Faculty Opinions recommendation of EB1 reveals mobile microtubule nucleation sites in Arabidopsis.

Neurons have highly polarized arrangements of microtubules, but it is incompletely understood how microtubule polarity is controlled in either axons or dendrites. To explore whether microtubule nucleation by γ-tubulin might contribute to polarity, we analyzed neuronal microtubules in Drosophila containing gain- or loss-of-function alleles of γ-tubulin. Both increased and decreased activity of γ-tubulin, the core microtubule nucleation protein, altered microtubule polarity in axons and dendrites, suggesting a close link between regulation of nucleation and polarity. To test whether nucleation might locally regulate polarity in axons and dendrites, we examined the distribution of γ-tubulin. Consistent with local nucleation, tagged and endogenous γ-tubulins were found in specific positions in dendrites and axons. Because the Golgi complex can house nucleation sites, we explored whether microtubule nucleation might occur at dendritic Golgi outposts. However, distinct Golgi outposts were not present in all dendrites that required regulated nucleation for polarity. Moreover, when we dragged the Golgi out of dendrites with an activated kinesin, γ-tubulin remained in dendrites. We conclude that regulated microtubule nucleation controls neuronal microtubule polarity but that the Golgi complex is not directly involved in housing nucleation sites.

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Microtubule nucleation by the isolated microtubule-organizing centre of Physarum polycephalum myxamoebae

Journal of Cell Science ◽

10.1242/jcs.55.1.365 ◽

1982 ◽

Vol 55 (1) ◽

pp. 365-381

Author(s):

A. Roobol ◽

J.C. Havercroft ◽

K. Gull

Keyword(s):

Physarum Polycephalum ◽

Rnase A ◽

Microtubule Nucleation ◽

Nucleation Sites ◽

Dnase 1 ◽

Pericentriolar Material

The nucleus—centrosome complex from Physarum polycephalum myxamoebae has been purified. The complex contained the centriole pair and pericentriolar material in association with the nucleus. Apart from some unusually stable microtubules, which appeared to be involved in maintaining the nucleus-centrosome association, endogenous microtubule arrays had been stripped from the complex during isolation. When the nucleus—centrosome complex was incubated with purified brain or myxamoebal tubulins the growth of 45–70 microtubules was initiated onto the pericentriolar material. The number and length of the nucleated microtubules was proportional to the tubulin concentration. Pretreatment of the nucleus—centrosome complex with DNase 1, RNase A, antitubulin antibody and anticentriolar antibody did not affect pericentriolar nucleation capacity, although pretreatment with DNase 1 did expose perinuclear nucleation sites that had a much lower minimal tubulin concentration for assembly than the pericentriolar site. After pretreatment with trypsin pericentriolar material and nucleation were destroyed, and microtubule elongation occurred directly onto the centriole microtubules.

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When Overexpressed, a Novel Centrosomal Protein, RanBPM, Causes Ectopic Microtubule Nucleation Similar to γ-Tubulin

The Journal of Cell Biology ◽

10.1083/jcb.143.4.1041 ◽

1998 ◽

Vol 143 (4) ◽

pp. 1041-1052 ◽

Cited By ~ 132

Author(s):

Masafumi Nakamura ◽

Hirohisa Masuda ◽

Johji Horii ◽

Kei-ichi Kuma ◽

Nobuhiko Yokoyama ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Sucrose Density Gradient ◽

Microtubule Nucleation ◽

Microtubule Network ◽

Sucrose Density Gradient Centrifugation ◽

Centrosomal Protein ◽

Nucleation Sites ◽

Two Hybrid

A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with γ-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to γ-tubulin was faded by the addition of GTPγS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

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An extended γ-tubulin ring functions as a stable platform in microtubule nucleation

The Journal of Cell Biology ◽

10.1083/jcb.201111123 ◽

2012 ◽

Vol 197 (1) ◽

pp. 59-74 ◽

Cited By ~ 39

Author(s):

Sarah Erlemann ◽

Annett Neuner ◽

Linda Gombos ◽

Romain Gibeaux ◽

Claude Antony ◽

...

Keyword(s):

Quantitative Analysis ◽

Fluorescence Recovery After Photobleaching ◽

Nucleation Site ◽

Fluorescence Recovery ◽

Microtubule Nucleation ◽

Nucleation Sites ◽

Small Complex ◽

Tubulin Binding

γ-Tubulin complexes are essential for microtubule (MT) nucleation. The γ-tubulin small complex (γ-TuSC) consists of two molecules of γ-tubulin and one molecule each of Spc97 and Spc98. In vitro, γ-TuSCs oligomerize into spirals of 13 γ-tubulin molecules per turn. However, the properties and numbers of γ-TuSCs at MT nucleation sites in vivo are unclear. In this paper, we show by fluorescence recovery after photobleaching analysis that γ-tubulin was stably integrated into MT nucleation sites and was further stabilized by tubulin binding. Importantly, tubulin showed a stronger interaction with the nucleation site than with the MT plus end, which probably provides the basis for MT nucleation. Quantitative analysis of γ-TuSCs on single MT minus ends argued for nucleation sites consisting of approximately seven γ-TuSCs with approximately three additional γ-tubulin molecules. Nucleation and anchoring of MTs required the same number of γ-tubulin molecules. We suggest that a spiral of seven γ-TuSCs with a slight surplus of γ-tubulin nucleates MTs in vivo.

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Stable kinetochore–microtubule attachments restrict MTOC position and spindle elongation in acentrosomal oocytes

10.1101/2020.05.07.081786 ◽

2020 ◽

Author(s):

Aurélien Courtois ◽

Shuhei Yoshida ◽

Tomoya S. Kitajima

Keyword(s):

Microtubule Nucleation ◽

Bipolar Spindle ◽

Mouse Oocytes ◽

Spindle Poles ◽

Microtubule Attachment ◽

Nucleation Sites ◽

Spindle Elongation ◽

The Stability

SummaryIn mouse oocytes, acentriolar MTOCs functionally replace centrosomes and act as microtubule nucleation sites. Microtubules nucleated from MTOCs initially assemble into an unorganized ball-like structure, which then transforms into a bipolar spindle carrying MTOCs at its poles, a process called spindle bipolarization. In mouse oocytes, spindle bipolarization is promoted by kinetochores but the mechanism by which kinetochore–microtubule attachments contribute to spindle bipolarity remains unclear. This study demonstrates that the stability of kinetochore–microtubule attachment is essential for confining MTOC positions at the spindle poles and for limiting spindle elongation. MTOC sorting is gradual and continues even in the metaphase spindle. When stable kinetochore–microtubule attachments are disrupted, the spindle is unable to restrict MTOCs at its poles and fails to terminate its elongation. Stable kinetochore fibers are directly connected to MTOCs and to the spindle poles, and thus may serve as a measure that defines proper spindle length. These findings reinforce the hypothesis that kinetochores act as scaffolds for acentrosomal spindle bipolarity.

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