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      In vivo mitotic spindle scaling can be modulated by changing the levels of a single protein: the microtubule polymerase XMAP215

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          Abstract

          In many organisms, early embryonic development is characterized by a series of reductive cell divisions that result in rapid increases in cell number and concomitant decreases in cell size. Intracellular organelles, such as the nucleus and mitotic spindle, also become progressively smaller during this developmental window, but the molecular and mechanistic underpinnings of these scaling relationships are not fully understood. For the mitotic spindle, changes in cytoplasmic volume are sufficient to account for size scaling during early development in certain organisms. This observation is consistent with models that evoke a limiting component, whereby the smaller absolute number of spindle components in smaller cells limits spindle size. Here we investigate the role of a candidate factor for developmental spindle scaling, the microtubule polymerase XMAP215. Microinjection of additional XMAP215 protein into Xenopus laevis embryos was sufficient to induce the assembly of larger spindles during developmental stages 6.5, 7, and 8, whereas addition of a polymerase-incompetent XMAP215 mutant resulted in a downward shift in the in vivo spindle scaling curve. In sum, these results indicate that even small cells are able to produce larger spindles if microtubule growth rates are increased and suggest that structural components are not limiting.

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          XMAP215 is a processive microtubule polymerase.

          Fast growth of microtubules is essential for rapid assembly of the microtubule cytoskeleton during cell proliferation and differentiation. XMAP215 belongs to a conserved family of proteins that promote microtubule growth. To determine how XMAP215 accelerates growth, we developed a single-molecule assay to visualize directly XMAP215-GFP interacting with dynamic microtubules. XMAP215 binds free tubulin in a 1:1 complex that interacts with the microtubule lattice and targets the ends by a diffusion-facilitated mechanism. XMAP215 persists at the plus end for many rounds of tubulin subunit addition in a form of "tip tracking." These results show that XMAP215 is a processive polymerase that directly catalyzes the addition of up to 25 tubulin dimers to the growing plus end. Under some circumstances XMAP215 can also catalyze the reverse reaction, namely microtubule shrinkage. The similarities between XMAP215 and formins, actin polymerases, suggest that processive tip tracking is a common mechanism for stimulating the growth of cytoskeletal polymers.
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            A TOG:αβ-tubulin complex structure reveals conformation-based mechanisms for a microtubule polymerase.

            Stu2p/XMAP215/Dis1 family proteins are evolutionarily conserved regulatory factors that use αβ-tubulin-interacting tumor overexpressed gene (TOG) domains to catalyze fast microtubule growth. Catalysis requires that these polymerases discriminate between unpolymerized and polymerized forms of αβ-tubulin, but the mechanism by which they do so has remained unclear. Here, we report the structure of the TOG1 domain from Stu2p bound to yeast αβ-tubulin. TOG1 binds αβ-tubulin in a way that excludes equivalent binding of a second TOG domain. Furthermore, TOG1 preferentially binds a curved conformation of αβ-tubulin that cannot be incorporated into microtubules, contacting α- and β-tubulin surfaces that do not participate in microtubule assembly. Conformation-selective interactions with αβ-tubulin explain how TOG-containing polymerases discriminate between unpolymerized and polymerized forms of αβ-tubulin and how they selectively recognize the growing end of the microtubule.
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              Nuclear size is regulated by importin α and Ntf2 in Xenopus.

              The size of the nucleus varies among different cell types, species, and disease states, but mechanisms of nuclear size regulation are poorly understood. We investigated nuclear scaling in the pseudotetraploid frog Xenopus laevis and its smaller diploid relative Xenopus tropicalis, which contains smaller cells and nuclei. Nuclear scaling was recapitulated in vitro using egg extracts, demonstrating that titratable cytoplasmic factors determine nuclear size to a greater extent than DNA content. Nuclear import rates correlated with nuclear size, and varying the concentrations of two transport factors, importin α and Ntf2, was sufficient to account for nuclear scaling between the two species. Both factors modulated lamin B3 import, with importin α increasing overall import rates and Ntf2 reducing import based on cargo size. Importin α also contributes to nuclear size changes during early X. laevis development. Thus, nuclear transport mechanisms are physiological regulators of both interspecies and developmental nuclear scaling. Copyright © 2010 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Role: Monitoring Editor
                Journal
                Mol Biol Cell
                Mol. Biol. Cell
                molbiolcell
                mbc
                mboc
                Molecular Biology of the Cell
                The American Society for Cell Biology
                1059-1524
                1939-4586
                01 June 2018
                : 29
                : 11
                : 1311-1317
                Affiliations
                [1]Department of Molecular Biology, University of Wyoming, Laramie, WY 82071
                [2]Molecular and Cellular Life Sciences Program, University of Wyoming, Laramie, WY 82071
                University of North Carolina
                Author notes

                The authors declare no competing financial interests.

                Author contributions: A.M.-J. and J.C.G. conceived the study. A.M.-J. and P.J. performed embryo microinjections and spindle length measurements/quantification. A.M.-J. and J.C.G. designed the experiments and drafted the manuscript. All authors participated in the revisions of manuscript drafts.

                *Address correspondence to: J. C. Gatlin ( jgatlin@ 123456uwyo.edu ).
                Article
                E18-01-0011
                10.1091/mbc.E18-01-0011
                5994900
                29851557
                a6be48a0-8e90-45ba-a884-4b5f4dafddae
                © 2018 Milunovic´-Jevtic´ et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

                This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.

                History
                : 05 January 2018
                : 26 March 2018
                : 30 March 2018
                Categories
                Brief Reports

                Molecular biology
                Molecular biology

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