FAM83D directs protein kinase CK1α to the mitotic spindle for proper spindle positioning

Abstract The concerted action of many protein kinases helps orchestrate the error‐free progression through mitosis of mammalian cells. The roles and regulation of some prominent mitotic kinases, such as cyclin‐dependent kinases, are well established. However, these and other known mitotic kinases alone cannot account for the extent of protein phosphorylation that has been reported during mammalian mitosis. Here we demonstrate that CK1α, of the casein kinase 1 family of protein kinases, localises to the spindle and is required for proper spindle positioning and timely cell division. CK1α is recruited to the spindle by FAM83D, and cells devoid of FAM83D, or those harbouring CK1α‐binding‐deficient FAM83D F283A/F283A knockin mutations, display pronounced spindle positioning defects, and a prolonged mitosis. Restoring FAM83D at the endogenous locus in FAM83D −/− cells, or artificially delivering CK1α to the spindle in FAM83D F283A/F283A cells, rescues these defects. These findings implicate CK1α as new mitotic kinase that orchestrates the kinetics and orientation of cell division.

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As you can see, both referees express interest in the proposed role of FAM83D and CK1α in mitotic spindle positioning. However, they also raise concerns that need to be addressed in full before we can consider publication of the manuscript here.
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Referee #1:
Fulcher and coworkers identify a mitosis-specific interaction between FAM83D and CK1α that recruits CK1α to the mitotic spindle where it phosphorylates FAM83D (and presumably other substrates) to regulate spindle alignment. The experiments are in general rigorous and done using state of the art elegant methods. The text is clear and easy to read. The figures are well assembled. The conclusions are interesting and provide an important insight into how the ubiquitous CK1α can do so many things.
A significant limitation of the manuscript is that virtually all studies were performed in a single cell type, U2OS. I would suggest that several key interaction studies be performed in other cell types as well to test if this is generalizable.
Other comments: Multiple antibodies against several of the proteins were used and it isn't clear which antibody was used in which experiment; e.g. for CK1α, there are three sources. For example, in Fig EV4, which of these was used in the various immunoblots? Anti-CK1α (cat.: A301-991A, 1:1000) was from Bethyl anti-CK1α (SA527, 3rd bleed, 1:1000) Dundee immunofluorescence, anti-CK1α (cat.: sc-6477, 1:100) was from Santa Cruz I strongly suggest the antibody section of the methods contain a table of the antibodies used listing the antigen (including epitope when known), source/catalog number, species (rat, mouse, rabbit, alpaca, etc.), dilution, which figure the antibody was used in, and importantly, a reference to demonstrate the validation of the specific lot of antibody. For example, how was this lot of Santa Cruz antibody validated? This may require an additional figure with validation of each antibody, or simply reference to loss of signal in a knockout or shift in molecular weight with addition of a tag (e.g. as in Fig EV4). This may sound picky but especially with a discontinued Santa Cruz antibody from a company with a sordid history of quality control problems, this is important. It would be helpful if the authors would speculate why CK1α only binds to FAM83D in mitosis. Could this be because of HMMR, or perhaps phosphorylation of FAM83D by a CDK?

Referee #2:
The correct positioning of the mitotic spindle is the key for error-free cell division and the proper development. In most instances, accurate spindle positioning requires either the interaction between the astral microtubules and the cortical dynein and/or robust subcortical actin cytoskeleton. In this manuscript, the authors uncovered that FAM83D (CHICA) specifically interacts with CK1α during mitosis, where CK1α localizes prominently enrich at the spindle pole with the help of FAM83D. Also, cells that lack either FAM83D, or FAM83D interaction with CK1α show a substantial delay in mitotic exit, and also reveal spindle positioning defects. Interestingly, artificial delivering CK1α in cells expressing a mutant form of FAM83D that is unable to interact with CK1α partly rescue both mitotic delays as well as spindle positioning. This manuscript by Fulcher et al., is interesting as the authors have attempted to characterize the function of CK1α in spindle orientation in mammalian cells. The strong point of this work is the discovery of a new kinase (CK1α) in spindle positioning in human cells. The weakness is that no attempt has been made to study how CK1α localization at the mitotic spindle influence spindle positioning (molecular mechanism; and see few major points related to that). Therefore, I feel that because of the already existing literature related with the FAM83D (CHICA) on mitotic delay, and spindle positioning, the scope of this study is somewhat limited until authors put substantial efforts in illustrating the mechanism by which CK1α regulates spindle positioning in mammalian cells.
Major points: 1. Control cells when grown on L-shape micro-patterns cell position their mitotic spindle along the hypotenuse and the authors observed significant spindle orientation defects in cells lacking FAM83D or expressing FAM83D(F283A). Interestingly the impact of FAM83D(F283A) on spindle orientation was rescued in cells where CK1α was targetted to the spindle with the help of FAM83D(F283A). CK1α-mediated phosphorylation may regulate spindle orientation in control cells downstream of FAM83D, can optogenetic targetting of CK1α in FAM83D knock out cells [not in FAM83D(F283A)] rescue spindle orientation as well as a mitotic delay? Also, since at many instances a delay in the mitotic progression (metaphase to anaphase transition) can impact spindle orientation, it may well be that the spindle orientation defect in the absence of CK1α at mitotic spindle is rather indirect. 3. Also, in Figure 4C and 4D can authors should analyze the stability of FAM83D upon mitotic exit in the presence of MG132 (RO-3306+MG132), this would be crucial to address whether proteasome alone or mitotic exit based posttranslational modification are at play for controlling FAM83D stability.
4. Authors show that CK1α is unable to directly phosphorylate FAM83D, however the CK1α interaction with the FAM83D is must for the electrophoretic mobility shift, and authors suggests that priming phosphorylation may be required for FAM83D phosphorylation by CK1α. Since several mitotic kinases localize to the spindle, and several specific inhibitors are available to test the function of such mitotic kinases, it would be straightforward to test which kinase-mediating priming is crucial for such mobility shift.

A significant limitation of the manuscript is that virtually all studies were performed in a single cell type, U2OS. I would suggest that several key interaction studies be performed in other cell types as well to test if this is generalizable.
Response: We thank the referee for highlighting the limitation. In the original manuscript, in addition to the U2OS cell data, we also showed that CK1α localises to the mitotic spindle in mouse embryonic fibroblasts derived from wild type but not HMMR knockout mice ( Figure  4I). We have now expanded endogenous FAM8D:CK1a interaction assays to three additional human cancer cell lines -HeLa, A549 and HaCaT -and show in all cases that FAM83D is phosphorylated (as shown by the mobility shift) in mitosis, and interacts with CK1a in mitosis. We have added this figure (new Figure 1K) to the manuscript, and insert it below for your perusal:   Fig EV4). This may sound picky but especially with a discontinued Santa Cruz antibody from a company with a sordid history of quality control problems, this is important. Response: We agree with the reviewer that this is important to clarify. As such, we have updated the Methods section to include a table describing all the antibodies used (new Table 1). The table also includes references for antibody specificity, and details which figures each antibody was used in. We thank the reviewer for this suggestion and we spent a significant time during revisions to address this. However, we encountered several technical challenges that have prevented us from providing a definitive answer. First, the anti-FAM83D and anti-CK1a antibodies which were raised against the respective human protein antigens failed to effectively immunoprecipitate endogenous FAM83D and CK1a from wild type and HMMR-/mouse embryonic fibroblasts. Second, these immortalised MEFs proved extremely resistant to transient transfections of plasmids encoding human FAM83D and CK1a. Third, the rates of synchronisation in these cells after STLC treatment were extremely low (possibly because of the transformation process), which translated into low yields of mitotic protein extracts, and subsequently, the typical protein concentration we would use for IPs from mitotic extracts was extremely challenging to achieve. Currently we do not have any HMMR knockout human cell line, which would mitigate these issues. Although we are in the process of generating such a cell line with CRISPR/Cas9, this has been challenging as well and as yet we do not have any HMMR knockout clones.
However, two sets of observations suggest that the FAM83D and CK1a interactions are not likely to occur in HMMR knockout cells: i. the FAM83D mitotic mobility shift evident in WT MEFs does not occur in HMMR knockout MEFs; and ii. the mitotic mobility shift of FAM83DGFP in FAM83DGFP/GFP knockin U2OS cells is also evident in asynchronous conditions when FAM83D-GFP is targeted with anti-GFP nanobody (aGFP.16) bound CK1α (new Figure  EV5A; see below in response to the next comment), suggesting that the interaction between FAM83D and CK1a is essential to cause the mobility shift in FAM83D.
In any case, whilst we cannot definitively say if FAM83D and CK1a still associate or not in the absence of HMMR, we can say that CK1a cannot localise to the spindle if HMMR or FAM83D are not present. The spindle recruitment of endogenous CK1a through FAM83D and HMMR is the main point of the set of experiments we have included in the manuscript. Fig 5B: the rescued mobility shift of FAM83D appears to be less than the mobility shift of wildtype. This should be commented upon. Why might this be? Response: We thank the reviewer for bringing this to our attention. Firstly, to rule out the possibility that aGFP.16-CK1α is phosphorylating FAM83D(F283A)-GFP on random sites, we artificially recruited aGFP.16-CK1α to the wild-type FAM83D-GFP knockin protein via retroviral infection, and compared the mobility shift between these cells and uninfected controls. The mitotic mobility-shift is the same in both the infected and non-infected cells, suggesting that the phosphorylation of FAM83D imparted by aGFP.16-CK1α is not random, and that, most likely, the same sites are phosphorylated by endogenous CK1α and aGFP.16-CK1α.
Thus, we speculate that the CK1-binding-deficient FAM83D(F283A) mutant does not display the same mobility shift as the wild-type FAM83D due to a potential structural change imparted by the F283A mutation. This F283A mutation may interfere with CK1α (or other downstream kinases) accessing some of the phospho-residues, and thus the full phosphorylation status is not achieved.
A new figure with the WT aGFP.16-CK1α is included as an extended view figure (new Fig.  EV5A) and we provide it below for your perusal. We have expanded the results section to reflect these changes and the interpretation. It would be helpful if the authors would speculate why CK1α only binds to FAM83D in mitosis. Could this be because of HMMR, or perhaps phosphorylation of FAM83D by a CDK? Response: We agree with the reviewer that this is an interesting and outstanding question.
Rather than just speculate, we set out to try and address this issue experimentally! When we express the FAM83D N-terminus (DUF1669) alone, we observe a robust interaction with CK1a. However, the C-terminus of FAM83D alone, which lacks the DUF1669, does not pull down CK1a. When we co-express the N-terminal and C-terminal fragments and pull down the N-terminus, we observe C-terminal fragments coimmunoprecipitating, suggesting that both termini associate with each other. In this case, due to excess N-terminal fragments, we can still detect CK1a in the N-terminal fragment IPs. However, when we co-express both the N-and C-terminal fragments and pull down the C-terminal fragment, we again observe robust association with the N-terminal fragment yet, in this case, no CK1a coimmunoprecipitates. Collectively these data suggest that the C-terminus of FAM83D inhibits the N-terminus from binding to CK1a.
Thus, we think that during mitosis FAM83D is modified in a way that relieves the inhibitory role of the C-terminus on the N-terminus, allowing the N-terminus access to CK1a binding. The underlying mechanisms for this could be either a post-translational modification (e.g. phosphorylation or dephosphorylation), or it could be due to mechanical forces when FAM83D associates with HMMR (and/or other proteins such as DYNLL1) on the spindle. We have explored the effects of mutating some of the identified mitotic FAM83D phosphosites to alanine, but these phospho-mutants, either alone or in combination, did not seem to impact the CK1a association. Thus, the exact mechanism regulating this remains an elusive, and our future endeavours will attempt to resolve this very interesting puzzle.
The new data are included as new panels in new Figs. EV2C&D, and are included below for your perusal:  We would like to respectfully emphasize that our novel findings include: • FAM83D interacts and co-localises with CK1a at the mitotic spindle during mitosis, all demonstrated at the endogenous level. • FAM83D-/-cells or those that harbour CK1α-binding deficient FAM83DF283A/F283A knockin mutation no longer recruit CK1α to the spindle, and display pronounced spindle positioning defects, and a delayed metaphase-to-anaphase transition. • We rescue these defects in FAM83D-/-cells by either restoring FAM83D at the endogenous locus using a novel CRISPR/Cas9 knockin strategy, or by using nanobody-directed recruitment to deliver wild-type CK1α to the spindle in FAM83DF283A/F283A knockin cells. • Importantly, nanobody-directed recruitment of catalytically-inactive CK1a in FAM83DF283A/F283A knockin cells did not restore these defects. • • HMMR directs FAM83D to the mitotic spindle and so, in HMMR-/-MEFs, both FAM83D and CK1α fail to localise to the mitotic spindle.
Of course, identifying key CK1α substrates at the mitotic spindle that define proper spindle orientation is desirable, but clearly beyond the scope of the current manuscript. For most mitotic kinases, even for some of the earliest ones to be identified decades ago, the mitotic substrate landscape is still not fully elucidated, and often, identification and validation of a single substrate for any mitotic kinase prompts publication in reputed journals. Our current work unequivocally places CK1α activity on the centrosomes and mitotic spindles, provides molecular mechanisms through which this happens, and highlights the spindle-misorientation and mitotic consequences of disrupting CK1α delivery to the spindle. Identifying FAM83D-dependent CK1α substrates in mitosis and how these ensure proper and timely mitotic progression is our next goal and we have included this in the Discussion.
On another note, CK1a and other CK1 isoforms have long been deemed undruggable due to their participation in multiple, diverse biological processes. Thus, the work presented here, in agreement with our published data on other FAM83 proteins, suggests that targeting individual FAM83:CK1 interactions might be an effective means of inhibiting CK1 in distinct physiological processes. Of relevance here, inhibiting the FAM83D-CK1a interaction would effectively block CK1a from localising to the spindle, whilst having minimal effect on the other CK1a-relevant cellular functions.
We have also incorporated additional mechanistic information into the manuscript which provides further insight into how the FAM83D-CK1a interaction is regulated in cells (see response to reviewer 1).
Major points:

Control cells when grown on L-shape micro-patterns cell position their mitotic spindle along the hypotenuse and the authors observed significant spindle orientation defects in cells lacking FAM83D or expressing FAM83D(F283A). Interestingly the impact of FAM83D(F283A) on spindle orientation was rescued in cells where CK1α was targetted to the spindle with the help of FAM83D(F283A). CK1α-mediated phosphorylation may regulate spindle orientation in control cells downstream of FAM83D, can optogenetic targetting of CK1α in FAM83D knock out cells [not in FAM83D(F283A)] rescue spindle orientation as well as a mitotic delay? Also, since at many instances a delay in the mitotic progression (metaphase to anaphase transition) can impact spindle orientation, it may well be that the spindle orientation defect in the absence of CK1α at mitotic spindle is rather indirect.
Response: We thank the reviewer for this suggestion. First, the best way to rescue defects in FAM83D knockout cells is by restoring wild-type FAM83D into these cells. Not only did we do this, but we ensured that the rescue was done at the cell-cycle-regulated native FAM83D locus by a novel CRISPR/Cas9 strategy. We believe this sets a new precedent on how rescue experiments should be performed for all future knockout studies. Second, as the referee correctly points out, we were able to identify a CK1α-interaction deficient mutant of FAM83D (i.e. a single amino acid, F283A, substitution), knock it in, again at the native FAM83D locus (albeit with a GFP-tag) with CRISPR/Cas9, and show that this mutant was defective in recruiting CK1α to the mitotic spindle and driving timely mitosis. Importantly, this mutant still localised to the mitotic spindle and by mass-spectrometry pulled down every other interactor of wild-type FAM83D except CK1α. It is in this context that we show that artificially taking aGFP.16-CK1α to the mitotic spindle on the FAM83D(F283A)-GFP mutant rescues the defects. Taking CK1α artificially to the mitotic spindle outside of the FAM83D context in FAM83D knockout cells would be potentially problematic in that seven other FAM83 proteins (and potentially their associated partners) that also interact with CK1α would be predicted to interfere with CK1α at the spindle, and might result in artefacts that are extremely hard to interpret. Therefore, we feel the optogenetic delivery of CK1a in FAM83D-knockout cells, while an attractive proposition, might produce data that are hard to interpret as it lacks the FAM83D context. Furthermore, we lacked the expertise and infrastructure to perform the suggested optogenetic targeting experiments within the given time frame and, as explained already, we believe we have done the rescue experiments in the best way possible.

It was not clear to me what is the impact of CK1α loss (RNAi or KO) on spindle positioning, mitotic progression and actin cytoskeleton?
Response: We thank the reviewer for bringing this to our attention. We were unable to generate CK1a-knockout cells by CRISPR/Cas9 (no homozygous knockout clones identified), and in line with this, a CRISPR study has previously identified CSNK1A1 (CK1a encoding gene) as an essential gene (Wang, Birsoy et al., 2015).
Therefore, we transiently depleted CK1a (~70% depletion compared to control cells) in U2OS cells using siRNAs, and under these conditions, we observed robust spindle positioning defects, as well as a delay in the metaphase-to-anaphase transition. We include these data in new Appendix Figure S1 and include them below for your perusal.
However, these data have to be taken cautiously as CK1a also interacts with seven other FAM83 members and has functions in multiple, diverse signalling processes beyond just mitosis. Therefore, the mitotic phenotypes observed with CK1a siRNAs, despite phenocopying FAM83D KO cells, could also be due to indirect consequence(s) of other CK1a function being affected. This is precisely why we believe that targeting regulatory subunits of CK1a (such as FAM83 proteins) present great promise for shutting down specific CK1a-dependent processes, and this is what we see as a big strength of the present study.
Appendix Figure S1: We thank the reviewer for raising this point. We think a likely explanation is that phosphorylation of specific substrates by CK1a is a key event in efficient mammalian cell division. In the absence of their phosphorylation, the spindle is unable to be effectively orientated. Currently, whether these substrates are localised on the mitotic spindle itself, or the subcortical actin clouds that also regulate spindle positioning, is unclear. We have added a sentence to this effect in the discussion to address this point, as we believe it is important to mention. (Connell et al., 2017, eLIFE). Therefore, I am wondering what the impact of FAM83D loss or FAM83D(F283A) mutation on active Plk1 levels at the centrosome as well as on the dynamic astral microtubules. Response: We thank the reviewer for this excellent suggestion. We were also considering this possibility, and indeed it was the Connell et al paper that prompted us to initiate collaboration with the Maxwell lab for the present study (Chris Maxwell, a co-author on this manuscript, is the corresponding author on the Connell et al paper). We had focussed more on the spindle positioning assays, but we performed the suggested experiments to see if FAM83D also acts in the PLK1-dependent spindle positioning pathway, like HMMR. However, it should be noted that HMMR is also involved in recruiting the TPX2-AuroraA kinase complex to the mitotic spindle. Thus, removing HMMR from cells would be predicted to have a more profound impact on spindle positioning and mitosis, than removing FAM83D (or TPX2) alone would.

Recently, FAM83D interacting partner HMMR was shown to affect spindle positioning, and it is involved in Plk1-dependent spindle positioning pathways. It was also shown in loss of HMMR affects levels of active Plk1 at the spindle pole, as well astral microtubules
That said, when CK1a is not able to localise to the spindle, we observe a reduction in active PLK1 at spindle poles and kinetochores, suggesting that CK1a may act in the PLK1dependent pathway. One likely explanation is that CK1a phosphorylates PLK1-docking proteins on the spindle pole to enable polo-box domain binding to the phosphoprotein, and hence PLK1 localisation. Alternatively, CK1a may be involved in the activation of PLK1 at spindle poles. However, in our FAM83D pulldowns, we did not detect any PLK1 by mass spectrometry, suggesting that any effects of FAM83D-bound CK1a on PLK1 are likely to be either indirect or involve transient interaction(s).
While these data are hugely promising, we believe they raise interesting questions on the mechanisms involved and clearly require a substantial body of further work to fully ascertain whether CK1a directly acts in the PLK1-dependent pathway, and where within the pathway CK1a acts. We will be following up on these observations in the future. We include these new data below for your perusal in rebuttal  We thank the referee for raising this query. The spindle is a very bright and prominent structure when viewed by immunofluorescence. Because of that, the optimal exposure of the images in Figure 2A was based on the spindle being visible and not saturated. When that happens, the cytoplasmic CK1α staining behind and surrounding the spindle appears faint. However, if one were to remove the spindle and increase the exposure, we would see that the cytoplasmic levels of CK1α are the same between WT and FAM83D KO cells. Additionally, in all of our immunoblotting figures, we do not observe any substantial decrease in CK1α protein levels between mitotic and asynchronous cells.
3. Also, in Figure 4C and 4D can authors should analyze the stability of FAM83D upon mitotic exit in the presence of MG132 (RO-3306+MG132), this would be crucial to address whether proteasome alone or mitotic exit based posttranslational modification are at play for controlling FAM83D stability. Response: We thank the referee for this excellent suggestion. We have now performed the suggested experiment and observe robust FAM83D degradation following forced mitotic exit with RO-3306. This degradation is halted in the presence of MG132. Thus, it appears that FAM83D is rapidly degraded via the proteasome, following mitotic exit. This data is included as a new figure (new Fig 4E) and is included below for your perusal:  Rebuttal Figure 2: An in vitro kinase assay was set up with recombinant FAM83D and the indicated kinases. Following 30 min incubation with radioactive ATP at 30¢XC, samples were denatured and separated by SDS-PAGE. The gel was stained with Coomassie blue, and exposed to x-ray films for 16 h overnight. FAM83G was included as a positive control for CK1α activity. The other kinases included in the screen were profiled by the Division of Signal Transduction Therapy (University of Dundee), and were used under conditions where they are catalytically active.

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Rebuttal Figure 3: An in vitro kinase assay was set up with recombinant FAM83D and the indicated kinases. Following 30 min incubation with radioactive ATP at 30¢XC, samples were denatured and separated by SDS-PAGE. The gel was stained with Coomassie blue, and exposed to x-ray films for 16 h overnight. FAM83G was included as a positive control for CK1α activity, and Histone H1 was included as a positive control for CDK1/Cyclin B activity. The other kinases included in the screen were profiled by the Division of Signal Transduction Therapy (University of Dundee), and were used under conditions where they are catalytically active.

References:
Wang Thank you for submitting the revised version of your manuscript. It has now been seen by two of the original referees.
As you can see, both referees find that the study is significantly improved during revision and recommend publication. Before I can accept the manuscript, I need you to address the below minor/editorial points: • Please address the remaining concerns of referee #2. Regarding point 1, I can see that you looked at actin cortex morphology in Appendix Figure S1 under CK1α kd conditions. As for the point 2, since this experiment was not recommended in the previous review, I don't deem necessary to address this point experimentally for publication. However, please respond to both points textually.
Thank you again for giving us to consider your manuscript for EMBO Reports, I look forward to your revision.

Referee #1:
The authors have addressed the issues raised. This is an important addition to our knowledge or CK1 and mitosis.

Referee #2:
Authors have substantially improved the revised version of the manuscript and have adequately addressed my previous concerns. I have now only a few remaining concerns: 1. Appendix Figure S1 revealing the siRNAs mediated depletion of siRNA CK1α must be included in the manuscript. Earlier, I also suggested the authors to analyse the impact of CK1α (RNAi) on the actin cytoskeleton; I guess that has not been addressed in the Appendix Figure S1. Corresponding Author Name: Gopal Sapkota a statement of how many times the experiment shown was independently replicated in the laboratory.
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