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Mid2p stabilizes septin rings during cytokinesis in fission yeast.

Berlin A, Paoletti A, Chang F - J. Cell Biol. (2003)

Bottom Line: In wild-type cells, septins assembled first during mitosis in a single ring and during septation developed into double rings that did not contract.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

ABSTRACT
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, mid2delta mutants had delays in cell-cell separation. mid2delta 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 mid2delta 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 mid2delta 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.

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The contractile ring appears to assemble and close normally in mid2Δ cells. (a) Wild-type and mid2Δ cells expressing cdc4-GFP were imaged live using confocal time-lapse microscopy. Z-stacks were rendered in three dimensions to produce a cross-sectional view of the ring. Bar, 2 μm. (B) Measurements of five individual ring diameters over time in wild-type and mid2Δ cells.
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fig3: The contractile ring appears to assemble and close normally in mid2Δ cells. (a) Wild-type and mid2Δ cells expressing cdc4-GFP were imaged live using confocal time-lapse microscopy. Z-stacks were rendered in three dimensions to produce a cross-sectional view of the ring. Bar, 2 μm. (B) Measurements of five individual ring diameters over time in wild-type and mid2Δ cells.

Mentions: Since mild defects in actin-myosin contractile ring organization can lead to cell–cell separation defects (unpublished data), we tested whether Mid2p is involved in the assembly or contraction of the actin ring during cytokinesis. mid2Δ cells stained for F-actin with Alexa Fluor phalloidin exhibited well-defined, normal actin rings (unpublished data). Using a Cdc4p (a myosin light chain) fusion to GFP (McCollum et al., 1995; Balasubramanian et al., 1997), we examined if the contraction of the actin-myosin ring might be perturbed. Confocal three-dimensional time-lapse images showed that wild-type Cdc4p-GFP rings contracted at the end of mitosis with a rate of 0.20 ± 0.017 μm/min (n = 5), consistent with previous reports (Bezanilla et al., 2000; Motegi et al., 2000; Pelham and Chang, 2002). In mid2Δ cells, Cdc4p-GFP rings appeared normal and contracted at rates similar to those of wild-type rings, 0.17 ± 0.048 μm/min (n = 5; P > 0.1) (Fig. 3). Therefore, neither the assembly nor the closure of the contractile ring was markedly perturbed in mid2Δ mutants.


Mid2p stabilizes septin rings during cytokinesis in fission yeast.

Berlin A, Paoletti A, Chang F - J. Cell Biol. (2003)

The contractile ring appears to assemble and close normally in mid2Δ cells. (a) Wild-type and mid2Δ cells expressing cdc4-GFP were imaged live using confocal time-lapse microscopy. Z-stacks were rendered in three dimensions to produce a cross-sectional view of the ring. Bar, 2 μm. (B) Measurements of five individual ring diameters over time in wild-type and mid2Δ cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172768&req=5

fig3: The contractile ring appears to assemble and close normally in mid2Δ cells. (a) Wild-type and mid2Δ cells expressing cdc4-GFP were imaged live using confocal time-lapse microscopy. Z-stacks were rendered in three dimensions to produce a cross-sectional view of the ring. Bar, 2 μm. (B) Measurements of five individual ring diameters over time in wild-type and mid2Δ cells.
Mentions: Since mild defects in actin-myosin contractile ring organization can lead to cell–cell separation defects (unpublished data), we tested whether Mid2p is involved in the assembly or contraction of the actin ring during cytokinesis. mid2Δ cells stained for F-actin with Alexa Fluor phalloidin exhibited well-defined, normal actin rings (unpublished data). Using a Cdc4p (a myosin light chain) fusion to GFP (McCollum et al., 1995; Balasubramanian et al., 1997), we examined if the contraction of the actin-myosin ring might be perturbed. Confocal three-dimensional time-lapse images showed that wild-type Cdc4p-GFP rings contracted at the end of mitosis with a rate of 0.20 ± 0.017 μm/min (n = 5), consistent with previous reports (Bezanilla et al., 2000; Motegi et al., 2000; Pelham and Chang, 2002). In mid2Δ cells, Cdc4p-GFP rings appeared normal and contracted at rates similar to those of wild-type rings, 0.17 ± 0.048 μm/min (n = 5; P > 0.1) (Fig. 3). Therefore, neither the assembly nor the closure of the contractile ring was markedly perturbed in mid2Δ mutants.

Bottom Line: In wild-type cells, septins assembled first during mitosis in a single ring and during septation developed into double rings that did not contract.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

ABSTRACT
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, mid2delta mutants had delays in cell-cell separation. mid2delta 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 mid2delta 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 mid2delta 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.

Show MeSH
Related in: MedlinePlus