Faculty Opinions recommendation of Meiosis-specific stable binding of augmin to acentrosomal spindle poles promotes biased microtubule assembly in oocytes.

2013 ◽  
Author(s):  
Ryoma Ohi
Keyword(s):  
Microtubule Assembly ◽  
Spindle Poles

scholarly journals BUB-1 promotes amphitelic chromosome biorientation via multiple activities at the kinetochore

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Frances Edwards ◽  
Gilliane Maton ◽  
Nelly Gareil ◽  
Julie C Canman ◽  
Julien Dumont
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Underlying Mechanism ◽  
Microtubule Assembly ◽  
Spindle Poles ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Chromatid Segregation ◽  
Associated Proteins ◽  
Chromosome Attachment

Accurate chromosome segregation relies on bioriented amphitelic attachments of chromosomes to microtubules of the mitotic spindle, in which sister chromatids are connected to opposite spindle poles. BUB-1 is a protein of the Spindle Assembly Checkpoint (SAC) that coordinates chromosome attachment with anaphase onset. BUB-1 is also required for accurate sister chromatid segregation independently of its SAC function, but the underlying mechanism remains unclear. Here we show that, in Caenorhabditis elegans embryos, BUB-1 accelerates the establishment of non-merotelic end-on kinetochore-microtubule attachments by recruiting the RZZ complex and its downstream partner dynein-dynactin at the kinetochore. In parallel, BUB-1 limits attachment maturation by the SKA complex. This activity opposes kinetochore-microtubule attachment stabilisation promoted by CLS-2CLASP-dependent kinetochore-microtubule assembly. BUB-1 is therefore a SAC component that coordinates the function of multiple downstream kinetochore-associated proteins to ensure accurate chromosome segregation.

scholarly journals Kinetochore microtubules shorten by loss of subunits at the kinetochores of prometaphase chromosomes

1991 ◽  
Vol 98 (2) ◽  
pp. 151-158 ◽  
Author(s):  
L. Cassimeris ◽  
E.D. Salmon
Keyword(s):  
Metaphase Plate ◽  
Force Generation ◽  
Microtubule Assembly ◽  
Immunofluorescent Staining ◽  
Chromosome Movement ◽  
Mitotic Spindles ◽  
Subunit Dissociation ◽  
Spindle Poles ◽  
Tubulin Subunit ◽  
A Site

The site of tubulin subunit dissociation was determined during poleward chromosome movement in prometaphase newt lung cell mitotic spindles using fluorescence photobleaching techniques and nocodazole-induced spindle shortening. Synchronous shortening of all kinetochore microtubules was produced by incubating cells in 17 microM nocodazole to block microtubule assembly. Under these conditions the spindle poles moved towards the metaphase plate at a rate of 3.6 +/− 0.4 microns min-1 (n = 3). On the basis of anti-tubulin immunofluorescent staining of cells fixed after incubation in nocodazole, we found that nonkinetochore microtubules rapidly disappeared and only kinetochore fibers were present after 60–90 s in nocodazole. To localize the site of tubulin subunit dissociation, a narrow bar pattern was photobleached across one half-spindle in prometaphase-metaphase cells previously microinjected with 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF)-labeled tubulin. Immediately after photobleaching, cells were perfused with 17 microM nocodazole to produce shortening of kinetochore microtubules. Shortening was accompanied by a decrease in the distance between the bleach bar and the kinetochores. In contrast, there was little or no decrease in the distance between the bleach bar and the pole. Compared to their initial lengths, the average kinetochore to pole distance shortened by 18%, the bleach bar to kinetochore distance shortened by 28% and the average bleached bar to pole distance shortened by 1.6%. The data provide evidence that tubulin subunits dissociate from kinetochore microtubules at a site near the kinetochore during poleward chromosome movement. These results are consistent with models of poleward force generation for chromosome movement in which prometaphase-metaphase poleward force is generated in association with the kinetochore.

scholarly journals Stability of microtubule attachment to metaphase kinetochores in PtK1 cells

1990 ◽  
Vol 96 (1) ◽  
pp. 9-15 ◽  
Author(s):  
L. Cassimeris ◽  
C.L. Rieder ◽  
G. Rupp ◽  
E.D. Salmon
Keyword(s):  
Attachment Site ◽  
Microtubule Assembly ◽  
Microtubule Depolymerization ◽  
Spindle Poles ◽  
Microtubule Attachment ◽  
Section Electron ◽  
The Mean ◽  
Serial Section Electron Microscopy ◽  
The Stability ◽  
Section Electron Microscopy

Kinetochore microtubules are known to be differentially stable to a variety of microtubule depolymerization agents compared to the non-kinetochore polar microtubules, but the dynamics of microtubule attachment to the kinetochore is currently controversial. We have examined the stability of kinetochore microtubules in metaphase PtK1 spindles at 23 degrees C when microtubule assembly is abruptly blocked with the drug nocodazole. Metaphase cells were incubated in medium containing 34 microM nocodazole for various times before fixation and processing either for immunofluorescence light microscopy or serial-section electron microscopy. Microtubules not associated with kinetochore fibers disappeared completely in less than 1 min. Kinetochore fibers persisted and shortened, as the spindle poles moved close to the chromosomes over a 10–20 min interval. During this shortening process, the number of kinetochore microtubules decreased slowly. The mean number of kinetochore microtubules was 24 +/− 5 in control cells and zero in cells incubated with nocodazole for 20 min. The half-time of microtubule attachment to the kinetochore was approximately 7.5 min. These results show that when microtubule assembly is blocked, kinetochore microtubules shorten more slowly and persist about 10 times longer than the labile polar microtubules. If kinetochore microtubules shorten by tubulin dissociation at their plus-ends like the non-kinetochore polar microtubules, then the microtubule surface lattice must be able to translocate through the kinetochore attachment site without frequent detachment occurring.

scholarly journals Mutants of the Drosophila ncd microtubule motor protein cause centrosomal and spindle pole defects in mitosis

1994 ◽  
Vol 107 (4) ◽  
pp. 859-867 ◽  
Author(s):  
S.A. Endow ◽  
R. Chandra ◽  
D.J. Komma ◽  
A.H. Yamamoto ◽  
E.D. Salmon
Keyword(s):  
Cell Cycle ◽  
Coiled Coil ◽  
Motor Protein ◽  
Spindle Pole ◽  
Opposite Polarity ◽  
Microtubule Assembly ◽  
Loss Of Function ◽  
Spindle Poles ◽  
Microtubule Motor ◽  
Microtubule Motor Protein

Nonclaret disjunctional (ncd) is a kinesin-related microtubule motor protein required for meiotic and early mitotic chromosome distribution in Drosophila. ncd translocates on microtubules with the opposite polarity to kinesin, toward microtubule minus ends, and is associated with spindles in chromosome/spindle preparations. Here we report a new mutant of ncd caused by partial deletion of the predicted coiled-coil central stalk. The mutant protein exhibits a velocity of translocation and ability to generate torque in motility assays comparable to near full-length ncd, but only partially rescues a null mutant for chromosome mis-segregation. Antibody staining experiments show that the partial loss-of-function and null mutants cause centrosomal and spindle pole defects, including centrosome splitting and loss of centrosomes from spindle poles, and localize ncd to centrosomes as well as spindles of wild-type embryos. Association of ncd with spindles and centrosomes is microtubule- and cell cycle-dependent: inhibition of microtubule assembly with colchicine abolishes ncd staining and centrosomal staining is observed in prometaphase, metaphase and anaphase, but diminishes in late anaphase/telophase. The cell cycle dependence of centrosomal staining and the defects of mutants provide clear evidence for activity of the ncd motor protein near or at the spindle poles in mitosis. The ncd motor may interact with centrosomal microtubules and spindle fibers to attach centrosomes to spindle poles, and mediate poleward translocation (flux) of kinetochore fibers, a process that may underlie poleward movement of chromosomes in mitosis. Together with previous work, our findings indicate that ncd is important in maintaining spindle poles in mitosis as well as in meiosis.

scholarly journals Aurora A site specific TACC3 phosphorylation regulates astral microtubule assembly by stabilizing γ-tubulin ring complex

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Resmi Rajeev ◽  
Puja Singh ◽  
Ananya Asmita ◽  
Ushma Anand ◽  
Tapas K. Manna
Keyword(s):  
Molecular Mechanisms ◽  
Coiled Coil ◽  
Time Lapse ◽  
Microtubule Assembly ◽  
Aurora A ◽  
Site Specific ◽  
Spindle Poles ◽  
Ring Complex ◽  
Division Axis ◽  
A Site

Abstract Background Astral microtubules emanating from the mitotic centrosomes play pivotal roles in defining cell division axis and tissue morphogenesis. Previous studies have demonstrated that human transforming acidic coiled-coil 3 (TACC3), the most conserved TACC family protein, regulates formation of astral microtubules at centrosomes in vertebrate cells by affecting γ-tubulin ring complex (γ-TuRC) assembly. However, the molecular mechanisms underlying such function were not completely understood. Results Here, we show that Aurora A site-specific phosphorylation in TACC3 regulates formation of astral microtubules by stabilizing γ-TuRC assembly in human cells. Mutation of the most conserved Aurora A targeting site, Ser 558 to alanine (S558A) in TACC3 results in robust loss of astral microtubules and disrupts localization of the γ-tubulin ring complex (γ-TuRC) proteins at the spindle poles. Under similar condition, phospho-mimicking S558D mutation retains astral microtubules and the γ-TuRC proteins in a manner similar to control cells expressed with wild type TACC3. Time-lapse imaging reveals that S558A mutation leads to defects in positioning of the spindle-poles and thereby causes delay in metaphase to anaphase transition. Biochemical results determine that the Ser 558- phosphorylated TACC3 interacts with the γ-TuRC proteins and further, S558A mutation impairs the interaction. We further reveal that the mutation affects the assembly of γ-TuRC from the small complex components. Conclusions The results demonstrate that TACC3 phosphorylation stabilizes γ- tubulin ring complex assembly and thereby regulates formation of centrosomal asters. They also implicate a potential role of TACC3 phosphorylation in the functional integrity of centrosomes/spindle poles.

scholarly journals The KinI kinesin Kif2a is required for bipolar spindle assembly through a functional relationship with MCAK

2004 ◽  
Vol 166 (4) ◽  
pp. 473-478 ◽  
Author(s):  
Neil J. Ganem ◽  
Duane A. Compton
Keyword(s):  
Cell Cycle Progression ◽  
Cultured Cells ◽  
Spindle Assembly ◽  
Microtubule Assembly ◽  
Cycle Progression ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Microtubule Attachment ◽  
Kinesin Eg5 ◽  
Dependent Pathway

Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochore–microtubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for spindle bipolarity. These treatments failed to restore bipolarity to cells lacking the activity of the kinesin Eg5. Thus, two independent pathways contribute to spindle bipolarity, with the Eg5-dependent pathway using motor force to drive spindle bipolarity and the Kif2a-dependent pathway relying on microtubule polymer dynamics to generate force for spindle bipolarity.

The checkpoint control for anaphase onset does not monitor excess numbers of spindle poles or bipolar spindle symmetry

1997 ◽  
Vol 110 (4) ◽  
pp. 421-429 ◽  
Author(s):  
G. Sluder ◽  
E.A. Thompson ◽  
F.J. Miller ◽  
J. Hayes ◽  
C.L. Rieder
Keyword(s):  
Sea Urchin ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Microtubule Assembly ◽  
Checkpoint Control ◽  
Animal Cells ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Monopolar Spindle

Exit from mitosis in animal cells is substantially delayed when spindle assembly is inhibited, spindle bipolarity is disrupted, or when a monopolar spindle is formed. These observations have led to the proposal that animal cells have a ‘spindle assembly’ checkpoint for the metaphase-anaphase transition that monitors bipolar spindle organization. However, the existence of such a checkpoint is uncertain because perturbations in spindle organization can produce unattached kinetochores, which by themselves are known to delay anaphase onset. In this study we have tested if cells monitor bipolar spindle organization, independent of kinetochore attachment, by analyzing the duration of mitosis in sea urchin zygotes and vertebrate somatic cells containing multipolar spindles in which all kinetochores are attached to spindle poles. We found that sea urchin zygotes containing tripolar or tetrapolar spindles progressed from nuclear envelope breakdown to anaphase onset with normal timing. We also found that the presence of supernumerary, unpaired spindle poles did not greatly prolong mitosis. Observation of untreated PtK1 cells that formed tripolar or tetrapolar spindles revealed that they progressed through mitosis, on average, at the normal rate. More importantly, the interval between the bipolar attachment of the last monooriented chromosome and anaphase onset was normal. Thus, neither of these cell types can detect the presence of gross aberrations in spindle architecture that inevitably lead to aneuploidy. We conclude that animal cells do not have a checkpoint for the metaphase-anaphase transition that monitors defects in spindle architecture independent of the checkpoint that monitors kinetochore attachment to the spindle. For dividing cells in which spindle microtubule assembly is not experimentally compromised, we propose that the completion of kinetochore attachment is the event which limits the time of the metaphase-anaphase transition.

scholarly journals Meiosis-Specific Stable Binding of Augmin to Acentrosomal Spindle Poles Promotes Biased Microtubule Assembly in Oocytes

PLoS Genetics ◽  
2013 ◽  
Vol 9 (6) ◽  
pp. e1003562 ◽  
Author(s):  
Nathalie Colombié ◽  
A. Agata Głuszek ◽  
Ana M. Meireles ◽  
Hiroyuki Ohkura
Keyword(s):  
Microtubule Assembly ◽  
Spindle Poles

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

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