Conformational transitions of the Spindly adaptor underlie its interaction with Dynein and Dynactin

  1. Ennio A. d’Amico
  2. Misbha Ud Din Ahmad
  3. Verena Cmentowski
  4. Mathias Girbig
  5. Franziska Müller
  6. Sabine Wohlgemuth
  7. Andreas Brockmeyer
  8. Stefano Maffini
  9. Petra Janning
  10. Ingrid R. Vetter
  11. Andrew P. Carter
  12. Anastassis Perrakis
  13. Andrea Musacchio

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Abstract

Cytoplasmic Dynein 1, or Dynein, is a microtubule minus end–directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein–Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein–Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein–Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. Thus, our work illustrates how specific Dynein activation at a defined cellular locale may require multiple factors.

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  1. Version published to 10.1083/jcb.202206131
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    Reply to the reviewers

    Reviewer #1

    In this article, Amico et al. explore how Spindly self-regulates its interaction with Dynein-Dynactin. They propose that Spindly adopts an auto-inhibited, closed conformation that blocks the CC1 box and Spindly motif, preventing its interaction with dynein-dynactin. The authors used a combination of X-ray crystallography, biochemistry, and structure predictions to detail the intramolecular interactions in Spindly that mediate this closed state. They then use analytical SEC to test their proposed auto-inhibition mechanism by monitoring Spindly binding to the pointed end complex. They suggest that auto-inhibited Spindly is unable to bind Dynein-Dynactin regardless of the presence or absence of Spindly's cargo, the RZZ complex. In contrast, by using mutagenesis to prevent this auto-inhibition, the authors show that uninhibited Spindly can interact with members of the Dynein-Dynactin complex. Finally, they use cellular experiments to show that relieving autoinhibition prevents the proper localization of Spindly and Dynein-Dynactin to kinetochores during mitosis, likely due to the formation of ectopic Spindly-Dynein-Dynactin complexes in these cells.

    This is an interesting paper that provides important insights into the mechanism of Spindly regulation and its associations with its interacting partners. However, additional work is necessary to support some of their conclusions. In addition, the text is at times quite dense and harder to follow, which prevents their findings as being impactful as could be possible for the bigger picture paradigms of kinetochore function.

    We thank the reviewer for a supportive assessment and for raising some concerns that we have now fully addressed in our revision.

    Major Points:

    The crosslinking and mass photometry experiments are done at highly differing concentrations (5 μM vs. 10 nM). The mass photometry should be performed at the same concentration as the crosslinking experiments to determine if Spindly forms a higher order oligomer at the higher concentration. These results will aid in the interpretation of the crosslinking mass spectrometry experiments, as the observed interactions could be intermolecular contacts rather than intramolecular contacts if Spindly is tetrameric at these concentrations, as is suggested in figure 4E for specific Spindly constructs.

    We thank the reviewer for raising this point. Mass photometry (MP) requires very low sample concentrations as it is essentially a single molecule technique, and therefore the particle density cannot be increased arbitrarily. To assess whether the Spindly construct is prevalently tetrameric at the concentration of the crosslinking experiment, we performed the crosslinking experiments at the standard concentration, and only then diluted the samples and performed MP measurements. The results, displayed in two new panels (Figure 1 – Supplement 1M-N), show that crosslinked samples are primarily dimeric, providing further evidence that we are looking at *bona fide *intra-dimer contacts.

    In figure 2, more conclusive evidence is needed to show that full length Spindly does not form a complex with Dynein-Dynactin. My interpretation of the gels in figure 2D suggests that full length Spindly does form a complex with Dynein-Dynactin, as in the final gel (red outline) it looks as if full length Spindly is indeed peaking with the rest of the Dynein-Dynactin proteins, albeit with excess Spindly eluting later. Figure legends containing protein concentrations used in SEC assays would aid in the interpretation of this data.To conclusively show that full length Spindly doesn't form a complex with Dynein-Dynactin, additional assays will be necessary, such as pull-down assays, or mass photometry.

    We have now added concentrations of binding species at the relevant points of the figure legends.

    The essence of the reviewer’s concern is that full length Spindly, like BicD2, binds the DD, which would invalidate our model that Spindly is auto-inhibited in absence of a second trigger (other than DD), or alternatively showing that auto-inhibition can be easily overcome. Our conclusion that Spindly remains auto-inhibited, however, is strongly supported by the gels in Figures 2D-G. There, the peak containing DD and BicD2 and eluting around 6.2 ml (panel D) is not visible when BicD2 is replaced with Spindly (panel E), and RZZ does not change this (panels F-G). Note that the peak at 6.6 ml appears to be a contaminant, possibly DNA, and it is visible also with individual Dynein and Dynactin samples. These experiments strongly support our point and we have tried to improve the presentation of the results by boxing relevant fractions of the displayed SDS-PAGEs.

    We have now also repeated these experiments with recombinant human Dynactin. The new results are displayed in Figure 2 – Supplement 2. Also in this case, we see minimal complex formation with Spindly and complex formation with BicD2, even if the trailing of Dynein, Dynactin, and Spindly in the earlier elution fractions (already in the absence of complex formation) makes the gels harder to interpret. We also note that these experiments are consistent with those with the isolated PE complex.

    Regretfully, we cannot gather additional information by mass photometry because even our positive control dissociates at the extremely low concentrations required to image this very large complex.

    In figure 3C, 3E, and figure 5C, there is a shift in the PE peaks in the presence of Spindly, but it isn't clear why doesn't the complex doesn't elute earlier than Spindly alone. If the complex is dissociating on the column, additional assays are necessary to confirm that these Spindly constructs stably interact with PE. If this shift is also accompanied by a major change in shape, thus allowing Spindly to elute later than it does alone, this needs to be explored or explained further.

    Elution from a size exclusion chromatography column is dominated by the hydrodynamic radius of the macromolecule. In this particular case, Spindly is highly elongated and essentially sets an upper limit for the elution volume of both the un-complexed and complexed protein. We have described this behavior in many other cases of highly elongated proteins (e.g. Huis in ‘t Veld et al. eLife 2019). We are aware that the binding affinity for the interaction of Spindly and the PE complex is low, and therefore are not surprised to observe dissociation of the complex during the SEC run, i.e. upon dilution of the sample after incubation. In these experiments, we have tried to focus on the shift in elution volume of the PE complex from its elution position in isolation.

    The authors should provide better a rationale for why the pointed-end complex is used in figure 3 in lieu of the complex used in figure 2.

    We now write that the Spindly motif of adaptors binds the pointed end complex with measurable affinity also in absence of Dynein (near line 294). We then clarify that “As the Spindly motif is predicted to sit within the autoinhibited portion of the protein, we hypothesized that the PE-Spindly motif interaction could be used as a proxy to measure the autoinhibition status of Spindly, bypassing the need to form the entire Dynein-Dynactin-Spindly complex.”

    In Figure 5I, WT Spindly also binds to LIC, although less WT Spindly is bound to LIC than Spindly CC2* or Spindly deltaRV. This should be addressed in the text.

    Thank you for pointing this out. We have now clarified this in the text near line 440.

    The authors claim that the mechanism they describe may be a paradigm for dynein activation by other adaptors at various cellular locations, but they aren't able to identify a mechanism for how Spindly converts from its auto-inhibited state to its permissive state. A more thorough examination of this mechanism is necessary to claim that this mechanism could be paradigmatic, or a revision of the text is needed.

    Following an additional concern by reviewer 3, we have now revised the text to meet this concern. So, both in the last sentence of the abstract, and in the last paragraph of the discussion, we do not any longer discuss our results as paradigmatic, although we have reasons to believe that they might be eventually recognized as such, after additional examples will have been analyzed.

    Minor Points:

    1. The manuscript could benefit from careful review of the text, captions, and figures, as a few minor typos and inconsistencies in the figures and text were present.

    We have now re-reviewed the text and figures to try eliminate residual inconsistencies.

    1. The list of common structural and functional features of Dynein-Dynactin adaptors could be indicated more clearly.

    We have re-written this part of the Introduction, where we now indicate more clearly the features of the DD complex

    1. Several times the authors use alpha fold predictions to confirm their data. Although the predictions support several of their conclusions, saying that predictions can confirm the data is an overstatement.

    We thank the reviewer for pointing this out. We now replaced “confirmed” with “also supported” on line 190, where we explicitly referred to AF2 predictions as “confirmatory”. We also re-wrote a statement in the Discussion where we had commented on the power of AF2 and indicate that it “became available in the late phases of our work as a guiding and validation tool” (line 524).

    1. Figure 1H would be improved by the addition of the amino acid numbers in the domain diagram.

    Fixed – we also added amino acid numbers in 1G for consistency.

    1. Concentrations used for each protein for the analytical SEC experiments should be listed in the figure or caption.

    Thank you for suggesting this. We have now added the protein concentrations for these experiments directly in the legends.

    1. In addition to the caption, it would be helpful to the reader to indicate which experiments use farnesylated Spindly.

    Done in legends wherever applicable.

    1. Error bars are missing from the WT sample in figure 5J. This figure would benefit from statistical analysis.

    Done – see also point 4, Reviewer 2.

    Significance:

    This paper builds on recent work from the Mussachio lab and others exploring the nature of the fibrous corona at kinetochores and the molecular basis for dynein recruitment. This paper is focused on the structural nature of the interactions that underlie Spindly recruitment to kinetochores and its interactions with dynein and other factors. Although reductionist in its approach, this paper has the potential to have broad implications for thinking about the control of corona assembly and dynein recruitment with an elegant auto-regulation of Spindly. Researchers interested in cell division, chromosome segregation, kinetochore function, dynein regulation, and the structural basis for core cellular processes should be interested in this paper.

    __Reviewer #2 __

    The study by d'Amico et al. presents an in-depth analysis of how intramolecular folding of the coiled-coil adaptor Spindly regulates its interaction with the motor dynein and its obligatory co-factor dynactin. Using biochemical reconstitution and diverse biophysical approaches (including cross-linking mass spectrometry, X-ray crystallography, AF2-based structure prediction, size exclusion chromatography, and analytical ultracentrifugation), the authors uncover and dissect an intricate Spindly autoinhibition mechanism. At kinetochores Spindly is known to co-oligomerize into filaments with the RZZ complex (its kinetochore receptor/cargo), which drives expansion of the outermost kinetochore region (the corona). Here the authors show that Spindly is a dimer in solution and that successive coiled-coil segments interact with each other in an asymmetric 'closed' conformation that is unable to form a complex with dynein and dynactin. Specifically, a 2-residue insertion in the middle of Spindly's first coiled-coil (CC1) creates a kink that allows CC1 to fold back on itself, which has two important structural consequences: it brings a key segment in CC2 (residues 276-309) in contact with a CC1 region called the CC1 box (previously shown to bind dynein light intermediate chain), and it blocks a motif at the beginning of CC2, called the Spindly motif, from accessing the pointed end complex that caps dynactin's minifilament. Mutations in either the CC1 box, the CC1 2-residue insertion, or the CC2(276-309) segment, 'open up' full-length Spindly and promote its interaction with the dynactin pointed end complex and, in case of the latter two types of mutants, with dynein light intermediate chain. CC1 box-deficient Spindly and the CC2 segment mutant (which corresponds to two charge-inverting point mutations) also support complex formation of Spindly and intact dynein-dynactin. Interestingly, while the CC2 mutant can bind to RZZ, the interaction between RZZ and wild-type Spindly is insufficient to make Spindly competent for dynein-dynactin binding (even when RZZ-Spindly are phosphorylated by mitotic kinases). The authors therefore propose that releasing Spindly from autoinhibition requires an additional trigger at the kinetochore, which likely involves an interaction between the Spindly CC2(276-309) segment and an as yet unidentified kinetochore component. The CC2 mutant is also shown to be defective in kinetochore recruitment and in Spindly-RZZ filament formation in vitro, suggesting kinetochore recruitment of Spindly is coupled to kinetochore expansion through a mechanism involving CC2(276-309).

    The experiments are of excellent technical quality and the results are presented in a logical and concise manner. There is clarity in the writing (the introduction deserves particular praise), and the authors' conclusions are fully supported by the data. Although there is no direct structural evidence for Spindly's closed conformation, as the authors themselves are careful to point out, the numerous Spindly mutants that are characterized (only some of which are mentioned in the summary above) in aggregate make a convincing case for the proposed autoinhibition mechanism.

    We are very grateful to the reviewer for supporting our work

    Minor comments:

    • Page 5: "605-residue adaptor Spindly". State that "605-residue" refers to the human protein.

    We have added this clarification

    • Page 8: "The region of Spindly downstream of the Spindly box (residues 281-322) is very conserved among Spindly orthologues, but not among other members of the BICD adaptor family (Figure 1 - Supplement 1L)." This is not very obvious from the alignment shown in the figure.

    We agree with the reviewer that the text, as written, was confusing. We have now rephrased it and write “Downstream of the Spindly box, sequences of Spindly orthologues and BICD family adaptors diverge”

    • Page 13: "...(A23V-A24V) mutant, which has been previously shown to inhibit the interaction with the LIC2 in a similar assay (Gama et al., 2017)." The LIC isoform used in the referenced study was LIC1.

    Thank you for identifying this error. We have corrected the text accordingly.

    -Figure 5J: Information about statistical significance should be added.

    Done. See also Minor point 7, Reviewer 1.

    -Figure 7B - D: Red on black is not an ideal color choice for these graphs.

    We now replaced red with yellow

    -Page 15: When discussing the recently discovered interphase functions of Spindly, also cite Clemente et al. (2018; doi:10.3390/jdb6020009) and Conte et al. (2018; doi:10.1242/bio.033233).

    We apologize for the involuntary omission of these two references, which have now been included in the revised manuscript.

    -Page 17: "Evidence supporting this idea is that mutations in the 276-306 region, including the deletion of this entire fragment or the introduction of charge-inverting point mutations at residues 295 and 297 respectively abolish or largely decrease the kinetochore recruitment of Spindly ((Raisch et al., 2021) and this study),...". Sacristan et al. (2018) should also be cited in this context, as this study established the importance of residues 274-287 for Spindly recruitment to kinetochores.

    We agree and apologize for the inadvertent omission. We have now included the Sacristan et al. reference in this context.

    • Page 17: "In vitro, the 276-306 region is also required for the assembly of RZZ-Spindly filaments (this study and (Raisch et al., 2021))." It could also be mentioned here that residues 274-287 of Spindly are necessary for RZZ-Spindly filament formation in cells, as shown by Sacristan et al. (2018).

    We have now reported this fact on lines 560-561.

    • Page 17: "Plausibly, the solution to this conundrum will require biochemical reconstitutions addressing the spectrum of interactions that this protein establishes at the kinetochore." Presumably, "this protein" refers to Spindly, but this is not clear since the subject of the preceding sentence is RZZ.

    Done – line 565

    Significance

    Cargo transport by cytoskeletal motors must be tightly regulated to establish and maintain intracellular organization and for faithful execution of development, including cell division. Much of this regulation occurs at the motor-cargo interface but remains poorly understood at the molecular level. In recent years it has become clear that adaptor proteins not only provide a physical link between motors and their cargo but also participate in motor activation. Adaptor-coupled activation is particularly important for dynein, because adaptors promote dynein's interaction with its essential co-factor dynactin.

    BICD2 (along with other Bicaudal D proteins) is the most intensely studied dynein adaptor and has long been known to be subject to autoinhibition with regard to dynein-dynactin binding, which is relieved by cargo binding to the BICD2 C-terminal region. A important question has been whether the same regulatory logic applies to other dynein adaptors. The study by d'Amico et al. presents the first evidence that conformational inhibition extends to adaptors other than Bicaudal D proteins. The study also reveals that Spindly's autoinhibition mechanism is more complex than that of BICD2. This likely reflects Spindly's dual function in dynein-dynactin recruitment and kinetochore expansion. The results of d'Amico et al. suggest that the Spindly autoinhibition mechanism has evolved to coordinate the two processes, and this idea is further supported by a recent study on the RZZ-Spindly interaction from the same group (Raisch et al. 2021; doi:10.1101/2021.12.03.471119). One of the most important insights from d'Amico et al. is that there must be another binding partner of Spindly at kinetochores besides the RZZ complex that participates in the relief of Spindly autoinhibition. The study has therefore identified an important future research direction. It will be interesting to investigate whether additional adaptors follow the multi-step activation model proposed here for Spindly.

    Regarding the technical aspects, the study illustrates that AF2-based structure prediction is a powerful tool for investigating conformational regulation, and it introduces an important innovation: the ability to generate recombinant human dynactin opens the door to the engineering of dynactin mutants, which promises to accelerate mechanistic dissection of this essential dynein co-factor. In conclusion, the study represents a significant step forward in our understanding of how dynein-cargo interactions are regulated by adaptor proteins and is therefore of general interest for researchers studying the molecular mechanisms of chromosome segregation as well as intracellular transport.

    Reviewer #3

    The Dynein-Dynactin (DD) complex interacts with different activating adaptors to assemble functional motor complexes capable of moving along microtubules while transporting various cargoes. However, it remains poorly understood how DD activation is precisely controlled so that Dynein-mediated transport is only stimulated at the appropriate time and place. DD adaptor regulation is likely a crucial piece of this puzzle. In this manuscript, the authors show that Spindly, a mitotic adaptor of DD complex, undergoes a series of conformational rearrangements that result in efficient Spindly autoinhibition and affect its ability to bind DD. The work from d'Amico et al includes an impressive amount of biochemical and biophysical data, supported by well-designed experiments that are carefully documented. Resorting to crosslinking experiments and protein structural modelling, the authors find that several intramolecular contacts occur between specialized domains within Spindly N-terminus. The resulting compact conformation occludes important DD-binding motifs in Spindly and, thus, limits the access of DD to the adaptor. By utilizing different Spindly mutants predicted to render the adaptor more elongated, the authors bypass Spindly autoinhibition and rescue binding to DD in vitro. Surprisingly, unlike other DD adaptors, Spindly autoinhibition is not relieve upon binding to its cargo (the RZZ complex) arguing that the interaction with an additional binding partner is require to fully unleash the potential of Spindly to bind DD. In line with this, the authors identify a Spindly mutant that is unable to localize to kinetochores from human cells, despite its open conformation. Collectively, this work provides significant advances in the understanding of Spindly regulation and brings a new perspective to the mechanism of DD adaptor activation and therefore should be of interest for a wide audience.

    We are very grateful to the reviewer for the support and for the thorough and constructive evaluation of our work.

    Major concerns:

    • The authors show that Spindly 33-605 is able to form a complex with DD which eventually enables the recruitment of Dynactin to kinetochores from Spindly 33-605-expressing cells. This result is unexpected since this Spindly mutant lacks CC1 box, which has been previously shown to be required for the kinetochore localization of Dynactin (Sacristan et al 2018). A more comprehensive discussion about this discrepancy would enrich the article and benefit the audience.

    We thank the reviewer for pointing this out. We now write (line 492): “This result was unexpected, because the CC1 box has been previously shown to be required for kinetochore localization of Dynactin (Sacristan et al., 2018)”

    • In Fig.7, the authors show that two Spindly mutants (Spindly CC2* and Spindly chimera) are unable to fully decorate the kinetochores from human cells. The same is true for Spindly AA/VV mutant. Do the authors know whether these mutants are expressed as stable proteins in cells for example by performing a western blot analysis?

    In this revised version of our manuscript, we have explained more clearly that in this experiment we electroporate recombinant proteins. These are essentially the same proteins that we use for the experiments in vitro. This provides an internal test in these experiments, because we can verify, through their successful expression and purification, that the proteins are stable. We cannot exclude, however, that the proteins are “treated differently” in cells, for instance because they interact differently with certain binding partners in ways that modifies their stability. As the proteins are not expressed continuously, but rather introduced in the cells in a single electroporation event several hours before imaging, the overall levels of these proteins may differ. We have now included a representative western blot (Figure 7 – Supplement 1C) that demonstrates the levels of electroporated proteins in the experiments in Figure 7. SpindlyCC2*appears to be present at somewhat lower levels than the other constructs. mChSpindly33-605 and Spindlychimera, on the other hand, were present at very similar levels, supporting our conclusion that a kinetochore-binding region is impaired in the latter. We now refer in the main text to the uncertainty created by the comparatively lower cellular levels of SpindlyCC2*. We have also chosen more representative kinetochores for the insets of CC2* and Chimera in Figure 7A.

    • In line with the previous point, could the authors tether each Spindly mutant to the kinetochore for example by fusing the construct to known kinetochores proteinssuch as Mis12 and test whether these fusion constructs are now able to recruit Dynactin to kinetochores?

    This would be a potentially interesting experiment. However, reasoning that Spindly is a strong dimer that needs to interact with another strong hexamer like the RZZ complex, discouraged us as these stoichiometries would almost certainly complicate the interpretation of these experiments. It is clear that further work will be required to define the complete picture for this complex system.

    • The authors conclude that the 2-step or multistep mechanism involved in the regulation of Spindly activation may be a common mechanism to different DD adaptors. However, the authors point out to existing differences between the conformational arrangement of Spindly and another DD adaptor, BICD2, arguing against a common mode of regulation for all adaptors. This needs to be clarified.

    The reviewer has a good point and we have indeed tuned this down. We have re-written the last sentence of the abstract and replaced it with “Thus, our work illustrates how Dynein can be specifically activated at a defined cellular locale.” We also write (line 592): Whether a similar 2-step or multistep mechanism applies to additional cargo-adaptor systems is an important question for future studies.

    Minor concerns:

    • In Fig.2D, full length Spindly does not bind DD in vitro. This is most likely to occur because Spindly N-terminus adopts a compacted conformation and hinders the access to DD-binding motifs. In Fig.2B, the authors show a structural prediction for Spindly 1-275 which should adopt a more elongated shape. According to prevailing model, this construct should now be able to bind DD in a similar biochemical assay.

    We agree with the reviewer that Spindly1-275 (and Spindly∆276-306) might be expected to be strong DD binders based on our model. Indeed, these proteins bind to the PE, albeit apparently weakly. Nevertheless, as explained in lines 350 and following, these mutants appear to form higher oligomers and we have not been able to show convincingly that they are fully open and available to bind DD.

    • In Gama et al 2017, LIC1 was able to pull down a wild-type N-terminal Spindly construct. How do the authors reconcile this with the data presented in this manuecript?

    We have expanded the discussion, also to answer major point 5, reviewer 1, on line 446 and following, where we also refer to the observation of Gama et al. 2017.

    • The section where the authors test point mutations to open Spindly ("Opening up Spindly with point mutations") should be better contextualized. The transition is difficult to follow as it is.

    We have now rephrased this part of the text to make are thoughts clearer.

    • In the text, it is not clear whether Mps1 kinase is required to promote RZZ oligomerization in the presence of Spindly chimera, an uninhibited Spindly mutant. According to the model, this mutant construct should drive oligomerization independently of Mps1 (as the N-terminal deletion construct from Sacristan et al 2018).

    The reviewer is correct and we have rephrased this part of the text to clarify

    • The nomenclature the authors adopt for the CC1 second conserved motif (SCM) and for the Spindly motif (SM) can be confusing at some point when identifying each mutant in the text and figures. Nomenclature should be standardized.

    We agree with the reviewer and have now adopted a different nomenclature for the CC2 box or second conserved motif, namely HBS1, for Heavy Chain Binding Site 1. This functional annotation derives from work of one of our laboratories (Carter) and has been discussed with, and approved by, Geert Kops, whose laboratory had originally proposed the name “CC2 box”, as well as Reto Gassmann, Erika Holzbaur, Roberto Dominguez, Sam Reck Peterson, Rick McKenney, and Ahmet Yildiz.

    • In Fig.6A, mCh-Spindly 33-605 and mCh-Spindly chimera lines have the same color.

    Thank you for spotting this subtle mistake. We have corrected the color line.

    Significance

    The work represents a significant advance in the field and it would be of interest for a wide range of audiences.

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    Referee #3

    Evidence, reproducibility and clarity

    The Dynein-Dynactin (DD) complex interacts with different activating adaptors to assemble functional motor complexes capable of moving along microtubules while transporting various cargoes. However, it remains poorly understood how DD activation is precisely controlled so that Dynein-mediated transport is only stimulated at the appropriate time and place. DD adaptor regulation is likely a crucial piece of this puzzle. In this manuscript, the authors show that Spindly, a mitotic adaptor of DD complex, undergoes a series of conformational rearrangements that result in efficient Spindly autoinhibition and affect its ability to bind DD. The work from d'Amico et al includes an impressive amount of biochemical and biophysical data, supported by well-designed experiments that are carefully documented. Resorting to crosslinking experiments and protein structural modelling, the authors find that several intramolecular contacts occur between specialized domains within Spindly N-terminus. The resulting compact conformation occludes important DD-binding motifs in Spindly and, thus, limits the access of DD to the adaptor. By utilizing different Spindly mutants predicted to render the adaptor more elongated, the authors bypass Spindly autoinhibition and rescue binding to DD in vitro. Surprisingly, unlike other DD adaptors, Spindly autoinhibition is not relieve upon binding to its cargo (the RZZ complex) arguing that the interaction with an additional binding partner is require to fully unleash the potential of Spindly to bind DD. In line with this, the authors identify a Spindly mutant that is unable to localize to kinetochores from human cells, despite its open conformation. Collectively, this work provides significant advances in the understanding of Spindly regulation and brings a new perspective to the mechanism of DD adaptor activation and therefore should be of interest for a wide audience.

    Major concerns:

    • The authors show that Spindly 33-605 is able to form a complex with DD which eventually enables the recruitment of Dynactin to kinetochores from Spindly 33-605-expressing cells. This result is unexpected since this Spindly mutant lacks CC1 box, which has been previously shown to be required for the kinetochore localization of Dynactin (Sacristan et al 2018). A more comprehensive discussion about this discrepancy would enrich the article and benefit the audience.
    • In Fig.7, the authors show that two Spindly mutants (Spindly CC2* and Spindly chimera) are unable to fully decorate the kinetochores from human cells. The same is true for Spindly AA/VV mutant. Do the authors know whether these mutants are expressed as stable proteins in cells for example by performing a western blot analysis?
    • In line with the previous point, could the authors tether each Spindly mutant to the kinetochore for example by fusing the construct to known kinetochores proteinssuch as Mis12 and test whether these fusion constructs are now able to recruit Dynactin to kinetochores?
    • The authors conclude that the 2-step or multistep mechanism involved in the regulation of Spindly activation may be a common mechanism to different DD adaptors. However, the authors point out to existing differences between the conformational arrangement of Spindly and another DD adaptor, BICD2, arguing against a common mode of regulation for all adaptors. This needs to be clarified.

    Minor concerns:

    • In Fig.2D, full length Spindly does not bind DD in vitro. This is most likely to occur because Spindly N-terminus adopts a compacted conformation and hinders the access to DD-binding motifs. In Fig.2B, the authors show a structural prediction for Spindly 1-275 which should adopt a more elongated shape. According to prevailing model, this construct should now be able to bind DD in a similar biochemical assay.
    • In Gama et al 2017, LIC1 was able to pull down a wild-type N-terminal Spindly construct. How do the authors reconcile this with the data presented in this manuecript?
    • The section where the authors test point mutations to open Spindly ("Opening up Spindly with point mutations") should be better contextualized. The transition is difficult to follow as it is.
    • In the text, it is not clear whether Mps1 kinase is required to promote RZZ oligomerization in the presence of Spindly chimera, an uninhibited Spindly mutant. According to the model, this mutant construct should drive oligomerization independently of Mps1 (as the N-terminal deletion construct from Sacristan et al 2018).
    • The nomenclature the authors adopt for the CC1 second conserved motif (SCM) and for the Spindly motif (SM) can be confusing at some point when identifying each mutant in the text and figures. Nomenclature should be standardized.
    • In Fig.6A, mCh-Spindly 33-605 and mCh-Spindly chimera lines have the same color.

    Significance

    The work represente a significant advance in the field and it would be of interest for a wide range of audiences.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The study by d'Amico et al. presents an in-depth analysis of how intramolecular folding of the coiled-coil adaptor Spindly regulates its interaction with the motor dynein and its obligatory co-factor dynactin. Using biochemical reconstitution and diverse biophysical approaches (including cross-linking mass spectrometry, X-ray crystallography, AF2-based structure prediction, size exclusion chromatography, and analytical ultracentrifugation), the authors uncover and dissect an intricate Spindly autoinhibition mechanism. At kinetochores Spindly is known to co-oligomerize into filaments with the RZZ complex (its kinetochore receptor/cargo), which drives expansion of the outermost kinetochore region (the corona). Here the authors show that Spindly is a dimer in solution and that successive coiled-coil segments interact with each other in an asymmetric 'closed' conformation that is unable to form a complex with dynein and dynactin. Specifically, a 2-residue insertion in the middle of Spindly's first coiled-coil (CC1) creates a kink that allows CC1 to fold back on itself, which has two important structural consequences: it brings a key segment in CC2 (residues 276-309) in contact with a CC1 region called the CC1 box (previously shown to bind dynein light intermediate chain), and it blocks a motif at the beginning of CC2, called the Spindly motif, from accessing the pointed end complex that caps dynactin's minifilament. Mutations in either the CC1 box, the CC1 2-residue insertion, or the CC2(276-309) segment, 'open up' full-length Spindly and promote its interaction with the dynactin pointed end complex and, in case of the latter two types of mutants, with dynein light intermediate chain. CC1 box-deficient Spindly and the CC2 segment mutant (which corresponds to two charge-inverting point mutations) also support complex formation of Spindly and intact dynein-dynactin. Interestingly, while the CC2 mutant can bind to RZZ, the interaction between RZZ and wild-type Spindly is insufficient to make Spindly competent for dynein-dynactin binding (even when RZZ-Spindly are phosphorylated by mitotic kinases). The authors therefore propose that releasing Spindly from autoinhibition requires an additional trigger at the kinetochore, which likely involves an interaction between the Spindly CC2(276-309) segment and an as yet unidentified kinetochore component. The CC2 mutant is also shown to be defective in kinetochore recruitment and in Spindly-RZZ filament formation in vitro, suggesting kinetochore recruitment of Spindly is coupled to kinetochore expansion through a mechanism involving CC2(276-309).

    The experiments are of excellent technical quality and the results are presented in a logical and concise manner. There is clarity in the writing (the introduction deserves particular praise), and the authors' conclusions are fully supported by the data. Although there is no direct structural evidence for Spindly's closed conformation, as the authors themselves are careful to point out, the numerous Spindly mutants that are characterized (only some of which are mentioned in the summary above) in aggregate make a convincing case for the proposed autoinhibition mechanism.

    Minor comments:

    • Page 5: "605-residue adaptor Spindly". State that "605-residue" refers to the human protein.
    • Page 88: "The region of Spindly downstream of the Spindly box (residues 281-322) is very conserved among Spindly orthologues, but not among other members of the BICD adaptor family (Figure 1 - Supplement 1L)." This is not very obvious from the alignment shown in the figure.
    • Page 13: "...(A23V-A24V) mutant, which has been previously shown to inhibit the interaction with the LIC2 in a similar assay (Gama et al., 2017)." The LIC isoform used in the referenced study was LIC1.
    • Figure 5J: Information about statistical significance should be added.
    • Figure 7B - D: Red on black is not an ideal color choice for these graphs.
    • Page 15: When discussing the recently discovered interphase functions of Spindly, also cite Clemente et al. (2018; doi:10.3390/jdb6020009) and Conte et al. (2018; doi:10.1242/bio.033233).
    • Page 17: "Evidence supporting this idea is that mutations in the 276-306 region, including the deletion of this entire fragment or the introduction of charge-inverting point mutations at residues 295 and 297 respectively abolish or largely decrease the kinetochore recruitment of Spindly ((Raisch et al., 2021) and this study),...". Sacristan et al. (2018) should also be cited in this context, as this study established the importance of residues 274-287 for Spindly recruitment to kinetochores.
    • Page 17: "In vitro, the 276-306 region is also required for the assembly of RZZ-Spindly filaments (this study and (Raisch et al., 2021))." It could also be mentioned here that residues 274-287 of Spindly are necessary for RZZ-Spindly filament formation in cells, as shown by Sacristan et al. (2018).
    • Page 17: "Plausibly, the solution to this conundrum will require biochemical reconstitutions addressing the spectrum of interactions that this protein establishes at the kinetochore." Presumably, "this protein" refers to Spindly, but this is not clear since the subject of the preceding sentence is RZZ.

    Significance

    Cargo transport by cytoskeletal motors must be tightly regulated to establish and maintain intracellular organization and for faithful execution of development, including cell division. Much of this regulation occurs at the motor-cargo interface but remains poorly understood at the molecular level. In recent years it has become clear that adaptor proteins not only provide a physical link between motors and their cargo but also participate in motor activation. Adaptor-coupled activation is particularly important for dynein, because adaptors promote dynein's interaction with its essential co-factor dynactin.

    BICD2 (along with other Bicaudal D proteins) is the most intensely studied dynein adaptor and has long been known to be subject to autoinhibition with regard to dynein-dynactin binding, which is relieved by cargo binding to the BICD2 C-terminal region. A important question has been whether the same regulatory logic applies to other dynein adaptors. The study by d'Amico et al. presents the first evidence that conformational inhibition extends to adaptors other than Bicaudal D proteins. The study also reveals that Spindly's autoinhibition mechanism is more complex than that of BICD2. This likely reflects Spindly's dual function in dynein-dynactin recruitment and kinetochore expansion. The results of d'Amico et al. suggest that the Spindly autoinhibition mechanism has evolved to coordinate the two processes, and this idea is further supported by a recent study on the RZZ-Spindly interaction from the same group (Raisch et al. 2021; doi:10.1101/2021.12.03.471119). One of the most important insights from d'Amico et al. is that there must be another binding partner of Spindly at kinetochores besides the RZZ complex that participates in the relief of Spindly autoinhibition. The study has therefore identified an important future research direction. It will be interesting to investigate whether additional adaptors follow the multi-step activation model proposed here for Spindly.

    Regarding the technical aspects, the study illustrates that AF2-based structure prediction is a powerful tool for investigating conformational regulation, and it introduces an important innovation: the ability to generate recombinant human dynactin opens the door to the engineering of dynactin mutants, which promises to accelerate mechanistic dissection of this essential dynein co-factor.

    In conclusion, the study represents a significant step forward in our understanding of how dynein-cargo interactions are regulated by adaptor proteins and is therefore of general interest for researchers studying the molecular mechanisms of chromosome segregation as well as intracellular transport.

    Reviewer expertise keywords: same as the keywords of this manuscript.

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    Referee #1

    Evidence, reproducibility and clarity

    In this article, Amico et al. explore how Spindly self-regulates its interaction with Dynein-Dynactin. They propose that Spindly adopts an auto-inhibited, closed conformation that blocks the CC1 box and Spindly motif, preventing its interaction with dynein-dynactin. The authors used a combination of X-ray crystallography, biochemistry, and structure predictions to detail the intramolecular interactions in Spindly that mediate this closed state. They then use analytical SEC to test their proposed auto-inhibition mechanism by monitoring Spindly binding to the pointed end complex. They suggest that auto-inhibited Spindly is unable to bind Dynein-Dynactin regardless of the presence or absence of Spindly's cargo, the RZZ complex. In contrast, by using mutagenesis to prevent this auto-inhibition, the authors show that uninhibited Spindly can interact with members of the Dynein-Dynactin complex. Finally, they use cellular experiments to show that relieving autoinhibition prevents the proper localization of Spindly and Dynein-Dynactin to kinetochores during mitosis, likely due to the formation of ectopic Spindly-Dynein-Dynactin complexes in these cells.

    This is an interesting paper that provides important insights into the mechanism of Spindly regulation and its associations with its interacting partners. However, additional work is necessary to support some of their conclusions. In addition, the text is at times quite dense and harder to follow, which prevents their findings as being impactful as could be possible for the bigger picture paradigms of kinetochore function.

    Major Points:

    1. The crosslinking and mass photometry experiments are done at highly differing concentrations (5 μM vs. 10 nM). The mass photometry should be performed at the same concentration as the crosslinking experiments to determine if Spindly forms a higher order oligomer at the higher concentration. These results will aid in the interpretation of the crosslinking mass spectrometry experiments, as the observed interactions could be intermolecular contacts rather than intramolecular contacts if Spindly is tetrameric at these concentrations, as is suggested in figure 4E for specific Spindly constructs.
    2. In figure 2, more conclusive evidence is needed to show that full length Spindly does not form a complex with Dynein-Dynactin. My interpretation of the gels in figure 2D suggests that full length Spindly does form a complex with Dynein-Dynactin, as in the final gel (red outline) it looks as if full length Spindly is indeed peaking with the rest of the Dynein-Dynactin proteins, albeit with excess Spindly eluting later. Figure legends containing protein concentrations used in SEC assays would aid in the interpretation of this data. To conclusively show that full length Spindly doesn't form a complex with Dynein-Dynactin, additional assays will be necessary, such as pull-down assays, or mass photometry.
    3. In figure 3C, 3E, and figure 5C, there is a shift in the PE peaks in the presence of Spindly, but it isn't clear why doesn't the complex doesn't elute earlier than Spindly alone. If the complex is dissociating on the column, additional assays are necessary to confirm that these Spindly constructs stably interact with PE. If this shift is also accompanied by a major change in shape, thus allowing Spindly to elute later than it does alone, this needs to be explored or explained further.
    4. The authors should provide better a rationale for why the pointed-end complex is used in figure 3 in lieu of the complex used in figure 2.
    5. In Figure 5I, WT Spindly also binds to LIC, although less WT Spindly is bound to LIC than Spindly CC2* or Spindly deltaRV. This should be addressed in the text.
    6. The authors claim that the mechanism they describe may be a paradigm for dynein activation by other adaptors at various cellular locations, but they aren't able to identify a mechanism for how Spindly converts from its auto-inhibited state to its permissive state. A more thorough examination of this mechanism is necessary to claim that this mechanism could be paradigmatic, or a revision of the text is needed.

    Minor Points:

    1. The manuscript could benefit from careful review of the text, captions, and figures, as a few minor typos and inconsistencies in the figures and text were present.
    2. The list of common structural and functional features of Dynein-Dynactin adaptors could be indicated more clearly.
    3. Several times the authors use alpha fold predictions to confirm their data. Although the predictions support several of their conclusions, saying that predictions can confirm the data is an overstatement.
    4. Figure 1H would be improved by the addition of the amino acid numbers in the domain diagram.
    5. Concentrations used for each protein for the analytical SEC experiments should be listed in the figure or caption.
    6. In addition to the caption, it would be helpful to the reader to indicate which experiments use farnesylated Spindly.
    7. Error bars are missing from the WT sample in figure 5J. This figure would benefit from statistical analysis.

    Significance

    This paper builds on recent work from the Mussachio lab and others exploring the nature of the fibrous corona at kinetochores and the molecular basis for dynein recruitment. This paper is focused on the structural nature of the interactions that underlie Spindly recruitment to kinetochores and its interactions with dynein and other factors. Although reductionist in its approach, this paper has the potential to have broad implications for thinking about the control of corona assembly and dynein recruitment with an elegant auto-regulation of Spindly. Researchers interested in cell division, chromosome segregation, kinetochore function, dynein regulation, and the structural basis for core cellular processes should be interested in this paper.

  6. Version published to 10.1101/2022.02.02.478874v1 on bioRxiv