scholarly journals The number of cytokinesis nodes in mitotic fission yeast scales with cell volume

2021 ◽  
Author(s):  
Wasim A Sayyad ◽  
Thomas D Pollard
Keyword(s):  
Plasma Membrane ◽  
Fission Yeast ◽  
Large Population ◽  
Yeast Cells ◽  
Wild Type ◽  
Contractile Ring ◽  
Node Number ◽  
Nmt1 Promoter ◽  
Wide Range ◽  
Mutant Cells

Cytokinesis nodes are assemblies of stoichiometric ratios of proteins associated with the plasma membrane, which serve as precursors for the contractile ring during cytokinesis by fission yeast. The total number of nodes is uncertain, because of the limitations of the methods used previously. Here we used the ~140 nm resolution of Airyscan confocal microscopy to resolve a large population of dim, unitary cytokinesis nodes in 3D reconstructions of whole fission yeast cells. Wild-type fission yeast cells make about 200 unitary cytokinesis nodes. Most, but not all of these nodes condense into a contractile ring. The number of cytokinesis nodes scales with cell size in four strains tested, although wide rga4Δ mutant cells form somewhat fewer cytokinesis nodes than expected from the overall trend. The surface density of Pom1 kinase on the plasma membrane around the equators of cells is similar with a wide range of node numbers, so Pom1 does not control cytokinesis node number. However, varying protein concentrations with the nmt1 promoter showed that the numbers of nodes increase above a baseline of about 200 with the total cellular concentration of either Pom1 or the kinase Cdr2.

scholarly journals Counting actin in contractile rings reveals novel contributions of cofilin and type II myosins to fission yeast cytokinesis

2021 ◽  
Author(s):  
Qian Chen ◽  
Mamata Malla ◽  
Thomas D. Pollard
Keyword(s):  
Fission Yeast ◽  
Actin Filaments ◽  
Yeast Cells ◽  
Type Ii ◽  
Wild Type ◽  
Contractile Ring ◽  
Peak Number ◽  
Ring Constriction ◽  
Proportional Increase ◽  
Mutant Cells

Cytokinesis by animals, fungi and amoebas depends on actomyosin contractile rings, which are stabilized by continuous turnover of actin filaments. Remarkably little is known about the amount of polymerized actin in contractile rings, so we used low concentration of GFP-Lifeact to count total polymerized actin molecules in the contractile rings of live fission yeast cells. Contractile rings of wild-type cells accumulated polymerized actin molecules at 4,900/min to a peak number of ~198,000 followed by a loss of actin at 5,400/min throughout ring constriction. In adf1-M3 mutant cells with cofilin that severs actin filaments poorly, contractile rings accumulated polymerized actin at twice the normal rate and eventually had almost two-fold more actin along with a proportional increase in type II myosins Myo2, Myp2 and formin Cdc12. Although 30% of adf1-M3 mutant cells failed to constrict their rings fully, the rest lost actin from the rings at the wild-type rates. Mutations of type II myosins Myo2 and Myp2 reduced contractile ring actin filaments by half and slowed the rate of actin loss from the rings.

scholarly journals Counting actin in contractile rings reveals novel contributions of cofilin and type II myosins to fission yeast cytokinesis

2021 ◽  
Author(s):  
Mamata Malla ◽  
Thomas D. Pollard ◽  
Qian Chen
Keyword(s):  
Fission Yeast ◽  
Actin Filaments ◽  
Yeast Cells ◽  
Type Ii ◽  
Wild Type ◽  
Contractile Ring ◽  
Peak Number ◽  
Ring Constriction ◽  
Proportional Increase ◽  
Mutant Cells

Cytokinesis by animals, fungi and amoebas depends on actomyosin contractile rings, which are stabilized by continuous turnover of actin filaments. Remarkably little is known about the amount of polymerized actin in contractile rings, so we used low concentration of GFP-Lifeact to count total polymerized actin molecules in the contractile rings of live fission yeast cells. Contractile rings of wild-type cells accumulated polymerized actin molecules at 4,900/min to a peak number of ∼198,000 followed by a loss of actin at 5,400/min throughout ring constriction. In adf1-M3 mutant cells with cofilin that severs actin filaments poorly, contractile rings accumulated polymerized actin at twice the normal rate and eventually had almost two-fold more actin along with a proportional increase in type II myosins Myo2, Myp2 and formin Cdc12. Although 30% of adf1-M3 mutant cells failed to constrict their rings fully, the rest lost actin from the rings at the wild-type rates. Mutations of type II myosins Myo2 and Myp2 reduced contractile ring actin filaments by half and slowed the rate of actin loss from the rings.

scholarly journals Anillin-related protein Mid1p coordinates the assembly of the cytokinetic contractile ring in fission yeast

2012 ◽  
Vol 23 (20) ◽  
pp. 3982-3992 ◽  
Author(s):  
Shambaditya Saha ◽  
Thomas D. Pollard
Keyword(s):  
Fission Yeast ◽  
Myosin Ii ◽  
Equatorial Region ◽  
Yeast Cells ◽  
Related Protein ◽  
Wild Type ◽  
Contractile Ring ◽  
Maturation Period ◽  
Ring Assembly ◽  
Open Questions

In fission yeast cells cortical nodes containing the protein Blt1p and several kinases appear early in G2, mature into cytokinetic nodes by adding anillin Mid1p, myosin-II, formin Cdc12p, and other proteins, and condense into a contractile ring by movements that depend on actin and myosin-II. Previous studies concluded that cells without Mid1p lack cytokinetic nodes and assemble rings unreliably from myosin-II strands but left open questions. Why do strands form outside the equatorial region? Why is ring assembly unreliable without Mid1p? We found in Δmid1 cells that Cdc12p accumulates in cytokinetic nodes scattered in the cortex and produces actin filaments that associate with myosin-II, Rng2p, and Cdc15p to form strands located between the nodes. Strands incorporate nodes, and in ∼67% of cells, strands slowly close into rings that constrict without the normal ∼25-min maturation period. Ring assembly is unreliable and slow without Mid1p because the scattered Cdc12p nodes generate strands spread widely beyond the equator, and growing strands depend on random encounters to merge with other strands into a ring. We conclude that orderly assembly of the contractile ring in wild-type cells depends on Mid1p to recruit myosin-II, Rng2p, and Cdc15p to nodes and to place cytokinetic nodes around the cell equator.

scholarly journals Actin filament severing by cofilin is more important for assembly than constriction of the cytokinetic contractile ring

2011 ◽  
Vol 195 (3) ◽  
pp. 485-498 ◽  
Author(s):  
Qian Chen ◽  
Thomas D. Pollard
Keyword(s):  
Fission Yeast ◽  
Computer Simulations ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Wild Type ◽  
Contractile Ring ◽  
Ring Constriction ◽  
Release Model ◽  
Mutant Cells

We created two new mutants of fission yeast cofilin to investigate why cytokinesis in many organisms depends on this small actin-binding protein. These mutant cofilins bound actin monomers normally, but bound and severed ADP-actin filaments much slower than wild-type cofilin. Cells depending on mutant cofilins condensed nodes, precursors of the contractile ring, into clumps rather than rings. Starting from clumped nodes, mutant cells slowly assembled rings from diverse intermediate structures including spiral strands containing actin filaments and other contractile ring proteins. This process in mutant cells depended on α-actinin. These slowly assembled contractile rings constricted at a normal rate but with more variability, indicating ring constriction is not very sensitive to defects in severing by cofilin. Computer simulations of the search-capture-pull and release model of contractile ring formation predicted that nodes clump when the release step is slow, so cofilin severing of actin filament connections between nodes likely contributes to the release step.

scholarly journals Amino acids whose intracellular levels change most during aging alter chronological lifespan of fission yeast

2020 ◽  
Author(s):  
Charalampos Rallis ◽  
Michael Mülleder ◽  
Graeme Smith ◽  
Yan Zi Au ◽  
Markus Ralser ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Fission Yeast ◽  
Yeast Cells ◽  
Total Amino Acid ◽  
Wild Type ◽  
Chronological Lifespan ◽  
Biomarkers Of Aging ◽  
Concentration Changes ◽  
Amino Acid Levels

AbstractAmino acid deprivation or supplementation can affect cellular and organismal lifespan, but we know little about the role of concentration changes in free, intracellular amino acids during aging. Here, we determine free amino-acid levels during chronological aging of non-dividing fission yeast cells. We compare wild-type with long-lived mutant cells that lack the Pka1 protein of the protein kinase A signalling pathway. In wild-type cells, total amino-acid levels decrease during aging, but much less so in pka1 mutants. Two amino acids strongly change as a function of age: glutamine decreases, especially in wild-type cells, while aspartate increases, especially in pka1 mutants. Supplementation of glutamine is sufficient to extend the chronological lifespan of wild-type but not of pka1Δ cells. Supplementation of aspartate, on the other hand, shortens the lifespan of pka1Δ but not of wild-type cells. Our results raise the possibility that certain amino acids are biomarkers of aging, and their concentrations during aging can promote or limit cellular lifespan.

Protein secretion in Tetrahymena thermophila: characterization of the secretory mutant strain SB281

1985 ◽  
Vol 78 (1) ◽  
pp. 49-65 ◽  
Author(s):  
N.J. Maihle ◽  
B.H. Satir
Keyword(s):  
Plasma Membrane ◽  
Mutant Strain ◽  
Protein Secretion ◽  
Tetrahymena Thermophila ◽  
Freeze Fracture ◽  
Size Class ◽  
Secretory Product ◽  
Wild Type ◽  
Intramembrane Particle ◽  
Mutant Cells

The ciliated protozoon Tetrahymena thermophila contains membrane-bounded secretory organelles termed mucocysts, the release of which has previously been characterized ultrastructurally as a model system for the events occurring during membrane fusion and protein secretion. Recently, a series of secretory mutant strains of Tetrahymena has been isolated following mutagenesis of a parental wild-type strain designated SB210. In this study, the correlates of non-release in one unique mutant strain of this series, designated SB281, are described. SB281 appears to express a diminished (undetectable) level of the major 34000 Mr proteinaceous secretory product of Tetrahymena, as determined by Western immunoblot analysis and indirect immunofluorescence labelling. Thin-section electron-microscopic studies of these cells reveal that they possess no docked or free mature mucocysts. In addition, freeze-fracture electron microscopy demonstrates that an intramembrane particle array termed the rosette, present in the plasma membrane of wild-type cells above sites of docked mucocysts, is absent in the plasma membrane of mutant SB281 cells. A morphometric analysis of intramembrane particles in the plasma membrane of both wild-type and mutant cells indicates that both strains have a similar intramembrane particle density in both leaflets of the the plasma membrane. Although assembled rosettes are missing in the plasma membrane of mutant cells, a 15 nm intramembrane particle size class does exist in the plasma membrane of the mutant, but this size class is significantly reduced in number relative to wild-type.

scholarly journals Mid2p stabilizes septin rings during cytokinesis in fission yeast

2003 ◽  
Vol 160 (7) ◽  
pp. 1083-1092 ◽  
Author(s):  
Ana Berlin ◽  
Anne Paoletti ◽  
Fred Chang
Keyword(s):  
Fission Yeast ◽  
Cell Separation ◽  
Sequence Similarity ◽  
Ring Closure ◽  
Pleckstrin Homology Domain ◽  
Wild Type ◽  
Contractile Ring ◽  
Single Ring ◽  
And Function ◽  
Cell Cell

Septins are filament-forming proteins with a conserved role in cytokinesis. In the fission yeast Schizosaccharomyces pombe, septin rings appear to be involved primarily in cell–cell separation, a late stage in cytokinesis. Here, we identified a protein Mid2p on the basis of its sequence similarity to S. pombe Mid1p, Saccharomyces cerevisiae Bud4p, and Candida albicans Int1p. Like septin mutants, mid2Δ mutants had delays in cell–cell separation. mid2Δ mutants were defective in septin organization but not contractile ring closure or septum formation. In wild-type cells, septins assembled first during mitosis in a single ring and during septation developed into double rings that did not contract. In mid2Δ cells, septins initially assembled in a single ring but during septation appeared in the cleavage furrow, forming a washer or disc structure. FRAP studies showed that septins are stable in wild-type cells but exchange 30-fold more rapidly in mid2Δ cells. Mid2p colocalized with septins and required septins for its localization. A COOH-terminal pleckstrin homology domain of Mid2p was required for its localization and function. No genetic interactions were found between mid2 and the related gene mid1. Thus, these studies identify a new factor responsible for the proper stability and function of septins during cytokinesis.

scholarly journals The size-wise nucleus: nuclear volume control in eukaryotes

2007 ◽  
Vol 179 (4) ◽  
pp. 583-584 ◽  
Author(s):  
Michael D. Huber ◽  
Larry Gerace
Keyword(s):  
Fission Yeast ◽  
Cell Size ◽  
Size Control ◽  
Growth Conditions ◽  
Nuclear Size ◽  
Yeast Cells ◽  
Volume Control ◽  
Wide Range ◽  
The Relationship

Eukaryotic cells have an “awareness” of their volume and organellar volumes, and maintain a nuclear size that is proportional to the total cell size. New studies in budding and fission yeast have examined the relationship between cell and nuclear volumes. It was found that the size of the nucleus remains proportional to cell size in a wide range of genetic backgrounds and growth conditions that alter cell volume and DNA content. Moreover, in multinucleated fission yeast cells, Neumann and Nurse (see p. 593 of this issue) found that the sizes of individual nuclei are controlled by the relative amount of cytoplasm surrounding each nucleus. These results highlight a role of the cytoplasm in nuclear size control.

scholarly journals Intramitotic controls in the fission yeast Schizosaccharomyces pombe: the effect of cell size on spindle length and the timing of mitotic events.

1990 ◽  
Vol 110 (5) ◽  
pp. 1617-1621 ◽  
Author(s):  
I M Hagan ◽  
P N Riddle ◽  
J S Hyams
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Average Length ◽  
Nuclear Migration ◽  
Yeast Cells ◽  
Reverse Direction ◽  
Wild Type ◽  
Spindle Elongation ◽  
Anaphase B ◽  
In Cells

We have used a new cinemicroscopy technique in combination with antitubulin immunofluorescence microscopy to investigate the timing of mitotic events in cells of the fission yeast Schizosaccharomyces pombe having lengths at division between 7 and 60 microns. Wild-type fission yeast cells divide at a length of 14 microns. Separation of daughter nuclei (anaphase B) proceeds at a rate of 1.6 +/- 0.2 microns min-1, until the spindle extends the length of the cell. Coincident with spindle depolymerization, the nuclei reverse direction and take up positions that will become the center of the two daughter cells. This post-mitotic nuclear migration occurs at a rate of 1.4 +/- 0.5 microns-1. In cells in which the weel+ gene is overexpressed fivefold and that have an average length at mitosis of 28 microns, the rate of nuclear separation was only slightly reduced but, as spindles in these cells measure 20-22 microns, the duration of anaphase B was extended by approximately 40%. By contrast, in the mutant weel.50, which divides at 7 microns, both the rate and duration of anaphase B were indistinguishable from wild type. Nuclei reach the ends of these cells earlier but remain there until a point corresponding to the time of postmitotic nuclear migration in wild type. Thus, the events of mitosis can be extended but not abbreviated. These results are discussed in terms of a mitotic termination control that monitors many different events, one of which is spindle elongation.

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