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doi: 10.1242/10.1242/jcs.00150

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The small GTPase Rho3 and the diaphanous/formin For3 function in polarized cell growth in fission yeast

Kentaro Nakano1,*, Jun Imai2, Ritsuko Arai1, Akio Toh-e2, Yasushi Matsui2 and Issei Mabuchi1,3

1 Division of Biology, Department of Life Sciences, Graduate Program of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan
2 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan
3 Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan



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  		Fig. 1. Aberrant morphology of rho3 null cells. (A) Growth of wild-type cells (WT) and rho3 null cells (rho3) on a YEA plate and on YEA containing 1 M sorbitol at the indicated temperatures for 3 days. (B) Wild-type cells and rho3 null cells grown in YEA medium at the indicated temperatures for 16 hours were stained with Calcofluor. (C) Wild-type cells and rho3 null cells grown in YEA medium at 25 or 37°C for 5 or 16 hours were stained with Rhodamine-phalloidin (red in merged images), TAT-1 (green in merged images), and DAPI (blue in merged images). Deconvoluted 3D images are shown. Arrows indicate cells with abnormal organization of cytoplasmic MTs. Arrowheads indicate nuclei in a cytokinesis-defective binucleate cell. Bars, 10 µm.

 


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  		Fig. 2. Localization of Rho3. (A) Rho3 was detected in the insoluble fraction of the cell homogenate. Homogenates of wild-type cells grown in YEA medium at 30°C were spun at 10,000 g to obtain the supernatant (S) and pellet (P) fractions. Each fraction was run on an SDS-15% polyacrylamide gel and subjected to CBB staining or immunoblot analysis using anti-Rho3p antibodies. Molecular weight markers (M) were run on the left lane: trypsin inhibitor (21 k), carbonic anhydrase (31 k), actin (42 k), BSA (68 k), phosphorylase b (94 k), ß-galactosidase (116 k), and myosin heavy chain (210 k). The arrowhead on the right indicates a band of Rho3. (B) Immunofluorescent localization of Rho3. rho3 null cells containing pREP81Rho3 grown in EMM medium without thiamine at 30°C were fixed and stained with Bodipy-phallacidin (green in merged images), anti-Rho3p antibodies (red in merged images), and DAPI (blue in merged images). Deconvoluted 3D images are shown. No Rho3 signal was detected in the cells grown in the presence of thiamine (data not shown). Bar, 5 µm.

 


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  		Fig. 3. Phenotype of Rho3Q71L-overexpressing cells. (A) Overexpression of Rho3Q71L produced large dumpy cells and mini-cells. Wild-type cells carrying pREP1Rho3Q71L were grown at 30°C in EMM without thiamine for 18 or 24 hours. DIC images are shown. (B) F-actin ring was abnormally formed in Rho3Q71L-overexpressing cells. The cells (18 hours in A) were fixed and stained with Rhodamine-phalloidin (top) and DAPI (bottom). Deconvoluted 3D images are shown. Bars, 10 µm. (C) Electron micrographs of Rho3Q71L-overexpressing cells (18 hours in A). Arrowheads indicate abnormally formed septa. N, nucleus. Mt, mitochondria. Bar, 0.2 µm.

 


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  		Fig. 4. Characterization of For3. (A) The N-terminus of For3 binds directly to constitutively active Rho3. L40 cells expressing the indicated proteins were cultured on an SD plate with (+) or without (-) histidine at 30°C for 3 days. `Control' means empty vector. (B,C) Localization of HA-For3. for3 null cells containing pREP41HA-for3 were grown in EMM without thiamine at 30°C for 18 hours. Before (-) and after (+) treatment with 10 µM Lat-A or 100 µM MBC for 10 minutes, the cells were fixed and stained with Bodipy-phallacidin or TAT-1 (green in merged images), anti-HA antibody Y-11 (red in merged images), and DAPI (blue in merged images). Deconvoluted 3D images are shown. Arrowheads in B indicate colocalization of HA-For3 with F-actin. No HA-For3 signal was detected in the cells grown with thiamine (data not shown). (D) Localization of HA-For3 in rho3 cdc42 double-deficient cells. rho3 null cells containing pREP41HA-for3 and pREP1cdc42 or pREP1cdc42T17N were grown in EMM without thiamine at 25°C for 20 hours and were fixed and stained with Bodipy-phallacidin (green in merged images), Y-11 (red in merged images), and DAPI (blue in merged images). Deconvoluted 3D images are shown. (E) Summary of the functional analysis of For3 and truncated proteins. (F) Localization of For3 and truncated proteins using their YFP fusion proteins. Wild-type cells containing pREP1YFP-for3, REP1YFP-for3NM, pREP1YFP-for3N2, or pREP1YFP-for3MC were grown in EMM containing thiamine until early log phase at 25°C. Arrowheads and arrows indicate the signal of YFP at the cell ends and the division site, respectively. Bars, 10 µm.

 


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  		Fig. 5. Overexpression of For3. (A) Effects of overexpression of For3 or truncated proteins on cell viability. Wild-type cells or rho3 null cells containing indicated vectors, respectively, were grown on an EMM plate with (+) or without (-) thiamine at 25°C for 4 days. (B) Effect of For3 overexpression on cell shape. Wild-type cells containing pREP1for3 grown in EMM without thiamine for the indicated periods were stained with Calcofluor. Arrows indicate bulges. Arrowheads indicate swollen cells. (C) MT bundles were formed in For3-overexpressing cells. The cells (0 and 20 hours in B) were fixed and stained with Rhodamine-phalloidin (top) and TAT-1 (bottom). Deconvoluted 3D images are shown. Arrows indicate thick bundles of cytoplasmic MTs. Bars, 5 µm.

 


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  		Fig. 6. Phenotype of for3 null cells. (A) Viability of for3 null cells. Indicated cells were grown at 25°C for 4 days on a YEA plate. (B) Aberrant morphology of for3 null cells and rho3 for3 double null cells. Cells grown in YEA medium at 25°C. DIC images are shown. (C) Defects in actin cytoskeleton and MTs in for3 null cells. for3 null cells grown in YEA medium at 25°C were stained with Bodipy-phallacidin (green), TAT-1 (red), and DAPI (blue). Deconvoluted 3D images are shown. Small arrowhead indicates disorganized cytoplasmic MTs in dumpy cells. Large arrowhead indicates curved MTs located on a convex side of a bent cell. Arrows indicate F-actin cables. (D) Interphase F-actin cables in for3 null cells. for3 null cells grown in YEA medium at 25°C were stained with Bodipy-phallacidin and DAPI. The pictures represent deconvoluted Z-axis sections (top, middle, and bottom sections from left to right) of three interphase cells. Arrows indicate F-actin cables. (E) Formation of thick F-actin cables by overexpression of Fim1A2. Wild-type cells or for3 null cells containing pREP1 fim1 A2 were grown in EMM without thiamine for 18 hours at 25°C. F-actin images are shown. (F) Observation of cells expressing For3-truncated proteins. (a) Deconvoluted 3D images of for3 {Delta}FH12C cells, for3 {Delta}FH2C cells, for3 {Delta}C cells, and for3 null cells containing pREP1 for3NC, pREP1 for3MC, pREP1 for3FH12, or pREP1 for3C are shown. The cells were stained as in C. (b) F-actin images of a for3 null cell containing pREP1 for3MC indicated by the large arrow in a. The pictures represent deconvoluted Z-axis sections (top, middle, and bottom sections from left to right). (G) A for3 rho3 null cell grown in YEA medium at 25°C was stained with Bodipy-phallacidin (left), and TAT-1 (right). Deconvoluted 3D images are shown. Bars, 5 µm.

 

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