Dataset Information

Mob4 plays a role in spindle focusing in Drosophila S2 cells.

ABSTRACT: The characteristic bipolar shape of the mitotic spindle is produced by the focusing of the minus ends of microtubules at the spindle poles. The focus is maintained by the centrosome, a microtubule-nucleating organelle, as well as by proteins that are capable of focusing kinetochore fibers (K fibers) even in the absence of a centrosome. Here, we have performed a small-scale RNA interference (RNAi) screen of known or suspected pole-related proteins in Drosophila S2 cells. An unexpected outcome of this screen was the finding that one of the four Drosophila Mob proteins (a family of kinase regulators) plays a role in spindle pole organization. Time-lapse microscopy of mitotic cells depleted of Drosophila Mob4 by RNAi revealed that the K fibers splay apart and do not maintain their focus either in the presence or absence of functional centrosomes. The Mob4 RNAi phenotype most closely resembles that observed after depletion of the protein encoded by abnormal spindle (Asp), although Asp localization is not substantially affected by Mob4 RNAi. Expression of a Drosophila Mob4-GFP fusion protein revealed its localization to the nucleus in interphase and to spindle poles and kinetochores during mitosis. We propose that Mob4 in Drosophila controls a mitotic kinase that in turn regulates downstream target proteins involved in K fiber focusing at the poles.

SUBMITTER: Trammell MA

PROVIDER: S-EPMC2851640 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Mob4 plays a role in spindle focusing in Drosophila S2 cells.

Trammell Matthew A MA Mahoney Nicole M NM Agard David A DA Vale Ronald D RD

Journal of cell science 20080401 Pt 8

The characteristic bipolar shape of the mitotic spindle is produced by the focusing of the minus ends of microtubules at the spindle poles. The focus is maintained by the centrosome, a microtubule-nucleating organelle, as well as by proteins that are capable of focusing kinetochore fibers (K fibers) even in the absence of a centrosome. Here, we have performed a small-scale RNA interference (RNAi) screen of known or suspected pole-related proteins in Drosophila S2 cells. An unexpected outcome of ...[more]

PMID: 18388316

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Project description:The CENP-W/T complex was previously reported to be required for mitosis. HeLa cells depleted of CENP-W displayed profound mitotic defects, with mitotic timing delay, disorganized prometaphases and multipolar spindles as major phenotypic consequences. In this study, we examined the process of multipolar spindle formation induced by CENP-W depletion. Depletion of CENP-W in HeLa cells labeled with histone H2B and tubulin fluorescent proteins induced rapid fragmentation of originally bipolar spindles in a high proportion of cells. CENP-W depletion was associated with depletion of Hec1 at kinetochores. The possibility of promiscuous centrosomal duplication was ruled out by immunofluorescent examination of centrioles. However, centrioles were frequently observed to be abnormally split. In addition, a large proportion of the supernumerary poles lacked centrioles, but were positively stained with different centrosomal markers. These observations suggested that perturbation in spindle force distribution caused by defective kinetochores could contribute to a mechanical mechanism for spindle pole disruption. 'Spindle free' nocodazole arrested cells did not exhibit pole fragmentation after CENP-W depletion, showing that pole fragmentation is microtubule dependent. Inhibition of centrosome separation by monastrol reduced the incidence of spindle pole fragmentation, indicating that Eg5 plays a role in spindle pole disruption. Surprisingly, CENP-W depletion rescued the monopolar spindle phenotype of monastrol treatment, with an increased frequency of bipolar spindles observed after CENP-W RNAi. We overexpressed the microtubule cross-linking protein TPX2 to create spindle poles stabilized by the microtubule cross-linking activity of TPX2. Spindle pole fragmentation was suppressed in a TPX2-dependent fashion. We propose that CENP-W, by influencing proper kinetochore assembly, particularly microtubule docking sites, can confer spindle pole resistance to traction forces exerted by motor proteins during chromosome congression. Taken together, our findings are consistent with a model in which centrosome integrity is controlled by the pathways regulating kinetochore-microtubule attachment stability.

Dataset Information

Mob4 plays a role in spindle focusing in Drosophila S2 cells.

Publications

Mob4 plays a role in spindle focusing in Drosophila S2 cells.

Similar Datasets

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