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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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Measurement of Spn4p-GFP dynamics using FRAP. (A) Wild-type and mid2Δ cells expressing Spn4p-GFP were photobleached in the zones marked by white rectangles. Recovery of fluorescence intensity was assayed over time (as described in Materials and methods). Representative images are shown. Top panels show cells just before photobleaching. 0 s represents time immediately after photobleaching. (B) Graph of mean t1/2 ± SD of Spn4p-GFP fluorescence recovery in seconds.
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fig5: Measurement of Spn4p-GFP dynamics using FRAP. (A) Wild-type and mid2Δ cells expressing Spn4p-GFP were photobleached in the zones marked by white rectangles. Recovery of fluorescence intensity was assayed over time (as described in Materials and methods). Representative images are shown. Top panels show cells just before photobleaching. 0 s represents time immediately after photobleaching. (B) Graph of mean t1/2 ± SD of Spn4p-GFP fluorescence recovery in seconds.

Mentions: This abnormal localization of Spn4p-GFP in mid2Δ cells suggested that septins may be flowing from the septin rings into the cleavage furrow during ring closure. Alternatively, new septin proteins may be deposited at the membrane in the furrow during this process. To distinguish between these two possibilities, we investigated the dynamics of septin proteins using FRAP. Portions of Spn4p-GFP rings were photobleached, and the rates of recovery of Spn4p-GFP fluorescence were assayed. In mid2+ cells, Spn4p-GFP in well established septin rings recovered relatively slowly with t1/2 = 350 ± 136 s (range 168–532 s; n = 7) (Fig. 5; see Materials and methods), showing that Spn4p is relatively stable. In contrast, in mid2Δ cells Spn4p-GFP fluorescence recovered over 30-fold more rapidly, with t1/2 = 10 ± 4 s (range 5–17 s; n = 6) (Fig. 5) showing that Spn4p is rapidly exchanging in the ring. Similar FRAP rates were observed in mid2Δ cells at all different stages of cleavage (n = 18), showing that this large difference between wild-type and mid2Δ cells was not due to cell cycle stage differences between the datasets. Since the rate of septin exchange is much faster than the rate of invagination, these dynamics show that Spn4p proteins in mid2Δ cells are not flowing in from the rings at the cell surface into the furrow but may be rapidly binding and exchanging with the invaginating membrane. Thus, Mid2p is required to stabilize septins.


Mid2p stabilizes septin rings during cytokinesis in fission yeast.

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

Measurement of Spn4p-GFP dynamics using FRAP. (A) Wild-type and mid2Δ cells expressing Spn4p-GFP were photobleached in the zones marked by white rectangles. Recovery of fluorescence intensity was assayed over time (as described in Materials and methods). Representative images are shown. Top panels show cells just before photobleaching. 0 s represents time immediately after photobleaching. (B) Graph of mean t1/2 ± SD of Spn4p-GFP fluorescence recovery in seconds.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Measurement of Spn4p-GFP dynamics using FRAP. (A) Wild-type and mid2Δ cells expressing Spn4p-GFP were photobleached in the zones marked by white rectangles. Recovery of fluorescence intensity was assayed over time (as described in Materials and methods). Representative images are shown. Top panels show cells just before photobleaching. 0 s represents time immediately after photobleaching. (B) Graph of mean t1/2 ± SD of Spn4p-GFP fluorescence recovery in seconds.
Mentions: This abnormal localization of Spn4p-GFP in mid2Δ cells suggested that septins may be flowing from the septin rings into the cleavage furrow during ring closure. Alternatively, new septin proteins may be deposited at the membrane in the furrow during this process. To distinguish between these two possibilities, we investigated the dynamics of septin proteins using FRAP. Portions of Spn4p-GFP rings were photobleached, and the rates of recovery of Spn4p-GFP fluorescence were assayed. In mid2+ cells, Spn4p-GFP in well established septin rings recovered relatively slowly with t1/2 = 350 ± 136 s (range 168–532 s; n = 7) (Fig. 5; see Materials and methods), showing that Spn4p is relatively stable. In contrast, in mid2Δ cells Spn4p-GFP fluorescence recovered over 30-fold more rapidly, with t1/2 = 10 ± 4 s (range 5–17 s; n = 6) (Fig. 5) showing that Spn4p is rapidly exchanging in the ring. Similar FRAP rates were observed in mid2Δ cells at all different stages of cleavage (n = 18), showing that this large difference between wild-type and mid2Δ cells was not due to cell cycle stage differences between the datasets. Since the rate of septin exchange is much faster than the rate of invagination, these dynamics show that Spn4p proteins in mid2Δ cells are not flowing in from the rings at the cell surface into the furrow but may be rapidly binding and exchanging with the invaginating membrane. Thus, Mid2p is required to stabilize septins.

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