions, which may affect different developmental and disease-associated processes.

C . elegans has four NIMA-related kinases, NEKL-1, NEKL-2, NEKL-3, and NEKL-4. NEKL-2, a homolog of human NEK8 and NEK9, contains a highly conserved kinase domain and a short disordered region at the C terminus [ 34 ]. NEKL-2 is expressed in a punctate pattern in the large epidermal syncytium, hyp7, where it colocalizes with the conserved ankyrin repeat proteins MLT-2 (ANKS6 homolog) and MLT-4 (INVS homolog). MLT-2 and MLT-4 are required for the proper localization of NEKL-2, and their physical association is conserved in vertebrates [ 34 – 36 ]. NEKL-3, a close homolog of human NEK6 and NEK7, contains a kinase domain only and is also expressed in a punctate pattern within hyp7. NEKL-3 forms a complex with a third conserved ankyrin repeat protein, MLT-3/ANKS3, which is required for the proper localization of NEKL-3 in C . elegans and NEK7 in vertebrates [ 37 ]. We have shown that NEKL-2 and NEKL-3, along with their MLT binding partners, are required for the completion of molting in C . elegans , as a strong loss of function in any of these proteins leads to molting defects in early larval stages [ 34 , 38 – 40 ]. Moreover, mutation of the kinase domain of nekl-2 or nekl-3 leads to uniform molting defects indicating that the catalytic activity of the NEKLs is essential for the promotion of molting [ 34 , 39 ].

Molting is an essential feature for C . elegans to develop through its four larval stages and to reach adulthood. During molting, the old cuticle, an apical extracellular matrix (aECM) made up of proteins and lipids, detaches from the underlying epidermis and is broken down as a new cuticle is synthesized underneath. This process of aECM remodeling requires the trafficking of macromolecules required for the synthesis, degradation, and recycling of cuticle components [ 41 , 42 ]. We previously reported that NEKL-2 and NEKL-3 regulate clathrin-mediated endocytosis (CME) at the apical membrane of hyp7. Our findings indicated that loss of NEKL-2 or NEKL-3 leads to a defect in the uncoating of apical clathrin-coated vesicles, thereby preventing the flow of apical cargo through the endocytic pathway. Consistent with this, an apically expressed low-density lipoprotein–like receptor, LRP-1/Megalin, was observed to be trapped at or near the apical membrane in NEKL-depleted animals [ 34 , 43 ]. LRP-1 internalization by hyp7 is essential for molting, as it is required for the uptake of cholesterol, a substrate required for hormonal signaling pathways that coordinate the molting cycle [ 41 , 44 , 45 ].

The involvement of NEKLs in CME is also strongly supported by our genetic screens, which identified subunits of the clathrin adapter protein complex, AP2, as strong suppressors of both molting and trafficking defects in nekl-2 and nekl-3 mutants. Likewise, loss of FCHO-1, an allosteric activator of AP2, alleviates molting and trafficking defects in nekl mutants [ 43 ]. Our collective data indicate that NEKL-2 and NEKL-3 directly or indirectly promote clathrin uncoating and suggest that AP2 activity can also affect the uncoating process. However, a possible role for NEKLs in other aspects of intracellular trafficking was not explored.

In this study, we further investigated the role of NEKLs at different membranes and in different compartments within the endocytic network. Importantly, we found that NEKL-2 and NEKL-3 have distinct functions within different endosome types and are required for basolateral trafficking and cargo recycling. Importantly, we extended our studies to human cell lines and demonstrated that the human NEKL-3 orthologs, NEK6 and NEK7, also appear to function at multiple points within the endocytic pathway in mammals. These studies provide new insights into the biological roles of NEK/NEKL family members, which may have relevance to their roles in human development and disease.

NEKL-2 and NEKL-3 colocalize with endosomal markers

Previously, we reported that NEKL-2 and NEKL-3 are specifically expressed and required within the large epidermal syncytium of C . elegans , hyp7 [ 34 , 38 ]. Moreover, both NEKL-2 and NEKL-3 are cytoplasmic and are localized to variably sized actin-rich puncta or aggregates, which are reminiscent of endosomal compartments [ 34 , 38 ]. To determine the subcellular compartments in which NEKLs may function, we carried out colocalization experiments using endogenously tagged NEKL strains, together with reporters for early (P rab-5 ::GFP::RAB-5) and late (P hyp7 ::GFP::RAB-7) endosomes [ 46 – 50 ]. In the case of NEKL-3, we observed robust colocalization of NEKL-3::mKate with GFP::RAB-5 and GFP::RAB-7 (~50% Manders’ overlap with each marker), such that a majority of detectable NEKL-3 may be associated with early-to-late endosomes ( Fig 1A–1F, 1A’–1F’ and 1M ). In contrast, whereas ~40% of NEKL-2::mKate colocalized with RAB-5::GFP, only ~10% colocalized with RAB-7::GFP, suggesting that NEKL-2 may play a greater role in early steps of endocytic trafficking ( Fig 1G–1L, 1G’–1L’ and 1M ). We also note that we previously reported the association of NEKL-2–MLT-2–MLT-4 with actin-rich puncta that form along the boundary of hyp7 and the seam cells, which do not appear to be endosomal and may represent adhesion junctions [ 38 , 39 ]. In addition, we have observed some diffuse reticulated signal from fluorescently tagged NEKL-3 and NEKL-2 in the cytoplasm of hyp7 ( Fig 1 ), which could correspond in part to the endoplasmic reticulum [ 39 ].

Colocalization assays were carried out in adult worms expressing either NEKL-2::mKate or NEKL-3::mKate with endosomal markers P rab-5 ::GFP::RAB-5 or P hyp7 ::GFP::RAB-7. (A–C, A’–C’, G–I, and G’–I’) Representative confocal images of adult worms expressing either NEKL-3::mKate (A–C and A’–C’) or NEKL-2::mKate (G–I and G’–I’), along with the early endosomal marker P rab-5 ::GFP::RAB-5. (D–F, D’–F’, J–L, and J’–L’) Representative confocal images of adult worms expressing either NEKL-3::mKate (D–F and D’–F’) or NEKL-2::mKate (J–L and J’–L’) and late endosomal marker P hyp7 ::GFP::RAB-7. Yellow arrowheads indicate overlap. Scale bar in A = 10 μm for A–L. Scale bar in A’ = 1 μm for A’–L’. (M) Manders’ coefficient was calculated for all the worms with the indicated backgrounds and plotted to determine the fraction of overlap between NEKL-2– or NEKL-3–positive pixels and the indicated markers. Error bars represent the 95% confidence intervals. p-Values were obtained by comparing means using an unpaired t-test: ****p < 0.0001, ***p < 0.001, *p < 0.05; ns, not significant (p > 0.05). Raw data are available in S1 File .

The differences observed between NEKL-2 and NEKL-3 localization are consistent with our previous report that NEKL-2 and NEKL-3, along with their MLT co-partners, exhibit distinct localization patterns in hyp7 [ 39 ]. Consistent with this, we find that the Manders’ overlap between fluorescently tagged NEKL-2 and NEKL-3 is <20% ( S1 Fig ), suggesting that the cellular functions of these kinases are distinct, despite having similar terminal molting-defective phenotypes. Notably, although a loss of NEKL-2 and NEKL-3 lead to defects in clathrin uncoating [ 43 ], neither showed strong colocalization with a marker for the AP2 clathrin-adapter protein complex, APA-2::mScarlet ( S2 Fig ). This suggests that the previously described impact of NEKLs on clathrin uncoating may be indirect or that levels of NEKLs at clathrin-coated vesicles are below detection. Notably, both NEKLs colocalize with RAB-5, which plays a role in the uncoating of clathrin-coated vesicles [ 51 ].

NEKLs are required for normal endosome morphologies

Given the observed colocalization of NEKLs with endosomes, we next wanted to determine if the loss of NEKLs led to an observable defect in the morphologies of endosomal compartments. During the process of endocytosis, endosomes undergo maturation, which is accompanied by changes in shape, molecular composition, and function. In the case of early/sorting and recycling endosomes, tubular extensions extend and pinch off from larger vesicles, allowing proteins to be recycled to specific destinations such as the plasma membrane and trans-Golgi [ 52 – 55 ]. Disruptions to endosomal functions can lead to a wide variety of defects including alterations in cargo localization and abundance.

To avoid indirect consequences caused by molting defects, we assessed the roles of NEKLs on endosomal morphology using the auxin-inducible degron (AID) system, in which endogenously tagged NEKL-2::AID and NEKL-3::AID were depleted in day-1 adults after the addition of auxin [ 43 , 56 , 57 ]. Depletion of NEKL-2::AID led to an increase in the overall size of the early endosome compartment (based on total intensity and area measurements; P rab-5 ::GFP::RAB-5) and, most strikingly, to an increase in the extent of tubulation ( Fig 2A, 2B and 2D–2F ). This phenotype suggests a requirement for NEKL-2 in the process of vesicle fission at early endosomes. In contrast, loss of NEKL-2 had more modest effects on the late endosome compartment (P hyp7 ::GFP::RAB-7), consistent with the observed strong colocalization of NEKL-2 to early, but not late, endosomes ( Fig 2G, 2H and 2J–2L ). Specifically, although the mean number of vesicles was decreased in NEKL-2–depleted worms as compared with wild type, the morphology of late endosomes was not strongly affected, and the observed changes could be due in part to gross alterations in the early endosome caused by NEKL-2 depletion.

(A–C, G–I, M–O) Confocal imaging was used to examine the effects of NEKL-2 (B, H, N) and NEKL-3 (C, I, O) loss relative to wild type (WT; A, G, M) in hyp7 of day-2 adult worms after auxin treatment. Representative images are shown. (A–C) Imaged worms expressed P rab-5 ::GFP::RAB-5 in the indicated backgrounds. (D–F) The mean intensity (D), vesicle area (E), and the roundness of the puncta (F) were plotted for worms expressing P rab-5 ::GFP::RAB-5. (G–I) Imaged worms expressed P hyp7 ::GFP::RAB-7 in the indicated backgrounds. (J–L) The mean intensity (J), vesicle area (K), and the number of vesicles (L) were plotted for worms expressing P hyp7 ::GFP::RAB-7. (M–O) Imaged worms expressed mScarlet::RAB-11, a marker for recycling endosomes, in the indicated backgrounds. (P–R) The mean intensity (P), vesicle area (Q), and perimeter of the vesicles (R) were plotted for individual worms expressing mScarlet::RAB-11. Area is in square pixels; perimeter is the number of pixels in the boundary of the object. Error bars represent the 95% confidence intervals. p-Values were obtained by comparing means using an unpaired t-test: ****p < 0.0001, ***p < 0.001, *p < 0.05; ns, not significant (p > 0.05). Raw data are available in S1 File .

In the case of NEKL-3::AID, depletion led to strong defects at both early and late endosomes, consistent with the localization of NEKL-3 to both compartments. In the case of early endosomes, however, the effects of NEKL-3 loss differed markedly from those after loss of NEKL-2 ( Fig 2B–2F ). Rather than increased tubulation, loss of NEKL-3 led to increased variability in the size and shape of early endosomes accompanied by an increase in total signal intensity. Thus, while NEKL-2 and NEKL-3 both act at early endosomes, their roles in controlling early endosome morphology appear to be distinct. Depletion of NEKL-3 also led to a strong overall decrease in the abundance of late endosomes, which was associated with a decrease in total intensity, vesicle size, and changes in vesicle morphology ( Fig 2G and 2I–2L ). These defects may be due to a combination of the roles for NEKL-3 in directly regulating late endosomes and the potential secondary effects associated with its effects on early endosomes. We also note that even in the absence of auxin, NEKL-3::AID worms are smaller than wild-type worms and show some arrest due to molting defects, which may be attributable to a partial LOF caused by the AID tag [ 43 ].

We also examined the effects of NEKL loss on recycling endosomes, which were marked with endogenously (CRISPR) tagged RAB-11. Whereas NEKL-2::AID depletion had little or no effect on mScarlet::RAB-11 localization, we observed a strong increase in tubulation after loss of NEKL-3:AID, which was accompanied by an increase in total intensity and vesicle size ( Fig 2M–2R ). For technical reasons, we were unable to carry out colocalization studies between the NEKLs and either mScarlet::RAB-11 or GFP::RAB-11. Our observations, however, suggest that NEKL-3 may also function at recycling endosomes, possibly to control vesicle fission. In contrast to endosomal compartments, depletion of NEKL-2::AID and NEKL-3::AID had little or no effect on the morphology of the Golgi compartment, based on a P hyp7 ::AMAN-2::mNeonGreen marker ( S3 Fig ). Nevertheless, the total number of AMAN-2–positive vesicles was variably reduced in these backgrounds, with strongest effects shown for NEKL-3::AID ( S3A–S3C Fig ), which could be caused by reduced endosome-to-Golgi recycling. Consistent with an indirect effect, a multi-copy NEKL-3::mCherry reporter [ 34 ] showed very low levels of colocalization with AMAN-2:: mNeonGreen ( S3D Fig ). Collectively, our studies demonstrate a role for NEKL-2 and NEKL-3 in regulating endosomal morphologies and further suggest direct functions within endosomal compartments.

NEKLs are required for basolateral cargo uptake by hyp7

We previously showed that NEKLs are required for CME at the apical membrane of hyp7 ( Fig 1A and 1B ) [ 44 , 45 , 58 ]. Specifically, depletion of NEKL-2 or NEKL-3 led to the accumulation of both clathrin and LRP-1, a low-density lipoprotein–like receptor, at the apical hyp7 surface [ 34 , 43 , 59 , 60 ]. Consistent with this, C . elegans LRP-1 endocytosis is dependent on clathrin and the clathrin adapter protein complex, AP2, analogous to findings in mammals [ 34 , 43 , 60 – 64 ]. To determine whether NEKL kinases are also required for trafficking at basolateral membranes of hyp7, we examined the internalization of two basolaterally localized BMP-family receptors. SMA-6 (Type I BMP receptor) and DAF-4 (Type II BMP receptor) form a heteromeric complex that binds to DBL-1 (BMP ligand), leading to the regulation of genes controlling C . elegans body size and male mating structures [ 65 ]. The internalization of SMA-6 depends on CME, as loss of AP2 function leads to the accumulation of SMA-6 at the basolateral membrane of intestinal cells [ 66 ]. In contrast, DAF-4 internalization does not require AP2, suggesting that it is endocytosed through a clathrin-independent mechanism [ 66 ].

To investigate possible roles for NEKLs in basolateral endocytosis, we expressed SMA-6::GFP and DAF-4::GFP using a hyp7-specific promoter and examined expression at the membrane of hyp7 adjacent to the seam cell (referred to hereafter as the lateral membrane) and at the membrane that forms the interior boundary of hyp7 (referred to hereafter as the basal membrane; Fig 3A and 3B ). Notably, the mean intensity of P hyp7 ::SMA-6::GFP was increased by ~1.3-fold or ~2.3-fold when NEKL-2 or NEKL-3 were depleted, respectively ( Fig 3C–3E and 3H ). Moreover, a strong increase (~4.8-fold) in the total level of P hyp7 ::DAF-4::GFP was observed after NEKL-3::AID depletion ( Fig 3F, 3G and 3I ), suggesting that NEKL-3 may also be required for non-clathrin–dependent endocytosis or at the downstream junction of these pathways. In contrast, P hyp7 ::DAF-4::GFP was not detectably affected by depletion of NEKL-2 ( Fig 3H and 3J ), suggesting that NEKL-2 may not be required for non-CME or may have a more limited role in endocytosis at the basolateral membrane relative to NEKL-3. Accumulation of these markers after NEKL-3 depletion was most evident at or near the basal hyp7 membrane, indicating a defect in plasma membrane uptake and/or processing through the early steps of endocytosis. In addition, some accumulation at or near the basolateral membrane with seam cells was also observed ( S4 Fig ). Together these results show that NEKL-2 and NEKL-3 play variable roles in the endocytosis of basolateral BMP receptors, although much stronger effects were observed for NEKL-3 in this process.

(A) Cross-sectional view of an adult worm depicting the position of the hyp7 syncytium (blue grey) and seam cell syncytium (red). Specific membrane domains of hyp7 are indicated: apical, facing externally directly underneath the cuticle (brown); basal, facing internally (purple); lateral, facing the sides and bottom of the seam cell (green). Dashed lines indicate the medial imaging plane used to acquire the images. (B) Top-down view of the long axis of the worm’s body indicating the positions of the hyp7 and seam cell syncytium along with indicated membranes as in A. (C–E) Representative confocal images of P hyp-7 ::SMA-6::GFP expression in auxin-treated wild-type (C), nekl-2 :: aid (D), and nekl-3 :: aid (E) day-2 adults. (F, G) Representative confocal images of P hyp-7 ::DAF-4::GFP expression in auxin-treated wild-type (G), nekl-2 :: aid (H), and nekl-3 :: aid (I) day-2 adults. Green and purple arrowheads (D,E,G) indicate seam and basal membranes (where detectable), respectively. Note that it is often not possible to identify the precise lateral/basolateral boundaries of hyp7 in the SMA-6::GFP and DAF-4::GFP lines in wild type because of the curved nature of the lateral membrane and because of low levels of these cargos marking the membrane. Scale bar in C = 10 μm for C–E, F, and G. (F, J) Mean intensity values for P hyp-7 ::SMA-6::GFP (F) and P hyp-7 ::DAF-4::GFP (J) expression were plotted for individual adults. The two highest datapoints in nekl-3 :: aid ; DAF-4::GFP (3J; 615 and 1233) were omitted for clarity of presentation. Error bars represent the 95% confidence intervals. Statistical significance was determined using a two-tailed, unpaired t-test: **p < 0.01, ****p < 0.0001. Raw data are available in S1 File .

NEKLs are required for cargo sorting

Membrane cargoes that are internalized by endocytosis are first delivered to sorting/early endosomes after which they may be recycled back to their original compartment(s) or routed to the lysosome for degradation. Previous studies have characterized the recycling routes for two conserved cargo proteins, MIG-14/Wntless and TGN-38/TGN38, both of which originate from the trans-Golgi [ 67 – 71 ]. MIG-14 is a transmembrane protein that binds to Wnt ligands in the Golgi and is responsible for delivering Wnts to the plasma membrane for secretion. Once at the plasma membrane, MIG-14 is endocytosed and recycled back to the Golgi through a retrograde pathway that requires the retromer complex [ 72 ]. In the absence of retromer function, MIG-14 is missorted to the lysosome and is degraded [ 67 , 68 ]. Likewise, TGN-38 is a trans-Golgi resident that is also recycled from the plasma membrane to the trans-Golgi by a retromer-associated retrograde pathway [ 67 , 73 , 74 ].

In wild-type adults, both P hyp7 ::MIG-14::GFP and P hyp7 ::TGN-38::GFP markers were observed in punctate structures of varying size throughout the cytoplasm, with some accumulation occurring at the lateral surface ( Fig 4A and 4E ). After depletion of NEKL-3::AID, P hyp7 ::MIG-14::GFP expression was decreased ~2.7-fold, whereas depletion of NEKL-2::AID resulted in at most a subtle decrease in total P hyp7 ::MIG-14::GFP levels (~1.2-fold; p = 0.09) ( Fig 4A–4D ). In the case of P hyp7 ::TGN-38::GFP, marker expression was decreased by ~2.8-fold after depletion of either NEKL-2::AID or NEKL-3::AID ( Fig 4E–4H ). Such findings further highlight differences between NEKL-2 and NEKL-3 with respect to their effects on specific cargoes and suggest that MIG-14::GFP and TGN-38::GFP may be missorted to the lysosomal degradative pathway after loss of NEKLs.

(A–C, E–G, and I–J) Representative confocal images of P hyp-7 ::MIG-14::GFP (A–C) and P hyp-7 ::TGN-38::GFP (E–G and I–J) expression within hyp7 in auxin-treated wild-type (A, E), NEKL-2::AID (B, F), NEKL-3::AID (C, G), NEKL-2::AID; cup-5 (I), and NEKL-3::AID; cup-5 (J) day-2 adults. Scale bar in A = 10 μm for A–C, E–G, I, and J. (D, H) Mean pixel intensity values of P hyp-7 ::MIG-14::GFP and P hyp-7 ::TGN-38::GFP expression for individual worms. Error bars represent the 95% confidence intervals. Statistical significance was determined using a two-tailed, unpaired t-test: ****p < 0.0001; ns, not significant (p > 0.05). Raw data are available in S1 File .

To test if NEKL depletion leads to the aberrant degradation of cargoes, we used CRISPR methods to introduce a partial loss-of-function mutation into cup-5/trpml1 , which encodes a conserved channel protein required for the maturation of lysosomes and whose loss leads to reduced lysosomal function [ 75 – 77 ]. Notably, reduction of CUP-5 activity led to an ~1.5-fold increase in the total expression of P hyp7 ::TGN-38::GFP in both NEKL-2::AID and NEKL-3::AID depleted strains ( Fig 4I–4K ). Moreover, accumulation of P hyp7 ::TGN-38::GFP was detected in moderate-to-large internal structures in these strains, which may correspond to defective lysosomes. Together these results indicate that, in addition to functioning in membrane cargo uptake, NEKL-2 and NEKL-3 are also required for the correct sorting and recycling of internalized cargo. We note that loss of cup-5 in an otherwise wild-type background also led to the accumulation of P hyp7 ::TGN-38::GFP, suggesting that some portion of TGN-38 is normally delivered to the lysosome in wild-type animals and that it’s degradation depends on lysosomal function ( S5 Fig ). TGN-38 accumulations, however, appeared larger in cup-5 single mutants relative to NEKL::AID; cup-5 strains (Figs ​ (Figs4 4 and S5 ). One possible explanation for this difference is that NEKL depletion may directly or indirectly affect lysosomal morphology or lysosome abundance.

Depletion of NEKL-3::AID but not NEKL-2::AID also led to a reduction in the levels of P hyp7 ::MIG-14::GFP at the hyp7 membrane surrounding the ALM and PLM neurons, which run longitudinally through hyp7 along the anterior-posterior axis ( S6 Fig ). Altogether our results indicate that loss of NEKLs leads to a range of defects affecting endosomal compartments as well as cargo internalization and sorting. Moreover, although nekl-2 and nekl-3 mutants appear superficially identical with respect to molting defects, they act in largely distinct subcellular compartments and lead to different subcellular phenotypes when depleted.

The mammalian homologs of NEKL-3, NEK6 and NEK7, are required for normal endocytic trafficking in human cells

Human NEK6 and NEK7 are both ~70% identical and ~85% similar to NEKL-3 and can rescue molting and trafficking-associated defects when expressed in C . elegans nekl-3 mutants [ 43 ]. Nevertheless, a systematic examination of potential roles for NEK6 and NEK7 in intracellular trafficking has not been undertaken. Using siRNA approaches, we were able to substantially reduce protein levels of NEK6 and NEK7 within 48 h of oligonucleotide transfection into cell lines ( Fig 5A ). As a first measure, we examined the effects of NEK6 and NEK7 knockdown on the cation-independent mannose 6-phosphate receptor (M6PR) in HeLa cells grown at steady state. M6PR is responsible for the delivery of mannose 6-phosphate–tagged lysosomal hydrolases from the trans-Golgi network (TGN), and thus cycles rapidly between the TGN and late endocytic organelles. Accordingly, M6PR is highly sensitive to disruptions in endocytic trafficking, which lead to alterations in its distribution.

(A) siRNA knockdown of NEK6 and NEK7 was validated by western blotting of HeLa cells that were mock-transfected or transfected with oligonucleotides specific for NEK6 (left panel) or NEK7 (right panel). GAPDH was used as the loading control (lower panels). (B–G) Mock-transfected cells (B and enlarged area in C), NEK6 siRNA–transfected cells (D and enlarged area in E), or NEK7 siRNA–transfected cells (F and enlarged area in G) were plated on coverslips and immunostained with antibodies against mannose 6-phosphate receptor (M6PR). (K, L) The mean intensity of M6PR immunostaining (K) and the mean area of M6PR distribution (L) of individual cells in mock-transfected, NEK6 siRNA–transfected, and NEK7 siRNA–transfected cells. (H–J) Saturated and zoomed micrographs demonstrating the distribution and intensity of mannose 6-phosphate receptor immunostaining in mock-transfected (H), NEK6 siRNA–transfected (I), and NEK7 siRNA–transfected cells (J). The dashed lines indicate individual cells. Scale bar in B = 10 μm for B, D, and F; scale bar in C = 10 μm for C, E, and G; scale bar in J = 10 μm for H–J. Statistical significance was determined using Student’s unpaired t-test. Raw data are available in S1 File .

Relative to control cells, the mean fluorescence intensity of M6PR-containing endosomes showed a significant decrease (~3-fold) in both NEK6 and NEK7 knockdown cells ( Fig 5B–5G and 5K ). These findings suggest that, analogous to observations for MIG-14 and TGN-38 in C . elegans , M6PR may be aberrantly targeted for lysosomal degradation after loss of the NEKL-3 orthologs. In addition, the distribution of M6PR in NEK6/7-depleted cells was altered, with M6PR showing a 2- to 3-fold increase in the area of dispersal ( Fig 5H–5J and 5L ). Although this increased M6PR dispersal might reflect slightly larger cell sizes in the NEK-depleted cells, M6PR appears to be more homogeneously distributed in the knockdown cells, an effect that has been associated with defects in endosome-to-Golgi transport via the retromer complex [ 78 ]. These data imply that both NEK6 and NEK7 are involved in the regulation of endosome-to-Golgi retrograde trafficking.

Given that loss of NEKLs led to a major impact on endosomes, we next assessed the impact of NEK6 and NEK7 depletion on EEA1, a well-characterized early endosomal protein that binds to endosomal PI3P via its FYVE domain [ 79 – 81 ]. Upon siRNA-mediated depletion of NEK6 or NEK7 ( Fig 6A ) and immunostaining with antibodies against EEA1, endosomal size was increased in cells depleted of NEK6 or NEK7, with those lacking NEK7 displaying especially large ring-like structures decorated by EEA1 ( Fig 6B–6G and 6H ). In contrast, the number of EEA1 endosomes was not significantly altered upon depletion of either NEK6 or NEK7 ( Fig 6I ). As a further test, we performed NEK6/7 siRNA knockdown in glioblastoma cells and observed an increase in the size of EEA1-marked vesicles after depletion of NEK7 but not NEK6 ( S7 Fig ), suggesting cell type–specific requirements for NEK functions. Together, these data suggest a role for NEK6 and NEK7 at endosomes, potentially in the regulation of fusion and/or fission events.

(A) siRNA knockdown of NEK6 (left panel) and NEK7 (right panel) in HeLa cells was confirmed by western blotting (B–G) Mock-transfected cells (B and enlarged area in C), NEK6 siRNA–transfected cells (D and enlarged area in E), or NEK7 siRNA–transfected cells (F and enlarged area in G) were plated on coverslips and immunostained with antibodies against the early/sorting endosome marker protein EEA1. Yellow boxes in B, D, and F indicate the area of higher magnification shown in C, E, and G, respectively. (H) The mean size of EEA1-containing endosomes in mock-transfected, NEK6 siRNA–transfected, and NEK7 siRNA–transfected cells. (I) The number of EEA1-containing endosomes in mock-transfected, NEK6 siRNA–transfected, and NEK7 siRNA–transfected cells. Scale bar in B = 10 μm for B, D, and F; Scale bar in C = 5 μm for C, E, and G. Statistical significance was determined using Student’s unpaired t-test. Raw data are available in S1 File .

To further examine the role of NEK6 and NEK7 at endosomes, we analyzed the effect of their depletion on tubular recycling endosomes marked by the protein MICAL-L1 [ 82 – 85 ]. After depletion of NEK6 or NEK7 with siRNA oligonucleotides ( Fig 7A ), HeLa cells were fixed and immunostained with antibodies against endogenous MICAL-L1. Inhibition of NEK6 or NEK7 led to an increase in the MICAL-L1 tubular endosomal surface area relative to controls ( Fig 7B–7E ). Similar observations were observed for glioblastoma cells, with depletion of either NEK6 or NEK7 leading to increased tubulation ( S8 Fig ). Together, these data indicate roles for NEK6 and NEK7 in endosomal morphology and function in mammalian cells and support the possibility of their involvement in fusion and/or fission activities.

(A) siRNA knockdown of NEK6 (left panel) and NEK7 (right panel) was validated by western blotting of HeLa cells. (B–D) Mock-transfected cells (B), NEK6 siRNA–transfected cells (C), or NEK7 siRNA–transfected cells (D) were plated on coverslips and immunostained with antibodies against the tubular recycling endosome marker protein MICAL-L1. (E) The surface area of MICAL-L1–containing endosomes in mock-transfected, NEK6 siRNA–transfected, and NEK7 siRNA–transfected cells. Scale bar in D = 10 μm for B–D. Statistical significance was determined using Student’s unpaired t-test. Raw data are available in S1 File .

NIMA kinases are required for proper endosome morphology

In this study we have demonstrated that C . elegans NEKLs and human NEKs are important for maintaining the proper morphology of endosomal compartments. Loss of NEKL-2 led to enlarged early endosomal compartments with noticeably longer tubular extensions. These changes could indicate a failure of early endosomes to undergo fission, an expansion of early endosomes caused by abnormal cargo retention, or both. Notably, the effect of NEKL-3 loss on early endosomes was distinct from that of NEKL-2, as no increase in tubular extensions was observed. The shape of early endosomes, however, became irregular, and the mean intensity of the early endosomal marker GFP::RAB-5 was increased. This suggests that NEKL-2 and NEKL-3 have distinct roles in regulating early endosome morphology. Notably, siRNA-mediated knockdown of human NEK6 or NEK7 also led to an increase in the size of EEA-1–positive early endosomes in HeLa cells. However, whereas knockdown of NEK7 led to an increase in the size of early endosomes in glioblastoma cells, inhibition of NEK6 did not show this effect, suggesting that NEK6 and NEK7 may regulate early endosomal morphology in a cell type–specific manner.

Consistent with their distinct localization patterns, depletion of NEKL-3 had much stronger effects on late endosomal compartments than did loss of NEKL-2. We note, however, that effects on late endosomes could be due in part to perturbations of early endosomes caused by loss of NEKL-2 and NEKL-3. For technical reasons, we could not determine the expression of NEKLs in recycling endosomes. However, an increase in membrane tubulation was observed with recycling endosomes in hyp7 after loss of NEKL-3 but not NEKL-2. Collectively, these results indicate that NEKL-2 and NEKL-3 have important but distinct roles in regulating endosomal morphology.

NIMA kinases may broadly affect cargo uptake and recycling

We examined two Golgi residents, MIG-14 and TGN-38, to better understand how NEKLs may influence the endocytic recycling process. We observed a dramatic reduction in the MIG-14 and TGN-38 signal after depletion of NEKL-3. In contrast, loss of NEKL-2 led to a strong reduction in TGN-38, but only minimal effects on MIG-14. These findings suggest that specific cargoes are variably missorted to the lysosomal degradative pathway when NEKL-2 or NEKL-3 is depleted, as was previously reported for mutants affecting actin assembly and the sorting nexin, SNX3 [ 67 , 68 ]. Consistent with this, expression of TGN-38 was rescued when lysosomal function was inhibited in NEKL::AID strains. Interestingly, defects consistent with abnormal recycling were also observed for M6PR when NEK6 and NEK7 were depleted in HeLa cells, consistent with M6PR being incorrectly targeted to lysosomes. Together these findings suggest that NIMA family kinases may have conserved functions in the sorting of cargoes from early endosomes.

Previously, we reported that NEKL-2 and NEKL-3 regulate endocytosis at the apical membrane of hyp7 [ 43 ]. In this study, we found that the internalization of a basolateral membrane cargo, SMA-6, was also affected in worms with NEKL-2 or NEKL-3 depletion. Our data also suggest that NEKL-3 may have a role in clathrin-independent endocytosis, as loss of NEKL-3 led to an accumulation of a putative clathrin-independent cargo, DAF-4, near the basolateral surface. One potential caveat to this interpretation is that in the presence of ligand, SMA-6 and DAF-4 would be predicted to form a complex [ 86 ], and thus SMA-6 or DAF-4 could potentially affect each other’s uptake or retention. For example, membrane retention of endogenous SMA-6 could impact the localization and uptake of DAF-4::GFP. However, SMA-6 is expressed at very low levels [ 87 , 88 ], and thus it seems unlikely that endogenous plasma-membrane associated SMA-6 could indirectly lead to the strong membrane/juxta-membrane retention of the more highly expressed DAF-4::GFP. Altogether, these findings point to roles for NEKL-3 in both CME and non-CME.

We note that neither SMA-6 nor DAF-4 appeared to be incorrectly targeted to lysosomes after NEKL depletion, which differs from what we observed for MIG-14 and TGN-38. We speculate that SMA-6 and DAF-4 may be trapped at or near the basolateral surface in NEKL-3::AID depleted worms, as our previous study failed to detect clathrin accumulation at basolateral membranes of hyp7 [ 43 ]. Moreover, we failed to detect GFP::RAB-5 accumulation at or near basolateral surface in NEKL-3::AID depleted worms, suggesting that the basolateral accumulation of SMA-6 and DAF-4 is not within a RAB-5–marked early endosomal compartment. This buildup SMA-6 and DAF-4 could be due plasma membrane accumulation, internalized juxta-membrane vesicles, or a combination of the two.

What are the core functions of NEKLs in intracellular trafficking?

In a previous study, we reported that NEKL-2 and NEKL-3 regulate CME at the apical membrane and that they promote the uncoating of clathrin from internalized vesicles [ 43 ]. Our current study, however, suggests that the primary sites for trafficking regulation by NEKL-2 and NEKL-3 may be endosomes, and we failed to observe strong colocalization of NEKL-2 or NEKL-3 to AP2–decorated clathrin coated pits and vesicles. Thus, the clathrin-uncoating defects we previously observed could in part be an indirect consequence of endosomal defects. In this context, it is worth noting that RAB-5, which colocalized with both NEKL-2 and NEKL-3, partially localizes to clathrin-coated pits and promotes clathrin and AP2 uncoating [ 51 , 89 ]. Moreover, human RAB5 has been detected at both early endosomes and clathrin-coated vesicles [ 46 , 90 ]. Thus, it remains possible that low levels of NEKL-2 and NEKL-3 may exert direct effects on clathrin uncoating, possibly in conjunction with RAB-5.

The effects we observed on multiple cargoes and compartments indicate that NEKL-2 and NEKL-3 may have widespread functions in intracellular trafficking. Likewise, effects on multiple compartments were observed after human NEK6 and NEK7 knockdowns. This could be due to NIMA kinases regulating distinct targets at different locations within the endocytic network. Alternatively, NIMAs may act on a smaller number of targets, which in turn have widespread functions in trafficking.

In this latter category, one compelling model is that NIMAs could regulate endosomal actin. Actin polymerization is deployed throughout endocytosis to provide mechanical forces needed to bend membranes, facilitate vesicle fission, and promote short-range vesicle transport [ 91 – 95 ]. For example, actin filaments are enriched at sorting endosomes and promote the fission of tubular extensions [ 52 , 96 ]. Actin assembly also plays a role in the sorting of macromolecules from early endosomes and their transport to the trans-Golgi [ 97 , 98 ]. Consistent with this idea, our previous studies showed that actin organization was strongly disrupted in nekl mutants and that nekl molting defects can be suppressed by loss of function in the conserved actin regulator CDC-42, along with one of its effectors, SID-3/ACK1/2 ( a ctivated C DC42 k inase) [ 38 ]. Moreover, activated (GTP-bound) CDC-42 was strongly upregulated in nekl mutants [ 38 ]. Interestingly, based on protein-interaction studies, CDC42 was reported to interact with both NEK6 and NEK7 and could therefore connect the NEKL–MLT network to actin polymerization and to their roles throughout the endocytic network. Future studies will seek to characterize the functional link between NIMA kinases and the actin cytoskeleton, along with their specific roles in regulating endocytosis.

Human NEKs in endocytosis and cancer

Although human homologs of NEKL-3 (NEK6, NEK7) and NEKL-2 (NEK9) have largely been studied for their roles in cell division, several studies have hinted at potential functions in endocytosis [ 99 – 104 ]. A high-throughput siRNA-based screen of mammalian protein kinases indicated that siRNA-mediated knockdown of NEK6 and NEK7 strongly disrupts CME in HeLa cells [ 99 ]. Moreover, genome-wide siRNA studies showed that knockdown of multiple NEK kinases, including NEK6, resulted in the abnormal uptake of endocytic cargoes [ 101 ]. A more recent study identified NEK6 as a potential downstream target of NRP-1, which is required for transferrin endocytosis by Trypanosoma brucei [ 105 ]. Moreover, proteomic studies of NEK6 and NEK7 identified interactions with several trafficking components including alpha and beta subunits of the AP2 adaptor complex (NEK7) [ 102 , 103 ]. Such reports are consistent with our current findings that NEK6 and NEK7 play roles in endocytic trafficking in human cells. Lastly, our ability to rescue both molting and trafficking defects through the expression of human NEKs in C . elegans nekl mutants suggests that these functions are conserved [ 43 ].

Finally, our studies provide evidence that NEKLs may affect two highly conserved signaling pathways, BMP and Wnt, which are misregulated in many human cancers [ 106 – 108 ]. Previous studies have described the role of endocytosis in cancers including how endocytosis adapts to the needs of cancer cells, a phenomenon known as “adaptive CME” [ 109 – 111 ]. For example, upregulation of clathrin light-chain (CLCb) leads to an increased rate of CME; this in turn alters the trafficking of oncogenic epidermal growth factor receptor (EGFR) and promotes cancer cell migration and metastasis [ 112 ]. Thus, it is possible that NIMA kinases, which are overexpressed in many tumors, could be driving cancer formation in part through their roles in trafficking as well as by their previously described functions in mitosis [ 19 , 113 – 117 ]. Future studies will elucidate how NIMA kinases control trafficking, which will aid in understanding their involvement in human disease.

Materials and methods

Strains and maintenance.

C . elegans strains were maintained according to standard protocols [ 118 ] and were propagated at 22°C unless stated otherwise. Strains used in this study are listed in S1 Table . Loss-of-function alleles of cup-5 ( fd395 and fd397 ) were generated by the CRISPR-Cas9 protocol described by Ghanta et al. [ 119 ]. Sequences for the sgRNA and repair template are given below. Capital letters represent the altered or inserted nucleotides.

sgRNA: 5′-aacgatgcgcttattatcat-3′

Repair Template: 5′-aacttaaatttttataaaaattacctgaaactcgatggatatttttgcaacGGTCTCaatgataataagcgcatcgttgaccacaatcatcacataccacaaattcag-3′

Reporter strain construction

Plasmids for C . elegans hyp7-specific expression used the promoter from semo-1/Y37A1B . 5 (P hyp-7 ) [ 120 ]. Vector details are available upon request. Cloning was performed using the Gateway system (Invitrogen) and modified versions of hygromycin-resistant and miniMos-enabled vector pCFJ1662 (gift from Erik Jorgensen, University of Utah; Addgene #51482). pDONR221 entry vectors containing coding regions for rab-5 , rab-7 , mig-14 , tgn-38 , aman-2 , daf-4 , and sma-6 were transferred into hyp7 destination vectors with the Gateway LR clonase II reaction to generate C-/N-terminal fusions. Single-copy integrations were obtained using miniMos technology [ 121 ].

Image acquisition

Confocal fluorescence images in Figs ​ Figs1 1 – 4 and S1 and S3 and S5 were acquired using an Olympus IX83 inverted microscope with a Yokogawa spinning-disc confocal head (CSU-W1). z-Stack images were acquired using a 100×, 1.35 N.A. silicone oil objective. cellSense 3.1 software (Olympus Corporation) was used for image acquisition. Fluorescence images in S2 Fig were acquired using an Olympus IX81 inverted microscope with a Yokogawa spinning-disc confocal head (CSU-X1). MetaMorph 7.7 software was used for image acquisition. z-Stack images were acquired using a 100×, 1.40 N.A. oil objective.

Confocal images for Figs ​ Figs5 5 – 7 and S6 and S7 were acquired using an Olympus IX83 inverted microscope with a Yokogawa spinning-disc confocal head (CSU-W1) or with a Zeiss LSM 800 microscope with a 63×, 1.40 N.A. objective with appropriate filters.

Image analysis

Mean intensity (measured in arbitrary units, a.u.), quantification of various shape parameters, and the colocalization analysis were performed using Fiji software [ 122 ]. To quantify mean intensity (Figs 2D, 2J, 2P , 3H, 3I , 4D, 4H, 4K and S3B ) for a z-plane of interest, rolling ball background subtraction was performed (radius = 50 pixels), and the polygon selection tool was used to choose the region of hyp7 in which the mean intensity was quantified.

To quantify shape parameters, a z-plane of interest was selected, and the minimum filter (radius = 10.0 pixels) was applied to the raw image. The filter-applied image was then subtracted from the raw image using the image calculator function. Images were then thresholded, and the Despeckle function was used to remove noise corresponding to 1 pixel in size. Finally, various shape parameters were quantified using the Shape Descriptor plugin.

For colocalization studies, the raw z-stack images were deconvoluted using the Wiener deconvolution algorithm (cellSense 3.1 software). The desired z-plane was extracted from both deconvoluted and raw z-stack images. Then, the Gaussian filter (radius = 10 pixels) was applied to the deconvoluted image, which was subtracted from the original deconvoluted image using the image calculator function. Next, these subtracted images were thresholded to obtain binary images to be used as masks. These binary masks were combined using the “AND” Boolean operation to the background-subtracted (rolling ball algorithm; radius = 50 pixels) raw images. Using the polygon tool, the region of hyp7 was selected in the combined images, and coloc2 plugin was used to calculate Manders’ coefficient.

The mean area and intensity of fluorescence in HeLa cells Fig 5K and 5L was obtained with Zeiss LSM Zen software, after outlining a region of interest. EEA1-positive endosome size ( Fig 6H ) was measured using Imaris software, and MICAL-L1 tubular endosome surface area ( Fig 7E ) was quantified using ImageJ software., or the number of MICAL-L1 tubular recycling endosomes were counted manually ( S8 Fig ), since number and area are generally correlated. Endosome size in the human cell studies was quantified using NIH ImageJ. Size parameters were set for 0 –infinity. Brightness parameters were selected to eliminate recognition of background by ImageJ’s particle counter while optimizing selection of true positive fluorescent pixels. Statistical analyses was performed in Prism by first testing the assumption of normal distribution with the D’Agostino and Pearson normality test. Statistical significance was then calculated with an unpaired two-tailed t-test.

Auxin treatment

Auxin (indole-3-acetic acid) was purchased from Alfa Aesar. A 100× stock auxin solution (0.4 M) was made by dissolving 0.7 g of auxin in 10 ml of 100% ethanol. A mixture of 25 μl of stock auxin solution and 225 μl of autoclaved deionized water was added to each plate containing day-1 adult worms.

Rabbit antibodies against NEK6 were obtained from Mybiosource (Cat# MBS94186), rabbit antibodies against NEK7 were obtained from Novus (Cat# 31110), mouse horseradish peroxidase (HRP)-conjugated antibodies against GAPDH were obtained from Proteintech (Cat# HRP-60004), rabbit antibodies against EEA1 were obtained from Cell Signaling (Cat# 3288), and mouse polyclonal antibodies against MICAL-L1 were obtained from Novus (Cat# H00085777-B01P).

Immunoblotting

HeLa cells were lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, and 0.5% deoxycholate. Proteins from the lysates were separated by SDS-PAGE, transferred to nitrocellulose filters, and immunoblotted with antibodies using standard methods.

siRNA knockdown

Cells were transfected with siRNA oligonucleotides from Sigma (600 nM NEK6 600 or 100 nM NEK7) using Dharmafect transfection reagent (Dharmacon) for 48 h, prior to validation of knockdown by immunoblotting using GAPDH as the loading control as described above.

All statistical tests were performed using software from Prism GraphPad.

Supporting information

Colocalization assays were carried out in adult worms expressing both NEKL-2::mNeonGreen and NEKL-3::mKate. (A–C, A’–C’, D–F, and D’–F’) Representative images of the apical region of hyp7 in adult worms expressing NEKL-2::mNeonGreen and NEKL-3::mKate (A–C and A’–C’) as well as the medial plane of hyp7 (D–F and D’–F’). Here the medial plane is considered the plane ~1 μm below the apical surface. Scale bar in A = 10 μm for A–F. Scale bar in A’ = 1 μm for A’–F’. (G, H) Manders’ coefficient was calculated and plotted for individual worms in nekl-2 :: mNeonGreen ; nekl-3 :: mKate strains. The fraction of NEKL-2::mNeonGreen puncta overlapping with NEKL-3::mKate puncta in the apical and medial plane (G) and vice versa (H) are shown. Raw data are available in S1 File .

(A–F) Colocalization assays were performed on strains expressing either NEKL-2::mNeonGreen (A–C) or NEKL-3::mNeonGreen (D–F) with APA-2::mScarlet, the alpha subunit of the AP2 adaptor complex, which is present in clathrin-coated pits. Note that these images were collected on a different confocal microscope and thus appear somewhat different than images in the paper (see Materials and Methods ). Scale bar in A = 10 μm in A–F.

(A) Representative confocal images of P hyp7 ::AMAN-2::mNeonGreen expression in the indicated backgrounds. (B, C) Mean intensity of P hyp7 ::AMAN-2::mNeonGreen expression (B) and the mean number of P hyp7 ::AMAN-2::mNeonGreen-positive vesicles (C) for individual worms were plotted in the graphs. (D) Representative confocal images of P hyp7 ::AMAN-2::mNeonGreen co-expressed with a functional multi-copy NEKL-3::mCherry reporter. Error bars represent the 95% confidence intervals. p-Values were obtained by comparing means using an unpaired t-test: ****p < 0.0001, ***p < 0.001; ns, not significant (p > 0.05). Raw data are available in S1 File .

(A–C) Three-dimensional plots showing the individual pixel intensity along the x and y planes for the fluorescent images presented in Fig 3C–3E . (D,E) Three-dimensional plots showing the pixel intensity value along the x and y planes for the fluorescent images presented in Fig 3F and 3G . We note that the basal boundary occurs in the x axes at approximately 40 μm and 10 μm whereas the seam cell boundary occurs between 20–30 μm.

Representative images of P hyp-7 ::TGN-38::GFP expression in auxin-treated day-2 adults in wildtype and cup-5 mutant background. Red arrows indicate accumulation of cargoes in vesicle-like structures. Mean intensity values of the P hyp-7 ::TGN-38::GFP expression for individual worms are plotted in the graph. Error bars represent the 95% confidence intervals. p-Values were obtained by comparing means using an unpaired t-test: ****p < 0.0001, ns, not significant (p > 0.05). Raw data are available in S1 File .

Representative images of P hyp-7 ::MIG-14::GFP expression in auxin-treated wild-type, nekl-2 :: aid , and nekl-3 :: aid day-2 adults in the apical/medial plane. Red arrows show the presence of MIG-14::GFP expression in wild-type and nekl-2 :: aid adults at the hyp7 membrane surrounding the ALM neuron. The red arrowhead indicates the absence of MIG-14::GFP expression in nekl-3 :: aid worms. Scale bar = 10 μm in the three lower-magnification images. The yellow bracket along the ALM neuron in wild type indicates the region show in the higher magnification image (yellow outline). The bar graph shows the percentage of worms exhibiting P hyp-7 ::MIG-14::GFP expression surrounding the ALM/PLM neurons in the indicated backgrounds. Numbers of worms are indicated for each bar. Error bars represent the 95% confidence interval. p-Values were obtained using Fisher’s exact test: ****p < 0.0001. Raw data are available in S1 File .

Mock-transfected cells, NEK6 siRNA-transfected cells, or NEK7 siRNA-transfected cells were plated on coverslips and immunostained with antibodies against the sorting endosome marker protein EEA1. Average sizes of early/sorting endosomes are shown for the three conditions, with significant differences observed between the mock-transfected cells and both siRNA treatments as well as between NEK6 siRNA and NEK7 siRNA-transfected cells. Yellow box in panels indicates region of enlarged inset. Scale bars = 10 μm.

Glioblastoma cells were mock transfected or were transfected with NEK6 siRNA or NEK7 siRNA. Transfected cells were then plated on coverslips and immunostained with antibodies against the tubular recycling endosome marker protein MICAL-L1. Average numbers of MICAL-L1 tubular endosomes per field were determined for the three conditions, with significant differences observed between the mock-transfected cells and both siRNA treatments. Yellow box in panels indicates region of enlarged inset. Scale bars = 10 μm.

Acknowledgments

We thank Amy Fluet for editing this manuscript.

Funding Statement

This project was supported by NIH R35 GM136236 to DSF, BBJ, SB, SC, LR and ROH (University of Wyoming), NIH R01 GM135326 to BDG, GB (Rutgers, State University of New Jersey), NIH R35 GM144102 to SC and NN (University of Nebraska Medical Center), and by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (P20GM103432) to DF, BBJ and SB (University of Wyoming). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability

• PLoS Genet. 2023 Apr; 19(4): e1010741.

Decision Letter 0

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Summary

The Fay lab previously demonstrated that the NIMA like kinases NEKL-2 and NEKL-3 localize to foci and regulate clathrin-mediated LRP-1 endocytosis at the apical membrane of the Hyp7 syncytium. Here Joseph et al further characterize the role NEKL-2 and NEKL-3 in endosome trafficking. They demonstrate that NEKL-3 colocalizes with RAB-5 and RAB-7 positive endosomes while NEKL-2 shows some colocalization with RAB-5 and much less with RAB-7. NEKL-2 and NEKL-3 do not grossly colocalize indicating that they are mostly on distinct compartments. Auxin mediated depletion of NEKL-2 resulted in tubulation of RAB-5 compartments and depletion of NEKL-3 resulted in tubulation of RAB-11 compartments. The basal recycling cargo SMA-6 (clathrin dependent) and DAF-4 (clathrin independent) accumulate at or near the plasma membrane in NEKL-2 and NEKL-3 depleted animals consistent with their role in trafficking. However, intracellular cargo receptors MIG-14 and TGN-38 which originate from the Golgi are reduced in NEKL-2 and NEKL-3 depleted animals to different degrees suggesting they are being degraded in the lysosome. Consistent with that hypothesis TGN-38 levels are increased in NEKL-2 and NEKL-3 depleted animals when endosome to lysosome trafficking is impeded in a cup-5 mutant. Consistent with the NEKL-2 and NEKL-3 results, siRNA depletion of NEK6 and NEK7 (human NEKL-3 homologs) in HeLa cells results in decreased levels of Mannose-6-phosphate receptor suggesting that it too might be targeted to the lysosome. siRNA of NEK6 and NEK7 increased EEA1 early endosome size in HeLa and cells and siNEK7, but not siNEK6 does the same in Glioblastoma cells. siNEK6 and siNEK increase the area (length?) of MICAL-L1 tubules in both HeLa and glioblastoma cells. These data suggest that there is a conserved role for NIMA like kinases in endosome fission or fusion.

This paper is well written and easy to read without notable typographical errors.

Most of the data is quantified and clearly displayed. All the image quantification data is available in an excel file.

The novelty of the study is that NIMA like kinases regulate endosome tubulation.

Experimentation in both C. elegans and human cell lines broadens the impact of the findings.

The experiments are well done.

The study is a bit of a look and see approach rather than hypothesis driven. I find the tubulation phenotypes are the most dramatic and interesting as they point toward a specific function for the NEKL-2 and 3. I would have liked to see more experiments focused on the tubulation defects. A specific role for the NIMA kinases is not determined.

Supplementary data on glioblastoma cells is not quantified.

Points of consideration

NEKL-3 appears to have a reticulate localization pattern with bright foci that colocalize with endosomes. Does NEKL-3 localize to the ER? The ER contacts many membrane-bound organelles and is implicated in fission of these compartments. The roles of NEKL-2 and NEKL-3 in membrane scission suggests that they could localize to ER::endosome contacts to regulate endosome fission. This would be an interesting avenue to pursue in the future.

Mutation of cup-5 increases TGN-38 levels in NEKL-2/3 depleted animals. Does it also increase TGN-38 levels in a wild-type background? In other words, is it specific to the NEKL depleted animals?

Is kinase activity required for NEKL-2 and 3 function?

Figure 3. Is it possible to discern the basolateral membrane in wild-type animals as a point of reference using arrows? There is clearly an accumulation of SMA-6 and DAF-4, however the boundary is not clear in wild-type.

Reviewer #2: This manuscript submitted to PLoS Genetics by Joseph et al. reports the functions of two conserved NIMA-related kinases, NEKL-2 (NEK8/9 homolog) and NEKL-3 (NEK6/7 homolog), in endocytic trafficking in both C. elegans and mammalian tissue culture cells. The Fay Lab previously reported that NEKL-2 and NEKL-3 are essential for C. elegans molting and that they regulate apical clathrin-mediated endocytosis in the epidermis. In this study, the authors found that NEKL-2 and NEKL-3 are differentially localized to, and regulate the morphology of, distinct endosomal compartments. They also provided evidence that NEKL-2 and NEKL-3 control the sorting of cargoes between different intracellular compartments, and the endocytosis of two BMP receptors from the basolateral surface. Furthermore, they showed that knocking down the NEKL-3 homologs in human cell lines also affects endosomal morphology, suggesting that the functions of these NIMA related kinases in regulating endocytic trafficking is conserved.

The manuscript is very well written. It has identified new roles for members of the NIMA family of kinases, which are important in human development and disease. I have one main concern. The authors showed that depletion of NEKL-2 or NEKL-3 affects the uptake of both SMA-6, a clathrin-dependent cargo, and DAF-4, a clathrin-independent cargo, from the basolateral surface of epidermal cells. The authors speculated that these two receptors may be trapped in early endosomes near the basolateral surface. In light of their previous findings that NEKL-2 and NEKL-3 regulate apical clathrin-mediated endocytosis in the epidermis, it will be important to determine whether additional clathrin-independent cargoes are also affected upon NEKL-2 or NEKL-3 depletion. Furthermore, the data (at least those presented in Figure 2) do not seem to show accumulation of early endosomes upon NEKL-2 or NEKL-3 depletion. Maybe the accumulation of SMA-6 and DAF-4 is not due to defects in intracellular trafficking upon NEKL-2 and NEKL-3 depletion, rather, due to some other un-identified role(s) of NEKL-2 and NEKL-3 on the cell surface.

I have several additional comments listed below:

1) I wonder if Figure 1 is somehow mis-labeled. The images and the quantifications do not seem to match. The authors concluded, based on the quantification shown in panel M, that NEKL-2 has substantial co-localization with the early endosome marker RAB-5, but not with the late endosome marker RAB-7, while NEKL-3 has substantial co-localization with both RAB-5 and RAB-7. However, based on the images shown in Figure 1, NEKL-2 (but not NEKL-3) seems to have substantial co-localization with both RAB-5 and RAB-7 (1I’ and 1L’), while NEKL-3 appears to primarily co-localize with RAB-7 (1C’ and 1F’).

2) The NEKL-2::mNeonGreen and NEKL-3::mNeonGreen signals shown in Figure S1 appear to be significantly brighter than those shown in Figure S2. Is this due to differences in different focal planes imaged or to other reasons?

3) In Figure 3, the diameters of NEKL-3::AID worms (panels E and G) seem to be much smaller than those of WT or NEKL-2::AID worms (panels C, D and F). Is this difference due to differences in focal planes and/or stages of the animals examined, or due to NEKL-3::AID worms being unhealthy after NEKL-3 depletion?

4) Based on the western blots shown for both NEK6 and NEK7 in Figures 5-7, there seems to be substantial variation in the efficiency of the siRNA knockdown experiments. Is there any correlation between the knockdown efficiency and the severity of the phenotypes observed? Along the same line, the antibodies for NEK7 used for the western blots shown in Figures 5-7 seem questionable. It is unclear which band is specifically NEK7. In Figure 5, NEK7 appears to be the bottom one of two bands, while in Figure 6, it appears to be the middle one of three. Surprisingly, the intensities of the bottom two bands are both drastically reduced in Figure 7. The authors may want to either repeat the western blots, or comment on the specificity of the antibody.

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy , and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

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Author response to Decision Letter 0

Submitted filename: Response to reviewers.pdf

Decision Letter 1

11 Apr 2023

We are pleased to inform you that your manuscript entitled "Conserved NIMA kinases regulate multiple steps of endocytic trafficking" has been editorially accepted for publication in PLOS Genetics. Congratulations! 

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23 Apr 2023

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Entrepreneurs

Nima yamini, pn & friends & fake paid articles.

“Nima Yamini is a TikToker, Co-Founder of P.N. & Friends, and Founder of Yamini LLC. Currently, he acts as a comedian content creator and self proclaimed Alpha King in various podcasts” – Bio from Paid Articles

Nima Yamini’s Paid PR articles and Paid success stories revolve predominantly around his tech startup journey, in collaboration with his partner, Pablo Heman . His SPONSORED articles primarily center on the promotion of P.N. & Friends , a tech startup dedicated to providing specialized training to individuals interested in the fields of stocks and cryptocurrency investment. Within these articles, Nima Yamini delves into the narrative of how he and Pablo Heman discovered their profound passion for cryptocurrency and stocks, which ultimately spurred them to share their wealth of knowledge and experiences through their innovative startup.

Nima Yamini and Pablo Heman have already amassed a substantial following and garnered trust through their educational tips and investment insights on TikTok and Instagram. Now, by incorporating a selection of sponsored success stories and featured articles into their profiles, they aim to bolster their credibility and further strengthen their self-promotion. Notably, they are strategically sharing these resources within their Discord Community, which they aptly label as “Featured” or “As Seen on” rather than saying the fact that ” it is paid for ” Please refer to the accompanying screenshot from their community for where the Moderator proudly shows the paid article as part of their organic media coverage.

While Nima Yamini undeniably possesses genuine skills and accomplishments, it’s important to note that the below-featured interviews and success stories below his profile are sponsored & paid. These articles are self-authored, fabricated, and are the product of paid PR placements, which can be secured for a fee ranging from $500 to $2500, depending on the publication or purchased in Package.

Furthermore, it’s worth mentioning that Nima Yamini’s TikTok account has been banned recently. Consequently, he has begun sharing content highlighting the toxic aspects of TikTok. Apart from that he also made sure that our old article was taken down on grounds of copyright violation. Our old article was removed on copyright grounds. But It is difficult to argue against facts and no one cannot refute what is true.

SOCIAL PRESENCE: https://www.instagram.com/nimayamini [ Verified due to below Paid Articles ] https://www.pnandfriends.com/ https://nimayamini.com/ https://www.instagram.com/pablo_heman/ https://www.linkedin.com/in/nima-yamini-3772a317a https://twitter.com/nimayamini

LET’S ANALYZE THE PAID ARTICLES PUBLICATIONS:

Recognizing the significance of self-branding, Nima Yamini, an active social media content creator, allocates a substantial budget towards personal branding endeavors which is a really good strategy if you are into Digital space that includes the acquisition of paid feature articles and success stories. The rationale is straightforward – these articles not only contribute to social media verification but also serve as valuable assets for promoting his image. Eventually, he achieved his IG Verification in April 2021 with the help of paid mentions.

Nima Yamini manages a thriving community comprising over 30,000 users, making these paid articles a savvy investment. They function as persuasive marketing collateral, reinforcing the credibility of their services among community members. Nima Yamini and Pablo Heman’s articles are prominently featured in reputable publications renowned for their branded content. Interested individuals can procure these paid placements directly from these publications. Additionally, some of these opportunities can be secured through freelancing platforms such as Fiverr.com or PeoplePerhour.com, typically ranging from $150 to $500.

These publications readily accept submitted content and publish it on their platforms with a disclaimer. To illustrate, the cost for an article on Flaunt is $800, on Khaleej Times it’s $600, and for Disrupt Magazine, the fee is just $25.

https://flaunt.com/content/nima-yamini [ Deleted ] https://www.khaleejtimes.com/kt-network/entrepreneur-nima-yamini-on-the-lessons-he-learnt-as-a-tech-co-founder-future-plans-and-more https://disruptmagazine.com/viral-tiktok-stars-nima-yamini-and-pablo-heman-launch-online-learning-company-to-teach-investment-cryptocurrency-and-nfts/ https://www.vesterholdings.com/news/learn-how-these-entrepreneurs-explained-to-the-world-the-intricacies-of-crypto-investing/ https://britonian.co.uk/2021/08/06/tech-startup-p-n-friends-co-founded-by-nima-yamini-and-pablo-heman-passes-30000-members/ https://celebmix.com/pablo-heman-and-nima-yamini-become-the-go-to-gurus-on-tiktok-for-financial-literacy/ https://miamiwire.com/esteemed-entrepreneur-and-finance-expert-nima-yamini-builds-impressive-educational-blockchain-based-community/ https://ceoweekly.com/nima-yamini-helping-young-people-carve-successful-financial-paths-through-his-company-pn-friends/ https://www.vizaca.com/nima-yamini-pablo-heman-success/ https://thriveglobal.com/stories/nima-yamini-managing-partner-yamini-llc-and-pablo-heman-tiktok-influencers/ [ Anyone can register and Post the content ]

NIMA YAMINI AND PABLO HEMAN ON ENTREPRENEUR.COM. HOW?

Securing a feature on Entrepreneur has become more accessible, provided you can cover the cost and have the appropriate connections in public relations (PR). Entrepreneur.com offers the opportunity for individuals to publish content within their BRAND CONTENT program for a fee ranging from $1500 to $2000, attributing it to the desired author’s name. It’s important to note that if the content pertains to cryptocurrency, it will include a disclaimer prominently displayed at the conclusion of the article.

https://www.entrepreneur.com/article/402787

NIMA YAMINI AND PABLO HEMAN ON FORBES. HOW?

Forbes boasts regional publications like Forbes India and Forbes Monaco, both of which offer the opportunity for sponsored content. In the case of Forbes Monaco, you can publish your advertising content for a fee of $3000 . Essentially, this avenue prioritizes financial investment over the presence of a genuine story or established credibility.

Notably, Forbes Monaco articles support verification, which has led many PR service providers to include Forbes Monaco in their packages. The association with Forbes Monaco contributes significantly to one’s credibility. Therefore, it’s a sensible choice to consider incorporating it into your profile. However, lately, Forbes Monaco has been shut down by Forbes.

https://forbes.mc/article/how-nima-yamini-pablo-heman-two-former-finance-professionals-built-an-online-investment-community

We look forward to more of their Paid Articles

DISCLAIMER: This article is submitted by the user registered on our website. The Website or Admin is not responsible for the content submitted by the users. Images or Links in this Article are collected from search Engines and public domains. All images are copyrighted to their respective owners. Content published in our publication is a journalistic opinion and the information on this site is for information purposes only. Please contact us for any copyright issues.

© 2020 Let me Expose. All Rights Reserved

Oluwaseun Adeyanju, Forbes Contributor & Paid PR Mentions

Mike Che Bugembe, Forbes Contributor & Paid PR Mentions

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Sped-Up Remixes Ruled TikTok in 2023

Up-tempo re-works of tracks by Justine Skye, FIFTY FIFTY, and Toosii dominated in the U.S., according to TikTok's year-end report

By Elias Leight

Elias Leight

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Sped-up remixes continue to resonate on TikTok : The four most popular songs on the platform in the U.S. in 2023 were all sped-up, according to TikTok’s year-end report. The leader of the pack was the “more sped-up” version of Justine Skye’s “Collide,” followed by sped-up renditions of FIFTY FIFTY’s “Cupid,” PARTYNEXTDOOR’s “Her Way,” and Toosii’s “Favorite Song.”

Sped-Up Songs Are Taking Over TikTok and Driving Songs Up the Charts

The sped-up remixes that zip around TikTok are usually made first by creators (sometimes funded by label marketing efforts). If they start to perform well, it’s become routine for labels to release their own official versions. 

“Back in the day, we used club remixes to diversify the visibility of a record,” Nima Nasseri , who then served as global head of A&R strategy for Universal Music Group’s music strategy and tactics team, explained in 2022. “The purpose was to bring back visibility to the main version. Now people are discovering the main version from the sped-up or slowed one. Instead of spending $50,000 for a remix from a big-name DJ, you’re spending relatively minimal amounts [on a sped-up rendition] and getting much more return and reach.”

With Sped-Up Songs Taking Over, Artists Feel the Need for Speed

Why have these simple remixes proved consistently effective? Steven Pardo , digital marketing director at Secretly Group, told Billboard in 2022 that “in a video platform that prioritizes catching attention immediately, being able to get the impact of the lyrics across more quickly is advantageous.” 

Scott Plagenhoef , global head of music programming at Apple Music, echoed this sentiment during an interview with Billboard in March: “Sped-up songs allow for more of a track to be heard within the time constraints of a TikTok video and mirror the pace at which users consume content online.”

In the last 15 months, sped-up remixes have spurred chart surges for Thundercat ‘s “Them Changes,” Miguel ’s “Sure Thing” (actually a resurge, as it first charted over a decade ago), The Weeknd ’s “Die for You,” Lady Gaga ’s “Bloody Mary,” Mariah Carey ’s “It’s a Wrap,” and more.

TikTok & Billboard Partner to Launch TikTok Billboard Top 50 Chart

Due to TikTok’s popularity and its ability to drive streaming activity, Billboard launched a TikTok Top 50 chart in September, ranking tracks on the platform according to a combination of creations, video views and user engagement in the U.S. “The chart gives a clear picture of the music that is being listened to on TikTok, and consequently starting to trend on DSPs and other services,” Ole Obermann , global head of music business development at TikTok, said in a statement.

Across the first two months of the chart, hip-hop proved to be by far the most popular genre, accounting for more than 35% of chart entries. Pop was next, hovering at 20%, largely thanks to Taylor Swift , who had nine different charting tracks in the first eight weeks. The third most popular genre was R&B (10%).  

U.S. listeners did listen to some music at its original tempo, according to TikTok’s year-end report. PinkPantheress and Ice Spice ‘s “Boy’s a Liar Pt. 2,” Ohboyprince’s “Bounce When She Walk,” Young Nudy and 21 Savage’s “Peaches & Eggplants,” Ice Spice’s “In Ha Mood,” Jain’s “Makeba,” and Swift’s “Cruel Summer” rounded out the rest of the top 10.

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What is NIMAS?

NIMAS refers to the National Instructional Materials Accessibility Standard. It is a provision of the Individuals with Disabilities Education Act (IDEA) and was endorsed by the U.S. Department of Education as the preferred approach for publishers to provide accessible curricular materials to students in grades K-12. NIMAS guides the production and electronic distribution of digital versions of textbooks and other instructional materials so they can be more easily converted into accessible formats.

By December 2006, all state and local education agencies needed to establish a means of purchasing and distributing these materials and the U.S. Department of Education established the National Instructional Materials Access Center (NIMAC) as a repository for files provided by publishers. This legislation was a step forward in providing accessible materials for students with disabilities in elementary and secondary educational settings. It was not mandated for public universities as students in postsecondary settings are protected under provisions of the Americans with Disabilities Act (ADA) and Section 504 of the Rehabilitation Act of 1973 , not IDEA.

In the past, instead of being provided in quantities that met the need of the community, accessible materials were often provided on an individual basis, frequently untimely (Pisha and Stahl, 2005). Previously, it was difficult for students with disabilities to anticipate in what format their materials might arrive. With NIMAS, there is a minimum standard for schools and publishers to follow when creating these educational materials.

Source: Pisha, B., & Stahl, S. (November 2005). The Promise of New Learning Environments for Students with Disabilities. Intervention in School and Clinic , 41 (2), 67-75.