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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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Time-lapse analysis of medial  ring formation in mutant cells. Wild-type (JB13), pom1-Δ1 (JB110), plo1-1  (YDM110), and mid1-18 (YDM296)  cells, all expressing GFP-Cdc4p fusion  protein, were grown at 25°C, then  shifted to 36°C for 1 h in liquid culture.  The living cells were then mounted on  a microscope slide overlaid with an  agar slab and viewed by fluorescence  microscopy at 36°C. For wild-type,  plo1-1, and mid1-18 cells, images were  collected every 0.5 μm at 5-min intervals and processed by deconvolution  methods to generate two-dimensional  projections of the three-dimensional  images (see Materials and Methods).  Images of pom1-Δ1 cells were taken in  a single focal plane. Asterisks and the  arrowhead indicate structures described in the text.
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Figure 3: Time-lapse analysis of medial ring formation in mutant cells. Wild-type (JB13), pom1-Δ1 (JB110), plo1-1 (YDM110), and mid1-18 (YDM296) cells, all expressing GFP-Cdc4p fusion protein, were grown at 25°C, then shifted to 36°C for 1 h in liquid culture. The living cells were then mounted on a microscope slide overlaid with an agar slab and viewed by fluorescence microscopy at 36°C. For wild-type, plo1-1, and mid1-18 cells, images were collected every 0.5 μm at 5-min intervals and processed by deconvolution methods to generate two-dimensional projections of the three-dimensional images (see Materials and Methods). Images of pom1-Δ1 cells were taken in a single focal plane. Asterisks and the arrowhead indicate structures described in the text.

Mentions: From the observations described above, it was not clear how the medial ring defects in the three mutants arose. For example, it seemed possible either that the mutants initiated medial ring formation at the wrong place or that they initiated medial ring formation at the right place but were unable to anchor the ring properly, so that it could drift away. In addition, in the cases of plo1 and mid1, it seemed possible either that the aberrant Cdc4p-containing structures formed de novo or that they formed by the breakdown of normally structured rings that had formed initially. To explore these possibilities, we used time-lapse video microscopy of living cells expressing the GFP-Cdc4p fusion protein at restrictive temperature. The medial rings formed in the pom1 mutant were observed readily in a single focal plane. However, the aberrant structures formed in the plo1 and mid1 mutants were difficult to observe in a single focal plane, so we used compiled images derived from serial optical sections (see above and Materials and Methods). Images were collected at 5-min intervals (the shortest convenient interval for the serial sectioning). For each strain, time courses were done on at least six cells, and representative series are shown in Fig. 3. In the pom1 mutant, medial rings first appeared at incorrect locations and subsequently did not move; except for their locations, the rings appeared normal. In contrast, in the plo1 and mid1 mutants, the structure as well as the placement of the medial rings was clearly aberrant from the beginning. Most cells began by forming Cdc4p-containing cables that ran along the long axis of the cell. These structures often appeared first at the cell ends (Fig. 3, asterisks) and then grew and moved into different regions of the cell. Occasionally, some cells managed to assemble a relatively normal looking medial ring (Fig. 3, arrowhead). It seemed possible that the deconvolution processing methods could have caused some structures present in the original images to be lost or could even have caused the appearance of structures that were not present in the original images. However, examination of the individual sets of raw images showed that all structures found in the deconvoluted images were present in the raw data, and all structures found in the raw images were represented in the deconvoluted images (data not shown). It also seemed possible that a crucial early stage of medial ring formation had been missed in these experiments because of the 5-min interval between images. However, capturing images of plo1-1 cells in a single focal plane every 30 s gave very similar results: of 22 cells examined, 18 had Cdc4p-containing cables appear first at their tips (data not shown). In addition, examination of medial ring formation in asynchronous populations of the plo1-1 and mid1-18 mutants 1 h after shift to restrictive temperature also showed that a majority of these cells (66 of 99 for mid1-18 and 79 of 110 for plo1-1) formed Cdc4p-containing cables at their ends (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)

Time-lapse analysis of medial  ring formation in mutant cells. Wild-type (JB13), pom1-Δ1 (JB110), plo1-1  (YDM110), and mid1-18 (YDM296)  cells, all expressing GFP-Cdc4p fusion  protein, were grown at 25°C, then  shifted to 36°C for 1 h in liquid culture.  The living cells were then mounted on  a microscope slide overlaid with an  agar slab and viewed by fluorescence  microscopy at 36°C. For wild-type,  plo1-1, and mid1-18 cells, images were  collected every 0.5 μm at 5-min intervals and processed by deconvolution  methods to generate two-dimensional  projections of the three-dimensional  images (see Materials and Methods).  Images of pom1-Δ1 cells were taken in  a single focal plane. Asterisks and the  arrowhead indicate structures described in the text.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132972&req=5

Figure 3: Time-lapse analysis of medial ring formation in mutant cells. Wild-type (JB13), pom1-Δ1 (JB110), plo1-1 (YDM110), and mid1-18 (YDM296) cells, all expressing GFP-Cdc4p fusion protein, were grown at 25°C, then shifted to 36°C for 1 h in liquid culture. The living cells were then mounted on a microscope slide overlaid with an agar slab and viewed by fluorescence microscopy at 36°C. For wild-type, plo1-1, and mid1-18 cells, images were collected every 0.5 μm at 5-min intervals and processed by deconvolution methods to generate two-dimensional projections of the three-dimensional images (see Materials and Methods). Images of pom1-Δ1 cells were taken in a single focal plane. Asterisks and the arrowhead indicate structures described in the text.
Mentions: From the observations described above, it was not clear how the medial ring defects in the three mutants arose. For example, it seemed possible either that the mutants initiated medial ring formation at the wrong place or that they initiated medial ring formation at the right place but were unable to anchor the ring properly, so that it could drift away. In addition, in the cases of plo1 and mid1, it seemed possible either that the aberrant Cdc4p-containing structures formed de novo or that they formed by the breakdown of normally structured rings that had formed initially. To explore these possibilities, we used time-lapse video microscopy of living cells expressing the GFP-Cdc4p fusion protein at restrictive temperature. The medial rings formed in the pom1 mutant were observed readily in a single focal plane. However, the aberrant structures formed in the plo1 and mid1 mutants were difficult to observe in a single focal plane, so we used compiled images derived from serial optical sections (see above and Materials and Methods). Images were collected at 5-min intervals (the shortest convenient interval for the serial sectioning). For each strain, time courses were done on at least six cells, and representative series are shown in Fig. 3. In the pom1 mutant, medial rings first appeared at incorrect locations and subsequently did not move; except for their locations, the rings appeared normal. In contrast, in the plo1 and mid1 mutants, the structure as well as the placement of the medial rings was clearly aberrant from the beginning. Most cells began by forming Cdc4p-containing cables that ran along the long axis of the cell. These structures often appeared first at the cell ends (Fig. 3, asterisks) and then grew and moved into different regions of the cell. Occasionally, some cells managed to assemble a relatively normal looking medial ring (Fig. 3, arrowhead). It seemed possible that the deconvolution processing methods could have caused some structures present in the original images to be lost or could even have caused the appearance of structures that were not present in the original images. However, examination of the individual sets of raw images showed that all structures found in the deconvoluted images were present in the raw data, and all structures found in the raw images were represented in the deconvoluted images (data not shown). It also seemed possible that a crucial early stage of medial ring formation had been missed in these experiments because of the 5-min interval between images. However, capturing images of plo1-1 cells in a single focal plane every 30 s gave very similar results: of 22 cells examined, 18 had Cdc4p-containing cables appear first at their tips (data not shown). In addition, examination of medial ring formation in asynchronous populations of the plo1-1 and mid1-18 mutants 1 h after shift to restrictive temperature also showed that a majority of these cells (66 of 99 for mid1-18 and 79 of 110 for plo1-1) formed Cdc4p-containing cables at their ends (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