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First published online 20 July 2004
doi: 10.1242/jcs.01244

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The Clp1p/Flp1p phosphatase ensures completion of cytokinesis in response to minor perturbation of the cell division machinery in Schizosaccharomyces pombe

Mithilesh Mishra^1,*, Jim Karagiannis^1,*, Susanne Trautmann², Hongyan Wang¹, Dannel McCollum² and Mohan K. Balasubramanian^1,3,

¹ Cell Division Laboratory, Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, NUS, Singapore 117604, Rep. of Singapore
³ Department of Biological Sciences, National University of Singapore, 1 Research Link, NUS, Singapore 117604, Rep. of Singapore
² Department of Microbiology and Molecular Genetics, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655-0122, USA

View larger version (68K): [in a new window]   Fig. 1. Clp1p is essential when cell division structures are mildly perturbed and ensures the completion of cytokinesis. (A) Cells of the indicated genotype were streaked to YES plates and assayed for colony formation after 3 days at 30°C (left) and 32°C (right). (B) Cells of indicated genotype were cultured to exponential growth phase at 24°C and then shifted to 32°C for 5 hours, fixed and then stained with aniline blue and DAPI to visualize cell wall/septa and nuclei respectively. Septa are indicated with arrowheads. Scale bar 10 µm. (C) Septum formation in group I cytokinesis mutants at semi-permissive temperatures in the presence or absence of Clp1p. Cells of the indicated genotype were cultured to exponential growth phase at 24°C, shifted to 32°C for 5 hours, fixed and then stained with aniline blue and DAPI to visualize cell wall/septa and nuclei respectively. (D) Wild-type and clp1 cells were cultured to exponential growth phase at 24°C, synchronized in early G2 by centrifugal elutriation and then treated with a low dose (0.2 µM) of Lat A or DMSO (solvent control) and cultured at 32°C. Cells were subsequently fixed at 30 minute intervals and stained with DAPI (nuclei) and aniline blue (cell wall/septa). (E) Wild-type and clp1 cells treated as in Fig. 1D (at t=240 minutes) and stained with both DAPI (nuclei) and aniline blue (cell wall/septa). Imperfect, but functional, septa are indicated with arrowheads. Scale bar 10 µm. (F) Tenfold serial dilutions of wild-type and clp1 cells grown on YES plates containing either 0.25 µM Lat A or DMSO. Images were captured after 3 days growth at 32°C. (G) Wild-type and clp1 cells were cultured to exponential growth phase at 24°C, synchronized in early G2 by centrifugal elutriation and then treated with a low dose (0.2 µM) of Lat A or DMSO (solvent control) and cultured at 32°C. Cell number was determined using a haemocytometer.

View larger version (73K): [in a new window]   Fig. 2. Nuclear cycle arrest is insufficient to allow completion of cytokinesis upon perturbation of the cell division machinery. (A) cdc25-22 and clp1 cdc25-22 cells were grown to early log phase at 24°C and then shifted to 36°C for three hours to arrest cells at the G2/M transition. Cells were subsequently shifted down to 24°C for 30 minutes, treated with a low dose of Lat A (0.2 µM) (t=0) to perturb the actomyosin ring and then shifted back to 36°C at t=30 minutes in the continuing presence of the drug. (B) Quantitative data for cdc25-22 and clp1cdc25-22 cells treated as in Fig. 2A and then fixed with methanol, washed twice with PBS, and stained with DAPI (nuclei) and aniline blue (septa) at the indicated time points. Septa were classified into three groups: normal (similar to septa formed in wild type cells during logarithmic growth); imperfect and complete (functional septa that appeared thicker and more disorganized but bisected the cell); and spotty and incomplete (non-functional deposits of septal material that failed to form a linear structure across the width of the cell). (C) cdc25-22 and clp1cdc25-22 cells treated as in Fig. 2A (at t=4 hours) and stained with both DAPI (nuclei) and aniline blue (cell wall/septa). Imperfect but functional, septa are indicated with arrowheads. (D) cdc25-22 and clp1cdc25-22 cells treated as in Fig. 2A (at t=4 hours) and stained with aniline blue (cell wall/septa). Z-series were obtained and deconvolved as described in Materials and Methods. Max projections of the entire cell are shown to the left of each panel, whereas three alternate views of 3D reconstructions of septa are shown to the right.

View larger version (44K): [in a new window]   Fig. 3. Clp1p is important for the maintenance of the actomyosin ring at the medial region of the cell. (A) cps1-191 and clp1cps1-191 were grown to exponential phase at 24°C, shifted to 36°C for four hours and stained with rhodamine-conjugated phalloidin to determine the distribution of actin. `Medial' refers to the localization of actin in a medial actomyosin ring structure. `Tip' refers to the localization of actin patches to the cell ends. (B) Wild-type and clp1 cells were cultured to exponential growth phase at 24°C, synchronized in early G2 by centrifugal elutriation and then treated with a low dose (0.2 µM) of Lat A or DMSO (solvent control). Cells were subsequently fixed at 30-minute intervals and stained with DAPI (nuclei) and ALEXA-488 conjugated phalloidin (actin). The graph shows the percent cells with actomyosin rings. (C) Images of cells treated as in B.

View larger version (88K): [in a new window]   Fig. 4. Dynamics of Clp1p-dependent actomyosin ring maintenance upon perturbation of cytokinetic machinery. (A) Cells of the indicated genotypes expressing Rlc1p-GFP were imaged under conditions shown on the left-hand side. The numbers on the top right of each frame indicates the time in minutes. Note that whereas the Rlc1p-GFp rings are maintained for a prolonged period of time, leading to the accomplishment of division septum assembly in wild-type cells upon treatment with a low dose of Lat A (0.2 µM), these rings fragment in similarly treated clp1 cells. Note also that SIN mutants display a phenotype similar to that observed in clp1 cells treated with a low dose of Lat A. (B) 3D-reconstructions of Rlc1p-GFP expressing cells of the indicated genotype treated with DMSO or 0.2 µM Lat A. Time (t) is indicated in minutes. Wild-type, clp1, and clp1 sid1-239 cells were imaged at 32°C. sid1-239 cells were imaged at 36°C.

View larger version (71K): [in a new window]   Fig. 5. SIN mutants are hypersensitive to mild perturbations of the cell division machinery whereas ectopic activation of the SIN compensates for the loss of Clp1p. (AI) cdc14-118, myo2-E1, and myo2-E1 cdc14-118 mutants were streaked to YES plates and assayed for colony formation after 3 days at the semi-permissive temperature of 30°C. (AII, III and IV) cdc14-118, myo2-E1 and myo2-E1 cdc14-118 cells were grown at 24°C to early-log phase, shifted to 30°C for six hours, and then fixed and stained with DAPI and aniline blue to visualize nuclei and division septa, respectively. Scale bar 10 µm. (B) Quantitative data for cells in AII-IV. (C) Cells of the indicated genotypes were grown to early-log phase at 24°C, shifted to 36°C, and then treated with 0.2 µM of Lat A or DMSO (solvent control) for 5 hours. Cells were subsequently fixed and stained with DAPI and aniline blue to visualize nuclei and division septa respectively. Scale bar 10 µm. (D) Quantitative data for cells treated with Lat A in C.

View larger version (52K): [in a new window]   Fig. 6. (A) Wild-type and clp1 cells carrying integrated copies of Sid1p-GFP and Cdc7p-GFP were cultured to exponential growth phase at 24°C, synchronized in early G2 by centrifugal elutriation and then treated with a low dose (0.2 µM) of Lat A or DMSO (solvent control) and cultured at 32°C. Cells were subsequently fixed at 30 minute intervals, stained with antibodies against GFP (see Materials and Methods), and scored for localization of Sid1p-GFP (left) and Cdc7p-GFP (right) to the SPB. (B) Wild-type and cdc14-118 cells carrying an integrated copy of Clp1p-GFP were treated with 0.2 µM Lat A or DMSO, shifted to 32°C for 3.5 hours, fixed and then stained with DAPI. Clp1p-GFP localization was monitored using GFP autofluorescence.

View larger version (24K): [in a new window]   Fig. 7. Summary and Model. (A) Summary of cellular behavior in differing genotypes and growth conditions. Actin patches and rings are shown in red. Nuclei are shown as black circles. Cdc7p localization to the spindle pole body (a marker of active SIN) is shown schematically as a blue circle. Wild-type or clp1 cells under normal growth conditions (column 1); wild-type or clp1 cells upon treatment with low doses of Lat A (columns 2 and 3, respectively); cps1-191 and clp1 cps1-191 cells at the restrictive temperature of 36°C (columns 4 and 5, respectively); a SIN mutant at the restrictive temperature of 36°C (column 6). (B) Model. Upon perturbation of the cytokinesis machinery Clp1p and the SIN function in a positive feedback loop to maintain the integrity of the actomyosin ring (see Discussion).

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