Live analysis of lagging chromosomes during anaphase and their effect on spindle elongation rate in fission yeast

2000 ◽  
Vol 113 (23) ◽  
pp. 4177-4191 ◽  
Author(s):  
A.L. Pidoux ◽  
S. Uzawa ◽  
P.E. Perry ◽  
W.Z. Cande ◽  
R.C. Allshire
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Time Lapse ◽  
Spindle Pole ◽  
Checkpoint Proteins ◽  
Spindle Dynamics ◽  
Lagging Chromosome ◽  
Cytoplasmic Microtubules ◽  
Spindle Elongation ◽  
Anaphase B

The fission yeast Schizosaccharomyces pombe is widely used as a model system for studies of the cell cycle and chromosome biology. To enhance these studies we have fused GFP to the chromodomain protein Swi6p, thus allowing nuclear and chromosome behaviour to be followed in living cells using time-lapse fluorescence microscopy. Like endogenous Swi6p, GFP-Swi6p localises to the nucleus and is concentrated at the heterochromatic centromeres and telomeres. The nucleus is highly dynamic during interphase: the clustered centromeres, in particular, are highly mobile. By expressing GFP-(α)2-tubulin and GFP-Swi6p in the same cells we observe that the clustered centromeres move in concert with the cytoplasmic microtubules, which is likely to reflect their association with the spindle pole body. Drug treatment indicates that this movement is dependent on intact cytoplasmic microtubules. We have also used GFP-Swi6p to investigate the properties of lagging chromosomes observed in mutants with defects in chromosome segregation. Lagging chromosomes display a variety of behaviours on anaphase spindles, most surprisingly, chromosomes appear to initiate microtubule interactions and move to the poles late in anaphase B. Interestingly, in cells displaying lagging chromosomes, the rate of spindle elongation is slowed by a factor of two. This suggests that cells are able to sense the presence of a lagging chromosome and slow anaphase B in order to allow it extra time to reach the pole. However, this mechanism is not dependent on the spindle checkpoint proteins Bub1p or Dma1p, raising the possibility that a novel checkpoint mechanism operates to retard spindle elongation if lagging chromosomes are detected. An alternative model is also discussed in which single defective kinetochores on lagging chromatids are able to interact simultaneously with microtubules emanating from both poles and affect spindle dynamics by counteracting the spindle elongation force.

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 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.

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 Mto2p, a Novel Fission Yeast Protein Required for Cytoplasmic Microtubule Organization and Anchoring of the Cytokinetic Actin Ring

2005 ◽  
Vol 16 (6) ◽  
pp. 3052-3063 ◽  
Author(s):  
Srinivas Venkatram ◽  
Jennifer L. Jennings ◽  
Andrew Link ◽  
Kathleen L. Gould
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Dependent Manner ◽  
Microtubule Organization ◽  
Actin Ring ◽  
Medial Region ◽  
Cytoplasmic Microtubule ◽  
Associated Proteins ◽  
Cytoplasmic Microtubules

Microtubules regulate diverse cellular processes, including chromosome segregation, nuclear positioning, and cytokinesis. In many organisms, microtubule nucleation requires γ-tubulin and associated proteins present at specific microtubule organizing centers (MTOCs). In fission yeast, interphase cytoplasmic microtubules originate from poorly characterized interphase MTOCs and spindle pole body (SPB), and during late anaphase from the equatorial MTOC (EMTOC). It has been previously shown that Mto1p (Mbo1p/Mod20p) function is important for the organization/nucleation of all cytoplasmic microtubules. Here, we show that Mto2p, a novel protein, interacts with Mto1p and is important for establishing a normal interphase cytoplasmic microtubule array. In addition, mto2Δ cells fail to establish a stable EMTOC and localize γ-tubulin complex members to this medial structure. As predicted from these functions, Mto2p localizes to microtubules, the SPB, and the EMTOC in an Mto1p-dependent manner. mto2Δ cells fail to anchor the cytokinetic actin ring in the medial region of the cell and under conditions that mildly perturb actin structures, these rings unravel in mto2Δ cells. Our results suggest that the Mto2p and the EMTOC are critical for anchoring the cytokinetic actin ring to the medial region of the cell and for proper coordination of mitosis with cytokinesis.

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.

A cytoplasmic dynein required for mitotic aster formation in vivo

1998 ◽  
Vol 111 (17) ◽  
pp. 2607-2614 ◽  
Author(s):  
S. Inoue ◽  
O.C. Yoder ◽  
B.G. Turgeon ◽  
J.R. Aist
Keyword(s):  
Spindle Pole Body ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Mitotic Spindles ◽  
Laser Microbeam ◽  
Filamentous Ascomycete ◽  
Spindle Elongation ◽  
Pulling Force ◽  
Anaphase B

An astral pulling force helps to elongate the mitotic spindle in the filamentous ascomycete, Nectria haematococca. Evidence is mounting that dynein is required for the formation of mitotic spindles and asters. Obviously, this would be an important mitotic function of dynein, since it would be a prerequisite for astral force to be applied to a spindle pole. Missing from the evidence for such a role of dynein in aster formation, however, has been a dynein mutant lacking mitotic asters. To determine whether or not cytoplasmic dynein is involved in mitotic aster formation in N. haematococca, a dynein-deficient mutant was made. Immunocytochemistry visualized few or no mitotic astral microtubules in the mutant cells, and studies of living cells confirmed the veracity of this result by revealing the absence of mitotic aster functions in vivo: intra-astral motility of membranous organelles was not apparent; the rate and extent of spindle elongation during anaphase B were reduced; and spindle pole body separation almost stopped when the anaphase B spindle in the mutant was cut by a laser microbeam, demonstrating unequivocally that no astral pulling force was present. These unique results not only provide a demonstration that cytoplasmic dynein is required for the formation of mitotic asters in N. haematococca; they also represent the first report of mitotic phenotypes in a dynein mutant of any filamentous fungus and the first cytoplasmic dynein mutant of any organism whose mitotic phenotypes demonstrate the requirement of cytoplasmic dynein for aster formation in vivo.

The use of cell division cycle mutants to investigate the control of microtubule distribution in the fission yeast Schizosaccharomyces pombe

1988 ◽  
Vol 89 (3) ◽  
pp. 343-357 ◽  
Author(s):  
I.M. Hagan ◽  
J.S. Hyams
Keyword(s):  
Cell Division ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cell Division Cycle ◽  
Microtubule Organization ◽  
Cytoplasmic Face ◽  
Cytoplasmic Microtubules ◽  
Positional Control

We have characterized the changes in microtubule organization that occur through the cell division cycle of the fission yeast Schizosaccharomyces pombe by indirect immunofluorescence microscopy. During interphase, groups of cytoplasmic microtubules, independent of the spindle pole body (SPB), form an array extending between the cell tips. These microtubules are involved in positioning the nucleus at the cell equator and in the establishment of cell polarity. At mitosis, the interphase array disappears and is replaced by an intranuclear spindle extending between the now duplicated SPBs. Elongation of the spindle sees the appearance of astral microtubules emanating from the cytoplasmic face of the SPBs. These persist until the end of anaphase whereupon the spindle microtubules depolymerize and two microtubule organizing centres (MTOCs) at the cell equator re-establish the interphase array. We have used the unique properties of various cell division cycle mutants to investigate further the function of these different microtubule arrays and their temporal and positional control.

scholarly journals Plo1 Kinase Recruitment to the Spindle Pole Body and Its Role in Cell Division inSchizosaccharomyces pombe

1999 ◽  
Vol 10 (8) ◽  
pp. 2771-2785 ◽  
Author(s):  
Daniel P. Mulvihill ◽  
Janni Petersen ◽  
Hiroyuki Ohkura ◽  
David M. Glover ◽  
Iain M. Hagan
Keyword(s):  
Cell Division ◽  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Multiple Roles ◽  
Anaphase Promoting Complex ◽  
Pole Body ◽  
Early Anaphase ◽  
Anaphase B ◽  
Mitotic Event

Polo kinases execute multiple roles during cell division. The fission yeast polo related kinase Plo1 is required to assemble the mitotic spindle, the prophase actin ring that predicts the site for cytokinesis and for septation after the completion of mitosis ( Ohkuraet al., 1995 ; Bahler et al., 1998 ). We show that Plo1 associates with the mitotic but not interphase spindle pole body (SPB). SPB association of Plo1 is the earliest fission yeast mitotic event recorded to date. SPB association is strong from mitotic commitment to early anaphase B, after which the Plo1 signal becomes very weak and finally disappears upon spindle breakdown. SPB association of Plo1 requires mitosis-promoting factor (MPF) activity, whereas its disassociation requires the activity of the anaphase-promoting complex. The stf1.1 mutation bypasses the usual requirement for the MPF activator Cdc25 ( Hudson et al., 1990 ). Significantly, Plo1 associates inappropriately with the interphase SPB of stf1.1 cells. These data are consistent with the emerging theme from many systems that polo kinases participate in the regulation of MPF to determine the timing of commitment to mitosis and may indicate that pole association is a key aspect of Plo1 function. Plo1 does not associate with the SPB when septation is inappropriately driven by deregulation of the Spg1 pathway and remains SPB associated if septation occurs in the presence of a spindle. Thus, neither Plo1 recruitment to nor its departure from the SPB are required for septation; however, overexpression ofplo1+activates the Spg1 pathway and causes transient Cdc7 recruitment to the SPB and multiple rounds of septation.

scholarly journals The Yeast Spindle Pole Body Component Spc72p Interacts with Stu2p and Is Required for Proper Microtubule Assembly

1998 ◽  
Vol 141 (5) ◽  
pp. 1169-1179 ◽  
Author(s):  
Xiaoyue Peter Chen ◽  
Hongwei Yin ◽  
Tim C. Huffaker
Keyword(s):  
Chromosome Segregation ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Temperature Sensitive ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Spindle Elongation

We have previously shown that Stu2p is a microtubule-binding protein and a component of the Saccharomyces cerevisiae spindle pole body (SPB). Here we report the identification of Spc72p, a protein that interacts with Stu2p. Stu2p and Spc72p associate in the two-hybrid system and can be coimmunoprecipitated from yeast extracts. Stu2p and Spc72p also interact with themselves, suggesting the possibility of a multimeric Stu2p-Spc72p complex. Spc72p is an essential component of the SPB and is able to associate with a preexisting SPB, indicating that there is a dynamic exchange between soluble and SPB forms of Spc72p. Unlike Stu2p, Spc72p does not bind microtubules in vitro, and was not observed to localize along microtubules in vivo. A temperature-sensitive spc72 mutation causes defects in SPB morphology. In addition, most spc72 mutant cells lack cytoplasmic microtubules; the few cytoplasmic microtubules that are observed are excessively long, and some of these are unattached to the SPB. spc72 cells are able to duplicate and separate their SPBs to form a bipolar spindle, but spindle elongation and chromosome segregation rarely occur. The chromosome segregation block does not arrest the cell cycle; instead, spc72 cells undergo cytokinesis, producing aploid cells and polyploid cells that contain multiple SPBs.

scholarly journals Saccharomyces cerevisiaeCells with Defective Spindle Pole Body Outer Plaques Accomplish Nuclear Migration via Half-Bridge–organized Microtubules

1998 ◽  
Vol 9 (5) ◽  
pp. 977-991 ◽  
Author(s):  
Arndt Brachat ◽  
John V. Kilmartin ◽  
Achim Wach ◽  
Peter Philippsen
Keyword(s):  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Time Lapse ◽  
Nuclear Migration ◽  
Spindle Pole ◽  
Microtubule Organizing Center ◽  
Multinucleated Cells ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Low Efficiency

Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion ofCNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Δ1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half- bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. AlthoughCNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy ofcnm67Δ1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.

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