Design principles of a microtubule polymerase

Geyer, Elisabeth A.; Miller, Matthew P.; Brautigam, Chad A.; Biggins, Sue; Rice, Luke M.

doi:10.7554/elife.34574

Cited by 43 publications

(105 citation statements)

References 45 publications

(94 reference statements)

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“…Our observation that the bZIP allele restores kinetochore binding and viability suggest that it is homodimerization of Stu2 and not sequence specific elements within the coiled-coil domain that is important for Stu2's kinetochore functions. While previous studies showed that Stu2's in vitro microtubule binding is reduced when dimerization is prevented (Al-Bassam et al, 2006;Geyer et al, 2018), we found that the stu2 cc∆ mutant cells maintained normal spindle length in vivo. Although we cannot rule out the possibility that the stu2 cc∆ mutant has an effect on microtubule dynamics in vivo, the spindles appeared to function normally because inactivation of the spindle checkpoint allowed the stu2 cc∆ mutant cells to enter anaphase and segregate chromosomes.…”

Section: Stu2 Utilizes Multiple Domains To Achieve Kinetochore Associcontrasting

confidence: 89%

“…Cells that expressed the stu2 cc∆ allele arrested with a spindle length (1.91 ± 0.54 μm) that was nearly indistinguishable from that of cells expressing a wild-type copy of STU2. Thus, while mutants that alter Stu2's dimerization exhibit polymerization defects in vitro (Al-Bassam et al, 2006;Geyer et al, 2018), our data indicate that this does not lead to a defect in spindle formation in vivo. Furthermore, the fact that the stu2 cc∆ mutant is defective in kinetochore binding yet supports a normal mitotic spindle provides a tool to examine Stu2's kinetochore functions in cells.…”

Section: Dimerization-deficient Mutant Of Stu2 Is Defective In Kinetomentioning

confidence: 64%

“…With the goal of identifying a mutant that disrupts Stu2's kinetochore localization but that supports normal mitotic spindle formation, we set out to 1) map the domain(s) required for its kinetochore association, and 5 2) examine the spindle length in cells expressing these mutants. XMAP215 family members are large proteins that contain variable numbers of highly conserved tumor over-expressed gene (TOG) domain arrays that bind to curved tubulin dimers (Al-Bassam et al, 2006;Ayaz et al, 2012Ayaz et al, , 2014Fox et al, 2014) as well as a basic linker domain that promotes binding to the microtubule lattice (Geyer et al, 2018;Wang and Huffaker, 1997). The budding yeast Stu2 protein contains two N-terminal TOG domains, followed by a basic linker domain (SK-rich), a homo-dimerization domain (coiled-coil), and a small Cterminal domain that is required for Stu2's association with the microtubule-associated proteins (MAPs) Bik1, Bim1 and Spc72 ( Figure 1A; (Al-Bassam et al, 2006;Usui et al, 2003;Wang and Huffaker, 1997;Wolyniak et al, 2006)).…”

Section: Dimerization-deficient Mutant Of Stu2 Is Defective In Kinetomentioning

confidence: 99%

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A Stu2-mediated intrinsic tension-sensing pathway promotes chromosome biorientation in vivo

Miller

Evans

Zelter

et al. 2018

Preprint

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Accurate segregation of chromosomes to daughter cells is a critical aspect of cell division. It requires the kinetochores on duplicated chromosomes to biorient, attaching to microtubules from opposite poles of the cell. Bioriented attachments come under tension, while incorrect attachments lack tension and must be destabilized. A well-studied error correction pathway is mediated by the Aurora B kinase, which destabilizes low tension-bearing attachments. We recently discovered that in vitro, kinetochores display an additional intrinsic tension-sensing pathway that utilizes Stu2. This pathway's contribution to error correction in cells, however, was unknown. Here, we identify a Stu2 mutant that abolishes its kinetochore function and show that it causes error correction defects in vivo. We also show that this intrinsic tensionsensing pathway functions in concert with the Aurora B-mediated pathway. Together, our work indicates that cells employ at least two pathways to ensure biorientation and the accuracy of chromosome segregation.

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Section: Stu2 Utilizes Multiple Domains To Achieve Kinetochore Associcontrasting

confidence: 89%

Section: Dimerization-deficient Mutant Of Stu2 Is Defective In Kinetomentioning

confidence: 64%

Section: Dimerization-deficient Mutant Of Stu2 Is Defective In Kinetomentioning

confidence: 99%

See 1 more Smart Citation

A Stu2-mediated intrinsic tension-sensing pathway promotes chromosome biorientation in vivo

Miller

Evans

Zelter

et al. 2018

Preprint

Self Cite

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“…Current models of TOG-catalysed tubulin polymerisation all rely on the presence of multiple TOG domains in a single polypeptide or a complex (Ayaz et al, 2014;Geyer et al, 2018;Nithianantham et al, 2018;Slep and Vale, 2007). MEKK1 only contains one TOG domain, suggesting that the MEKK1 TOG domain mechanism-of-action is different to that of other TOG domains.…”

Section: Discussionmentioning

confidence: 99%

A cryptic tubulin-binding domain links MEKK1 to microtubule remodelling

Filipčík

Latham

Cadell

et al. 2020

Preprint

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The MEKK1 protein is a pivotal kinase activator of responses to cellular stress.Activation of MEKK1 can trigger various responses, including mitogen activated protein (MAP) kinases, NF-κB signalling, or cell migration. Notably, MEKK1 activity is triggered by microtubule-targeting chemotherapies, amongst other stressors. Here we show that MEKK1 contains a previously unidentified tumour overexpressed gene (TOG) domain.The MEKK1 TOG domain binds to tubulin heterodimers-a canonical function of TOG domains-but is unusual in that it appears alone rather than as part of a multi-TOG array, and has structural features distinct from previously characterised TOG domains. MEKK1 TOG demonstrates a clear preference for binding curved tubulin heterodimers, which exist in soluble tubulin and at sites of microtubule polymerisation and depolymerisation. Mutations disrupting tubulin-binding lead to destabilisation of the MEKK1 protein in cells, and ultimately a decrease in microtubule density at the leading edge of polarised cells. We also show that MEKK1 mutations at the tubulin-binding interface of the TOG domain recur in patient derived tumour sequences, suggesting selective enrichment of tumour cells with disrupted MEKK1-microtubule association.Together, these findings provide a direct link between the MEKK1 protein and tubulin, which is likely to be relevant to cancer cell migration and response to microtubulemodulating therapies. SIGNIFICANCE STATEMENTThe protein kinase MEKK1 activates stress response pathways in response to various cellular stressors, including chemotherapies that disrupt dynamics of the tubulin cytoskeleton. Filipčík et al., show that MEKK1 contains a previously uncharacterised domain that can preferentially bind to the curved tubulin heterodimer-which is found at sites of microtubule assembly and disassembly. Mutations that interfere with MEKK1tubulin binding disrupt microtubule networks in migrating cells and are enriched in patient-derived tumour sequences. These results suggest that MEKK1-tubulin binding may be relevant to cancer progression, and the efficacy of microtubule-disrupting chemotherapies that require the activity of MEKK1.

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“…Among the 23 mutants or RNAi knockdowns tested in our survey for regulators of the fat body ncMTOC, minispindles (msps) RNAi knockdown ( Figure 14B, C) singularly caused a severe defect in nuclear centricity ( Figure 14D), indicating that Msps is essential for fat body ncMTOC activity. Msps, the homolog of XMAP215/Stu2/Dis1/Alp14/ZYG-9/ch-TOG, functions at the growing MT plus end as a processive MT polymerase (Brouhard et al, 2008;Al-Bassam and Chang, 2011;Wieczorek et al, 2015;Geyer et al, 2018). Msps localizes to the fat body perinuclear MTOC ( Figure 14A, Table 1) and msps knockdown had a severe impact on radial MT assembly at the fat body MTOC.…”

Section: Msps Is Required For the Fat Body Ncmtoc Assembly Independenmentioning

confidence: 99%

A perinuclear microtubule-organizing center controls nuclear positioning and basement membrane secretion

Zheng¹,

Buchwalter²,

Zheng³

et al. 2019

Preprint

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Non-centrosomal microtubule-organizing centers (ncMTOCs) have a variety of roles presumed to serve the diverse functions of the range of cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization, and function. Here we report a novel perinuclear MTOC in Drosophila fat body cells that is anchored by Msp300/Nesprin at the cytoplasmic surface of the nucleus. Msp300 recruits the MT minus-end protein Patronin/CAMSAP, which functions redundantly with Ninein to further recruit the MT polymerase Msps/XMAP215 to assemble noncentrosomal MTs and does so independently of the widespread MT nucleation factor g-tubulin.Functionally, the fat body ncMTOC and the radial MT arrays it organizes is essential for nuclear positioning and for secretion of basement membrane components via retrograde dynein-dependent endosomal trafficking that restricts plasma membrane growth. Together, this study identifies a perinuclear ncMTOC with unique architecture and MT regulation properties that serves vital functions. Highlights• A novel perinuclear MTOC in differentiated fat body cells • The predominant nucleator, g-tubulin, is not required at the fat body ncMTOC • Msp300/Nesprin organizes the ncMTOC at the nuclear surface by recruiting Patronin/CAMSAP and the spectraplakin Shot • Patronin cooperates with Ninein to control MT assembly at the fat body ncMTOC by recruiting Msps • Msps, a MT polymerase, is essential for radial MT elongation from the fat body ncMTOC • Patronin and Msps associate • The ncMTOC and radial MTs, but not actin, control nuclear positioning in the fat body • The fat body MTOC controls retrograde endocytic trafficking to regulate plasma membrane growth and secretion of basement membrane proteins Here we report the discovery of an ncMTOC that is assembled on the surface of nuclei in Drosophila larval fat body cells, a differentiated cell type that has critical secretory functions and serves the metabolic needs of the organism. Assembly of this perinuclear ncMTOC requires the Msp300/Nesprin (nuclear envelope spectrin repeat protein) as a primary organizer/anchor. Msp300 recruits Patronin, which functions redundantly with Ninein for MT assembly at the ncMTOC. Patronin and Ninein cooperate to recruit Msps, which is essential for the elongation of MTs from the fat body ncMTOC. Recruitment of Msps and MT assembly at the ncMTOC are independent of g-tubulin. Functionally, this ncMTOC is necessary for: 1) the regulation of nuclear positioning, 2) controlling plasma membrane growth by facilitating dynein/Rab5-mediated retrograde endosomal trafficking of excess plasma membrane, and 3) the secretion of collagen IV and other basement membrane (BM) proteins. Together, this study identifies a unique perinuclear ncMTOC with novel MT assembly mechanisms that controls physiological roles of fat body cells by supporting nuclear positioning and vital secretory functions of fat body cells.

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Design principles of a microtubule polymerase

Cited by 43 publications

References 45 publications

A Stu2-mediated intrinsic tension-sensing pathway promotes chromosome biorientation in vivo

A Stu2-mediated intrinsic tension-sensing pathway promotes chromosome biorientation in vivo

A cryptic tubulin-binding domain links MEKK1 to microtubule remodelling

A perinuclear microtubule-organizing center controls nuclear positioning and basement membrane secretion

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