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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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Timing of Mid1p  ring formation and F-actin  ring formation in wild-type  cells. (A) Mid1p-GFP expressing cells (strain YDM403)  growing exponentially at  32°C were triple stained for  Mid1p (using GFP-specific  antibodies), spindles, and  DNA. Representative interphase, pre-metaphase, and  anaphase cells are shown.  Very similar results were  obtained when strain 972  (wild-type) cells were  stained with Mid1p-specific  antibodies or when Mid1p-13Myc-expressing cells  (strain YDM603) were  stained with antibodies to  the Myc epitope. (B) Strain  972 cells growing exponentially at 36°C were triple  stained for F-actin, spindles,  and DNA. The fixation protocols used visualize Mid1p,  Mid1p-fusion proteins, F-actin, and spindle microtubules well, but preserve cytoplasmic microtubules poorly. Large arrowheads,  pre-metaphase cells with short spindles; arrow, cell with an anaphase spindle.
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Figure 5: Timing of Mid1p ring formation and F-actin ring formation in wild-type cells. (A) Mid1p-GFP expressing cells (strain YDM403) growing exponentially at 32°C were triple stained for Mid1p (using GFP-specific antibodies), spindles, and DNA. Representative interphase, pre-metaphase, and anaphase cells are shown. Very similar results were obtained when strain 972 (wild-type) cells were stained with Mid1p-specific antibodies or when Mid1p-13Myc-expressing cells (strain YDM603) were stained with antibodies to the Myc epitope. (B) Strain 972 cells growing exponentially at 36°C were triple stained for F-actin, spindles, and DNA. The fixation protocols used visualize Mid1p, Mid1p-fusion proteins, F-actin, and spindle microtubules well, but preserve cytoplasmic microtubules poorly. Large arrowheads, pre-metaphase cells with short spindles; arrow, cell with an anaphase spindle.

Mentions: The formation of a medial ring during mitosis is the earliest known step of cytokinesis and appears to determine the site at which division will occur (see Introduction). However, the precise timing of the appearance of various medial ring components relative to each other and to the stages of mitosis has not been well characterized. To study this timing in more detail, we first stained cells simultaneously for Mid1p, mitotic spindles, and DNA. As reported previously (Sohrmann et al., 1996), Mid1p was nuclear in all cells without mitotic spindles (interphase cells; Fig. 5 A, Interphase) and was visible as a discrete medial ring in metaphase or anaphase cells with well separated spindle poles (Fig. 5 A, Anaphase). However, in 46 of 50 cells with short spindles (pre-metaphase cells), although some Mid1p typically remained in the nucleus, most Mid1p was found in a diffuse band at the cell cortex in the nuclear region (Fig. 5 A, Pre-metaphase). Thus, Mid1p leaves the nucleus at an early stage of mitosis but does not organize into a tight ring until the spindle is fully formed at metaphase or anaphase onset.


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)

Timing of Mid1p  ring formation and F-actin  ring formation in wild-type  cells. (A) Mid1p-GFP expressing cells (strain YDM403)  growing exponentially at  32°C were triple stained for  Mid1p (using GFP-specific  antibodies), spindles, and  DNA. Representative interphase, pre-metaphase, and  anaphase cells are shown.  Very similar results were  obtained when strain 972  (wild-type) cells were  stained with Mid1p-specific  antibodies or when Mid1p-13Myc-expressing cells  (strain YDM603) were  stained with antibodies to  the Myc epitope. (B) Strain  972 cells growing exponentially at 36°C were triple  stained for F-actin, spindles,  and DNA. The fixation protocols used visualize Mid1p,  Mid1p-fusion proteins, F-actin, and spindle microtubules well, but preserve cytoplasmic microtubules poorly. Large arrowheads,  pre-metaphase cells with short spindles; arrow, cell with an anaphase spindle.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Timing of Mid1p ring formation and F-actin ring formation in wild-type cells. (A) Mid1p-GFP expressing cells (strain YDM403) growing exponentially at 32°C were triple stained for Mid1p (using GFP-specific antibodies), spindles, and DNA. Representative interphase, pre-metaphase, and anaphase cells are shown. Very similar results were obtained when strain 972 (wild-type) cells were stained with Mid1p-specific antibodies or when Mid1p-13Myc-expressing cells (strain YDM603) were stained with antibodies to the Myc epitope. (B) Strain 972 cells growing exponentially at 36°C were triple stained for F-actin, spindles, and DNA. The fixation protocols used visualize Mid1p, Mid1p-fusion proteins, F-actin, and spindle microtubules well, but preserve cytoplasmic microtubules poorly. Large arrowheads, pre-metaphase cells with short spindles; arrow, cell with an anaphase spindle.
Mentions: The formation of a medial ring during mitosis is the earliest known step of cytokinesis and appears to determine the site at which division will occur (see Introduction). However, the precise timing of the appearance of various medial ring components relative to each other and to the stages of mitosis has not been well characterized. To study this timing in more detail, we first stained cells simultaneously for Mid1p, mitotic spindles, and DNA. As reported previously (Sohrmann et al., 1996), Mid1p was nuclear in all cells without mitotic spindles (interphase cells; Fig. 5 A, Interphase) and was visible as a discrete medial ring in metaphase or anaphase cells with well separated spindle poles (Fig. 5 A, Anaphase). However, in 46 of 50 cells with short spindles (pre-metaphase cells), although some Mid1p typically remained in the nucleus, most Mid1p was found in a diffuse band at the cell cortex in the nuclear region (Fig. 5 A, Pre-metaphase). Thus, Mid1p leaves the nucleus at an early stage of mitosis but does not organize into a tight ring until the spindle is fully formed at metaphase or anaphase onset.

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