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Role of polo kinase and Mid1p in determining the site of cell division in fission yeast.

Bähler J, Steever AB, Wheatley S, Wang Yl, Pringle JR, Gould KL, McCollum D - J. Cell Biol. (1998)

Bottom Line: Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells.Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation.Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA.

ABSTRACT
The fission yeast Schizosaccharomyces pombe divides symmetrically using a medial F-actin- based contractile ring to produce equal-sized daughter cells. Mutants defective in two previously described genes, mid1 and pom1, frequently divide asymmetrically. Here we present the identification of three new temperature-sensitive mutants defective in localization of the division plane. All three mutants have mutations in the polo kinase gene, plo1, and show defects very similar to those of mid1 mutants in both the placement and organization of the medial ring. In both cases, ring formation is frequently initiated near the cell poles, indicating that Mid1p and Plo1p function in recruiting medial ring components to the cell center. It has been reported previously that during mitosis Mid1p becomes hyperphosphorylated and relocates from the nucleus to a medial ring. Here we show that Mid1p first forms a diffuse cortical band during spindle formation and then coalesces into a ring before anaphase. Plo1p is required for Mid1p to exit the nucleus and form a ring, and Pom1p is required for proper placement of the Mid1p ring. Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells. Genetic and two-hybrid analyses suggest that Plo1p and Mid1p act in a common pathway distinct from that involving Pom1p. Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation. Taken together, the data indicate that Plo1p plays a role in the positioning of division sites by regulating Mid1p. Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.

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Medial ring and septum defects in plo1-1 mutant cells  and comparison of the plo1-1 and mid1-18 defects. Wild-type  strain JB13 (A and B), plo1-1 mutant strain YDM110 (C–E), and  mid1-18 strain YDM296 (F), all expressing GFP-Cdc4p fusion  protein, were grown at 25°C and shifted to 36°C for 2 h before examination. (A–D) Cells were fixed and stained with Calcofluor to  visualize septal material; GFP-Cdc4p fluorescence (A and C) and  Calcofluor fluorescence (B and D) of the same cells are shown.  (E and F) Living cells were mounted on a microscope slide and  overlaid with an agar slab, and GFP-Cdc4p was viewed by fluorescence microscopy at 36°C. Images were collected every 0.25  μm and processed by deconvolution methods to generate a two-dimensional projection of the three-dimensional image (see Materials and Methods).
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Figure 1: Medial ring and septum defects in plo1-1 mutant cells and comparison of the plo1-1 and mid1-18 defects. Wild-type strain JB13 (A and B), plo1-1 mutant strain YDM110 (C–E), and mid1-18 strain YDM296 (F), all expressing GFP-Cdc4p fusion protein, were grown at 25°C and shifted to 36°C for 2 h before examination. (A–D) Cells were fixed and stained with Calcofluor to visualize septal material; GFP-Cdc4p fluorescence (A and C) and Calcofluor fluorescence (B and D) of the same cells are shown. (E and F) Living cells were mounted on a microscope slide and overlaid with an agar slab, and GFP-Cdc4p was viewed by fluorescence microscopy at 36°C. Images were collected every 0.25 μm and processed by deconvolution methods to generate a two-dimensional projection of the three-dimensional image (see Materials and Methods).

Mentions: We identified three temperature-sensitive mutants that displayed defects in septum placement (see Materials and Methods). Complementation and linkage analysis (see Materials and Methods and data not shown) revealed that all three mutants had mutations in the same gene. Molecular cloning, mapping of an integrated plasmid, and rescue with a plo1+ cDNA plasmid showed that the three mutants harbored alleles (plo1-1, plo1-24C, and plo1-25) of the previously described plo1 gene (see Materials and Methods). At permissive temperature (25°C), mutant cells carrying any of the three alleles were wild-type in appearance and divided symmetrically (data not shown). However, at restrictive temperature (36°C), the mutants, although viable, exhibited a variety of defects in septum positioning and structure, with the plo1-1 mutant displaying the most severe defects. In contrast to the septa of wild-type cells (Fig. 1 B), the plo1 mutant septa were often misplaced from the cell center and/or not perpendicular to the long axis of the cell; in extreme cases, the mutant septa ran longitudinally through the cell (Fig. 1 D). Because the medial ring is thought to guide the placement of the septum, we examined the localization of the medial ring in plo1-1 mutant cells using a GFP-Cdc4p fusion protein. Cdc4p encodes a putative myosin light chain which localizes to the medial ring (McCollum et al., 1995; Balasubramanian et al., 1997). At restrictive temperature, the mutant cells displayed misplaced and disorganized medial rings (Fig. 1 C) whose positions corresponded approximately to those of the misplaced septa (Fig. 1 D). Similar results were obtained by staining cells with antibodies against either actin or Cdc4p (data not shown). These data suggest that the underlying plo1 mutant defect is in medial ring formation.


Role of polo kinase and Mid1p in determining the site of cell division in fission yeast.

Bähler J, Steever AB, Wheatley S, Wang Yl, Pringle JR, Gould KL, McCollum D - J. Cell Biol. (1998)

Medial ring and septum defects in plo1-1 mutant cells  and comparison of the plo1-1 and mid1-18 defects. Wild-type  strain JB13 (A and B), plo1-1 mutant strain YDM110 (C–E), and  mid1-18 strain YDM296 (F), all expressing GFP-Cdc4p fusion  protein, were grown at 25°C and shifted to 36°C for 2 h before examination. (A–D) Cells were fixed and stained with Calcofluor to  visualize septal material; GFP-Cdc4p fluorescence (A and C) and  Calcofluor fluorescence (B and D) of the same cells are shown.  (E and F) Living cells were mounted on a microscope slide and  overlaid with an agar slab, and GFP-Cdc4p was viewed by fluorescence microscopy at 36°C. Images were collected every 0.25  μm and processed by deconvolution methods to generate a two-dimensional projection of the three-dimensional image (see Materials and Methods).
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Related In: Results  -  Collection

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Figure 1: Medial ring and septum defects in plo1-1 mutant cells and comparison of the plo1-1 and mid1-18 defects. Wild-type strain JB13 (A and B), plo1-1 mutant strain YDM110 (C–E), and mid1-18 strain YDM296 (F), all expressing GFP-Cdc4p fusion protein, were grown at 25°C and shifted to 36°C for 2 h before examination. (A–D) Cells were fixed and stained with Calcofluor to visualize septal material; GFP-Cdc4p fluorescence (A and C) and Calcofluor fluorescence (B and D) of the same cells are shown. (E and F) Living cells were mounted on a microscope slide and overlaid with an agar slab, and GFP-Cdc4p was viewed by fluorescence microscopy at 36°C. Images were collected every 0.25 μm and processed by deconvolution methods to generate a two-dimensional projection of the three-dimensional image (see Materials and Methods).
Mentions: We identified three temperature-sensitive mutants that displayed defects in septum placement (see Materials and Methods). Complementation and linkage analysis (see Materials and Methods and data not shown) revealed that all three mutants had mutations in the same gene. Molecular cloning, mapping of an integrated plasmid, and rescue with a plo1+ cDNA plasmid showed that the three mutants harbored alleles (plo1-1, plo1-24C, and plo1-25) of the previously described plo1 gene (see Materials and Methods). At permissive temperature (25°C), mutant cells carrying any of the three alleles were wild-type in appearance and divided symmetrically (data not shown). However, at restrictive temperature (36°C), the mutants, although viable, exhibited a variety of defects in septum positioning and structure, with the plo1-1 mutant displaying the most severe defects. In contrast to the septa of wild-type cells (Fig. 1 B), the plo1 mutant septa were often misplaced from the cell center and/or not perpendicular to the long axis of the cell; in extreme cases, the mutant septa ran longitudinally through the cell (Fig. 1 D). Because the medial ring is thought to guide the placement of the septum, we examined the localization of the medial ring in plo1-1 mutant cells using a GFP-Cdc4p fusion protein. Cdc4p encodes a putative myosin light chain which localizes to the medial ring (McCollum et al., 1995; Balasubramanian et al., 1997). At restrictive temperature, the mutant cells displayed misplaced and disorganized medial rings (Fig. 1 C) whose positions corresponded approximately to those of the misplaced septa (Fig. 1 D). Similar results were obtained by staining cells with antibodies against either actin or Cdc4p (data not shown). These data suggest that the underlying plo1 mutant defect is in medial ring formation.

Bottom Line: Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells.Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation.Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA.

ABSTRACT
The fission yeast Schizosaccharomyces pombe divides symmetrically using a medial F-actin- based contractile ring to produce equal-sized daughter cells. Mutants defective in two previously described genes, mid1 and pom1, frequently divide asymmetrically. Here we present the identification of three new temperature-sensitive mutants defective in localization of the division plane. All three mutants have mutations in the polo kinase gene, plo1, and show defects very similar to those of mid1 mutants in both the placement and organization of the medial ring. In both cases, ring formation is frequently initiated near the cell poles, indicating that Mid1p and Plo1p function in recruiting medial ring components to the cell center. It has been reported previously that during mitosis Mid1p becomes hyperphosphorylated and relocates from the nucleus to a medial ring. Here we show that Mid1p first forms a diffuse cortical band during spindle formation and then coalesces into a ring before anaphase. Plo1p is required for Mid1p to exit the nucleus and form a ring, and Pom1p is required for proper placement of the Mid1p ring. Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells. Genetic and two-hybrid analyses suggest that Plo1p and Mid1p act in a common pathway distinct from that involving Pom1p. Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation. Taken together, the data indicate that Plo1p plays a role in the positioning of division sites by regulating Mid1p. Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.

Show MeSH
Related in: MedlinePlus