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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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Localization of  Plo1p-GFP in wild-type,  cdc25-22, and cdc25-22 mid1-ΔF cells. (A) plo1-GFP strain  JB206 was grown at 30°C, and  cells were photographed at  various stages in the cell cycle  as described in the text. (B  and C) plo1-GFP cdc25-22  cells (strain YDM457; B) and  plo1-GFP cdc25-22 mid1-ΔF  cells (strain JB214; C) were  blocked at 36°C for 4 h and  then shifted to 25°C for 45  min. The arrowheads indicate  medial Plo1p rings.
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Figure 9: Localization of Plo1p-GFP in wild-type, cdc25-22, and cdc25-22 mid1-ΔF cells. (A) plo1-GFP strain JB206 was grown at 30°C, and cells were photographed at various stages in the cell cycle as described in the text. (B and C) plo1-GFP cdc25-22 cells (strain YDM457; B) and plo1-GFP cdc25-22 mid1-ΔF cells (strain JB214; C) were blocked at 36°C for 4 h and then shifted to 25°C for 45 min. The arrowheads indicate medial Plo1p rings.

Mentions: To determine the localization of Plo1p, a strain was constructed that expressed a Plo1p-GFP fusion protein from the normal chromosomal plo1 locus (see Materials and Methods). Examination of asynchronous cultures of living cells showed that shorter cells displayed no detectable signal (Fig 9 A, cell 1). However, in longer cells, which were presumably in late G2 or early mitosis, a faint nuclear staining was observed along with a brighter spot at the periphery of the nucleus that probably corresponded to the SPB (Fig. 9 A, cell 2), as judged by observations on cells with spindles. During spindle formation, there was a strong Plo1-GFP signal at both spindle poles and a faint signal along the length of the spindle (Fig. 9 A, cells 3–6). At the onset of anaphase, there was a strong diminution in the signal, and detectable signal was lost before cells divided. The loss of Plo1p-GFP signal during anaphase was also observed in living cells using real-time video microscopy and by examining cells synchronized by cdc25-22 block and release (data not shown). In some cells, a faint cortical ring could be observed in the middle of the cell (data not shown). This ring was only observed just before anaphase, as judged from spindle length, and was hard to document.


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)

Localization of  Plo1p-GFP in wild-type,  cdc25-22, and cdc25-22 mid1-ΔF cells. (A) plo1-GFP strain  JB206 was grown at 30°C, and  cells were photographed at  various stages in the cell cycle  as described in the text. (B  and C) plo1-GFP cdc25-22  cells (strain YDM457; B) and  plo1-GFP cdc25-22 mid1-ΔF  cells (strain JB214; C) were  blocked at 36°C for 4 h and  then shifted to 25°C for 45  min. The arrowheads indicate  medial Plo1p rings.
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Related In: Results  -  Collection

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

Figure 9: Localization of Plo1p-GFP in wild-type, cdc25-22, and cdc25-22 mid1-ΔF cells. (A) plo1-GFP strain JB206 was grown at 30°C, and cells were photographed at various stages in the cell cycle as described in the text. (B and C) plo1-GFP cdc25-22 cells (strain YDM457; B) and plo1-GFP cdc25-22 mid1-ΔF cells (strain JB214; C) were blocked at 36°C for 4 h and then shifted to 25°C for 45 min. The arrowheads indicate medial Plo1p rings.
Mentions: To determine the localization of Plo1p, a strain was constructed that expressed a Plo1p-GFP fusion protein from the normal chromosomal plo1 locus (see Materials and Methods). Examination of asynchronous cultures of living cells showed that shorter cells displayed no detectable signal (Fig 9 A, cell 1). However, in longer cells, which were presumably in late G2 or early mitosis, a faint nuclear staining was observed along with a brighter spot at the periphery of the nucleus that probably corresponded to the SPB (Fig. 9 A, cell 2), as judged by observations on cells with spindles. During spindle formation, there was a strong Plo1-GFP signal at both spindle poles and a faint signal along the length of the spindle (Fig. 9 A, cells 3–6). At the onset of anaphase, there was a strong diminution in the signal, and detectable signal was lost before cells divided. The loss of Plo1p-GFP signal during anaphase was also observed in living cells using real-time video microscopy and by examining cells synchronized by cdc25-22 block and release (data not shown). In some cells, a faint cortical ring could be observed in the middle of the cell (data not shown). This ring was only observed just before anaphase, as judged from spindle length, and was hard to document.

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