Arl2- and Msps-dependent microtubule growth governs asymmetric division

Chen, Keng; Koe, Chwee Tat; Xing, Zhanyuan Benny; Tian, Xiaolin; Rossi, Fabrizio; Wang, Cheng; Tang, Quan; Zong, Wenhui; Hong, Wan Jin; Taneja, Reshma; Yu, Fengwei; González, Cayetano; Wu, Chunlai; Endow, Sharyn A.; Wang, Hongyan

doi:10.1083/jcb.201503047

Cited by 26 publications

(56 citation statements)

References 73 publications

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“…Mini spindles (Msps) is a XMAP215/ch-TOG family protein and a key microtubule polymerase that promotes microtubule growth in dividing NSCs (Chen et al, 2016;Lee et al, 2001). However, the function of Msps in regulating microtubule assembly is poorly understood in non-dividing cells.…”

Section: Resultsmentioning

confidence: 99%

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Msps Governs Acentrosomal Microtubule Assembly and Reactivation of Quiescent Neural Stem Cells

Wang

Deng

Tan

et al. 2020

Preprint

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Running titleMsps promotes microtubule growth and NSC reactivation SUMMARYThe ability of stem cells to switch between quiescence and proliferation is crucial for tissue homeostasis and regeneration. Drosophila quiescent neural stem cells (NSCs) extend a primary cellular protrusion from the cell body prior to their reactivation. However, the structure and function of this protrusion are not well established. In this study, we show that in the primary protrusion of quiescent NSCs microtubules are predominantly acentrosomal and oriented plus-end-out, distal to the cell body. We have identified Mini Spindles (Msps)/XMAP215 as a key regulator of NSC reactivation and acentrosomal microtubule assembly in quiescent NSCs. We show that E-cadherin, a cell adhesion molecule, is localized to NSC-neuropil contact points, in a Msps-dependent manner, and is intrinsically required for NSC reactivation. Our study demonstrates a novel mechanism by which Msps-dependent microtubule assembly in the primary protrusion of quiescent NSCs targets E-cadherin to NSCneuropil contact sites to promote NSC reactivation. We propose that the neuropil functions as a new niche for promoting NSC reactivation, which may be a general paradigm in mammalian systems.

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Section: Resultsmentioning

confidence: 99%

“…Mini spindles (Msps), a XMAP215/ch-TOG/Msps family protein is a key regulator of microtubule growth in dividing cells (Chen et al, 2016;Lee et al, 2001). Msps functions as a microtubule polymerase by binding to microtubule plus-ends (Lee et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Msps Governs Acentrosomal Microtubule Assembly and Reactivation of Quiescent Neural Stem Cells

Wang

Deng

Tan

et al. 2020

Preprint

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“…When combined with the results from the Cowan lab showing direct effects on tubulin folding, there is compelling evidence for TBCD in this pathway. Other screens found mutations in ARL2 orthologs in C. elegans [20], T. brucei [21], and D. melanogaster [22] that further support a role for this regulatory GTPase in tubulin biology. However, ARL2 was never identified in the earlier tubulin folding assays.…”

Section: Introductionmentioning

confidence: 88%

Nucleotide Binding to ARL2 in the TBCD ∙ ARL2 ∙ β-Tubulin Complex Drives Conformational Changes in β-Tubulin

Francis

Goswami

Novick

et al. 2017

Journal of Molecular Biology

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Microtubules are highly dynamic tubulin polymers that are required for a variety of cellular functions. Despite the importance of a cellular population of tubulin dimers, we have incomplete information about the mechanisms involved in the biogenesis of αβ-tubulin heterodimers. In addition to prefoldin and the TCP-1 Ring Complex, five tubulin-specific chaperones, termed cofactors A-E (TBCA-E), and GTP are required for the folding of α- and β-tubulin subunits and assembly into heterodimers. We recently described the purification of a novel trimer, TBCD•ARL2•β-tubulin. Here, we employed hydrogen/deuterium exchange coupled with mass spectrometry to explore the dynamics of each of the proteins in the trimer. Addition of guanine nucleotides resulted in changes in the solvent accessibility of regions of each protein that led to predictions about each’s role in tubulin folding. Initial testing of that model confirmed that it is ARL2, and not β-tubulin, that exchanges GTP in the trimer. Comparisons of the dynamics of ARL2 monomer to ARL2 in the trimer suggested that its protein interactions were comparable to those of a canonical GTPase with an effector. This was supported by the use of nucleotide binding assays that revealed an increase in the affinity for GTP by ARL2 in the trimer. We conclude that the TBCD•ARL2•β-tubulin complex represents a functional intermediate in the β-tubulin folding pathway whose activity is regulated by the cycling of nucleotides on ARL2. The co-purification of guanine nucleotide on the β-tubulin in the trimer is also shown, with implications to modeling the pathway.

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“…The same genetic screens that identified mutations in CIN1/Alp1/TTN1 (TBCD orthologs) that altered microtubules also identified mutations in CIN4/ Alp41/TTN5, the orthologs of ARL2 in S. cerevisiae, S. pombe, and A. thaliana, respectively (8,9). Genetic screening similarly identified effects of mutations in ARL2 orthologs in Caenorhabditis elegans (21), Trypanosoma brucei (22), and Drosophila melanogaster (23). Thus, there is strong evidence that ARL2 orthologs play essential roles in tubulin and microtubule biology.…”

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confidence: 99%

A Trimer Consisting of the Tubulin-specific Chaperone D (TBCD), Regulatory GTPase ARL2, and β-Tubulin Is Required for Maintaining the Microtubule Network

Francis

Newman

Cunningham

et al. 2017

Journal of Biological Chemistry

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Edited by Velia M. FowlerMicrotubule dynamics involves the polymerization and depolymerization of tubulin dimers and is an essential and highly regulated process required for cell viability, architecture, and division. The regulation of the microtubule network also depends on the maintenance of a pool of ␣␤-tubulin heterodimers. These dimers are the end result of complex folding and assembly events, requiring the TCP1 Ring Complex (TriC or CCT) chaperonin and five tubulin-specific chaperones, tubulin binding cofactors A-E (TBCA-TBCE). However, models of the actions of these chaperones are incomplete or inconsistent. We previously purified TBCD from bovine tissues and showed that it tightly binds the small GTPase ARL2 but appears to be inactive. Here, in an effort to identify the functional form of TBCD and using non-denaturing gels and immunoblotting, we analyzed lysates from a number of mouse tissues and cell lines to identify the quaternary state(s) of TBCD and ARL2. We found that both proteins co-migrated in native gels in a complex of ϳ200 kDa that also contained ␤-tubulin. Using human embryonic kidney cells enabled the purification of the TBCD⅐ARL2⅐␤-tubulin trimer found in cell and tissue lysates as well as two other novel TBCD complexes. Characterization of ARL2 point mutants that disrupt binding to TBCD suggested that the ARL2-TBCD interaction is critical for proper maintenance of microtubule densities in cells. We conclude that the TBCD⅐ARL2⅐␤-tubulin trimer represents a functional complex whose activity is fundamental to microtubule dynamics.Microtubules are highly dynamic polymers that are best known for their roles as the central cytoskeletal structure in cells and in mitotic spindles during cell division. They are also the tracks on which organelles traffic, particularly important in polarized cells that generate great distances between parts of the cell. Additionally, they are the core of sensory and motile cilia or flagella. The formation and destruction of microtubules and microtubule bundles are orchestrated by a large number of proteins that include the microtubule-associated proteins. Microtubules are polymers of the ␣␤-tubulin dimer, with several genes encoding each ␣-or ␤-tubulin subunit (e.g. see Lewis et al. (1)), resulting in some diversity in composition. Tubulins can also be modified by posttranslational modifications, including acetylation, tyrosination, and phosphorylation, which can alter the dynamics of the polymerization and depolymerization reactions. Because of the essential role of microtubules in cell division, they have also been the target of many antitumor therapies, e.g. the taxanes and Vinca alkaloids (2). However, despite their importance to cells and in the clinic and decades of research, we still lack a complete molecular-level understanding of the biosynthesis and regulation of the formation of ␣␤-tubulin.Tubulins are typically the most abundant proteins in mammalian cells, but the generation of the ␣␤-tubulin dimer requires a complex series of biosynthetic steps to ...

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Arl2- and Msps-dependent microtubule growth governs asymmetric division

Abstract: Drosophila Arl2 governs neuroblast asymmetric cell division through regulation of microtubule growth and localization of Msps to centrosomes.

Cited by 26 publications

References 73 publications

Msps Governs Acentrosomal Microtubule Assembly and Reactivation of Quiescent Neural Stem Cells

Msps Governs Acentrosomal Microtubule Assembly and Reactivation of Quiescent Neural Stem Cells

Nucleotide Binding to ARL2 in the TBCD ∙ ARL2 ∙ β-Tubulin Complex Drives Conformational Changes in β-Tubulin

A Trimer Consisting of the Tubulin-specific Chaperone D (TBCD), Regulatory GTPase ARL2, and β-Tubulin Is Required for Maintaining the Microtubule Network

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