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      Microtubule minus-end stability is dictated by the tubulin off-rate

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          Abstract

          Dynamic microtubule minus ends are more stable than plus ends when controlling for growth rate. Strothman et al. find that minus-end stability is not determined by the size of the stabilizing GTP cap, but rather the GTP-tubulin off-rate. Furthermore, the mitotic kinesins MCAK and HSET antagonistically regulate minus-end catastrophe by modulation of the tubulin off-rate.

          Abstract

          Dynamic organization of microtubule minus ends is vital for the formation and maintenance of acentrosomal microtubule arrays. In vitro, both microtubule ends switch between phases of assembly and disassembly, a behavior called dynamic instability. Although minus ends grow slower, their lifetimes are similar to those of plus ends. The mechanisms underlying these distinct dynamics remain unknown. Here, we use an in vitro reconstitution approach to investigate minus-end dynamics. We find that minus-end lifetimes are not defined by the mean size of the protective GTP-tubulin cap. Rather, we conclude that the distinct tubulin off-rate is the primary determinant of the difference between plus- and minus-end dynamics. Further, our results show that the minus-end–directed kinesin-14 HSET/KIFC1 suppresses tubulin off-rate to specifically suppress minus-end catastrophe. HSET maintains its protective minus-end activity even when challenged by a known microtubule depolymerase, kinesin-13 MCAK. Our results provide novel insight into the mechanisms of minus-end dynamics, essential for our understanding of microtubule minus-end regulation in cells.

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          Control of microtubule organization and dynamics: two ends in the limelight.

          Microtubules have fundamental roles in many essential biological processes, including cell division and intracellular transport. They assemble and disassemble from their two ends, denoted the plus end and the minus end. Significant advances have been made in our understanding of microtubule plus-end-tracking proteins (+TIPs) such as end-binding protein 1 (EB1), XMAP215, selected kinesins and dynein. By contrast, information on microtubule minus-end-targeting proteins (-TIPs), such as the calmodulin-regulated spectrin-associated proteins (CAMSAPs) and Patronin, has only recently started to emerge. Here, we review our current knowledge of factors, including microtubule-targeting agents, that associate with microtubule ends to control the dynamics and function of microtubules during the cell cycle and development.
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            Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer.

            Microtubules can be assembled in vitro from purified alpha/beta tubulin heterodimers in the presence of GTP. Tubulin is routinely obtained from animal brain tissue through repetitive cycles of polymerization-depolymerization, followed by ion-exchange chromatography to remove any contaminating microtubule-associated proteins and motors. Here, we show that only two cycles of polymerization-depolymerization of pig brain tubulin in the presence of a high-molarity PIPES buffer allow the efficient removal of contaminating proteins and production of a high-concentration tubulin solution. The proposed protocol is rapid and yields more active tubulin than the traditional ion-exchange chromatography-based procedures.
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              Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau.

              Microtubule-associated proteins (MAP), such as tau, modulate the extent and rate of microtubule assembly and play an essential role in morphogenetic processes, such as axonal growth. We have examined the mechanism by which tau affects microtubule polymerization by examining the kinetics of microtubule assembly and disassembly through direct observation of microtubules using dark-field microscopy. Tau increases the rate of polymerization, decreases the rate of transit into the shrinking phase (catastrophe), and inhibits the rate of depolymerization. Tau strongly suppresses the catastrophe rate, and its ability to do so is independent of its ability to increase the elongation rate. Thus, tau generates a partially stable but still dynamic state in microtubules. This state is perturbed by phosphorylation by MAP2 kinase, which affects all three activities by lowering the affinity of tau for the microtubule lattice.
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                Author and article information

                Journal
                J Cell Biol
                J. Cell Biol
                jcb
                jcb
                The Journal of Cell Biology
                Rockefeller University Press
                0021-9525
                1540-8140
                02 September 2019
                16 August 2019
                : 218
                : 9
                : 2841-2853
                Affiliations
                [1 ]Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN
                [2 ]Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI
                [3 ]Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN
                [4 ]Department of Biochemistry, Vanderbilt University, Nashville, TN
                Author notes
                Correspondence to Marija Zanic: marija.zanic@ 123456vanderbilt.edu

                M. Podolski’s present address is Department of Bioengineering, University of California, Berkeley, Berkeley, CA.

                Author information
                https://orcid.org/0000-0001-7849-0626
                https://orcid.org/0000-0002-6893-2678
                https://orcid.org/0000-0003-2711-9581
                https://orcid.org/0000-0002-5127-5819
                Article
                201905019
                10.1083/jcb.201905019
                6719460
                31420452
                d78039ff-1752-418b-8adf-aeff801f19ab
                © 2019 Strothman et al.

                This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

                History
                : 03 May 2019
                : 11 July 2019
                : 23 July 2019
                Page count
                Pages: 13
                Funding
                Funded by: National Institutes of Health, DOI https://doi.org/10.13039/100000002;
                Award ID: R35GM119552
                Award ID: R01 GM086610
                Funded by: National Institutes of Health, DOI https://doi.org/10.13039/100000002;
                Award ID: 5T32GM008554-21
                Funded by: Human Frontier Science Program, DOI https://doi.org/10.13039/501100000854;
                Funded by: Searle Scholars Program
                Funded by: Vanderbilt University, DOI https://doi.org/10.13039/100006537;
                Categories
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                Cell biology
                Cell biology

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