Cytoplasmic Microtubule Organizing Centers Regulate Meiotic Spindle Positioning in Mouse Oocyte
2
Citation
51
Reference
10
Related Paper
Citation Trend
Abstract:
ABSTRACT During oocyte meiosis, migration of the spindle and its positioning must be tightly regulated to ensure elimination of the polar bodies and provide developmentally competent euploid eggs. Although the role of F-actin in regulating these critical processes has been studied extensively, little is known whether microtubules (MTs) participate in regulating these processes. Here, we characterize a pool of MTOCs in the oocyte that does not contribute to spindle assembly but instead remains free in the cytoplasm during metaphase I (metaphase cytoplasmic MTOCs; mcMTOCs). In contrast to spindle pole MTOCs, which primarily originate from the perinuclear region in prophase I, the mcMTOCs are found near the cortex of the oocyte. At nuclear envelope breakdown, they exhibit robust nucleation of MTs, which diminishes during polar body extrusion before returning robustly during metaphase II. The asymmetric positioning of the mcMTOCs provides the spindle with a MT-based anchor line to the cortex opposite the site of polar body extrusion. Depletion of mcMTOCs, by laser ablation, or manipulating their numbers, through autophagy inhibition, revealed that the mcMTOCs are required to regulate the timely migration and positioning of the spindle in meiosis. We discuss how forces exerted by F-actin in mediating movement of the spindle to the oocyte cortex are balanced by MT-mediated forces from the mcMTOCs to ensure spindle positioning and timely spindle migration.Keywords:
Polar body
Microtubule organizing center
Spindle pole body
Multipolar spindles
Microtubule nucleation
Spindle pole body
Microtubule nucleation
Microtubule organizing center
Astral microtubules
Cite
Citations (56)
ABSTRACT During oocyte meiosis, migration of the spindle and its positioning must be tightly regulated to ensure elimination of the polar bodies and provide developmentally competent euploid eggs. Although the role of F-actin in regulating these critical processes has been studied extensively, little is known whether microtubules (MTs) participate in regulating these processes. Here, we characterize a pool of MTOCs in the oocyte that does not contribute to spindle assembly but instead remains free in the cytoplasm during metaphase I (metaphase cytoplasmic MTOCs; mcMTOCs). In contrast to spindle pole MTOCs, which primarily originate from the perinuclear region in prophase I, the mcMTOCs are found near the cortex of the oocyte. At nuclear envelope breakdown, they exhibit robust nucleation of MTs, which diminishes during polar body extrusion before returning robustly during metaphase II. The asymmetric positioning of the mcMTOCs provides the spindle with a MT-based anchor line to the cortex opposite the site of polar body extrusion. Depletion of mcMTOCs, by laser ablation, or manipulating their numbers, through autophagy inhibition, revealed that the mcMTOCs are required to regulate the timely migration and positioning of the spindle in meiosis. We discuss how forces exerted by F-actin in mediating movement of the spindle to the oocyte cortex are balanced by MT-mediated forces from the mcMTOCs to ensure spindle positioning and timely spindle migration.
Polar body
Microtubule organizing center
Spindle pole body
Multipolar spindles
Microtubule nucleation
Cite
Citations (2)
Microtubule organizing center
Spindle pole body
Microtubule nucleation
Interphase
Cite
Citations (181)
Abstract Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran‐mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC‐ and Ran‐mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo‐like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the Ran GTP gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC‐ and Ran‐mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.
Microtubule organizing center
Spindle pole body
Microtubule nucleation
Germinal vesicle
Multipolar spindles
Ran
Cite
Citations (6)
Errors during cell division in oocytes and early embryos are linked to birth defects in mammals. Bipolar spindle assembly in early mouse embryos is unique in that three or more acentriolar microtubule-organizing centers (MTOCs) are initially formed and are then clustered into two spindle poles. Using a knockout mouse and live imaging of spindles in embryos, we demonstrate that MTOC clustering during the blastocyst stage requires augmin, a critical complex for MT-dependent MT nucleation within the spindle. Functional analyses in cultured cells with artificially increased numbers of centrosomes indicate that the lack of intra-spindle MT nucleation, but not loss of augmin per se or overall reduction of spindle MTs, is the cause of clustering failure. These data suggest that onset of mitosis with three or more MTOCs is turned into a typical bipolar division through augmin-dependent intra-spindle MT assembly.
Microtubule organizing center
Spindle pole body
Microtubule nucleation
Multipolar spindles
Cite
Citations (29)
gamma-Tubulin is a conserved component of microtubule-organizing centers and is thought to be involved in microtubule nucleation. A recently discovered Saccharomyces cerevisiae gene (TUB4) encodes a tubulin that is related to, but divergent from, gamma-tubulins. TUB4 is essential for cell viability, and epitope-tagged Tub4 protein (Tub4p) is localized to the spindle pole body (Sobel, S.G., and M. Snyder. 1995.J. Cell Biol. 131:1775-1788). We have characterized the expression of TUB4, the association of Tub4p with the spindle pole body, and its role in microtubule organization. Tub4p is a minor protein in the cell, and expression of TUB4 is regulated in a cell cycle-dependent manner. Wild-type Tub4p is localized to the spindle pole body, and a Tub4p-green fluorescent protein fusion is able to associate with a preexisting spindle pole body, suggesting that there is dynamic exchange between cytoplasmic and spindle pole body forms of Tub4p. Perturbation of Tub4p function, either by conditional mutation or by depletion of the protein, results in spindle as well as spindle pole body defects, but does not eliminate the ability of microtubules to regrow from, or remain attached to, the spindle pole body. The spindle pole bodies in tub4 mutant cells duplicate but do not separate, resulting in a monopolar spindle. EM revealed that one spindle pole body of the duplicated pair appears to be defective for the nucleation of microtubules. These results offer insight into the role of gamma-tubulin in microtubule-organizing center function.
Spindle pole body
Microtubule organizing center
Microtubule nucleation
Basal body
Multipolar spindles
Cite
Citations (156)
Spindle pole body
Microtubule organizing center
Microtubule nucleation
Multipolar spindles
Spindle checkpoint
Astral microtubules
Cite
Citations (371)
Microtubule organizing center
Spindle pole body
Microtubule nucleation
Cite
Citations (0)
Spindle pole body
Microtubule nucleation
Microtubule organizing center
Multipolar spindles
Astral microtubules
Cite
Citations (51)
Spindle pole body
Microtubule organizing center
Multipolar spindles
Mitotic exit
Cite
Citations (17)