scholarly journals Molecular mechanism of myosin-II assembly at the division site in Schizosaccharomyces pombe

2000 ◽  
Vol 113 (10) ◽  
pp. 1813-1825 ◽  
Author(s):  
F. Motegi ◽  
K. Nakano ◽  
I. Mabuchi
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Myosin Ii ◽  
Loss Of Function ◽  
Contractile Ring ◽  
Actin Ring ◽  
Medial Region ◽  
Division Site ◽  
Medial Cortex ◽  
Actin Cables ◽  
Mutant Cells

Schizosaccharomyces pombe cells divide by virtue of the F-actin-based contractile ring (F-actin ring). Two myosin-II heavy chains, Myo2 and Myp2/Myo3, have been localized to the F-actin ring. Here, we investigated the mechanism of myosin-II assembly at the division site in S. pombe cells. First, we showed that Cdc4, an EF-hand protein, appears to be a common myosin light chain associated with both Myo2 and Myo3. Loss of function of both Myo2 and Myo3 caused a defect in F-actin assembly at the division site, like the phenotype of cdc4 null cells. It is suggested that Myo2, Myo3 and Cdc4 function in a cooperative manner in the formation of the F-actin ring during mitosis. Next, we investigated the dynamics of myosin-II during mitosis in S. pombe cells. In early mitosis when accumulation of F-actin cables in the medial region was not yet observed, Myo2 was detected primarily as dots widely located in the medial cortex. Myo2 fibers also became visible following the appearance of the dots. The Myo2 dots and fibers then fused with each other to form a medial cortical network. Some Myo2 dots appeared to be localized with F-actin cables which are also accumulated in the medial region. Finally these structures were packed into a thin contractile ring. In mutant cells that cannot form the F-actin ring such as cdc3(ts), cdc8(ts) and cdc12(ts), Myo2 was able to accumulate as dots in the medial cortex, whereas no accumulation of Myo2 dots was detected in cdc4(ts) cells. Moreover, disruption of F-actin in the cell by applying latrunculin-A did not affect the accumulation of Myo2 dots, suggesting that F-actin is not required for their accumulation. A truncated Myo2 which lacks putative Cdc4-binding sites (Myo2dIQs) was able to rescue myo2 null cells, myo3 null cells, cdc4(ts) mutant cells and cdc4 null cells. The Myo2dIQs could assemble into a normal-shaped ring in these cells. Therefore, its assembly at the division site does not require the function of either Cdc4 or Myo3.

scholarly journals Myosin-II reorganization during mitosis is controlled temporally by its dephosphorylation and spatially by Mid1 in fission yeast

2004 ◽  
Vol 165 (5) ◽  
pp. 685-695 ◽  
Author(s):  
Fumio Motegi ◽  
Mithilesh Mishra ◽  
Mohan K. Balasubramanian ◽  
Issei Mabuchi
Keyword(s):  
Fission Yeast ◽  
Myosin Ii ◽  
Phosphorylation Site ◽  
Terminal Region ◽  
Contractile Ring ◽  
Actin Ring ◽  
Division Site ◽  
Ring Assembly ◽  
Medial Cortex ◽  
Early Mitosis

Cytokinesis in many eukaryotes requires an actomyosin contractile ring. Here, we show that in fission yeast the myosin-II heavy chain Myo2 initially accumulates at the division site via its COOH-terminal 134 amino acids independently of F-actin. The COOH-terminal region can access to the division site at early G2, whereas intact Myo2 does so at early mitosis. Ser1444 in the Myo2 COOH-terminal region is a phosphorylation site that is dephosphorylated during early mitosis. Myo2 S1444A prematurely accumulates at the future division site and promotes formation of an F-actin ring even during interphase. The accumulation of Myo2 requires the anillin homologue Mid1 that functions in proper ring placement. Myo2 interacts with Mid1 in cell lysates, and this interaction is inhibited by an S1444D mutation in Myo2. Our results suggest that dephosphorylation of Myo2 liberates the COOH-terminal region from an intramolecular inhibition. Subsequently, dephosphorylated Myo2 is anchored by Mid1 at the medial cortex and promotes the ring assembly in cooperation with F-actin.

scholarly journals The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis.

1994 ◽  
Vol 125 (6) ◽  
pp. 1289-1301 ◽  
Author(s):  
M K Balasubramanian ◽  
B R Hirani ◽  
J D Burke ◽  
K L Gould
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Null Allele ◽  
Temperature Shift ◽  
Multicopy Plasmid ◽  
Contractile Ring ◽  
Actin Ring ◽  
Medial Region ◽  
Encoding Gene

The fission yeast Schizosaccharomyces pombe divides by medial fission and, like many higher eukaryotic cells, requires the function of an F-actin contractile ring for cytokinesis. In S. pombe, a class of cdc- mutants defective for cytokinesis, but not for DNA replication, mitosis, or septum synthesis, have been identified. In this paper, we present the characterization of one of these mutants, cdc3-124. Temperature shift experiments reveal that mutants in cdc3 are incapable of forming an F-actin contractile ring. We have molecularly cloned cdc3 and used the cdc3+ genomic DNA to create a strain carrying a cdc3 null mutation by homologous recombination in vivo. Cells bearing a cdc3-null allele are inviable. They arrest the cell cycle at cytokinesis without forming a contractile ring. DNA sequence analysis of the cdc3+ gene reveals that it encodes profilin, an actin-monomer-binding protein. In light of recent studies with profilins, we propose that Cdc3-profilin plays an essential role in cytokinesis by catalyzing the formation of the F-actin contractile ring. Consistent with this proposal are our observations that Cdc3-profilin localizes to the medial region of the cell where the F-actin contractile ring forms, and that it is essential for F-actin ring formation. Cells overproducing Cdc3-profilin become elongated, dumbbell shaped, and arrest at cytokinesis without any detectable F-actin staining. This effect of Cdc3-profilin overproduction is relieved by introduction of a multicopy plasmid carrying the actin encoding gene, act1+. We attribute these effects to potential sequestration of actin monomers by profilin, when present in excess.

scholarly journals The F-BAR Cdc15 promotes contractile ring formation through the direct recruitment of the formin Cdc12

2015 ◽  
Vol 208 (4) ◽  
pp. 391-399 ◽  
Author(s):  
Alaina H. Willet ◽  
Nathan A. McDonald ◽  
K. Adam Bohnert ◽  
Michelle A. Baird ◽  
John R. Allen ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Binding Proteins ◽  
Molecular Mechanisms ◽  
Ring Formation ◽  
Scaffolding Protein ◽  
Actin Binding ◽  
Contractile Ring ◽  
Bar Domain ◽  
Medial Cortex ◽  
N Terminus

In Schizosaccharomyces pombe, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring (CR). Nucleation of F-actin for the CR requires a single formin, Cdc12, that localizes to the cell middle at mitotic onset. Although genetic requirements for formin Cdc12 recruitment have been determined, the molecular mechanisms dictating its targeting to the medial cortex during cytokinesis are unknown. In this paper, we define a short motif within the N terminus of Cdc12 that binds directly to the F-BAR domain of the scaffolding protein Cdc15. Mutations preventing the Cdc12–Cdc15 interaction resulted in reduced Cdc12, F-actin, and actin-binding proteins at the CR, which in turn led to a delay in CR formation and sensitivity to other perturbations of CR assembly. We conclude that Cdc15 contributes to CR formation and cytokinesis via formin Cdc12 recruitment, defining a novel cytokinetic function for an F-BAR domain.

scholarly journals Characterization of novel mutations at the Schizosaccharomyces pombe cdc2 regulatory phosphorylation site, tyrosine 15.

1996 ◽  
Vol 7 (10) ◽  
pp. 1573-1586 ◽  
Author(s):  
K L Gould ◽  
A Feoktistova
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Phosphorylation Site ◽  
Loss Of Function ◽  
Contractile Ring ◽  
Mutant Proteins ◽  
Regulatory Phosphorylation ◽  
Binding Loop

The cdc2 protein kinase family is regulated negatively by phosphorylation in the glycine ATP-binding loop at a conserved tyrosine residue, Y15, alone or in combination with T14 phosphorylation. In Schizosaccharomyces pombe and other systems, substitution of these residues with structurally similar but nonphosphorylatable amino acids has generated proteins (Y15F or T14AY15F) that behave as constitutively tyrosine-dephosphorylated proteins or threonine and tyrosine-dephosphorylated proteins. Here we report the characteristics of three additional mutants at Y15--Y15E, Y15S, and Y15T--in S. pombe cdc2p. All three mutant proteins are active in in vitro kinase assays, but are unable to functionally complement cdc2 loss-of-function mutations in vivo. Additionally, all three mutants are dominant negatives. A more detailed analysis of the Y15T mutant indicates that it can initiate chromosome condensation and F-actin contractile ring formation, but is unable to drive the reorganization of microtubules into a mitotic spindle.

Isolation and characterization of fission yeast mutants defective in the assembly and placement of the contractile actin ring

1996 ◽  
Vol 109 (1) ◽  
pp. 131-142 ◽  
Author(s):  
F. Chang ◽  
A. Woollard ◽  
P. Nurse
Keyword(s):  
Fission Yeast ◽  
Ring Formation ◽  
Cell Cycle Checkpoint ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Actin Ring ◽  
Division Site ◽  
Isolation And Characterization ◽  
Cycle Checkpoint ◽  
Actin Cables

Fission yeast cells divide by medial cleavage using an actin-based contractile ring. We have conducted a genetic screen for temperature-sensitive mutants defective in the assembly and placement of this actin ring. Six genes necessary for actin ring formation and one gene necessary for placement of the actin ring have now been identified. The genes can be further organized into different phenotypic groups, suggesting that the gene products may have different functions in actin ring formation. Mutants of cdc3 and cdc8, which encode profilin and tropomyosin respectively, display disorganized actin patches in all cells. cdc12 and cdc15 mutants display disorganized actin patches during mitosis, but normal interphase actin patterns. cdc4 and rng2 mutants display disorganized actin cables during mitosis, but normal interphase actin patterns. In mid1 mutants, the actin ring and septum are positioned at random locations and angles on the cell surface, although the nucleus is positioned normally, indicating that the mid1 gene product is required to couple the division site to the position of the nucleus. mid1 mutant cells may reveal a new cell cycle checkpoint in telophase that coordinates cell division and the proper distribution of nuclei. The actin ring forms medially in a beta-tubulin mutant, showing that actin ring formation and placement are not dependent on the mitotic spindle.

scholarly journals The Multiprotein Exocyst Complex Is Essential for Cell Separation in Schizosaccharomyces pombe

2002 ◽  
Vol 13 (2) ◽  
pp. 515-529 ◽  
Author(s):  
Hongyan Wang ◽  
Xie Tang ◽  
Jianhua Liu ◽  
Susanne Trautmann ◽  
David Balasundaram ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Schizosaccharomyces Pombe ◽  
Cell Separation ◽  
Contractile Ring ◽  
Medial Region ◽  
Independent Manner ◽  
Daughter Cells ◽  
Exocyst Complex ◽  
Interphase Cells ◽  
Ring Constriction

Schizosaccharomyces pombe cells divide by medial fission through the use of an actomyosin-based contractile ring. A mulitlayered division septum is assembled in concert with ring constriction. Finally, cleavage of the inner layer of the division septum results in the liberation of daughter cells. Although numerous studies have focused on actomyosin ring and division septum assembly, little information is available on the mechanism of cell separation. Here we describe a mutant, sec8-1, that is defective in cell separation but not in other aspects of cytokinesis.sec8-1 mutants accumulate ∼100-nm vesicles and have reduced secretion of acid phosphatase, suggesting that they are defective in exocytosis. Sec8p is a component of the exocyst complex. Using biochemical methods, we show that Sec8p physically interacts with other members of the exocyst complex, including Sec6p, Sec10p, and Exo70p. These exocyst proteins localize to regions of active exocytosis—at the growing ends of interphase cells and in the medial region of cells undergoing cytokinesis—in an F-actin–dependent and exocytosis-independent manner. Analysis of a number of mutations in various exocyst components has established that these components are essential for cell viability. Interestingly, all exocyst mutants analyzed appear to be able to elongate and to assemble division septa but are defective for cell separation. We therefore propose that the fission yeast exocyst is involved in targeting of enzymes responsible for septum cleavage. We further propose that cell elongation and division septum assembly can continue with minimal levels of exocyst function.

scholarly journals Drc1p/Cps1p, a 1,3-β-Glucan Synthase Subunit, Is Essential for Division Septum Assembly in Schizosaccharomyces pombe

Genetics ◽  
1999 ◽  
Vol 153 (3) ◽  
pp. 1193-1203
Author(s):  
Jianhua Liu ◽  
Hongyan Wang ◽  
Dannel McCollum ◽  
Mohan K Balasubramanian
Keyword(s):  
Cell Growth ◽  
Schizosaccharomyces Pombe ◽  
Essential Role ◽  
Temperature Sensitive ◽  
Contractile Ring ◽  
Interphase Nuclei ◽  
Glucan Synthase ◽  
Ring Constriction ◽  
Mutant Cells ◽  
Null Mutants

Abstract Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin-based contractile ring. A division septum is formed centripetally, concomitant with ring constriction. Although several genes essential for cytokinesis have been described previously, enzymes that participate in the assembly of the division septum have not been identified. Here we describe a temperature-sensitive mutation, drc1-191, that prevents division septum assembly and causes mutant cells to arrest with a stable actomyosin ring. Unlike the previously characterized cytokinesis mutants, which undergo multiple mitotic cycles, drc1-191 is the first cytokinesis mutant that arrests with two interphase nuclei. Interestingly, unlike drc1-191, drc1-null mutants proceed through multiple mitotic cycles, leading to the formation of large cells with many nuclei. drc1 is allelic to cps1, which encodes a 1,3-β-glucan synthase subunit. We conclude that Drc1p/Cps1p is not required for cell elongation and cell growth, but plays an essential role in assembly of the division septum. Furthermore, it appears that constriction of the actomyosin ring might depend on assembly of the division septum. We discuss possible mechanisms that account for the differences in the phenotypes of the drc1-191 and the drc1-null mutants and also reflect the potential links between Drc1p and other cytokinesis regulators.

F-actin ring formation and the role of F-actin cables in the fission yeastSchizosaccharomyces pombe

2002 ◽  
Vol 115 (5) ◽  
pp. 887-898 ◽  
Author(s):  
Ritsuko Arai ◽  
Issei Mabuchi
Keyword(s):  
Three Dimensional ◽  
Spindle Pole ◽  
Ring Formation ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Actin Ring ◽  
Medial Region ◽  
Dimensional Reconstruction ◽  
Actin Cables

Cells of the fission yeast Schizosaccharomyces pombe divide by the contraction of the F-actin ring formed at the medial region of the cell. We investigated the process of F-actin ring formation in detail using optical sectioning and three-dimensional reconstruction fluorescence microscopy. In wild-type cells, formation of an aster-like structure composed of F-actin cables and accumulation of F-actin cables were recognized at the medial cortex of the cell during prophase to metaphase. The formation of the aster-like structure seemed to initiate from branching of the longitudinal F-actin cables at a site near the spindle pole bodies, which had been duplicated but not yet separated. A single cable extended from the aster and encircled the cell at the equator to form a primary F-actin ring during metaphase. During anaphase,the accumulated F-actin cables were linked to the primary F-actin ring, and then all of these structures seemed to be packed to form the F-actin ring. These observations suggest that formation of the aster-like structure and the accumulation of the F-actin cables at the medial region of the cell during metaphase may be required to initiate the F-actin ring formation. In the nda3 mutant, which has a mutation in ß-tubulin and has been thought to be arrested at prophase, an F-actin ring with accumulated F-actin cables similar to that of anaphase wild-type cells was formed at a restrictive temperature. Immediately after shifting to a permissive temperature, this structure changed into a tightly packed ring. This suggests that the F-actin ring formation progresses beyond prophase in the nda3 cells once the cells enter prophase. We further examined F-actin structures in both cdc12 and cdc15 early cytokinesis mutants. As a result,Cdc12 seemed to be required for the primary F-actin ring formation during prophase, whereas Cdc15 may be involved in both packing the F-actin cables to form the F-actin ring and rearrangement of the F-actin after anaphase. In spg1, cdc7 and sid2 septum initiation mutants, the F-actin ring seemed to be formed in order.

scholarly journals Immobile myosin-II plays a scaffolding role during cytokinesis in budding yeast

2013 ◽  
Vol 200 (3) ◽  
pp. 271-286 ◽  
Author(s):  
Carsten Wloka ◽  
Elizabeth A. Vallen ◽  
Lydia Thé ◽  
Xiaodong Fang ◽  
Younghoon Oh ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Budding Yeast ◽  
Myosin Ii ◽  
Small Region ◽  
Myosin V ◽  
Actin Ring ◽  
Essential Light Chain ◽  
Division Site ◽  
Ring Assembly ◽  
Core Components

Core components of cytokinesis are conserved from yeast to human, but how these components are assembled into a robust machine that drives cytokinesis remains poorly understood. In this paper, we show by fluorescence recovery after photobleaching analysis that Myo1, the sole myosin-II in budding yeast, was mobile at the division site before anaphase and became immobilized shortly before cytokinesis. This immobility was independent of actin filaments or the motor domain of Myo1 but required a small region in the Myo1 tail that is thought to be involved in higher-order assembly. As expected, proteins involved in actin ring assembly (tropomyosin and formin) and membrane trafficking (myosin-V and exocyst) were dynamic during cytokinesis. Strikingly, proteins involved in septum formation (the chitin synthase Chs2) and/or its coordination with the actomyosin ring (essential light chain, IQGAP, F-BAR, etc.) displayed Myo1-dependent immobility during cytokinesis, suggesting that Myo1 plays a scaffolding role in the assembly of a cytokinesis machine.

scholarly journals Nonmedially assembled F-actin cables incorporate into the actomyosin ring in fission yeast

2012 ◽  
Vol 199 (5) ◽  
pp. 831-847 ◽  
Author(s):  
Junqi Huang ◽  
Yinyi Huang ◽  
Haochen Yu ◽  
Dhivya Subramanian ◽  
Anup Padmanabhan ◽  
...  
Keyword(s):  
De Novo ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Animal Cells ◽  
Myosin V ◽  
Contractile Ring ◽  
Division Site ◽  
Ring Assembly ◽  
Actin Cables

In many eukaryotes, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring. Despite the central role of this ring in cytokinesis, the mechanism of F-actin assembly and accumulation in the ring is not fully understood. In this paper, we investigate the mechanism of F-actin assembly during cytokinesis in Schizosaccharomyces pombe using lifeact as a probe to monitor actin dynamics. Previous work has shown that F-actin in the actomyosin ring is assembled de novo at the division site. Surprisingly, we find that a significant fraction of F-actin in the ring was recruited from formin-Cdc12p nucleated long actin cables that were generated at multiple nonmedial locations and incorporated into the ring by a combination of myosin II and myosin V activities. Our results, together with findings in animal cells, suggest that de novo F-actin assembly at the division site and directed transport of F-actin cables assembled elsewhere can contribute to ring assembly.

Sign in / Sign up

Export Citation Format

Share Document