A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance
Authors: Daniel T Montoro
Martin S Taylor
Gerard W Dougherty
Hanan E Shamseldin
Evgeni M Frenkel
Lida P Hariri
Raghu R Chivukula
Ibrahim Almogarri
Patrick R Sears
Vladimir Vinarsky
Maimoona A Zariwala
Guillermo J Tearney
Hui Min Leung
Jason Yang
M Leigh Anne Daniels
Michael R Knowles
Heymut Omran
David M Sabatini
Katharine E Black
Fowzan S Alkuraya
Johnny Carson
Journal: Nature Medicine (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Mucociliary clearance; Bronchiectasis; NEK10; Ciliary cells; Respiratory disorders
Affiliations: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. ., Whitehead Institute for Biomedical Research, Cambridge, MA, USA. ., Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. ., Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA. ., Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. ., Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA., Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA., Harvard Medical School, Boston, MA, USA., Whitehead Institute for Biomedical Research, Cambridge, MA, USA., Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA., Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA., Department of Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia., Department of Pathology, Massachusetts General Hospital, Boston, MA, USA., Department of General Pediatrics, University Children's Hospital Muenster, Münster, Germany., Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA., Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA., Division of Pulmonary Diseases and Critical Care Medicine, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA., Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA., Division of Pulmonary and Critical Care Medicine, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA., Department of Pediatrics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia., Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA., Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA., Department of Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia. .

Abstract:  Mucociliary clearance, the physiological process by which mammalian conducting airways expel pathogens and unwanted surface materials from the respiratory tract, depends on the coordinated function of multiple specialized cell types, including basal stem cells, mucus-secreting goblet cells, motile ciliated cells, cystic fibrosis transmembrane conductance regulator (CFTR)-rich ionocytes, and immune cells1,2. Bronchiectasis, a syndrome of pathological airway dilation associated with impaired mucociliary clearance, may occur sporadically or as a consequence of Mendelian inheritance, for example in cystic fibrosis, primary ciliary dyskinesia (PCD), and select immunodeficiencies3. Previous studies have identified mutations that affect ciliary structure and nucleation in PCD4, but the regulation of mucociliary transport remains incompletely understood, and therapeutic targets for its modulation are lacking. Here we identify a bronchiectasis syndrome caused by mutations that inactivate NIMA-related kinase 10 (NEK10), a protein kinase with previously unknown in vivo functions in mammals. Genetically modified primary human airway cultures establish NEK10 as a ciliated-cell-specific kinase whose activity regulates the motile ciliary proteome to promote ciliary length and mucociliary transport but which is dispensable for normal ciliary number, radial structure, and beat frequency. Together, these data identify a novel and likely targetable signaling axis that controls motile ciliary function in humans and has potential implications for other respiratory disorders that are characterized by impaired mucociliary clearance. 

Retracted: No

PubPeer comments:
 Extended Fig. 5b (NEK10) doesnu2019t match with the raw figure provided by the authors. It is very surprising that Nature Editors Reviewers didnu2019t check the raw data provided by authors... Could the authors check? **Itu2019s important to note that not only the pictures donu2019t match, but itu2019s pattern also not. In the published Fig. 5b NEK10 expression is decreased in de-ciliated ALI in the NEK10G C condition, while on the original scans itu2019s expressed in the same levels as de-ciliated ALI WT.** ![file] , 
 Dear Puniceicoccus, Thanks for your message and question. Iu2019ve now had a chance to look into the point you raised. Extended Data Figure 5b is correct as presented. The associated raw data file (uncropped image) does in fact depict the correct source blot but is a shorter film exposure than in the Figure. The bands in question in the uncropped blot are indeed different in morphology and intensity than in the Figure, as they represent an abundant but non-specific protein with which this antibody cross-reacts (as in Extended Data Figure 1d, for example). The longer exposure uncropped blot makes clear that the specific bands (correctly sized at ~133kD) are identical to those in Extended Data Figure 5b and illustrates that NEK10 is severely depleted in G C mutant lysates. A figure highlighting this is attached, as is an updated raw data file - both have been downsampled in line with Pubpeer image size limits. I do apologize that this mismatched exposure was included as raw data and will reach out to amend the file online with Nature Medicine. Thanks again for your interest in our work. Best wishes, Raghu ![file] ![file] , 
 The bands in the figure do not match the ones in the new raw data provided. , 
 #2 The intensity of the lowest  non-specific bands clearly show that there was unequal loading of the lanes being compared, with the left having 2X as much protein as the right one. , 
 #2 Acetylated tubulin and NEK10 are about the same size - 55KD. Raptor is 140KD. Matching up the marker dots on the blots shown here reveals that the original poster is likely correct. The exposure shown in the original raw data is correct in showing NEK10, for which there is no discernible difference in expression between the samples shown. The author is urged to reflect on this matter. , 
 Salsola Zygophylla is incorrect. Nek10 exists in several different isoforms, with the full length NEK10 being a 1173aa protein. https: www.ncbi.nlm.nih.gov protein Q6ZWH5 However, most antibodies are raised against a partial NEK10 protein, and detect the isoform variant which is only ~55kD: https: www.ncbi.nlm.nih.gov protein NP_001026911.1 And this isoform can be very abundant, hence the ease of detection. I do not know where Raghu s argument that the band detected and shown in the original extended data is non-specific. It could be a NEK10 protein isoform as there are several. Was the non-specific band ever extracted and submitted for mass spec? If not or if the antibody has not undergone more thorough testing in a line with large-scale deletion of NEK10 or at least the portion of NEK10 correesponding to the antibody s targeted epitope I would argue that the comment  abundant but non-specific protein is indefensible. The conclusion of the paper may be unaffected if NEK10 longest isoform is responsible and of interest. , 
 Dear u201cFlexamiau201d and u201cSalsolau201d, Respectfully, the bands in the uncropped blot do, in fact, match those in the figure. In the interests of further clarifying this, the attached figure includes all 3 images (cropped Figure image from journal .JPG, uncropped short exposure scan, and uncropped long exposure scan) resized and annotated for comparison. The region corresponding to full length NEK10 in the uncropped blot is highlighted in red, making it clear where the bands in Extended Data Fig 5b originate. With regard to the size of full-length NEK10 in human airway, this is an interesting question we spent a significant amount of time and effort on. In particular, given the less than stellar quality of available NEK10 antibody reagents, we were careful to validate their performance. Based on multiple lines of evidence outlined below, we believe that full length NEK10 in the airway is a ~133kD protein. In order of appearance in the paper, these data include: 1. Quantitative reverse-transcriptase PCR (qRT-PCR) on human airway tissue as well as HBECs and ALI, with independent amplicons targeting all previously annotated transcript variants. These data demonstrate that all portions of the NEK10 locus are transcribed in airway and indicate roughly equal abundance of 5u2019, 3u2019, and central exons in cDNA - arguing against short isoforms being present and strongly suggesting the full length (4.2kb) transcript is the sole or dominant species (Fig 1d). The cDNAs we cloned predict a 1172 amino acid protein with estimated mass of 133kD (ED Fig 1c). 2. Exactly as predicted by (1), immunoblotting reveals a ~133kD band in ALI lysates but not in HBEC lysates (Fig 1e, ED Fig 1d). All bands in the HBEC condition are, by definition, non-specific cross-reacting proteins since no NEK10 transcript (of any isoform) is detectable in these cells (Fig 1d). Therefore, the only band induced by transcription of the NEK10 locus is the 133kD one and this species is detectable with 2 independent antibodies raised against completely different epitopes (rabbit anti-N-terminus, mouse anti-C-terminus) within the NEK10 protein. 3. NEK10G C mutant ALI lysates (in which NEK10 function is lost as evidenced by multiple functional assays and complementation by NEK10 add-back) show a specific loss of the ~133kD band with no consistent changes in any of the non-specific lower molecular weight bands with either of 2 antibodies (Fig 1e). 4. Exogenous expression of epitope-tagged NEK10 increases the abundance of the ~133kD species as detected by immunoblotting against NEK10 and against the FLAG epitope but not of non-specific bands (Fig 1g). 5. CRISPR Cas9-mediated NEK10 inactivation with 3 independently targeting guide RNAs results in the loss of the ~133kD species but no change in any of the non-specific lower molecular weight bands (ED Fig 3d). In summary, in airway epithelium NEK10 appears to be expressed predominantly as a 4.2kb transcript encoding a ~133kD polypeptide, the identity of which we have validated with multiple antibodies, correlation with transcript expression and cDNA cloning, patient data, experimental loss-of-function, and experimental gain of function. Such data cannot, of course, completely rule out a minor isoform of lower molecular weight or a biologically active NEK10 proteolytic cleavage product, but we have not uncovered evidence for these. We have also not explored whether other cell type and tissue contexts might utilize alternate transcriptional start and stop sites within this locus, but this would be an interesting area for future work. Best wishes, Raghu ![file] *Edited to add in-line citations , 
 There seems to be sufficient confusion regarding what forms of NEK10 were present in the cells studied here to throw the conclusions into considerable doubt. According to the Methods, the primary antibodies used for WBs in this study were: 1) Rabbit anti-NEK10 (Atlas Sigma HPA03894157), which is not validated by the manufacturers for WB. The target antigen sequences used to raise this antibody is reported by the manufacturer to be aa338-465 of NCBI Reference Sequence NP_689747.3 (i.e. predicted full-length NEK10, 1172aa, 133kD). This sequence is shared by several reported predicted isoforms; and this antibody has been reported to detect a 53kDa protein in HEK cell lysates (Garcia-Diez et al. Genes Chromosomes Cancer 2019; 58:164u2013174). 2) Mouse anti-NEK10 Sigma WH0152110M1, which is described by the manufacturer as being raised against aa 211-300 of NP_689747. Blots on the manufacturer site show this antibody detecting proteins with kD sizes of 40, 50, 55, and 70. In Extended Figure 1d of this paper, we are shown probings with two antibodies: ![file] The legend states these blots show:  Immunoblotting against indicated NEK10 epitopes, representative of three experiments. HBEC bands are non-specific. Full-length 133u2009kDa NEK10 protein is indicated with a dashed box. From the Methods and descriptions by the antibody manufacturer, I cannot determine which antibody is specific for which terminus, nor can I see how all of the other bands can be dismissed as being non-specific, especially since two different antibodies appear to be lighting up several of the same size. The lack of protein loading controls is also problematic. , 
 Dear u201cSalsolau201d, The NEK10 RefSeq transcript NM_152534 corresponds to the cDNA we cloned from human airway. The 3519bp open reading frame would be translated to yield a 1172 AA protein. As already mentioned, we used 2 independently raised antibodies to probe for this protein: A. Rabbit polyclonal anti-NEK10, Sigma HPA038941 (not HPA03894157, not sure what this refers to). Immunogen: LHILQGDRNFVSDHSSIGSLSSANAAGRIQQLHLSEDLSPREIQENTFSLQAACCAALTELVLNDTNAHQVVQENGVYTIAKLILPNKQKNAAKSNLLQCYAFRALRFLFSMERNRPLFKRLFPTDLF An alignment reveals this peptide to map to residues 338-465 of full length NEK10. B. Mouse monoclonal anti-NEK10, Sigma WH0152110M1. Immunogen: RYFMEANRNTVTCHHELAVLSHETFEKASLSSSSSGAASLKSELSESADLPPEGFQASYGKDEDRACDEILSDDNFNLENAEKDTYSEVD Alignment reveals this peptide to map to residues 814-903 of full length NEK10, not residues 211-300 as proposed. I wonder if an annotation mixup might have led to this error (?), but this would need to be discussed with Sigma. At any rate, the point remains that the rabbit antibody is raised against an epitope within the putative N-terminal u201cisoformu201d and the mouse antibody against the putative C-terminal u201cisoformu201d, a fact which can be checked easily by any interested reader using the above sequences. To make this even more clear, the attached figure illustrates where each immunogen maps in the locus and indicates expected molecular weights for the putative proteins. This should put to rest any possible lingering confusion about antibodies used in the paper, though everything I have just outlined is either in the Methods or available publicly. In addition, contrary to your comment, protein loading control for the cited blot is indeed present within Figure 1 itself; ED Fig 1d is simply the uncropped, long exposure of Fig 1e (also making rather illogical the unfounded insinuation by others that we would intentionally omit a nearly identical uncropped long exposure blot later in the paper). ![file] This point aside, we can return again to the question of full-length NEK10, though I have rather extensively covered this in a prior response. Briefly, the gold standards for validating antibodies are (1) examining immunoblotting in biological negative controls versus samples expressing the gene and (2) experimentally disrupting the gene and examining change in immunoblotting pattern. Simply assuming that the most abundant or reactive band is specific is of course unwise, as the intended target of an antibody may be unabundant, post-translationally modified, etc. Indeed, both NEK10 antibodies recognize several bands u2013 clearly not all of them are NEK10, and one must experimentally identify which are specific. While no such validation appears to have been done by the manufacturer (not uncommon), we have performed both methods: (1) HBEC cells do not express any NEK10 transcript as measured by qRT-PCR and the gene is induced transcriptionally during airway differentiation. We therefore have a very convenient biological negative control with which to evaluate these antibodies. Examination reveals that all bands other than the 133kD species are present in cells which do not transcribe this gene. Therefore, any claims that alternate sized species represent full-length NEK10 would need to explain how this protein could possibly be generated in the absence of its mRNA (as well as why a species of exactly the predicted molecular weight would appear upon mRNA expression). It is certainly possible that other cell and tissue types express alternate forms of this protein, though these would also need to be validated as well. Our data are completely agnostic to this possibility as we have only studied airway epithelium in this work. That said, I would direct the interested reader to examine the exons which comprise the supposed transcription start site of the C-terminal isoform and the supposed transcription stop site of the N-terminal isoform - both are very poorly conserved, whereas the exons of full-length (133kD) NEK10 are highly conserved among vertebrates as expected for a genuine, biologically relevant coding sequence. (2) When the NEK10 locus is targeted by any of 3 CRISPR Cas9 guides (but not irrelevant control guide RNA), it is the 133kD band which disappears while lower molecular weight species remain unchanged (ED Figure 3). These CRISPR knockouts fully phenocopy the patient LOF phenotype both morphologically and functionally and disrupt the ciliary proteome. The 133kD species is therefore biologically relevant and dependent on the intact NEK10 locus, whereas the lower molecular weight ones are not. Claims that alternate sized species represent full-length NEK10 in airway would need to explain how their abundance could remain unchanged in the setting of extensively phenotypically validated knockout of the gene. I hope this is helpful and clarifying. I am somewhat surprised by skepticism that commercial antibodies might perform poorly; this is the rule rather than the exception in my experience. In any case, in the interests of avoiding a third lengthy and largely repetitive reply I am going to refrain from further posts on this topic unless new, substantive discussion about the work arises which incorporates the above points. I do genuinely appreciate being made aware of the original oversight on our part (inclusion of a mismatched blot exposure in the uncropped file). I certainly donu2019t claim to be perfect and am happy to engage in good faith dialogue about our work, but the Internet can be a challenging venue for this at times. Best wishes, Raghu , 
 #8, #9 It is understandable that there might have been some confusion about Extended Fig 1d, as the figure legend is quite brief and does not spell out all of the details necessary for the interpretation. Thankfully, I find that the author s response in #9 has thoroughly and convincingly clarified the uncertainties expressed by Salsola in #8. The logic is sound, and the basis for assigning the 133 kDa band as the full-length protein (and other bands as non-specific) is now abundantly clear. I appreciate the author s efforts to clarify matters here, and I agree that the matter seems settled. , 
 The folks at the Human Proteome Atlas take antibody validation rather seriously, hence my reluctance to accept that HPA038941 performed so poorly in your hands. FYI, their validation page is here: https: www.proteinatlas.org ENSG00000163491-NEK10 antibody#WB I find it a bit odd that an antibody validated for IHC IF was not used for that purpose in your study, but I guess according to you those results would not be trustworthy anyway. As for the mouse monoclonal, it does look like Sigma got that one wrong. Odd that BLASTing either epitope sequence only turns up NEK10 putative isoforms. ,