Interzonal microtubules are dynamic during spindle elongation

1990 ◽  
Vol 97 (2) ◽  
pp. 273-281 ◽  
Author(s):  
E. Shelden ◽  
P. Wadsworth
Keyword(s):  
Microtubule Assembly ◽  
Late Anaphase ◽  
Confocal Fluorescence ◽  
Fluorescence Light Microscopy ◽  
Fluorescence Light ◽  
Marked Decline ◽  
Microtubule Growth ◽  
Spindle Elongation ◽  
Anaphase B ◽  
Short Times

The pattern and extent of microtubule assembly during spindle elongation has been examined in PtK1 cells by microinjection of biotin-tubulin and immunolocalization of biotin-tubulin-containing microtubules using antibodies to biotin. PtK1 cells were microinjected at 30 degrees C, incubated for various intervals to allow incorporation of biotin-tubulin into microtubules, then lysed, fixed and stained for biotin-tubulin and total tubulin. When mid- to late anaphase cells were examined at short times post-injection, using conventional fluorescence light microscopy, rapid incorporation of biotin-tubulin was detected throughout the interzonal region, between the separating chromosomes, and in the spindle asters. Using confocal fluorescence microscopy, the segments of biotin-labeled microtubules in the interzonal region were found to be continuous with the distal, or plus-ends, of unlabeled microtubules. When teleophase cells were examined, a marked decline in the extent of incorporation was apparent. Quantitative analysis of the total length of labeled polymer in the interzonal region of cells from mid-anaphase through telophase further reveals that the extent of incorporation was maximal during late anaphase, and decreased during telophase. The measured rate of interzonal microtubule growth remained relatively constant during this period. Our results provide direct evidence for plus-end elongation of interzonal microtubules during spindle elongation and further reveal that interzonal microtubules are highly dynamic during late anaphase spindle elongation. The implications of these results for the mechanism of anaphase B are discussed.

scholarly journals Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium.

1985 ◽  
Vol 101 (5) ◽  
pp. 1966-1976 ◽  
Author(s):  
J B Tucker ◽  
S A Mathews ◽  
K A Hendry ◽  
J B Mackie ◽  
D L Roche
Keyword(s):  
Nuclear Envelope ◽  
Early Stage ◽  
Microtubule Assembly ◽  
Membrane Association ◽  
Paramecium Tetraurelia ◽  
Spindle Microtubule ◽  
Spatial Control ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Anaphase B

Spindles underwent a 12-fold elongation before anaphase B was completed during the closed mitoses of micronuclei in Paramecium tetraurelia. Two main classes of spindle microtubules have been identified. A peripheral sheath of microtubules with diameters of 27-32 nm was found to be associated with the nuclear envelope and confined to the midportion of each spindle. Most of the other microtubules had diameters of approximately 24 nm and were present along the entire lengths of spindles. Nearly all of the 24-nm microtubules were eliminated from spindle midportions (largely because of microtubule disassembly) at a relatively early stage of spindle elongation. Disassembly of some of these microtubules also occurred at the ends of spindles. About 60% of the total microtubule content of spindles was lost at this stage. Most, perhaps all, peripheral sheath microtubules remained intact. Many of them detached from the nuclear envelope and regrouped to form a compact microtubule bundle in the spindle midportion. There was little, if any, further polymerization of 24-nm microtubules after the disassembly phase. Polymerization of microtubules with diameters of 27-32 nm continued as spindle elongation progressed. Most microtubules in the midportions of well-elongated spindles were constructed from 14-16 protofilaments. A few 24-nm microtubules with 13 protofilaments were also present. The implications of these findings for spatial control of microtubule assembly, disassembly, positioning, and membrane association, that apparently discriminate between microtubules with different protofilament numbers have been explored. The possibility that microtubule sliding occurs during spindle elongation has also been considered.

scholarly journals Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Lara Katharina Krüger ◽  
Matthieu Gélin ◽  
Liang Ji ◽  
Carlos Kikuti ◽  
Anne Houdusse ◽  
...  
Keyword(s):  
Dual Function ◽  
Tubulin Subunit ◽  
Microtubule Growth ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Spindle Stability ◽  
Spindle Function ◽  
Anaphase B

Mitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation in S. pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, creating a link between the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.

scholarly journals Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth

2021 ◽  
Author(s):  
Lara K. Krüger ◽  
Matthieu Gélin ◽  
Liang Ji ◽  
Carlos Kikuti ◽  
Anne Houdusse ◽  
...  
Keyword(s):  
Dual Function ◽  
Tubulin Subunit ◽  
Microtubule Growth ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Spindle Stability ◽  
Spindle Function ◽  
Anaphase B

AbstractMitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation in S. pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, to link the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.

scholarly journals The Drosophila kinesin-like protein KLP3A is a midbody component required for central spindle assembly and initiation of cytokinesis.

1995 ◽  
Vol 129 (3) ◽  
pp. 709-723 ◽  
Author(s):  
B C Williams ◽  
M F Riedy ◽  
E V Williams ◽  
M Gatti ◽  
M L Goldberg
Keyword(s):  
Motor Protein ◽  
Spindle Assembly ◽  
Male Meiosis ◽  
Cleavage Furrow ◽  
Critical Component ◽  
Central Spindle ◽  
Late Anaphase ◽  
Spindle Elongation ◽  
Anaphase B ◽  
Kinesin Superfamily

We describe here a new member of the kinesin superfamily in Drosophila, KLP3A (Kinesin-Like-Protein-at-3A). The KLP3A protein localizes to the equator of the central spindle during late anaphase and telophase of male meiosis. Mutations in the KLP3A gene disrupt the interdigitation of microtubules in spermatocyte central spindles. Despite this defect, anaphase B spindle elongation is not obviously aberrant. However, cytokinesis frequently fails after both meiotic divisions in mutant testes. Together, these findings strongly suggest that the KLP3A presumptive motor protein is a critical component in the establishment or stabilization of the central spindle. Furthermore, these results imply that the central spindle is the source of signals that initiate the cleavage furrow in higher cells.

scholarly journals Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae.

1995 ◽  
Vol 130 (3) ◽  
pp. 687-700 ◽  
Author(s):  
E Yeh ◽  
R V Skibbens ◽  
J W Cheng ◽  
E D Salmon ◽  
K Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Translocation ◽  
Spindle Pole ◽  
Cell Cortex ◽  
Wild Type ◽  
Immunofluorescent Localization ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spindle Dynamics ◽  
Spindle Elongation ◽  
Anaphase B

We have used time-lapse digital- and video-enhanced differential interference contrast (DE-DIC, VE-DIC) microscopy to study the role of dynein in spindle and nuclear dynamics in the yeast Saccharomyces cerevisiae. The real-time analysis reveals six stages in the spindle cycle. Anaphase B onset appears marked by a rapid phase of spindle elongation, simultaneous with nuclear migration into the daughter cell. The onset and kinetics of rapid spindle elongation are identical in wild type and dynein mutants. In the absence of dynein the nucleus does not migrate as close to the neck as in wild-type cells and initial spindle elongation is confined primarily to the mother cell. Rapid oscillations of the elongating spindle between the mother and bud are observed in wild-type cells, followed by a slower growth phase until the spindle reaches its maximal length. This stage is protracted in the dynein mutants and devoid of oscillatory motion. Thus dynein is required for rapid penetration of the nucleus into the bud and anaphase B spindle dynamics. Genetic analysis reveals that in the absence of a functional central spindle (ndcl), dynein is essential for chromosome movement into the bud. Immunofluorescent localization of dynein-beta-galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies and the cell cortex: with spindle pole body localization dependent on intact microtubules. A kinetic analysis of nuclear movement also revealed that cytokinesis is delayed until nuclear translocation is completed, indicative of a surveillance pathway monitoring nuclear transit into the bud.

scholarly journals Cdc14-regulated midzone assembly controls anaphase B

2007 ◽  
Vol 177 (6) ◽  
pp. 981-993 ◽  
Author(s):  
Anton Khmelinskii ◽  
Clare Lawrence ◽  
Johanna Roostalu ◽  
Elmar Schiebel
Keyword(s):  
Cell Cycle ◽  
Protein Phosphatase ◽  
Cross Linking ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Spindle Midzone ◽  
A Cell ◽  
Spindle Elongation ◽  
And Function ◽  
Anaphase B

Spindle elongation in anaphase of mitosis is a cell cycle–regulated process that requires coordination between polymerization, cross-linking, and sliding of microtubules (MTs). Proteins that assemble at the spindle midzone may be important for this process. In this study, we show that Ase1 and the separase–Slk19 complex drive midzone assembly in yeast. Whereas the conserved MT-bundling protein Ase1 establishes a midzone, separase–Slk19 is required to focus and center midzone components. An important step leading to spindle midzone assembly is the dephosphorylation of Ase1 by the protein phosphatase Cdc14 at the beginning of anaphase. Failure to dephosphorylate Ase1 delocalizes midzone proteins and delays the second, slower phase of anaphase B. In contrast, in cells expressing nonphosphorylated Ase1, anaphase spindle extension is faster, and spindles frequently break. Cdc14 also controls the separase–Slk19 complex indirectly via the Aurora B kinase. Thus, Cdc14 regulates spindle midzone assembly and function directly through Ase1 and indirectly via the separase–Slk19 complex.

scholarly journals Alp7-Mto1 and Alp14 synergize to promote interphase microtubule regrowth from the nuclear envelope

2019 ◽  
Vol 11 (11) ◽  
pp. 944-955 ◽  
Author(s):  
Wenyue Liu ◽  
Fan Zheng ◽  
Yucai Wang ◽  
Chuanhai Fu
Keyword(s):  
Nuclear Envelope ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Microtubule Assembly ◽  
Dependent Manner ◽  
Microtubule Nucleation ◽  
Microtubule Organizing Centers ◽  
Evolutionarily Conserved ◽  
Microtubule Growth ◽  
In Cells

Abstract Microtubules grow not only from the centrosome but also from various noncentrosomal microtubule-organizing centers (MTOCs), including the nuclear envelope (NE) and pre-existing microtubules. The evolutionarily conserved proteins Mto1/CDK5RAP2 and Alp14/TOG/XMAP215 have been shown to be involved in promoting microtubule nucleation. However, it has remained elusive as to how the microtubule nucleation promoting factors are specified to various noncentrosomal MTOCs, particularly the NE, and how these proteins coordinate to organize microtubule assembly. Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe, efficient interphase microtubule growth from the NE requires Alp7/TACC, Alp14/TOG/XMAP215, and Mto1/CDK5RAP2. The absence of Alp7, Alp14, or Mto1 compromises microtubule regrowth on the NE in cells undergoing microtubule repolymerization. We further demonstrate that Alp7 and Mto1 interdependently localize to the NE in cells without microtubules and that Alp14 localizes to the NE in an Alp7 and Mto1-dependent manner. Tethering Mto1 to the NE in cells lacking Alp7 partially restores microtubule number and the efficiency of microtubule generation from the NE. Hence, our study delineates that Alp7, Alp14, and Mto1 work in concert to regulate interphase microtubule regrowth on the NE.

scholarly journals Hepatoma Up-Regulated Protein Is Required for Chromatin-induced Microtubule Assembly Independently of TPX2

2008 ◽  
Vol 19 (11) ◽  
pp. 4900-4908 ◽  
Author(s):  
Claudia M. Casanova ◽  
Sofia Rybina ◽  
Hideki Yokoyama ◽  
Eric Karsenti ◽  
Iain W. Mattaj
Keyword(s):  
Xenopus Laevis ◽  
Detailed Analysis ◽  
Spindle Assembly ◽  
Microtubule Assembly ◽  
Spindle Microtubule ◽  
Microtubule Stabilization ◽  
Egg Extracts ◽  
Spindle Midzone ◽  
Microtubule Growth ◽  
Spindle Microtubules

The production of RanGTP around chromosomes is crucial for spindle microtubule assembly in mitosis. Previous work has shown that hepatoma up-regulated protein (HURP) is a Ran target, required for microtubule stabilization and spindle organization. Here we report a detailed analysis of HURP function in Xenopus laevis mitotic egg extracts. HURP depletion severely impairs bipolar spindle assembly around chromosomes: the few spindles that do form show a significant decrease in microtubule density at the spindle midzone. HURP depletion does not interfere with microtubule growth from purified centrosomes, but completely abolishes microtubule assembly induced by chromatin beads or RanGTP. Simultaneous depletion of the microtubule destabilizer MCAK with HURP does not rescue the phenotype, demonstrating that the effect of HURP is not to antagonize the destabilization activity of MCAK. Although the phenotype of HURP depletion closely resembles that reported for TPX2 depletion, we find no evidence that TPX2 and HURP physically interact or that they influence each other in their effects on spindle microtubules. Our data indicate that HURP and TPX2 have nonredundant functions essential for chromatin-induced microtubule assembly.

scholarly journals Proper timing of cytokinesis is regulated by Schizosaccharomyces pombe Etd1

2009 ◽  
Vol 186 (5) ◽  
pp. 739-753 ◽  
Author(s):  
Juan Carlos García-Cortés ◽  
Dannel McCollum
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Yeast Protein ◽  
Late Anaphase ◽  
Pole Body ◽  
Spindle Elongation ◽  
Cell Asymmetry ◽  
Guanosine Triphosphatase ◽  
Asymmetric Activation

Cytokinesis must be initiated only after chromosomes have been segregated in anaphase and must be terminated once cleavage is completed. We show that the fission yeast protein Etd1 plays a central role in both of these processes. Etd1 activates the guanosine triphosphatase (GTPase) Spg1 to trigger signaling through the septum initiation network (SIN) pathway and onset of cytokinesis. Spg1 is activated in late anaphase when spindle elongation brings spindle pole body (SPB)–localized Spg1 into proximity with its activator Etd1 at cell tips, ensuring that cytokinesis is only initiated when the spindle is fully elongated. Spg1 is active at just one of the two SPBs during cytokinesis. When the actomyosin ring finishes constriction, the SIN triggers disappearance of Etd1 from the half of the cell with active Spg1, which then triggers Spg1 inactivation. Asymmetric activation of Spg1 is crucial for timely inactivation of the SIN. Together, these results suggest a mechanism whereby cell asymmetry is used to monitor cytoplasmic partitioning to turn off cytokinesis signaling.

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