Phosphorylation-dependent interactions of VAPB and ELYS contribute to the temporal progression of mitosis

  1. Christina James
  2. Ulrike Möller
  3. Sabine König
  4. Henning Urlaub
  5. Ralph H. Kehlenbach

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Abstract

ELYS is a nucleoporin that localizes to the nuclear side of the nuclear envelope in interphase cells. In mitosis, it serves as an assembly platform that interacts with chromatin and then with nucleoporin subcomplexes to initiate the formation of novel nuclear pore complexes. Here we describe the interaction of ELYS with the membrane protein VAPB. In mitosis, ELYS becomes phosphorylated at many sites, including a predicted FFAT (two phenylalanines in an acidic tract) motif, which is shown to mediate interaction with the MSP (major sperm protein)-domain of VAPB. Phosphorylation-dependent binding of VAPB to ELYS is demonstrated by peptide binding assays and co-immunoprecipitation experiments. In anaphase, the two proteins co-localize to the non-core region of the newly forming nuclear envelope. Depletion of VAPB resulted in prolonged mitosis and slow progression from meta-to anaphase and also to chromosome segregation defects. Together, our results suggest an active role of VAPB in recruiting membrane fragments to chromatin and in the biogenesis of a novel nuclear envelope during mitosis.

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    The authors do not wish to provide a response at this time.

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    Referee #3

    Evidence, reproducibility and clarity

    This MS contains carefully carried out and well controlled experiments describing a new pFFAT in ELYS. There is a similarly convincing demonstration of functionally relevant colocalisation by proximity ligation assay (PLA), particularly that both ELYS and VAP are nuclear envelope proteins in interphase without interacting (neg control in Fig 4D).

    Major Issue: Functional significance

    A key conclusion is that experiments prove that "ELYS serves as the crucial initiation factor for post-mitotic NPC-assembly" (p5). However, evidence for this is lacking as this would require reconstitution of NPC assembly with a mutant form of ELYS carefully changing the FFAT motif (e.g. 1321A 1324E) and exclusion of other probable VAP targets in experiments with mutant VAP. VAPs are among the proteins with the highest number of documented interactors (see Huttlin 2015/7 etc, e.g. PMID 26186194), so knocking down VAP may have pleiotropic effects and quite indirect read-outs in many aspects of cell function. In addition, for this work specifically there are other NE proteins that are known interactors of VAP: Emerin (EMD) and LBR both interact with VAP (high-throughput data, VAPA and VAPB). EMD has a motif similar to the canonical phospho-FFAT: 98 SYFTTRT 104. LBR has no motif. These findings should not be overlooked in this work. For example, was the interaction with emerin (page 4) sensitive to mutating VAP or ELYS? Could the effect seen in Figure 5 result from interactions with proteins other them ELYS?

    Further experiments should be carried out to justify all statements in the current MS of functional significance. Instead of doing more experiments, an alternative for the authors would be to describe the current set of results more cautiously. However, that would require changing much of the impact of the current MS, from the title onwards.

    Moderate Issue: VAPA

    From the start of the Introduction and some elements of the Discussion, include VAPA in equal measure with VAPB. When describing interactions of ELYS with VAP note that Huttlin et al., reported interactions twice for each of VAPA and VAPB. When describing own results (James et al. 2019) and those of others (Saiz-Ros et al., 2019) that focused on VAPB, clarify if the authors' view is that VAPA would (or would not) have the same interaction.

    Is there any evidence that only VAPB is on NE? Note that some refs in the Introduction relate to VAPA: Mesmin (not VAPB); ACBD5: although article titles refer to VAPB, early work (10.1083/jcb.201607055) showed almost identical involvement of VAPA. Also, this redundancy likely explains "function of VAPB in mitosis is not essential," (in Discussion). The lack of effect of VAPA knock-down may indicate that in these cells VAPB is dominant, but does not exclude a role for VAPA when VAPB is reduced. That might be tested by depleting both. Even following that, there is MOSPD2 to consider

    Other aspects of the writing

    "two amino acid residues are crucial for the interaction (VAPB K87 and M89)." This is wrong. Many residues are critical, these are merely 2 of possibly >10 that were chosen by Kaiser et al (2005) to create their non-binder.. Others have used different mutations to block FFAT binding.

    "They may exhibit a certain binding preference to specific members of the VAP ... family...". I cannot think of any example. I note no citation is given.

    When listing many or all MSP proteins, the text should state that MOSPD2 is uniquely close to VAPA/B. CFAP65 is typically not mentioned in the VAP-like lists as it does not have any of the conserved sequence that binds FFAT. If however the authors wish to include all human MSP domain protein, they should also include Hydin.

    Slightly wrong to cite De Vos et al., 2012 about PTPIP51's FFAT as that paper makes no mention of the motif. Better pick Di Mattia (again)

    On VAPB (and also A) on INM: there are references to be cited esp. relating to intranuclear Scs2 in yeast (Brickner et al 2004, Ptak et al 2021)

    Citations for VAP at ER-mito contacts "De Vos et al., 2012; Gómez-Suaga et al., 2019; Stoica et al., 2014)". These all refer to the same bridging protein, PTPIP51. Reduce to one citation. Then mention other proteins at the same site VPS13A, mitoguardin(MIGA)-2 ...

    "The domain interacts with characteristic peptide sequences ..." add citation to this sentence

    "Several variants of such motifs have been described: (i)" ... "(ii)": (i) and (ii) are entirely unlinked. Delete these and also "Several variants of such motifs have been described." Which is repeated later

    "FFAT-like motifs come in different flavors and may even lack the two phenylalanine residues (Murphy and Levine, 2016)": while motifs can tolerate variation at both positions, this text is misleading as it implies much more variation than is known. The 1st F can only be conservatively substituted (Y).

    Minor aspects in Results:

    ORP1L peptide as positive control: cite Kaiser 2005

    Was phosphoproteomics done in such a way as to find peptides that have both S1314 and S1326?

    Figure 4D, row 2: Comment on intranuclear staining in Prophase (at approx 4 o'clock) of both ELYS & VAP that is PLA positive

    Referees cross-commenting

    I agree with this point from Reviewer #1. We all agree that the main issue can be resolved experimentally to determine the effect of subtle point mutations in ELYS. Both other reviewers have done a good job in finding issues with the experiments that can also be addressed.

    Significance

    This work documents an interaction between the protein ELYS, that is involved in the reformation of nuclear pore complexes after mitosis, and the ER membrane protein VAPB. The interactions was previously known through high-throughput studies, along with many 100's of others for VAP, but here it is studied in detail and with care, identifying how the motif is induced by phosphorylation of ELYS. The two proteins are co-localised using convincing proximity ligation assays. This biochemistry and cell biological localisation is well done.

    Functional experiments then show that VAP (in this case VAPB) knock-down affects mitosis and chromosome segregation. While the result is incontrovertible, it has many possible interpretations, mainly because VAP has hundreds of interactions, including with multiple proteins involved in mitosis beyond just ELYS. This means that there are major limitations on how the interaction and co-localisation should be interpreted, reducing the advance associated with the current manuscript to incremental, and the limiting the audience to specialized.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this study, James et al. follow up on their prior discovery that the ER contact site protein VAPB localizes to the nuclear envelope and is a putative binding partner of the nucleoporin ELYS, which coordinates nuclear envelope reformation (NER) with nuclear pore complex (NPC) biogenesis at mitotic exit and is also a constituent of the nuclear facing Y-complexes of mature NPCs. Using a series of complementary biochemical approaches the authors 1) demonstrate that VAPB and ELYS directly interact, 2) map the binding sites on ELYS that are sufficient to bind VAPB, 3) show that mutations that disrupt VAPB-FFAT motif binding also abrogate binding to ELYS including of the full-length protein, 4) define mitotic phosphorylation sties on VAPB-bound ELYS, 5) demonstrate that phosphorylation of ELYS, specifically at the FFAT2 motif, is required for binding to VAPB, and 6) demonstrate that the phosphorylation of ELYS that regulates VAPB binding occurs in mitosis. Turning to cell biology, the authors find that VAPB, which is an established ER protein, has some preference for non-core regions during NER (like ELYS). In addition, PLA analysis suggests that the interaction of VAPB and ELYS is most robust during anaphase and is somewhat disrupted when the binding of VAPB to FFAT motifs is lost due to targeted mutation. Last, the authors demonstrate that depletion of VAPB leads to metaphase delay and lagging chromosomes.

    Major comments:

    The data supporting direct binding of peptides encoding the FFAT 1 and FFAT2 motif derived from ELYS to VAPB in a manner similar to other FFAT sequences is strong, as is the effect of phosphorylation of FFAT1 on the strength of this interaction.

    The evidence supporting the mitotic-specific nature of the ELYS-VAPB interaction is strong, and that this interaction is direct, is also strong, and was rigorously tested using a combination of endogenous expression, heterologous expression, and recombinant protein approaches. Moreover, the sensitivity of this interaction to established mutations in VAPB abrogating FFAT interactions reinforces the outlined underlying biochemical interaction mechanism. The essentiality of ELYS phosphorylation (and therefore the mechanism underlying the mitotic specificity of the interaction) is also strongly supported by the data using phosphatase treatment. Although it is an intuitive model, whether the cell biological evidence support the simplest view that the ELYS-VAPB complex bridges the nuclear envelope to chromatin during NER in late anaphase / at mitotic exit is far less solid and, at a minimum, alternative models should be considered/discussed. For example, how a delay in metaphase in the siVAPB condition is consistent with a role in NER, which occurs exclusively post-metaphase, is unclear. Is it not possible that the VAPB-ELYS complex is regulated by phosphorylation during mitotic progression such that VAPB and/or ELYS can only exert their biological effects when released from the complex? In other words, might ELYS be licensed to act in NER only when it is released from VAPB, which could prevent premature NER/NPC biogenesis? Subtleties of when during mitosis the phosphorylation occurs is challenging, and it could be that the anaphase A to anaphase B transition, when many mitotic entry phosphorylation events begin to be reversed, could be relevant here. Along these lines, in Fig. 5 how the VAPB knock-down does or does not recapitulate the phenotype of an ELYS knock-down in this cell type (and the effect of the combination, to address epistasis) is needed for context, as is whether VAPB knock-down affects ELYS distribution in mitosis. ELYS knock-down would also be very beneficial for the PLA analysis to establish the "floor" of measurable signal. Last, it is also possible that VAPB has other roles in mitosis that should be acknowledged - for example although it is in yeast, it is relevant that a VAPB orthologue Scs2 is required for normal nuclear envelope expansion in mitosis by regulating SUMOylation (Ptak, Saik et al., JCB, 2021 and Saik et al., JCB, 2023) - this work should be referenced as well. Of course, the ideal experiment would be one in which an ELYS knock-down is complemented with a resistant form that encodes the S to A mutations in the FFAT2 region to assess its localization and to see if it can complement the knock-out function of ELYS in post-mitotic NPC assembly or, as suggested by a sequestration model, it can drive the same metaphase delay seen upon VAPB knock-down. This is technically challenging for sure, particularly given the size of the ELYS gene, but it would address the cell biological function of this interaction in the most direct manner. Several other observations that could warrant further comment or study include 1) is there a VAPB signal at the metaphase poles as suggested by Fig. 4A and, if so, could this represent aa distinct mitotic function?; 2) Does the HA-VAPB KD/MD mutant localize differently in mitosis compared to the WT - it appears that it might be less enriched in non-core regions (Fig. 4E)?; 3) does VAPB alter post-mitotic NPC biogenesis/number?

    Minor comments:

    I would suggest avoiding the use of "novel" when describing newly assembled NPCs or post-mitotic nuclear envelope reformation, as its other meaning of "non-standard" makes this wording confusing. It is unclear whether when the authors state that ELYS localizes "to the nuclear side of the nuclear envelope" they are referring to the nuclear aspect of the NPC and/or a separate pool at the INM - please edit to clarify. More descriptive y-axes for the plots in Fig. 4F and 5F and related legends would be useful; although the details are in the methods section, it would be nice not to have to hunt them down. Also, please clarify the meaning of blue and orange points in Fig. 4F.

    Significance

    General assessment: The biochemical analysis is rigorous and compelling and establishes the mitotic-specific interaction of VAPB and ELYS including detailed information about the binding interface and its regulation by phosphorylation. The new insight provided into the function of this VAPB-ELYS interaction is somewhat less well developed as the current manuscript, in its current form, does not yet mechanistically define the function of the VAPB-ELYS interaction in mitosis.

    Advance: Conceptually, to the best of my knowledge, the idea that VAPB contributes to mitosis in mammalian cells is novel and is therefore impactful and will motivate further work. As the authors connect VAPB biochemically to ELYS, an established factor that promotes the coordination of NER and NPC biogenesis, this interaction is likely to be mechanistically important, although the specific details by which this interaction facilitate normal mitotic progression is not yet clear.

    Audience: This work will be of interest to a broad swath of cell biologists including those interested in NPCs, the nuclear envelope, the ER, membrane remodeling, and chromosome segregation.

    My expertise is in nuclear envelope dynamics, nuclear pore complexes, and chromatin organization.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    The VAP proteins are well established as tail anchored proteins of the ER membrane. VAPs mediates co-operation between the ER and other organelles by creating a transient molecular tether with binding partners on opposing organelles to form a membrane contact site over which lipids and metabolites are exchanged. Proteins which bind VAPs generally contain a short FFAT motif, of varying sequence which binds the MSP domain of VAP. More recently the FFAT motif has been more extensively analysed in multiple different proteins and differential phosphorylation of the FFAT motif has been shown to either enhance or block VAP binding depending on the position of the phosphosite.

    Recent work conducted by the authors demonstrated that a small population of VAPB is not exclusively localised to the ER and can also reach the inner nuclear membrane. They also identified ELYS as a potential interaction partner of VAPB in a screening approach. ELYS is a nucleoporin that can be found at the nuclear side of the nuclear envelope where it forms part of nuclear pore complexes. During mitosis, ELYS serves as an assembly platform that bridges an interaction between decondensing chromosomes and recruited nucleoporin subcomplexes to generate new nuclear pore complexes for post-mitotic daughter cells. In this manuscript, James et al seek to explore this enigmatic potential interaction between ELYS and VAPB to address why VAPB may be found at the inner nuclear membrane.

    Peptide binding assays and some co-immunoprecipitation experiments are used to demonstrate that interactions occur via the MSP-domain of VAPB and FFAT-like motifs within ELYS. In addition, it is demonstrated that, for the ELYS FFAT peptides, the interaction is dependent on the phosphorylation status of serine residues of a particular FFAT-motif that can either promote or reduce its affinity to VAPB. Of most relevance is a serine in the acidic tract (1314) which, when phosphorylated increases VAPB binding. This is completely in line with what is already known about the FFAT motif and so is not surprising, in particular when using a peptide in an in vitro assay.

    The authors then utilise cell synchronisation techniques to provide evidence that both phosphorylation of ELYS and its binding to VAPB are heightened during mitosis. Immunofluorescence and proximity ligation assays are used to demonstrate that the proteins co-localise specifically during anaphase and at the non-core regions of segregating chromosomes.

    The manuscript is concluded by investigating the effect of VAPB depletion on mitosis with some evidence to suggest that transition from meta-anaphase is delayed and defects such as lagging chromosomes are observed.

    Major comments

    Overall, this manuscript is well written and the data presented in Figures 1-3 convincingly show the nature of the interaction between ELYS and VAPB. Clearly the proteins interact via FFAT motifs and this interaction appears to be enhanced during mitosis. However, the work as is, relies heavily on peptide binding assays and would benefit from additional experiments to further support the results. The authors need to more clearly show that this specific phosphorylation happens during mitosis, they may have this data but it is not clearly explained. In addition, the data that VAPB-ELYS interaction contributes to temporal progression of mitosis (as per the title) is not sufficiently clear. VAPB silencing appears to have some impact on mitosis but this is not the same thing. So this section needs to be strengthened before this statement can be made.

    The authors claim that the study "suggests an active role of VAPB in recruiting membrane fragments to chromatin and in the biogenesis of a novel nuclear envelope during mitosis". Given the data presented in Figures 4 and 5, this appears to be rather speculative with little evidence to support it, so data should be provided or this statement toned down. Currently, without additional supporting data the authors may wish to revise the overarching conclusions of the study and change the title.

    Specific points.

    Peptide pull down assays clearly show which FFAT-like motifs are important in facilitating binding. The co-immunoprecipitation systems used in Figure 2 also provide useful information on the interaction in a cell context. The authors should combine these findings by introducing full length ELYS mutants with altered FFAT-like motifs into their stably expressing GFP-VAPB HeLa cell line and then performing Co-IPs to help identify which FFAT motif/s drive the mitotic interaction. Other mutants of ELYS harbouring either phosphomimetic or phospho-resistant residues may also be introduced to further investigate mechanisms of the molecular switch in a cellular environment to support the work currently done with peptides alone. This is an obvious gap in the work which, based on the other data the authors have shown, should presumably be straightforward and would also lead directly into the next major point.

    • Whilst silencing VAPB does appear to delay mitosis, no reference is made to ELYS throughout Figure 5 nor as part of its associated discussion. Given that VAPB has more than 250 proposed binding partners, the observed aberration of mitotic progression could result from a huge number of indirect processes. Further work is needed to link the experiment specifically to the VAPB-ELYS interaction and not just loss of VAPB. We would suggest generating a complementation system where ELYS is either knocked out or silenced and then wild-type ELYS and an ELYS FFAT mutant (which cannot interact with VAPB),and/or a phospho mutant (whose interaction cannot be regulated during mitosis) are introduced. Then the observed effects can be better attributed to the VAPB-ELYS interaction and not just loss of VAPB.
    • The immunofluorescence and PLA results in Figure 4 could be strengthened by including other ER markers. This would show that co-localisation of ELYS at the non-core region is specific to VAPB protein, not any ER protein or rather than an artefact of the ER being pushed out of the organelle exclusion zone during mitosis and therefore 'bunching' at the periphery of the nuclear envelope. It would be worthwhile repeating these experiments with candidates such as VAPA, other ER membrane proteins or at least GFP-KDEL, to make this phenomenon more convincing. As part of this the authors should ideally generate a complemented ELYS KO (see point above) to avoid the residual activity attributed to endogenous background in the PLA Figure 4E.
    • Authors should clarify if the phosphorylation events (in particular S1314) only occur or are increased during mitosis. This may be data they have from the MS experiment in Figure 3 or it could also be shown using a phospho-antibody (although this can be challenging if a suitable antibody cannot be made).
    • The authors should clarify why they need to do these semi in-vitro assays with purified GST-VAPB-MSP on beads and then lysates added and not just a standard co-IP. If this is simply signal intensity due to a very small proportion of VAPB binding to ELYS then this is fine but this should be stated and it should be made clear that ELYS is not a major binding partner - most of VAPB is on the ER. Otherwise, this is misleading.

    I estimate that the suggested alterations above would incur approximately 3-6 months of additional experimental work, depending on if KO cell lines were required.

    Minor comments

    • To show that the observed interactions and potential role of VAPB-ELYS interaction is universal it would be useful to have at least a subset of experiments also shown in another cell line or system - this is now also a requirement for some journals.
    • Consider re-wording the title of the manuscript to better reflect the data presented within the study. Alternatively, provide further evidence that VAPB-ELYS interactions directly affect temporal progression of mitosis to validate this claim, as discussed above.
    • Quantification of blots in Figure 2A could allow measurement of relative binding affinities between VAPB-ELYS throughout the cell cycle. The same could be applied to the effect of phosphorylation on binding affinity in Figure 2D.
    • The cells used are never clearly mentioned in the text - I assume this is always in HeLa but this should be added in all cases for clarity
    • Page 8: "As shown in Fig. 2A,a large proportion of GFP-VAPB was precipitated under our experimental conditions." - I don't understand how this is shown in this figure as the non-bound fraction is not shown?
    • Please provide some controls to demonstrate the extent to which the samples used are asyn, G1/M or M.
    • Page 9 - why are Phos-tag gels not shown as this would make this result more convincing?
    • Figure 3A - I find the SDS-PAGE gel confusing. Why not show the whole gel and why is the band size apparently reduced in the mitotic fraction when previously it was increased (by phosphorylation)? It would also be useful to see if there were any other band shifts.
    • "FFAT-2 of ELYS is regulated by phosphorylation" The way you have setup the experiment leads the reader to think you are going to show which sites are differentially phosphorylated in mitosis, but then this is not the case - so there seems no purpose to doing the experiment this way. If you used TMT MS approach you would be able to potentially quantify the change in phosphorylation at the FFAT motif sites in mitosis. Otherwise what is the purpose of using these 2 samples, mitotic and AS?
    • For all of the antibodies used, in particular for the PLA, please provide evidence of validation of the antibodies.
    • Just a minor point to consider - In the methods for your lysis buffer you use 400mM NaCl - might this slightly reduce the VAPB-FFAT interaction? Worth considering reducing this?
    • "The rather small difference observed between the wild-type and the mutant protein observed in this experiment probably results from the presence of endogenous VAPB in the stable cell lines, which could form dimers with the exogeneous HA-tagged versions." If this is the case then please demonstrate that this is happening, or use the KO approach in the major points above.
    • "we now show that the proteins can indeed interact with each other, without the need for additional bridging factors (Figs. 1 and 3)." You show that the peptides can bind - but this is not the same thing as the peptide in the full context of the protein - so this should be toned down or removed.
    • "Remarkably, this region is highly conserved between species, suggesting that it is important for protein functions (data not shown)". Please show the alignments so the reader can judge for themselves. It is conserved in ALL species and the phosphosites are also conserved??
    • "In our experiments, knockdown of VAPA alone did not lead to a delay in mitosis (data not shown). " Why not show this data - as this is a very interesting and potentially important observation? Also add the validation of knockdown of VAPA.
    • I find the end to the discussion to the paper rather abrupt. It would be interesting to discuss further how VAPB, but not apparently VAPA reaches the INM and if so why this function is required of an ER adaptor and not another more obvious adaptor protein. In short - why would VAPB be performing this role?

    Referees cross-commenting

    I agree with the comments of the other reviewers, and they are very much in line with my own review. We all seem convinced that VAPB binds ELYS via a pFFAT, and that this interaction is enhanced during mitosois. However the role of this interaction in mitotic progression remains unclear and based on this data should not be claimed in the title or discussion of the paper.

    Significance

    Overall, if the manuscript could be improved with the suggested changes, then this could be a considerable conceptual advance in how we understand the VAP proteins, showing functions beyond those as an ER adaptor. This would be significant for the field.

    In the context of the existing literature the work does not advance our knowledge of FFAT-VAP interactions, this has already been shown, but it would give a nice example of how this can be regulated during mitosis and how VAP can contribute beyond just as an ER adaptor at membrane contact sites.

    There would be a wide audience in the cell biology field and more widely as mutations in VAPB cause a form of ALS, and many people are working in this area.

    My field of expertise is in organelle cell biology and membrane contact sites.

  5. Version published to 10.1101/2023.07.03.547506v1 on bioRxiv