In vitro Assays for Mitotic Spindle Assembly and Function

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.

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Mitotic Spindle Assembly In Vitro

Current Protocols in Cell Biology ◽

10.1002/0471143030.cb1113s09 ◽

2001 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

John Merlie ◽

Rebecca Heald

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly ◽

Mitotic Spindle Assembly

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Nup98 regulates bipolar spindle assembly through association with microtubules and opposition of MCAK

Molecular Biology of the Cell ◽

10.1091/mbc.e10-06-0478 ◽

2011 ◽

Vol 22 (5) ◽

pp. 661-672 ◽

Cited By ~ 44

Author(s):

Marie K. Cross ◽

Maureen A. Powers

Keyword(s):

Nuclear Pore Complex ◽

Mitotic Spindle ◽

Spindle Assembly ◽

Microtubule Dynamics ◽

Nuclear Pore ◽

Bipolar Spindle ◽

Terminal Domain ◽

C Terminus ◽

Mitotic Spindle Assembly

During mitosis, the nuclear pore complex is disassembled and, increasingly, nucleoporins are proving to have mitotic functions when released from the pore. We find a contribution of the nucleoporin Nup98 to mitotic spindle assembly through regulation of microtubule dynamics. When added to Xenopus extract spindle assembly assays, the C-terminal domain of Nup98 stimulates uncontrolled growth of microtubules. Conversely, inhibition or depletion of Nup98 leads to formation of stable monopolar spindles. Spindle bipolarity is restored by addition of purified, recombinant Nup98 C-terminus. The minimal required region of Nup98 corresponds to a portion of the C-terminal domain lacking a previously characterized function. We show association between this region of the C-terminus of Nup98 and both Taxol-stabilized microtubules and the microtubule-depolymerizing mitotic centromere–associated kinesin (MCAK). Importantly, we demonstrate that this domain of Nup98 inhibits MCAK depolymerization activity in vitro. These data support a model in which Nup98 interacts with microtubules and antagonizes MCAK activity, thus promoting bipolar spindle assembly.

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The Kinesin-Related Protein, Hset, Opposes the Activity of Eg5 and Cross-Links Microtubules in the Mammalian Mitotic Spindle

The Journal of Cell Biology ◽

10.1083/jcb.147.2.351 ◽

1999 ◽

Vol 147 (2) ◽

pp. 351-366 ◽

Cited By ~ 219

Author(s):

Vicki Mountain ◽

Calvin Simerly ◽

Louisa Howard ◽

Asako Ando ◽

Gerald Schatten ◽

...

Keyword(s):

Mitotic Spindle ◽

Cultured Cells ◽

Spindle Assembly ◽

Meiotic Spindle ◽

Cross Linking ◽

Simultaneous Inhibition ◽

Balance Of Forces ◽

And Function

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.

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Human Ovarian Granulosa Cells Isolated during an IVF Procedure Exhibit Differential Expression of Genes Regulating Cell Division and Mitotic Spindle Formation

Journal of Clinical Medicine ◽

10.3390/jcm8122026 ◽

2019 ◽

Vol 8 (12) ◽

pp. 2026

Author(s):

Maciej Brązert ◽

Wiesława Kranc ◽

Błażej Chermuła ◽

Katarzyna Kowalska ◽

Maurycy Jankowski ◽

...

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Granulosa Cells ◽

Mitotic Spindle ◽

Spindle Assembly ◽

In Vitro Cultivation ◽

Cellular Ultrastructure ◽

Mitotic Spindle Assembly

Granulosa cells (GCs) are a population of somatic cells whose role after ovulation is progesterone production. GCs were collected from patients undergoing controlled ovarian stimulation during an in vitro fertilization procedure, and they were maintained for 1, 7, 15, and 30 days of in vitro primary culture before collection for further gene expression analysis. A study of genes involved in the biological processes of interest was carried out using expression microarrays. To validate the obtained results, Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) was performed. The direction of changes in the expression of the selected genes was confirmed in most of the examples. Six ontological groups (“cell cycle arrest”, “cell cycle process”, “mitotic spindle organization”, “mitotic spindle assembly checkpoint”, “mitotic spindle assembly”, and “mitotic spindle checkpoint”) were analyzed in this study. The results of the microarrays obtained by us allowed us to identify two groups of genes whose expressions were the most upregulated (FAM64A, ANLN, TOP2A, CTGF, CEP55, BIRC5, PRC1, DLGAP5, GAS6, and NDRG1) and the most downregulated (EREG, PID1, INHA, RHOU, CXCL8, SEPT6, EPGN, RDX, WNT5A, and EZH2) during the culture. The cellular ultrastructure showed the presence of structures characteristic of mitotic cell division: a centrosome surrounded by a pericentric matrix, a microtubule system, and a mitotic spindle connected to chromosomes. The main goal of the study was to identify the genes involved in mitotic division and to identify the cellular ultrastructure of GCs in a long-term in vitro culture. All of the genes in these groups were subjected to downstream analysis, and their function and relation to the ovarian environment are discussed. The obtained results suggest that long-term in vitro cultivation of GCs may lead to their differentiation toward another cell type, including cells with cancer-like characteristics.

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Mitotic spindle assembly in vitro

Nature ◽

10.1038/250185a0 ◽

1974 ◽

Vol 250 (5463) ◽

pp. 185-185

Author(s):

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly ◽

Mitotic Spindle Assembly

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NuMA regulates mitotic spindle assembly, structural dynamics and function via phase separation

Nature Communications ◽

10.1038/s41467-021-27528-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Mengjie Sun ◽

Mingkang Jia ◽

He Ren ◽

Biying Yang ◽

Wangfei Chi ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Structural Dynamics ◽

Mitotic Spindle ◽

Spindle Assembly ◽

Spindle Poles ◽

Mitotic Spindle Assembly ◽

Dynamics And Function ◽

And Function ◽

Liquid Liquid Phase Separation

AbstractA functional mitotic spindle is essential for accurate chromosome congression and segregation during cell proliferation; however, the underlying mechanisms of its assembly remain unclear. Here we show that NuMA regulates this assembly process via phase separation regulated by Aurora A. NuMA undergoes liquid-liquid phase separation during mitotic entry and KifC1 facilitates NuMA condensates concentrating on spindle poles. Phase separation of NuMA is mediated by its C-terminus, whereas its dynein-dynactin binding motif also facilitates this process. Phase-separated NuMA droplets concentrate tubulins, bind microtubules, and enrich crucial regulators, including Kif2A, at the spindle poles, which then depolymerizes spindle microtubules and promotes poleward spindle microtubule flux for spindle assembly and structural dynamics. In this work, we show that NuMA orchestrates mitotic spindle assembly, structural dynamics and function via liquid-liquid phase separation regulated by Aurora A phosphorylation.

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