The γ-tubulin-specific inhibitor gatastatin reveals temporal requirements of microtubule nucleation during the cell cycle

Chinen, Takumi; Liu, Peng; Shioda, Shuya; Pagel, Judith; Cerikan, Berati; Lin, Tien-chen; Gruß, Oliver J.; Hayashi, Yoshiki; Takeno, Haruka; Shima, Tomohiro; Okada, Yasushi; Hayakawa, Ichiro; Hayashi, Yoshio; Kigoshi, Hideo; Usui, Takeo; Schiebel, Elmar

doi:10.1038/ncomms9722

Cited by 51 publications

(62 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Given this lack of minus-end-specific binding in vitro, we hypothesized that NuMA is indirectly recruited to spindle minus-ends by one of three known direct minus-end binders active at mitosis: g-TuRC (Zheng et al, 1995), CAMSAP1 (Hendershott and Vale, 2014;Jiang et al, 2014) (Figure 1; CAMSAP2 and 3 are phosphorylated at mitosis and no longer interact with microtubules ), and KANSL1/3 (Meunier et al, 2015). To test this hypothesis, we treated RPE1 cells with 30 mM gatastatin to block g-TuRC binding (Chinen et al, 2015) ( Figure 5A; Figure 5-figure supplement 1A,B). We also made inducible CRISPR/Cas9 RPE1 cell lines to knock out CAMSAP1 or KANSL1 ( Figure 5-figure supplement 1C,D), as KANSL1 depletion has been shown to delocalize the entire KANSL complex (Meunier et al, 2015).…”

Section: Numa Localizes To Minus-ends Independently Of Known Minus-enmentioning

confidence: 99%

“…To inhibit g-tubulin, 30 mM gatastatin (gift of Takeo Usui and Ichiro Hayakawa, University of Tsukuba and Okayama University, respectively) (Chinen et al, 2015) was added 25-60 min before imaging ( Figure 5A-C, Figure 5-figure supplement 1A). To test microtubule re-growth in the presence of gatastatin ( Figure 5-figure supplement 1B), all cells were treated with 10 mM STLC for 6 hr to create monopolar spindles.…”

Section: Drug Treatment and Microtubule Re-growthmentioning

confidence: 99%

See 1 more Smart Citation

NuMA Recruits Dynein Activity to Microtubule Minus-Ends at Mitosis

Hueschen

Kenny

et al. 2018

Biophysical Journal

View full text Add to dashboard Cite

To build the spindle at mitosis, motors exert spatially regulated forces on microtubules. We know that dynein pulls on mammalian spindle microtubule minus-ends, and this localized activity at ends is predicted to allow dynein to cluster microtubules into poles. How dynein becomes enriched at minus-ends is not known. Here, we use quantitative imaging and laser ablation to show that NuMA targets dynactin to minus-ends, localizing dynein activity there. NuMA is recruited to new minus-ends independently of dynein and more quickly than dynactin; both NuMA and dynactin display specific, steady-state binding at minus-ends. NuMA localization to minus-ends involves a C-terminal region outside NuMA's canonical microtubule-binding domain and is independent of minus-end binders g-TuRC, CAMSAP1, and KANSL1/3. Both NuMA's minus-endbinding and dynein-dynactin-binding modules are required to rescue focused, bipolar spindle organization. Thus, NuMA may serve as a mitosis-specific minus-end cargo adaptor, targeting dynein activity to minus-ends to cluster spindle microtubules into poles.

show abstract

Section: Numa Localizes To Minus-ends Independently Of Known Minus-enmentioning

confidence: 99%

Section: Drug Treatment and Microtubule Re-growthmentioning

confidence: 99%

NuMA Recruits Dynein Activity to Microtubule Minus-Ends at Mitosis

Hueschen

Kenny

et al. 2018

Biophysical Journal

View full text Add to dashboard Cite

show abstract

“…This causes errors in centriole duplication and spindle assembly, often inducing monopolar spindles (21). These phenotypes resemble defects from impaired recruitment or function of ␥-tubulin (23)(24)(25).…”

mentioning

confidence: 99%

Functional Analysis of γ-Tubulin Complex Proteins Indicates Specific Lateral Association via Their N-terminal Domains

Farache

Jauneau²,

Chemin

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

Edited by Velia FowlerMicrotubules are nucleated from multiprotein complexes containing ␥-tubulin and associated ␥-tubulin complex proteins (GCPs). Small complexes (␥TuSCs) comprise two molecules of ␥-tubulin bound to the C-terminal domains of GCP2 and GCP3. ␥TuSCs associate laterally into helical structures, providing a structural template for microtubule nucleation. In most eukaryotes ␥TuSCs associate with additional GCPs (4, 5, and 6) to form the core of the so-called ␥-tubulin ring complex (␥TuRC). GCPs 2-6 constitute a family of homologous proteins. Previous structural analysis and modeling of GCPs suggest that all family members can potentially integrate into the helical structure. Here we provide experimental evidence for this model. Using chimeric proteins in which the N-and C-terminal domains of different GCPs are swapped, we show that the N-terminal domains define the functional identity of GCPs, whereas the C-terminal domains are exchangeable. FLIM-FRET experiments indicate that GCP4 and GCP5 associate laterally within the complex, and their interaction is mediated by their N-terminal domains as previously shown for ␥TuSCs. Our results suggest that all GCPs are incorporated into the helix via lateral interactions between their N-terminal domains, whereas the C-terminal domains mediate longitudinal interactions with ␥-tubulin. Moreover, we show that binding to ␥-tubulin is not essential for integrating into the helical complex.In all eukaryotes, microtubules are nucleated from specialized multiprotein complexes containing ␥-tubulin and associated proteins (1-3). These complexes resemble small rings by electron microscopy and are thus called ␥-tubulin ring complexes (␥TuRCs) 4 (4 -7). Closer inspection revealed that these ␥TuRCs are helices of one turn, with the two ends overlapping. They are ubiquitous and essential for viability: the growth of new microtubules is crucial to drive mitotic spindle formation and cell division. ␥TuRCs are mainly composed of ␥-tubulin and of proteins of the GCP (␥-tubulin complex protein) family. GCPs are characterized by sequence homology in two specific regions, also referred to as the grip1 and grip2 motifs (8). Five members of this family are known: GCPs 2, 3, 4, 5, and 6. GCPs 2 and 3 associate with ␥-tubulin to form a V-shaped subcomplex, called ␥-tubulin small complex (␥TuSC). GCPs 2 and 3 constitute the arms of the V, interacting laterally via their N-terminal domains (Fig. 1, A and B). The C-terminal domains are located at the two tips of the V, each binding one molecule of ␥-tubulin (9). In the budding yeast Saccharomyces cerevisiae, ␥TuSCs are directly recruited to the spindle pole body (yeast centrosome equivalent) by the protein Spc110. Oligomers of Spc110 interact with the basis of the V-shaped ␥TuSCs and stabilize their lateral association (10 -13). Likely, seven ␥TuSCs assemble stepwise into a helix of one turn plus a small overlap (14). In this helical array, the ␥-tubulin molecules are exposed to form a platform from which ␣/␤-tubulin dimers assemble into...

show abstract

“…Microtubules are mainly composed of α-tubulin and β-tubulin heterodimers that turn over continuously (Kavallaris, 2010;Pasquier & Kavallaris, 2008). Tubulin has multiple cellular functions, for instance, maintaining cell morphology as cytoskeleton, participating in material transportation and signal transduction and playing an important role in the process of mitosis by constructing a bipolar spindle (Chinen et al, 2015;2015a;Yan et al, 2016). The importance of tubulin in cell division makes it as an important target for antitumor agents (Risinger, Giles, & Mooberry, 2009).…”

Section: Introductionmentioning

confidence: 99%

Design, synthesis, and biological evaluation of 2,3‐diphenyl‐cycloalkyl pyrazole derivatives as potential tubulin polymerization inhibitors

et al. 2019

View full text Add to dashboard Cite

Several novel cycloalkyl‐fused 2,3‐diaryl pyrazole derivatives were designed, synthesized, and evaluated as potential anti‐tubulin agents. Compound A10 exhibited the most potent antiproliferative activity against a panel of cancer lines (IC50 = 0.78–2.42 μM) and low cytotoxicity against 293T & L02 (CC50 values of 131.74 and 174.89 μM, respectively). Moreover, A10 displayed inhibition of tubulin polymerization in vitro, arrested the G2/M phase of the cell cycle, changed morphology of tubulin, increased intracellular reactive oxygen species, and induced apoptosis of HeLa cells. Docking simulation and 3D‐QSAR models were performed to elaborate on the anti‐tubulin mechanism of the derivatives. The inhibition of monoclonal colony formation provided more intuitional data to verify the possibility of A10 as a novel tubulin assembling inhibitor.

show abstract

The γ-tubulin-specific inhibitor gatastatin reveals temporal requirements of microtubule nucleation during the cell cycle

Cited by 51 publications

References 43 publications

NuMA Recruits Dynein Activity to Microtubule Minus-Ends at Mitosis

NuMA Recruits Dynein Activity to Microtubule Minus-Ends at Mitosis

Functional Analysis of γ-Tubulin Complex Proteins Indicates Specific Lateral Association via Their N-terminal Domains

Design, synthesis, and biological evaluation of 2,3‐diphenyl‐cycloalkyl pyrazole derivatives as potential tubulin polymerization inhibitors

Contact Info

Product

Resources

About