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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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Spindle formation defects in plo1-24C mutant cells.  Cells of strain YDM114 were grown at 25°C to exponential  phase, shifted to 36°C for 2 h, and triple stained for tubulin (A),  DNA (B), and Cdc4p (C). The upper two cells were blocked at  an early stage of spindle formation with highly condensed chromatin and no Cdc4p rings, whereas the lower three cells had  leaked past the block and formed spindles and aberrant medial  rings.
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Figure 2: Spindle formation defects in plo1-24C mutant cells. Cells of strain YDM114 were grown at 25°C to exponential phase, shifted to 36°C for 2 h, and triple stained for tubulin (A), DNA (B), and Cdc4p (C). The upper two cells were blocked at an early stage of spindle formation with highly condensed chromatin and no Cdc4p rings, whereas the lower three cells had leaked past the block and formed spindles and aberrant medial rings.

Mentions: Previous analysis has shown that plo1 mutants are defective in forming a bipolar spindle as well as in cytokinesis (Ohkura et al., 1995). To ask if the new plo1 mutations also cause defects in spindle formation, the mutant strains were stained with anti-tubulin and anti-Cdc4p antibodies after 2 h of growth at 36°C. Both plo1-24C and plo1-25 mutants showed spindle defects similar to those described previously for the mutant; in both cases, a majority of the cells that had entered mitosis (as judged by the breakdown of cytoplasmic microtubules) displayed highly condensed chromosomes and either a small dot of tubulin staining in the nucleus or what appeared to be a short monopolar spindle (Fig. 2, A and B, upper 2 cells). Similar defects were observed in a small percentage of plo1-1 mutant cells. The block in spindle formation appeared to be transient: after 2 h at 36°C, some cells had formed seemingly normal spindles and proceeded through anaphase (Fig. 2, A and B, lower 3 cells), and after 4 h at 36°C, almost all plo1-24C and plo1-25 mutant cells had completed nuclear division at least once and contained two to four nuclei (data not shown). Cells that were blocked in spindle formation did not form medial rings (Fig. 2 C, upper 2 cells), but aberrant medial ring structures were observed after spindle formation (Fig. 2 C, lower 3 cells).


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)

Spindle formation defects in plo1-24C mutant cells.  Cells of strain YDM114 were grown at 25°C to exponential  phase, shifted to 36°C for 2 h, and triple stained for tubulin (A),  DNA (B), and Cdc4p (C). The upper two cells were blocked at  an early stage of spindle formation with highly condensed chromatin and no Cdc4p rings, whereas the lower three cells had  leaked past the block and formed spindles and aberrant medial  rings.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132972&req=5

Figure 2: Spindle formation defects in plo1-24C mutant cells. Cells of strain YDM114 were grown at 25°C to exponential phase, shifted to 36°C for 2 h, and triple stained for tubulin (A), DNA (B), and Cdc4p (C). The upper two cells were blocked at an early stage of spindle formation with highly condensed chromatin and no Cdc4p rings, whereas the lower three cells had leaked past the block and formed spindles and aberrant medial rings.
Mentions: Previous analysis has shown that plo1 mutants are defective in forming a bipolar spindle as well as in cytokinesis (Ohkura et al., 1995). To ask if the new plo1 mutations also cause defects in spindle formation, the mutant strains were stained with anti-tubulin and anti-Cdc4p antibodies after 2 h of growth at 36°C. Both plo1-24C and plo1-25 mutants showed spindle defects similar to those described previously for the mutant; in both cases, a majority of the cells that had entered mitosis (as judged by the breakdown of cytoplasmic microtubules) displayed highly condensed chromosomes and either a small dot of tubulin staining in the nucleus or what appeared to be a short monopolar spindle (Fig. 2, A and B, upper 2 cells). Similar defects were observed in a small percentage of plo1-1 mutant cells. The block in spindle formation appeared to be transient: after 2 h at 36°C, some cells had formed seemingly normal spindles and proceeded through anaphase (Fig. 2, A and B, lower 3 cells), and after 4 h at 36°C, almost all plo1-24C and plo1-25 mutant cells had completed nuclear division at least once and contained two to four nuclei (data not shown). Cells that were blocked in spindle formation did not form medial rings (Fig. 2 C, upper 2 cells), but aberrant medial ring structures were observed after spindle formation (Fig. 2 C, lower 3 cells).

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