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First published online 15 November 2005
doi: 10.1242/jcs.02664

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Cell wall remodeling at the fission yeast cell division site requires the Rho-GEF Rgf3p

Jennifer L. Morrell-Falvey^*,, Liping Ren^*, Anna Feoktistova, Greg Den Haese and Kathleen L. Gould

Howard Hughes Medical Institute and Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA

View larger version (80K): [in a new window]   Fig. 1. Phenotype of the lad1-1 mutant strain. (A) lad1-1 cells (KGY2030) were grown at 25°C and replica-plated overnight to 36°C. Cells were photographed on the plate. (B-D) lad1-1 (KGY2030) was grown to mid-exponential phase at 25°C and a synchronous population was isolated by centrifugal elutriation. (B) One portion of the culture was incubated at 25°C and the other at 36°C. The septation index of both cultures was determined microscopically as was the percentage of lysed doublets at the indicated time points. (C) Cells from B incubated at 36°C were stained with calcofluor at 20 (a), 60 (b), 100 (c), 140 (d), 160 (e) and 180 (f) minutes after shifting to 36°C. Arrows indicate extruding or lysing areas of the cells. (D) From the same elutriated culture, samples were collected at 120 (a) or 160 minutes (b and c), fixed with methanol and stained with antibodies to actin (a and b) or Arp3 (c). (E) A double-mutant strain, cdc25-22 lad1-1 (KGY2041), was grown to mid-exponential phase, shifted to 36°C for 4 hours, and stained with calcofluor. (F) A double mutant strain, lad1-1 spn3::ura4 (KGY 3944), was shifted to 36°C for 5 hours and stained with calcofluor. Arrows indicate multiseptate cells (a) and multi-septated cells beginning to lyse as cell division proceeds (b and c).

View larger version (79K): [in a new window]   Fig. 2. Electron microscopic analysis of lad1-1 cells. Electron micrographs of representative wild-type cells (KGY246) (B) and lad1-1 mutant (KGY2030) cells (A and C) after shifting to 36°C. The arrows in B indicate areas in which the primary septum was degraded. The arrows in C indicate regions lacking cell wall. Bar, 0.9 µm (A,C); 1.0 µm (B).

View larger version (103K): [in a new window]   Fig. 3. Identification and characterization of lad1-1 suppressors. (A) lad1-1 cells containing pUR19 (vector), pUR19 rho1 or pUR19gyp10 were isolated at 25°C and struck to YE plates at 36°C. Colonies were allowed to form for 3 days. (B) The pREP81-GFPgyp10 plasmid was transformed into wild-type cells (KGY246). After growth of transformants in the absence of thiamine for 16 hours, images of live cells were captured, and a composite of representative cells is shown. (C) Wild-type (KGY246), gyp10 (KGY2111), exo70 (MBY919) and gyp10 exo70 (KGY3945) cells were struck to plates for 3 days at the indicated temperatures. (D) gyp10 cells (KGY2011) were grown at 36°C for 6 hours, fixed, and stained with DAPI and Aniline Blue to visualize DNA and cell wall material, respectively. (E) Wild-type cells (KGY246) were transformed with the indicated plasmids and colonies recovered at 25°C. These were then grown in liquid medium at 36°C in the absence of thiamine for 18 hours. Cells were fixed with ethanol and stained with Aniline Blue and DAPI.

View larger version (77K): [in a new window]   Fig. 4. Localization of rgf1+ and rgf3+. (A) Schematic representation of Rgf1 and Rgf3 protein domain structures. Both proteins contain RhoGEF, pleckstrin homology (PH) and citron homology (CNH) domains. In addition, Rgf1 contains a DEP (dishevelled, Egl-10 and pleckstrin) domain. The asterisk denotes the F867S mutation found within rgf3 in the lad1-1 strain. (B-D) Localization of Rgf1p-GFP in live cells (B,C) and Rgf3p-GFP in fixed cells (D) (KGY4404 and KGY4407, respectively). (C) The ring of Rgf1p-GFP viewed from the side and en face. (E) Model of Rgf1p and Rgf3p localization during cell division.

View larger version (71K): [in a new window]   Fig. 5. Localization of Rgf3p but not Rgf1p or Rho1p is disrupted in lad1-1 cells. (A,B) rgf1-GFP lad1-1 cells (KGY1116) were grown at 25°C (A) and then shifted to 36°C for 4 hours (B). (C,D) rgf3-GFP lad1-1 (KGY1117) (C) and rgf3-GFP cells (KGY4407) (D) cells were grown at 25°C and then shifted to 36°C for 4 hours. Images of live cells are shown in each panel. (D) The same strains grown in parts A-D were grown at 25°C and shifted to 36°C for 0 or 2 hours and equal amounts of cell lysates were probed for Cdc2p levels using anti-PSTAIRE, or immunoprecipitated and immunoblotted with antibodies to GFP. (F) lad1-1 cells containing pREP1 (vector) or pREP1rgf3+ were isolated at 25°C and struck to YE plates at 36°C. Colonies were allowed to form for 3 days. (G) rgf3-GFP lad1-1 cells (KGY1117) were transformed with pREP1gyp10 and colonies were allowed to form at 25°C. Transformants were grown in liquid medium lacking thiamine for 18 hours and then shifted to 36°C for 3 hours. Live cell images were captured. The arrowhead indicates the Rgf3-GFP medial ring.

View larger version (51K): [in a new window]   Fig. 6. Rgf3 is independent of the SIN. (A) The rgf1::ura4+ strain (KGY 5385) was grown at 32°C in YE, fixed with ethanol, and stained with DAPI. (B) pARTGFP-Rho1 was transformed into lad1-1 cells. Transformants were grown at 25°C, filtered and resuspended in YE medium and shifted to 36°C for 4 hours. GFP-Rho1p localization was visualized in live cells. Arrowheads indicate medially localized GFP-Rho1p. (C) rgf3-GFP sid2-250 cells (KGY5326) were synchronized in G2 phase by lactose gradient centrifugation, filtered, resuspended in fresh medium at 36°C and followed throughout a cell cycle. The percentage of binucleate (anaphase) cells, septated cells and cells with Rgf3p-GFP rings were determined every 20 minutes.

View larger version (41K): [in a new window]   Fig. 7. Rgf3p localizes early in mitosis. (A) Rgf3p-Myc13 cells (KGY4401) were grown to mid-log phase at 32°C in YE medium, and synchronized in early G2 phase by centrifugal elutriation. Samples were collected every 15 minutes and analyzed for the abundance of Rgf3p-Myc13 by immunoblotting of denatured cell protein lysates. Blotting with anti-PSTAIRE to Cdc2p from the same immunoblot served as a loading control. The septation index and the percentage of binucleate (anaphase) cells were also measured at each time point. (B) The level of Rgf3p-Myc13 was determined in log-phase wild type cells (KGY246) (lane 1), rgf3-myc13 (KGY4401) (lane 2), rgf3-myc13 ace2::kanR (KGY5160) (lane 3) and rgf3-myc13 overproducing Ace2p from the nmt1 promoter (lane 4) was determined by immunoblotting of denatured protein lysates. The abundance of Cdc2p determined by immunoblotting the same protein gel with anti-PSTAIRE served as a loading control. (C) rgf3-GFP cells (KGY4407) synchronized in G2 phase by lactose gradient centrifugation and followed throughout a cell cycle. The percentage of binucleate (anaphase) cells, septated cells, and cells with Rgf3p-GFP rings were determined every 20 minutes. (D and E) rgf3-GFP nda3-km311 cells (KGY5400) were arrested by incubation at 19°C for 6 hours and released at time 0 by shift to 32°C. Samples were fixed in ethanol every 5 minutes to determine the percentage of cells with Rgf3p-GFP rings and a septum (D). (E) A representative image of these cells at the arrest point. (F) rgf1-GFP cells (KGY4407) were synchronized in G2 phase by lactose gradient centrifugation and followed throughout a cell cycle. The percentage of binucleate (anaphase) cells, septated cells and cells with Rgf1p-GFP rings were determined every 20 minutes.

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