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First published online 13 June 2006
doi: 10.1242/jcs.02998

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The fission yeast Chs2 protein interacts with the type-II myosin Myo3p and is required for the integrity of the actomyosin ring

Departamento de Microbiología y Genética/Instituto de Microbiología Bioquímica, Universidad de Salamanca/CSIC, Edificio Departamental, Laboratorio 231, Campus Miguel de Unamuno, 37007 Salamanca, Spain

View larger version (67K): [in a new window]   Fig. 1. chs2+ overexpression affects the distribution of some CAR proteins. (A) WT and chs2+-overexpressing cells carrying the Cdc15-GFP protein showing cell wall and nuclear staining (left panels; Cf: Calcofluor) or GFP fluorescence (right panels). The asterisk marks the asymmetric distribution of the Cdc15-GFP ring in a cell. The arrow points to a cell in which the asymmetric Cdc15-GFP ring displays abnormal strong fluorescence. (B) The same analysis as in A was performed in cells carrying Cdc4-GFP. The asterisk indicates a WT cell to show that Cdc4p cannot be observed after the septum has been completed. The arrowhead points to a cell with two Cdc4-GFP rings close together. The diamond marks a cell with a double septum in which the Cdc4-GFP ring did not disassemble after septation. (C) Cells carrying Cdc4-GFP were imaged by confocal microscopy. The same cells are shown from different views. The arrow points to the midzone of a cell in which there are two Cdc4-GFP rings, slightly displaced, that are at different stages of contraction. The asterisks indicates a cell with two rings apparently tilted with respect to each other. Bar, 10µm.

View larger version (62K): [in a new window]   Fig. 2. Localisation of Chs2p depends on the general system for polarised growth, CAR assembly, and a functional SIN pathway. (A) cdc25-22 cells carrying the Chs2-GFP fusion protein were arrested at 37°C and then released at 25°C in the presence of DMSO (upper panels) or 100 µM latrunculin A (lower panels); the cells were imaged after 75 minutes at 25°C. The micrographs show DAPI and Calcofluor (Cf) staining (left panels), Chs2-GFP (central panels) or Rhodamine-phalloidin staining (right panels; F-actin). (B) An asynchronous culture was treated with DMSO (upper panels) or with 100 µM latrunculin A (lower panels). The cells shown were imaged after 20 minutes; cell wall and nuclei (left panels); Chs2-GFP (central panels); F-actin (right panels). (C-H) Analysis of nuclei and cell walls (DAPI/Cf; left panels) and the distribution of the Chs2-GFP (right panels) in the indicated strains incubated at the corresponding restrictive temperatures or at the temperature indicated for specific cases. In all cases the asterisks indicate the midzone of cells in which a new septum is being synthesized but in which the Chs2-GFP ring cannot be observed. (C) WT strain incubated at 37°C. (D) Myosin V mutant myo52. The arrowhead points to the midzone of a septating cell in which the Chs2-GFP signal is observed but shows an aberrant thread-like distribution. (E) Exocyst mutant exo70. The arrowhead points to a cell (incubated at 37°C) in which there is a weak Chs2-GFP signal in a forming ring. The diamond marks Chs2-GFP abnormal maintenance at the medial region of a cell in which the septum has been completed. (F) Analysis of mutants defective in type-II myosins: cdc4-8, rlc1, myo3 or myo2-E1 myo3. The arrowhead in the right myo3 panel indicates a spot of Chs2-GFP intracellular localisation. (G) cdc15-140 mutant, defective in CAR assembly. (H) SIN mutants cdc11-119 or cdc16-116. Bar, 10 µm.

View larger version (63K): [in a new window]   Fig. 3. chs2 mutants interact genetically with mutants affected in CAR assembly and/or function. (A) Upper panels: yeast extract with supplements (YES) plates with the indicated strains were incubated at 32°C for 2 days. Lower panels: imp2 or imp2 chs2 strains were stained with DAPI and Calcofluor (Cf) or with Rhodamine-phalloidin to visualize nuclei, cell walls and F-actin (indicated with asterisks). (B) Analysis of the genetic interaction between chs2 and the myosin-II heavy-chain mutants. A YES plate with the indicated strains was incubated at 32°C (upper left panel). The lower left panels show plates of Edinburgh minimal medium (EMM) and EMM supplemented with 1 M KCl on which serial dilutions of the indicated strains were spotted that were incubated at 25°C. The right panels show Calcofluor staining of cells from the indicated strains grown in liquid YES medium at 28°C. Bar, 10 µm.

View larger version (56K): [in a new window]   Fig. 4. Analysis of the Rlc1-GFP rings in different strains. (A) Left panels: WT, myo3, and myo3 chs2 cells were stained with Calcofluor and photographed under a conventional microscope. The position of Rlc1-GFP or Rlc1-GFP and the septum is shown. The arrows point to secondary rings that are slightly displaced from the septum. Right panels: different confocal images of a three-dimensional reconstruction of the Rlc1-GFP rings in WT, chs2, myo3 and myo3 chs2 strains. The arrows point to deformed rings. (B-D) Time-lapse analysis of CAR contraction in different strains. Cells from WT (B; bar 5 µm), myo3 (C), and myo3 chs2 (D) strains were photographed at the indicated times (in minutes). The arrows and diamond indicate cells in which Rlc1-GFP can be visualized from the first stages of ring assembly. The asterisk and the spots mark cells in which the ring was already assembled. Bar, 10 µm. (E) A graphical representation of the average time for ring assembly or ring contraction and disassembly in different cells from the indicated strains.

View larger version (31K): [in a new window]   Fig. 5. Chs2p interacts physically with Myo3p and is required for its proper distribution at the ring. Cell extracts from strains carrying the Chs2-HA and/or Myo3-GFP (A and B) and/or the Myo2-GFP (C) fusion proteins were analysed by western blot using the anti-HA (-HA) or anti-GFP (-GFP) antibodies before (Extracts) or after (IP) immunoprecipitation with the indicated antibody. (D) Distribution of Myo3-GFP in WT or chs2 cells; the left panels show the transmitted images of the cells and the central panels show a 2D average projection of the series (overlaid to the transmitted images). The graphs show the intensity of relative fluorescence (arbitrary units) plotted against the distance along a line drawn through the cell midzone. Asterisks mark the dots with the strongest relative fluorescence values at both sides of the ring. Myo3-GFP rings are shown in the insets as a 3D reconstruction of the maximum projection of the same z-series.

View larger version (35K): [in a new window]   Fig. 6. Chs2p is required for proper CAR contraction and stability. (A,B) Cdc15-GFP was used to visualize the CAR in either a WT or chs2 cells using conventional (A) or confocal (B) microscopy. (A) The Hht2-GFP histone was used to compare the progression of the nuclear cycle and the images shown were taken every 7 minutes after anaphase completion. (B) Time-lapse images of WT and chs2 cells carrying Cdc15-GFP, taken every 3 minutes. Lateral and frontal views of the three-dimensional reconstructions are shown. Numbers indicate the order of the time-point series taken after the initiation of CAR contraction. Arrowheads point to abnormal structures detected in the Cdc15p ring in the chs2 strain. (C) cdc25-22 or cdc25-22 chs2 cultures were arrested at 37°C and released at 25°C. Samples were collected at the indicated times, stained with DAPI and Calcofluor, and scored for the presence of nuclei and complete septa.