Emergent properties of a mitotic Kif18b-MCAK-EB network

  1. Toni McHugh
  2. Julie P.I. Welburn

  • Curated by eLife

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    Evaluation Summary:

    The authors investigate the mechanisms underlying the regulation of microtubule dynamics and length regulation in cells. Understanding how microtubule-binding proteins synergize to affect microtubule behavior is important, and resolving the seemingly contradictory effects of Kif18a on microtubules in mitotic cells vs. in vitro microtubule assays is a worthy endeavor. A major conclusion is that on dynamic microtubules, combining EB3, MCAK, and Kif18b increases microtubule catastrophe compared to other single or double protein combinations. This is in principle a new and interesting finding, but additional evidence would help to more strongly support this conclusion.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

The precise regulation of microtubule length during mitosis is essential to assemble and position the mitotic spindle and segregate chromosomes. Prior work has identified key molecular players in this process, including the kinesin-18 Kif18b and the kinesin-13 Kif2C/MCAK, which both promote microtubule depolymerization. MCAK acts as a potent microtubule depolymerase diffusing short distances on microtubules, while Kif18b is a mitotic processive motor with weak depolymerase activity. However the individual activities of these factors cannot explain the dramatic increase in microtubule dynamics in mitosis. Using in vitro reconstitution and single molecule imaging, we demonstrate that Kif18b, MCAK and the plus-end tracking protein EB3 act in an integrated manner to potently promote microtubule depolymerization. We find Kif18b acts as a microtubule plus end delivery factor for its cargo MCAK, and that Kif18b also promotes EB accumulation to plus ends independently of lattice nucleotide state. Together, our work defines the mechanistic basis for a cooperative Kif18b-EB-MCAK network with emergent properties, that acts to efficiently shorten microtubules in mitosis.

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  1. eLife

    Evaluation Summary:

    The authors investigate the mechanisms underlying the regulation of microtubule dynamics and length regulation in cells. Understanding how microtubule-binding proteins synergize to affect microtubule behavior is important, and resolving the seemingly contradictory effects of Kif18a on microtubules in mitotic cells vs. in vitro microtubule assays is a worthy endeavor. A major conclusion is that on dynamic microtubules, combining EB3, MCAK, and Kif18b increases microtubule catastrophe compared to other single or double protein combinations. This is in principle a new and interesting finding, but additional evidence would help to more strongly support this conclusion.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  2. eLife

    Reviewer #1 (Public Review):

    The study by McHugh et al. uses a combination of in vitro methodologies in an attempt to reconstitute some of the complicated biochemistry that is typically found at the plus end of dynamic microtubules in cells. A myriad of effector molecules play important roles in modulating microtubule dynamics, which is critical to ensure microtubule-mediated processes occur with appropriate spatial and temporal precision (e.g., assembly of the mitotic spindle). Whereas many groups have assessed the role of individual factors in affecting microtubule dynamics (MCAK, Kif18b, etc.), how these molecules function in concert to affect microtubule dynamics is less well understood. This study attempts to do so, with a particular focus on the plus end-directed kinesin, Kif18b, the plus end-binding protein, EB3, and the microtubule-depolymerizing kinesin, MCAK. Although the study yields some interesting insight into the concerted action of these three molecules, the work is confounded by various factors, including findings that conflict with previously published studies, somewhat ambiguous and unconvincing results, and at least one significant unsubstantiated statement/conclusion (see note about MCAK impacting Kif18b processivity). Notably, it seems that the most novel and important contribution of this study is limited to a single finding (that the EB3/Kif18b/MCAK complex is a more potent effector of microtubule dynamics that each protein alone), limiting the overall impact and scope of this study.

    The authors demonstrate the following: (1) that Kif18b binds to EB3 (this was shown previously by Stout et al., MBoC 2011); (2) that Kif18b binds to MCAK (this was shown by Tanenbaum et al., Curr Biol 2011), and can use it as cargo via the MCAK N-terminus; (3) that Kif18b may form a tripartite complex with EB3 and MCAK, at least on stabilized MTs; (4) that Kif18b protects the plus ends of stabilized MTs from MCAK-mediated depolymerization (note this contrasts with findings on dynamic MTs; see point #7 below); (5) that the Kif18b tail reduces MCAK depolymerization activity, although the mechanism may simply be due to lattice binding-mediated stabilization (they note the same with CAMSAP); (6) that EB3/MCAK are slightly better at affecting catastrophe frequencies than MCAK alone (note this contrasts with previously published work (Montenegro Gouveia et al, Curr Biol 2010); (7) that Kif18b increases the frequency of MCAK-mediated catastrophe on dynamic MTs (albeit to a modest effect); and, (8) that EB3, MCAK and Kif18b act cooperatively to induce catastrophe on dynamic MTs (this is the most interesting and novel of their observations). My specific concerns are detailed below.

    Major points:

    1. "Our data indicate that Kif18b promotes EB3 and MCAK plus end targeting and that Kif18b and EB3 proteins function cooperatively to facilitate MCAK microtubule plus end accumulation." To be clear, the authors showed plus end accumulation along GMPCPP-stabilized MTs. This statement should be changed to reflect this important difference. Data presented in Figure 1 suggest that the three proteins interact, but do not indicate how the proteins will interplay along a dynamic microtubule with a native plus end. In fact, given that EB3 exhibits very low affinity for these microtubules, it is unclear how permitting EB-plus end binding would impact these interactions. Moreover, as noted below (point 2), a clear interaction between EB3 and MCAK is not observed by the authors when dynamic MTs are used (Figure 4), thus challenging this statement.

    2. The results in Figure 4 seem to contrast with published data describing the combined effects of MCAK and EB3. For one, Montenegro Gouveia et al (Curr Biol 2010) showed that EB3 targets MCAK to the plus ends of dynamic microtubules in vitro, which the authors of this study do not observe (thus challenging their statement noted in point 1 above; also note that MCAK localizes to plus ends in cells). Secondly, the Montenegro Gouveia et al study also showed that EB3/MCAK dramatically increases catastrophe frequency (in fact, they observed no growth at all above 6 nM MCAK). The reasons for these notable discrepancies are unclear; however, the lack of plus end association of MCAK in their example images (kymographs in Figure 4e, right) raise concerns about the functionality of their protein. Alternatively, the buffer conditions may be sufficiently different from those used by the other study, which then raises the question of which buffer is the best to recapitulate in vivo phenomena? I am more likely to believe positive data than negative data, given the latter could be a consequence of functionally impaired protein, inappropriate buffer conditions, or otherwise.

    3. The authors postulate that the MCAK N-terminus "impacts the processive behavior of Kif18b on the lattice" (this point is also stated in the conclusion paragraph of the introduction); however, they did not report on motor processivity in any condition (e.g., with or without MCAK/EB3). Rather, they only reported a qualitative description of diffusive motion, and pointed to the effects of this behavior on velocity. The authors should either remove the statement concerning processivity modulation, or directly measure it (I would suggest the latter since it may report on something of import). However, the authors would need to analyze many more particles to report an accurate read-out of processivity.

    4. Conceptually speaking, I'm unclear how much information to extract from the interaction studies on stabilized MTs (Figs. 1 and 2). For example, given the weak affinity of EB3 for these (GMPCPP) MTs, how would the Kif18b-EB3 interaction occur when EB3 is near its high affinity-binding site at the plus end? If the authors think that Kif18b can transport EB3 to plus ends, then why didn't they look for this in their dynamic MT experimental scheme (Figs. 4 and 5)?

  3. eLife

    Reviewer #2 (Public Review):

    In this study by McHugh and Welburn, the authors investigate why release of Kif18b into cytoplasm at nuclear envelope breakdown (NEB) results in robust astral MT shortening when the molecule's own MT depolymerizing activity in isolation is comparatively modest. The authors demonstrate, using live reconstitution of EB3, Kif18b and MCAK motors on both GMP-PNP stabilized and live MTs that these proteins interact to form a putative complex in the context of the MT. Kif18b convincingly promoted transport of MCAK and EB3 toward the MT end. Also, accumulation of these proteins at MT tips was enhanced when all three proteins were present. Both MCAK and Kif18b possess consensus sequences (SxIP) for association with EB proteins. Counterintuitively, increased doses of Kif18b protected the MT plus end from depolymerization by MCAK but enhanced catastrophe of live MTs in combination with MCAK. Collectively, these data suggest that in the context of the MT, EB3 and Kif18b synergize with MCAK via weak interactions to promote the targeting of MCAK to MT ends and regulate astral MT length by increasing MT catastrophes in mitosis.

    This manuscript confirms and extends, with more extensive data, the previous work of Tanenbaum et al. Curr. Biol. (2011) suggesting that Kif18b complexes with MCAK to facilitate MT depolymerization and MT length regulation. These data also underscore the concept that weak interactions, in the context of MT binding, can refine diffusive behavior, MT dwell time and MT end targeting of MT regulators. The authors define a system in mammalian cells in which weak interactions between a plus-end directed kinesin and EB proteins can influence the extent and distribution of EB proteins at MT ends. This concept has previously been demonstrated in yeast via reconstitution of Mal3, Tip2 and Tea2 by Bieling et al. Nature (2007). Finally, similar to Montenegro Gouveia et al. Curr Biol (2010), these data show how MCAK activity (with respect to catastrophes and MT length regulation) is potentiated by plus end targeting by accessory proteins. Thus, although none of the principles guiding the behavior of Kif18b, MCAK and EB3 are completely novel, I find the data to be of good quality and generally supportive of the authors' conclusions. Also, despite the previously published studies cited above, we still do not fully understand how the many proteins that are recruited to MT ends are coordinated in their activity. This manuscript represents a solid contribution toward understanding how these proteins synergize functionally at MT ends although there are still some unanswered questions regarding the mechanism of coordination between Kif18b's observed inhibition of MCAK-dependent depolymerization rates while facilitating MCAK-dependent catastrophe.

  4. eLife

    Reviewer #3 (Public Review):

    In this manuscript, McHugh and Welburn, address how the collective activity of three mitotic proteins that act at microtubule ends, MCAK, Kif18b and EB, results in potent microtubule depolymerization, at levels greater than either depolymerase (Kif18b or MCAK) in the system individually. In the cell biological context, their findings provide a biochemical basis for the observed cellular phenotypes of Kif18b depletion despite its relatively weak depolymerase activity in vitro. Prior in vitro and cellular studies have suggested interactions between pairs of proteins in this 3-protein module, but their collective activity has not been reconstituted in vitro, and biochemical models for how they act together are not tested. The findings in this manuscript suggest that Kif18b promotes the microtubule tip localization of both EB and MCAK, where they act collectively to promote depolymerization. The work also adds to our understanding of how MCAK can be effectively localized at microtubule ends when length scales are incompatible with the proposed "diffusion and capture" mechanism or EB-MCAK interactions are not sufficient to explain the observations. The findings presented will be of interest to researchers in the fields of mitosis and microtubule organization. There are some questions as detailed below, which need to be addressed.

  5. Version published to 10.1101/2020.08.09.242602v1 on bioRxiv