scholarly journals Direct experimental evidence for the existence, structural basis and function of astral forces during anaphase B in vivo

1991 ◽  
Vol 100 (2) ◽  
pp. 279-288 ◽  
Author(s):  
J.R. Aist ◽  
C.J. Bayles ◽  
W. Tao ◽  
M.W. Berns
Keyword(s):  
Critical Mass ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Structural Basis ◽  
Mitotic Apparatus ◽  
Pole Body ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
And Function ◽  
Anaphase B

The existence, structural basis and function of astral forces that are active during anaphase B in the fungus, Nectria haematococca, were revealed by experiments performed on living cells. When one of the two asters of a mitotic apparatus was damaged, the entire mitotic apparatus migrated rapidly in the direction of the opposing astral forces, showing that the force that accelerated spindle pole body separation in earlier experiments is located in the asters. When a strong solution of the antimicrotubule drug, MBC, was applied at anaphase A, tubulin immunocytochemistry showed that both astral and spindle microtubules were destroyed completely in less than a minute. As a result, separation of the spindle pole bodies during anaphase B almost stopped. By contrast, disrupting only the spindle microtubules with a laser microbeam increased the rate of spindle pole body separation more than fourfold. Taken together, these two experiments show that the astral forces are microtubule-dependent. When only one of the two or three bundles of spindle microtubules was broken at very early anaphase B, most such diminished spindles elongated at a normal rate, whereas others elongated at an increased rate. This result suggests that only a critical mass or number of spindle microtubules needs be present for the rate of spindle elongation to be fully governed, and that astral forces can accelerate the elongation of a weakened or diminished spindle.

scholarly journals Key phosphorylation events in Spc29 and Spc42 guide multiple steps of yeast centrosome duplication

2018 ◽  
Vol 29 (19) ◽  
pp. 2280-2291 ◽  
Author(s):  
Michele Haltiner Jones ◽  
Eileen T. O’Toole ◽  
Amy S. Fabritius ◽  
Eric G. Muller ◽  
Janet B. Meehl ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Centrosome Duplication ◽  
Phosphorylation Sites ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Pole Body ◽  
Core Components ◽  
Spindle Elongation

Phosphorylation modulates many cellular processes during cell cycle progression. The yeast centrosome (called the spindle pole body, SPB) is regulated by the protein kinases Mps1 and Cdc28/Cdk1 as it nucleates microtubules to separate chromosomes during mitosis. Previously we completed an SPB phosphoproteome, identifying 297 sites on 17 of the 18 SPB components. Here we describe mutagenic analysis of phosphorylation events on Spc29 and Spc42, two SPB core components that were shown in the phosphoproteome to be heavily phosphorylated. Mutagenesis at multiple sites in Spc29 and Spc42 suggests that much of the phosphorylation on these two proteins is not essential but enhances several steps of mitosis. Of the 65 sites examined on both proteins, phosphorylation of the Mps1 sites Spc29-T18 and Spc29-T240 was shown to be critical for function. Interestingly, these two sites primarily influence distinct successive steps; Spc29-T240 is important for the interaction of Spc29 with Spc42, likely during satellite formation, and Spc29-T18 facilitates insertion of the new SPB into the nuclear envelope and promotes anaphase spindle elongation. Phosphorylation sites within Cdk1 motifs affect function to varying degrees, but mutations only have significant effects in the presence of an MPS1 mutation, supporting a theme of coregulation by these two kinases.

Fine structure of the haplosporidan Kernstab, a persistent, intranuclear mitotic apparatus

1975 ◽  
Vol 18 (2) ◽  
pp. 327-346
Author(s):  
F.O. Perkins
Keyword(s):  
Fine Structure ◽  
Nuclear Envelope ◽  
Light Microscope ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Interphase Nuclei ◽  
Mitotic Apparatus ◽  
Pole Body

The fine structure of the haplosporidan mitotic apparatus is described from observations of plasmodial nuclei of Minchinia nelsoni, M. costalis, Minchinia sp., and Urosporidium crescens. The apparatus, which is the Kernstab of light-microscope studies, consists of a bundle of microtubules terminating in a spindle pole body (SPB) at each end of the bundle. A few microtubules extend from SPB to SPB, but most either extend from an SPB and terminate in the nucleoplasm or lie in the nucleoplasm, free of either SPB. The bundle lengthens during mitosis, increasing the SPB-to-SPB distance by a factor of 2 to 3 as compared to interphase nuclei. SPBs are not in contact with the nuclear envelope, being found always in the nucleoplasm which is delimited by the nuclear envelope throughout mitosis. The mitotic apparatus is persistent through interphase, at least in a form which is not significantly different from that found in mitotic nuclei.

scholarly journals A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast

2017 ◽  
Vol 28 (25) ◽  
pp. 3647-3659 ◽  
Author(s):  
Masashi Yukawa ◽  
Tomoki Kawakami ◽  
Masaki Okazaki ◽  
Kazunori Kume ◽  
Ngang Heok Tang ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Pole Body ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Bipolar Spindle ◽  
Pole Body ◽  
Cross Linker ◽  
Spindle Elongation

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly.

The influence of the microtubule inhibitor, methyl benzimidazol-2-yl-carbamate (MBC) on nuclear division and the cell cycle in Saccharomyces cerevisiae

1980 ◽  
Vol 46 (1) ◽  
pp. 341-352
Author(s):  
R.A. Quinlan ◽  
C.I. Pogson ◽  
K. Gull
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Ultrastructural Examination ◽  
Microtubule Inhibitor ◽  
Microtubule Polymerization ◽  
Pole Body ◽  
Outer Component ◽  
Spindle Microtubules

Methyl benzimidazol-2-yl-carbamate (MBC), at a concentration of 100 microM, has a pronounced effect on the growth of Saccharomyces cerevisiae, resulting in the accumulation of cells as large doublets. We have determined a specific execution point for the effect of MBC on the yeast cell cycle, and have shown that this execution point is between the cycle events of spindle pole body duplication and spindle pole body separation. An ultrastructural examination of the MBC-treated cells revealed the absence of cytoplasmic and spindle microtubules. MBC treatment also produced an altered spindle pole body morphology, causing the disappearance of the outer component. Nuclear size was also markedly increased in the MBC-induced doublet cells, although the septa were completely absent from these doublet cells. It is proposed that MBC inhibits microtubule polymerization, rather than causing the depolymerization of stable microtubules.

Ultrastructure of somatic mitosis in a diploid strain of the plant pathogenic fungus Cochliobolus sativus

1975 ◽  
Vol 53 (4) ◽  
pp. 403-414 ◽  
Author(s):  
H. C. Huang ◽  
R. D. Tinline ◽  
L. C. Fowke
Keyword(s):  
Nuclear Envelope ◽  
Ultrastructural Study ◽  
Pathogenic Fungus ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cochliobolus Sativus ◽  
Diploid Strain ◽  
Interphase Nuclei ◽  
Pole Body ◽  
Spindle Microtubules

An ultrastructural study of mitosis in a diploid strain of Cochliobolus sativus showed the event to be intranuclear. Two nucleoli occasionally were present in interphase nuclei. During division the spindle pole body peripheral to the nuclear envelope divided; spindle microtubules radiated into the nucleoplasm from the amorphous granular region abutting the nuclear envelope beneath the bodies; chromosomes condensed at prophase, approached the equatorial plane at metaphase, and moved asynchronously at anaphase; single microtubules appeared attached to kinetochore-like structures. At telophase, nuclei exhibited maximal elongation; fissures of the nuclear envelope appeared in the interzonal region; the nucleolus dispersed. The polar nuclear areas became new daughter nuclei with nucleoli.

Ultrastructure of mitosis and the spindle pole body cycle in the smut fungus, Tilletia foetida

1985 ◽  
Vol 63 (1) ◽  
pp. 86-96 ◽  
Author(s):  
James A. Hoffmann ◽  
Blair J. Goates
Keyword(s):  
Nuclear Membrane ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Dense Layer ◽  
Double Structure ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Spindle Microtubules ◽  
Dense Chromatin ◽  
Tilletia Foetida

The interphase nucleus in secondary sporidia of Tilletia foetida consists of mostly diffuse chromatin, one or two nucleoli, and an area of heterochromatin located opposite an electron-dense, extranuclear spindle pole body (SPB). The interphase SPB is an oval- to bar-shaped, double-structured disc that has a crystallinelike substructure. During nuclear migration into nascent sporidia, SPBs and nucleoli are randomly oriented. At the onset of division, chromatin begins to condense and the SPB becomes located on a nuclear protuberance. Cytoplasmic microtubules terminate at the SPBs and multivesicular bodies surround the SPBs from the early stages of SPB division to early postdivision. SPB discs become spheroid and each develops a medial, dense layer. Then, a basal, dense layer develops and elongates as the SPBs separate and become positioned on opposite sides of the nuclear protuberance. The nuclear membrane opens opposite the SPB during SPB division. The nucleolus is extruded into a nuclear bleb and degenerates. SPBs migrate to opposing sides of the nucleus and become diffuse as a microtubular spindle develops between them. Some spindle microtubules terminate at dense chromatin patches that are contiguous with the major mass of chromatin surrounding the spindle. During late division stages, spindle microtubules often appear to be closely juxtaposed. Except for polar openings adjacent to the SPBs, the nuclear membrane is entire until late division when it degenerates in the midregion of the nucleus. During early postdivision, the SPB condenses into a small, dense sphere as the chromatin and heterochromatin opposite the SPB become diffuse. The SPB then elongates into a dense bar and SPB material increases, except at the midportion, reforming the double structure of interphase.

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.

scholarly journals BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin.

1990 ◽  
Vol 111 (6) ◽  
pp. 2573-2586 ◽  
Author(s):  
V Berlin ◽  
C A Styles ◽  
G R Fink
Keyword(s):  
Synthetic Lethality ◽  
Nuclear Fusion ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Chromosome Loss ◽  
Physical Interaction ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
And Function

BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells. Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function. Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules. Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules. Subsequently, cells lose all microtubule structures, coincident with the arrest of division. Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.

scholarly journals Relief of the Dma1-mediated checkpoint requires Dma1 autoubiquitination and dynamic localization

2018 ◽  
Vol 29 (18) ◽  
pp. 2176-2189 ◽  
Author(s):  
Christine M. Jones ◽  
Jun-Song Chen ◽  
Alyssa E. Johnson ◽  
Zachary C. Elmore ◽  
Sierra N. Cullati ◽  
...  
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Ubiquitin Ligase ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Checkpoint Activation ◽  
Pole Body ◽  
Septation Initiation Network ◽  
Anaphase B

Chromosome segregation and cell division are coupled to prevent aneuploidy and cell death. In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) promotes cytokinesis, but upon mitotic checkpoint activation, the SIN is actively inhibited to prevent cytokinesis from occurring before chromosomes have safely segregated. SIN inhibition during the mitotic checkpoint is mediated by the E3 ubiquitin ligase Dma1. Dma1 binds to the CK1-phosphorylated SIN scaffold protein Sid4 at the spindle pole body (SPB), and ubiquitinates it. Sid4 ubiquitination antagonizes the SPB localization of the Pololike kinase Plo1, the major SIN activator, so that SIN signaling is delayed. How this checkpoint is silenced once spindle defects are resolved has not been clear. Here we establish that Dma1 transiently leaves SPBs during anaphase B due to extensive autoubiquitination. The SIN is required for Dma1 to return to SPBs later in anaphase. Blocking Dma1 removal from SPBs by permanently tethering it to Sid4 prevents SIN activation and cytokinesis. Therefore, controlling Dma1’s SPB dynamics in anaphase is an essential step in S. pombe cell division and the silencing of the Dma1-dependent mitotic checkpoint.

scholarly journals Brr6 drives the Schizosaccharomyces pombe spindle pole body nuclear envelope insertion/extrusion cycle

2011 ◽  
Vol 195 (3) ◽  
pp. 467-484 ◽  
Author(s):  
Tiina Tamm ◽  
Agnes Grallert ◽  
Emily P.S. Grossman ◽  
Isabel Alvarez-Tabares ◽  
Frances E. Stevens ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Microtubule Organizing Center ◽  
Pole Body ◽  
Cytoplasmic Face ◽  
Organizing Center ◽  
Nuclear Component ◽  
Anaphase B

The fission yeast interphase spindle pole body (SPB) is a bipartite structure in which a bulky cytoplasmic domain is separated from a nuclear component by the nuclear envelope. During mitosis, the SPB is incorporated into a fenestra that forms within the envelope during mitotic commitment. Closure of this fenestra during anaphase B/mitotic exit returns the cytoplasmic component to the cytoplasmic face of an intact interphase nuclear envelope. Here we show that Brr6 is transiently recruited to SPBs at both SPB insertion and extrusion. Brr6 is required for both SPB insertion and nuclear envelope integrity during anaphase B/mitotic exit. Genetic interactions with apq12 and defective sterol assimilation suggest that Brr6 may alter envelope composition at SPBs to promote SPB insertion and extrusion. The restriction of the Brr6 domain to eukaryotes that use a polar fenestra in an otherwise closed mitosis suggests a conserved role in fenestration to enable a single microtubule organizing center to nucleate both cytoplasmic and nuclear microtubules on opposing sides of the nuclear envelope.

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