Differential pathways of recruitment for centrosomal antigens to the mitotic poles during bipolar spindle formation

As cells enter mitosis, centrosomes undergo many transformations and become associated with different molecules in a stage-specific manner. We have developed a protocol for immunofluorescence staining with four antibody probes that can help us to follow the interaction of centrosomal components during mitosis. The cells were first stained with a human autoimmune serum (5051); a monoclonal anti-phosphocentrosomal antibody (CHO3); and an antitubulin antibody. Localization of the antibodies was detected using rhodamine-, fluorescein- and AMCA-conjugated second antibodies, respectively. After photographing marked mitotic cells, coverslips were soaked with 0.2 M glycine-HCl at pH 1.0 for 1 h to release all antibodies bound to the structures. The same cells were re-stained with a human autoantibody (SP-H) specific for spindle poles and a fluorescein-conjugated second antibody. This allowed us to compare the subcellular distribution of three kinds of centrosomal antigens in a single cell. Mitotic PtK1 cells treated with either nocodazole or taxol included microtubule-containing cytoplasmic foci and parallel bundles of short microtubules at the cell periphery. All the centrosomal antibodies stained the same one or two dots corresponding to structures labeled by the tubulin antibody. CHO3 also revealed extra cytoplasmic foci, whereas the SP-H antigen was additionally localized at one end of the free microtubule bundles. As the microtubules reorganized into bipolar spindles during the recovery from drug treatment, the CHO3 and SP-H antigens coalesced into the spindle poles where the 5051 antigen was located, suggesting that centrosomal antigens become associated with spindle poles through very different recruitment pathways.

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Aster-free spindle poles in insect spermatocytes: evidence for chromosome-induced spindle formation?

The Journal of Cell Biology ◽

10.1083/jcb.102.5.1679 ◽

1986 ◽

Vol 102 (5) ◽

pp. 1679-1687 ◽

Cited By ~ 57

Author(s):

W Steffen ◽

H Fuge ◽

R Dietz ◽

M Bastmeyer ◽

G Müller

Keyword(s):

Spindle Pole ◽

Serial Sections ◽

Spindle Formation ◽

Microtubule Polymerization ◽

Spindle Poles ◽

Pericentriolar Material ◽

Lysis Buffer ◽

Bipolar Spindles

Tipulid spermatocytes form normally functioning bipolar spindles after one of the centrosomes is experimentally dislocated from the nucleus in late diakinesis (Dietz, R., 1959, Z. Naturforsch., 14b:749-752; Dietz, R., 1963, Zool. Anz. Suppl., 23:131-138; Dietz, R., 1966, Heredity, 19:161-166). The possibility that dissociated pericentriolar material (PCM) is nevertheless responsible for the formation of the spindle in these cells cannot be ruled out based on live observation. In studying serial sections of complete cells and of lysed cells, it was found that centrosome-free spindle poles in the crane fly show neither pericentriolar-like material nor aster microtubules, whereas the displaced centrosomes appear complete, i.e., consist of a centriole pair, aster microtubules, and PCM. Exposure to a lysis buffer containing tubulin resulted in an increase of centrosomal asters due to aster microtubule polymerization. Aster-free spindle poles did not show any reaction, also indicating the absence of PCM at these poles. The results favor the hypothesis of chromosome-induced spindle pole formation at the onset of prometaphase and the dispensability of PCM in Pales.

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Kinesin-5 forms a stable bipolar spindle in a fast, irreversible snap

10.1101/452821 ◽

2018 ◽

Author(s):

Allen Leary ◽

Elena Nazarova ◽

Shannon Sim ◽

Kristy Shulist ◽

Paul Francois ◽

...

Keyword(s):

Mitotic Spindle ◽

Electron Tomography ◽

Maximal Extension ◽

Spindle Formation ◽

Bipolar Spindle ◽

Spindle Poles ◽

Equilibrium Length ◽

Bipolar Spindles

SUMMARYGRAPHICAL ABSTRACTSeparation of duplicated spindle poles is the first step in forming the mitotic spindle. Kinesin-5 crosslinks and slides anti-parallel microtubules, but it is unclear how these two activities contribute to the first steps in spindle formation. In this study we report that in monopolar spindles, the duplicated spindle poles snap apart in a fast and irreversible step that produces a nascent bipolar spindle. Using mutations in Kinesin-5 that inhibit microtubule sliding, we show crosslinking alone drives the fast, irreversible pole separation. Electron tomography revealed microtubule pairs in monopolar spindles have short overlaps that intersect at high angles and are unsuited for ensemble Kinesin-5 sliding. However, maximal extension of a subset of microtubule pairs approaches the length of nascent bipolar spindles and is consistent with a Kinesin-5 crosslinking driven transition. Finally, stochastic microtubule sliding by Kinesin-5 stabilizes the nascent spindle and sets a stereotyped equilibrium length.

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Aurora A Phosphorylates MCAK to Control Ran-dependent Spindle Bipolarity

Molecular Biology of the Cell ◽

10.1091/mbc.e08-02-0198 ◽

2008 ◽

Vol 19 (7) ◽

pp. 2752-2765 ◽

Cited By ~ 82

Author(s):

Xin Zhang ◽

Stephanie C. Ems-McClung ◽

Claire E. Walczak

Keyword(s):

Phosphorylation Site ◽

Aurora B ◽

Aurora A ◽

Spindle Formation ◽

Chromatin Region ◽

Spindle Poles ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

Bipolar Spindles

During mitosis, mitotic centromere-associated kinesin (MCAK) localizes to chromatin/kinetochores, a cytoplasmic pool, and spindle poles. Its localization and activity in the chromatin region are regulated by Aurora B kinase; however, how the cytoplasmic- and pole-localized MCAK are regulated is currently not clear. In this study, we used Xenopus egg extracts to form spindles in the absence of chromatin and centrosomes and found that MCAK localization and activity are tightly regulated by Aurora A. This regulation is important to focus microtubules at aster centers and to facilitate the transition from asters to bipolar spindles. In particular, we found that MCAK colocalized with NuMA and XMAP215 at the center of Ran asters where its activity is regulated by Aurora A-dependent phosphorylation of S196, which contributes to proper pole focusing. In addition, we found that MCAK localization at spindle poles was regulated through another Aurora A phosphorylation site (S719), which positively enhances bipolar spindle formation. This is the first study that clearly defines a role for MCAK at the spindle poles as well as identifies another key Aurora A substrate that contributes to spindle bipolarity.

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Development of single-cell-level microfluidic technology for long-term growth visualization of living cultures of Mycobacterium smegmatis

Microsystems & Nanoengineering ◽

10.1038/s41378-021-00262-1 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Han Wang ◽

Gloria M. Conover ◽

Song-I Han ◽

James C. Sacchettini ◽

Arum Han

Keyword(s):

Drug Treatment ◽

Single Cell ◽

Mycobacterium Smegmatis ◽

Culture Media ◽

Low Cost ◽

Bacterial Pathogen ◽

Growth Dynamics ◽

Time Lapse ◽

Cell Phenotype

AbstractAnalysis of growth and death kinetics at single-cell resolution is a key step in understanding the complexity of the nonreplicating growth phenotype of the bacterial pathogen Mycobacterium tuberculosis. Here, we developed a single-cell-resolution microfluidic mycobacterial culture device that allows time-lapse microscopy-based long-term phenotypic visualization of the live replication dynamics of mycobacteria. This technology was successfully applied to monitor the real-time growth dynamics of the fast-growing model strain Mycobacterium smegmatis (M. smegmatis) while subjected to drug treatment regimens during continuous culture for 48 h inside the microfluidic device. A clear morphological change leading to significant swelling at the poles of the bacterial membrane was observed during drug treatment. In addition, a small subpopulation of cells surviving treatment by frontline antibiotics was observed to recover and achieve robust replicative growth once regular culture media was provided, suggesting the possibility of identifying and isolating nonreplicative mycobacteria. This device is a simple, easy-to-use, and low-cost solution for studying the single-cell phenotype and growth dynamics of mycobacteria, especially during drug treatment.

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Bimodal Interaction of Mammalian Polo-Like Kinase 1 and a Centrosomal Scaffold, Cep192, in the Regulation of Bipolar Spindle Formation

Molecular and Cellular Biology ◽

10.1128/mcb.00068-15 ◽

2015 ◽

Vol 35 (15) ◽

pp. 2626-2640 ◽

Cited By ~ 23

Author(s):

Lingjun Meng ◽

Jung-Eun Park ◽

Tae-Sung Kim ◽

Eun Hye Lee ◽

Suk-Youl Park ◽

...

Keyword(s):

Xenopus Laevis ◽

Spindle Assembly ◽

Human Cells ◽

Mitotic Progression ◽

Spindle Formation ◽

Content Type ◽

Bipolar Spindle ◽

Egg Extracts ◽

Cultured Human Cells ◽

Bipolar Spindles

Serving as microtubule-organizing centers, centrosomes play a key role in forming bipolar spindles. The mechanism of how centrosomes promote bipolar spindle assembly in various organisms remains largely unknown. A recent study withXenopus laevisegg extracts suggested that the Plk1 ortholog Plx1 interacts with the phospho-T46 (p-T46) motif ofXenopusCep192 (xCep192) to form an xCep192-mediated xAurA-Plx1 cascade that is critical for bipolar spindle formation. Here, we demonstrated that in cultured human cells, Cep192 recruits AurA and Plk1 in a cooperative manner, and this event is important for the reciprocal activation of AurA and Plk1. Strikingly, Plk1 interacted with Cep192 through either the p-T44 (analogous toXenopusp-T46) or the newly identified p-S995 motif via its C-terminal noncatalytic polo-box domain. The interaction between Plk1 and the p-T44 motif was prevalent in the presence of Cep192-bound AurA, whereas the interaction of Plk1 with the p-T995 motif was preferred in the absence of AurA binding. Notably, the loss of p-T44- and p-S995-dependent Cep192-Plk1 interactions induced an additive defect in recruiting Plk1 and γ-tubulin to centrosomes, which ultimately led to a failure in proper bipolar spindle formation and mitotic progression. Thus, we propose that Plk1 promotes centrosome-based bipolar spindle formation by forming two functionally nonredundant complexes with Cep192.

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Eg5 is static in bipolar spindles relative to tubulin

The Journal of Cell Biology ◽

10.1083/jcb.200106011 ◽

2001 ◽

Vol 154 (6) ◽

pp. 1125-1134 ◽

Cited By ~ 111

Author(s):

Tarun M. Kapoor ◽

Timothy J. Mitchison

Keyword(s):

Motor Activity ◽

Half Life ◽

Microtubule Dynamics ◽

Spindle Matrix ◽

Spindle Poles ◽

Kinesin Eg5 ◽

Mitotic Kinesin Eg5 ◽

Bipolar Spindles

We used fluorescent speckle microscopy to probe the dynamics of the mitotic kinesin Eg5 in Xenopus extract spindles, and compared them to microtubule dynamics. We found significant populations of Eg5 that were static over several seconds while microtubules flux towards spindle poles. Eg5 dynamics are frozen by adenylimidodiphosphate. Bulk turnover experiments showed that Eg5 can exchange between the spindle and the extract with a half life of <55 s. Eg5 distribution in spindles was not perturbed by inhibition of its motor activity with monastrol, but was perturbed by inhibition of dynactin with p50 dynamitin. We interpret these data as revealing the existence of a static spindle matrix that promotes Eg5 targeting to spindles, and transient immobilization of Eg5 within spindles. We discuss alternative interpretations of the Eg5 dynamics we observe, ideas for the biochemical nature of a spindle matrix, and implications for Eg5 function.

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Kinetochore-mediated outward force promotes spindle pole separation in fission yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e19-07-0366 ◽

2019 ◽

Vol 30 (22) ◽

pp. 2802-2813 ◽

Cited By ~ 4

Author(s):

Yutaka Shirasugi ◽

Masamitsu Sato

Keyword(s):

Fission Yeast ◽

Motor Proteins ◽

Spindle Assembly ◽

Spindle Pole ◽

Double Knockout ◽

Bipolar Spindle ◽

Spindle Poles ◽

Bipolar Spindles ◽

Meiosis I

Bipolar spindles are organized by motor proteins that generate microtubule-dependent forces to separate the two spindle poles. The fission yeast Cut7 (kinesin-5) is a plus-end-directed motor that generates the outward force to separate the two spindle poles, whereas the minus-end-directed motor Pkl1 (kinesin-14) generates the inward force. Balanced forces by these antagonizing kinesins are essential for bipolar spindle organization in mitosis. Here, we demonstrate that chromosomes generate another outward force that contributes to the bipolar spindle assembly. First, it was noted that the cut7 pkl1 double knockout failed to separate spindle poles in meiosis I, although the mutant is known to succeed it in mitosis. It was assumed that this might be because meiotic kinetochores of bivalent chromosomes joined by cross-overs generate weaker tensions in meiosis I than the strong tensions in mitosis generated by tightly tethered sister kinetochores. In line with this idea, when meiotic mono-oriented kinetochores were artificially converted to a mitotic bioriented layout, the cut7 pkl1 mutant successfully separated spindle poles in meiosis I. Therefore, we propose that spindle pole separation is promoted by outward forces transmitted from kinetochores to spindle poles through microtubules.

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The association of Plk1 with the astrin–kinastrin complex promotes formation and maintenance of a metaphase plate

Journal of Cell Science ◽

10.1242/jcs.251025 ◽

2020 ◽

Vol 134 (1) ◽

pp. jcs251025

Author(s):

Zoë Geraghty ◽

Christina Barnard ◽

Pelin Uluocak ◽

Ulrike Gruneberg

Keyword(s):

Molecular Mechanisms ◽

Metaphase Plate ◽

Direct Interaction ◽

Mitotic Chromosomes ◽

Spindle Formation ◽

Bipolar Spindle ◽

Mitotic Kinase ◽

Spindle Poles ◽

Chromosome Congression ◽

Chromosome Alignment

ABSTRACTErrors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin–kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule–kinetochore attachment. However, the molecular mechanisms by which astrin–kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule–kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule–kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.

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