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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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Mid1p levels in plo1 and pom1 mutants, and the effect  of Plo1p overproduction on Mid1p. (A) Strains expressing either  normal Mid1p (no Myc) (YDM105) or Mid1p-13Myc (wt,  YDM603; pom1, YDM609; plo1-1, YDM607; plo1-25, YDM604;  and plo1-24C, YDM608) were grown at 25°C, shifted to 36°C for  2.5 h, and harvested. Protein lysates were prepared from each  strain and equal amounts of protein were analyzed by Western  blotting using anti-Myc antibodies. (B-D) Mid1p-13Myc-expressing cells (YDM603) containing a plasmid (pREP1Plo1) for expression of plo1 from the thiamine-repressible nmt1 promoter,  were grown at 30°C in the presence of thiamine. This culture was  then used to inoculate two new cultures either with (promoter  off) or without (promoter on) thiamine, and growth was continued for 16 h. Portions of each culture were then either fixed and  stained for Mid1p-13Myc using Myc-specific antibodies or used  to prepare lysates for analysis by western blotting. (B and C)  Mid1p-13Myc staining in cells either not overexpressing (B) or  overexpressing (C) Plo1p. (D) Western blot analysis of Mid1p-13Myc in cells either not overexpressing (+T) or overexpressing  (−T) Plo1p.
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Figure 8: Mid1p levels in plo1 and pom1 mutants, and the effect of Plo1p overproduction on Mid1p. (A) Strains expressing either normal Mid1p (no Myc) (YDM105) or Mid1p-13Myc (wt, YDM603; pom1, YDM609; plo1-1, YDM607; plo1-25, YDM604; and plo1-24C, YDM608) were grown at 25°C, shifted to 36°C for 2.5 h, and harvested. Protein lysates were prepared from each strain and equal amounts of protein were analyzed by Western blotting using anti-Myc antibodies. (B-D) Mid1p-13Myc-expressing cells (YDM603) containing a plasmid (pREP1Plo1) for expression of plo1 from the thiamine-repressible nmt1 promoter, were grown at 30°C in the presence of thiamine. This culture was then used to inoculate two new cultures either with (promoter off) or without (promoter on) thiamine, and growth was continued for 16 h. Portions of each culture were then either fixed and stained for Mid1p-13Myc using Myc-specific antibodies or used to prepare lysates for analysis by western blotting. (B and C) Mid1p-13Myc staining in cells either not overexpressing (B) or overexpressing (C) Plo1p. (D) Western blot analysis of Mid1p-13Myc in cells either not overexpressing (+T) or overexpressing (−T) Plo1p.

Mentions: The behavior of Mid1p differed in a pom1 deletion strain. In this case, interphase cells displayed nuclear Mid1p staining that was reproducibly weaker and more diffuse than that in wild-type cells (Fig. 7, A and B). During mitosis, the pom1 mutant cells did form Mid1p rings (Fig. 7, A and B), but these rings were frequently misplaced (Fig. 7, A and C, arrows) like the medial rings and septa seen in pom1 mutants (Bähler and Pringle, 1998; see also above). Double staining for Mid1p and actin showed that the mislocalized Mid1p and actin rings coincided, as expected (Fig. 7, C and D, arrows). The weaker nuclear staining of interphase cells might have resulted from a reduction in Mid1p levels in the pom1 mutant strain. However, when we determined Mid1p levels in wild-type, plo1, and pom1 mutant backgrounds using strains that expressed Mid1p-13Myc from the normal chromosomal mid1 locus (see Materials and Methods), all strains displayed similar levels of protein after incubation at 36°C (Fig. 8 A). Thus, Pom1p appears to be necessary both for normal nuclear localization of Mid1p in interphase cells and for normal placement of the Mid1p ring in mitotic cells, but not for the actual formation of the Mid1p ring.


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)

Mid1p levels in plo1 and pom1 mutants, and the effect  of Plo1p overproduction on Mid1p. (A) Strains expressing either  normal Mid1p (no Myc) (YDM105) or Mid1p-13Myc (wt,  YDM603; pom1, YDM609; plo1-1, YDM607; plo1-25, YDM604;  and plo1-24C, YDM608) were grown at 25°C, shifted to 36°C for  2.5 h, and harvested. Protein lysates were prepared from each  strain and equal amounts of protein were analyzed by Western  blotting using anti-Myc antibodies. (B-D) Mid1p-13Myc-expressing cells (YDM603) containing a plasmid (pREP1Plo1) for expression of plo1 from the thiamine-repressible nmt1 promoter,  were grown at 30°C in the presence of thiamine. This culture was  then used to inoculate two new cultures either with (promoter  off) or without (promoter on) thiamine, and growth was continued for 16 h. Portions of each culture were then either fixed and  stained for Mid1p-13Myc using Myc-specific antibodies or used  to prepare lysates for analysis by western blotting. (B and C)  Mid1p-13Myc staining in cells either not overexpressing (B) or  overexpressing (C) Plo1p. (D) Western blot analysis of Mid1p-13Myc in cells either not overexpressing (+T) or overexpressing  (−T) Plo1p.
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Figure 8: Mid1p levels in plo1 and pom1 mutants, and the effect of Plo1p overproduction on Mid1p. (A) Strains expressing either normal Mid1p (no Myc) (YDM105) or Mid1p-13Myc (wt, YDM603; pom1, YDM609; plo1-1, YDM607; plo1-25, YDM604; and plo1-24C, YDM608) were grown at 25°C, shifted to 36°C for 2.5 h, and harvested. Protein lysates were prepared from each strain and equal amounts of protein were analyzed by Western blotting using anti-Myc antibodies. (B-D) Mid1p-13Myc-expressing cells (YDM603) containing a plasmid (pREP1Plo1) for expression of plo1 from the thiamine-repressible nmt1 promoter, were grown at 30°C in the presence of thiamine. This culture was then used to inoculate two new cultures either with (promoter off) or without (promoter on) thiamine, and growth was continued for 16 h. Portions of each culture were then either fixed and stained for Mid1p-13Myc using Myc-specific antibodies or used to prepare lysates for analysis by western blotting. (B and C) Mid1p-13Myc staining in cells either not overexpressing (B) or overexpressing (C) Plo1p. (D) Western blot analysis of Mid1p-13Myc in cells either not overexpressing (+T) or overexpressing (−T) Plo1p.
Mentions: The behavior of Mid1p differed in a pom1 deletion strain. In this case, interphase cells displayed nuclear Mid1p staining that was reproducibly weaker and more diffuse than that in wild-type cells (Fig. 7, A and B). During mitosis, the pom1 mutant cells did form Mid1p rings (Fig. 7, A and B), but these rings were frequently misplaced (Fig. 7, A and C, arrows) like the medial rings and septa seen in pom1 mutants (Bähler and Pringle, 1998; see also above). Double staining for Mid1p and actin showed that the mislocalized Mid1p and actin rings coincided, as expected (Fig. 7, C and D, arrows). The weaker nuclear staining of interphase cells might have resulted from a reduction in Mid1p levels in the pom1 mutant strain. However, when we determined Mid1p levels in wild-type, plo1, and pom1 mutant backgrounds using strains that expressed Mid1p-13Myc from the normal chromosomal mid1 locus (see Materials and Methods), all strains displayed similar levels of protein after incubation at 36°C (Fig. 8 A). Thus, Pom1p appears to be necessary both for normal nuclear localization of Mid1p in interphase cells and for normal placement of the Mid1p ring in mitotic cells, but not for the actual formation of the Mid1p ring.

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