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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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Failure of Mid1p  nuclear exit and ring formation in the plo1-1 mutant. (A)  mid1p-GFP plo1-1 cells  (strain JB250) growing exponentially at 25°C were fixed  and triple stained for Mid1p,  spindles, and DNA as in Fig.  5. Very similar results were  obtained when plo1-1 cells  (strain YDM110) were stained  with Mid1p-specific antibodies or when mid1-13Myc  plo1-1 cells (strain YDM607)  were stained with Myc-specific antibodies. (B) plo1-1  cells expressing Mid1p-GFP  (strain JB250) or Mid1p-13Myc (strain YDM607) cells  growing exponentially at  25°C were shifted to 36°C for  2.5 h before fixation. The  fixed cells were triple stained  for Mid1p-GFP (top) or  Mid1p-13Myc (bottom) using GFP-specific or Myc-specific antibodies as appropriate, for spindles, and for DNA as in Fig. 5. Very similar results were obtained when plo1-1 cells (strain YDM110) were stained with Mid1p-specific antibodies. Note that the anti-tubulin antibodies used to stain the Mid1p-13Myc cells (see Materials and Methods) do not give the background nuclear staining like the TAT-1  anti-tubulin monoclonal antibody which is used above.
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Figure 6: Failure of Mid1p nuclear exit and ring formation in the plo1-1 mutant. (A) mid1p-GFP plo1-1 cells (strain JB250) growing exponentially at 25°C were fixed and triple stained for Mid1p, spindles, and DNA as in Fig. 5. Very similar results were obtained when plo1-1 cells (strain YDM110) were stained with Mid1p-specific antibodies or when mid1-13Myc plo1-1 cells (strain YDM607) were stained with Myc-specific antibodies. (B) plo1-1 cells expressing Mid1p-GFP (strain JB250) or Mid1p-13Myc (strain YDM607) cells growing exponentially at 25°C were shifted to 36°C for 2.5 h before fixation. The fixed cells were triple stained for Mid1p-GFP (top) or Mid1p-13Myc (bottom) using GFP-specific or Myc-specific antibodies as appropriate, for spindles, and for DNA as in Fig. 5. Very similar results were obtained when plo1-1 cells (strain YDM110) were stained with Mid1p-specific antibodies. Note that the anti-tubulin antibodies used to stain the Mid1p-13Myc cells (see Materials and Methods) do not give the background nuclear staining like the TAT-1 anti-tubulin monoclonal antibody which is used above.

Mentions: It was shown previously that the relocalization of Mid1p from the nucleus to the medial ring is correlated with an increase in Mid1p phosphorylation (Sohrmann et al., 1996). Since both plo1 and pom1 encode putative protein kinases that are involved in medial ring formation, we asked if these kinases were required for Mid1p localization to the medial ring. At permissive temperature, the localization of Mid1p in both interphase and mitotic cells of the plo1-1 mutant was indistinguishable from that in wild-type cells (Fig. 6 A, and data not shown). However, at restrictive temperature, Mid1p appeared to remain in the nucleus throughout mitosis and failed to form a discrete ring at the cell cortex (Fig. 6 B). In plo1-25 and plo1-24C strains, at least some Mid1p appeared to leave the nucleus during mitosis, but Mid1p rings were never observed (data not shown). Control experiments showed that Mid1p rings formed normally at 36°C in wild-type cells (data not shown).


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)

Failure of Mid1p  nuclear exit and ring formation in the plo1-1 mutant. (A)  mid1p-GFP plo1-1 cells  (strain JB250) growing exponentially at 25°C were fixed  and triple stained for Mid1p,  spindles, and DNA as in Fig.  5. Very similar results were  obtained when plo1-1 cells  (strain YDM110) were stained  with Mid1p-specific antibodies or when mid1-13Myc  plo1-1 cells (strain YDM607)  were stained with Myc-specific antibodies. (B) plo1-1  cells expressing Mid1p-GFP  (strain JB250) or Mid1p-13Myc (strain YDM607) cells  growing exponentially at  25°C were shifted to 36°C for  2.5 h before fixation. The  fixed cells were triple stained  for Mid1p-GFP (top) or  Mid1p-13Myc (bottom) using GFP-specific or Myc-specific antibodies as appropriate, for spindles, and for DNA as in Fig. 5. Very similar results were obtained when plo1-1 cells (strain YDM110) were stained with Mid1p-specific antibodies. Note that the anti-tubulin antibodies used to stain the Mid1p-13Myc cells (see Materials and Methods) do not give the background nuclear staining like the TAT-1  anti-tubulin monoclonal antibody which is used above.
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Figure 6: Failure of Mid1p nuclear exit and ring formation in the plo1-1 mutant. (A) mid1p-GFP plo1-1 cells (strain JB250) growing exponentially at 25°C were fixed and triple stained for Mid1p, spindles, and DNA as in Fig. 5. Very similar results were obtained when plo1-1 cells (strain YDM110) were stained with Mid1p-specific antibodies or when mid1-13Myc plo1-1 cells (strain YDM607) were stained with Myc-specific antibodies. (B) plo1-1 cells expressing Mid1p-GFP (strain JB250) or Mid1p-13Myc (strain YDM607) cells growing exponentially at 25°C were shifted to 36°C for 2.5 h before fixation. The fixed cells were triple stained for Mid1p-GFP (top) or Mid1p-13Myc (bottom) using GFP-specific or Myc-specific antibodies as appropriate, for spindles, and for DNA as in Fig. 5. Very similar results were obtained when plo1-1 cells (strain YDM110) were stained with Mid1p-specific antibodies. Note that the anti-tubulin antibodies used to stain the Mid1p-13Myc cells (see Materials and Methods) do not give the background nuclear staining like the TAT-1 anti-tubulin monoclonal antibody which is used above.
Mentions: It was shown previously that the relocalization of Mid1p from the nucleus to the medial ring is correlated with an increase in Mid1p phosphorylation (Sohrmann et al., 1996). Since both plo1 and pom1 encode putative protein kinases that are involved in medial ring formation, we asked if these kinases were required for Mid1p localization to the medial ring. At permissive temperature, the localization of Mid1p in both interphase and mitotic cells of the plo1-1 mutant was indistinguishable from that in wild-type cells (Fig. 6 A, and data not shown). However, at restrictive temperature, Mid1p appeared to remain in the nucleus throughout mitosis and failed to form a discrete ring at the cell cortex (Fig. 6 B). In plo1-25 and plo1-24C strains, at least some Mid1p appeared to leave the nucleus during mitosis, but Mid1p rings were never observed (data not shown). Control experiments showed that Mid1p rings formed normally at 36°C in wild-type cells (data not shown).

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