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cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin.

Chang F, Drubin D, Nurse P - J. Cell Biol. (1997)

Bottom Line: Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes.Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested.Finally, we found that cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

View Article: PubMed Central - PubMed

Affiliation: Imperial Cancer Research Fund, London, United Kingdom. fc99@columbia.edu

ABSTRACT
As in many other eukaryotic cells, cell division in fission yeast depends on the assembly of an actin ring that circumscribes the middle of the cell. Schizosaccharomyces pombe cdc12 is an essential gene necessary for actin ring assembly and septum formation. Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes. Using indirect immunofluorescence, we show that cdc12p is located in the cell division ring and not in other actin structures. When overexpressed, cdc12p is located at a medial spot in interphase that anticipates the future ring site. cdc12p localization is altered in actin ring mutants. cdc8 (tropomyosin homologue), cdc3 (profilin homologue), and cdc15 mutants exhibit no specific cdc12p staining during mitosis. cdc4 mutant cells exhibit a medial cortical cdc12p spot in place of a ring. mid1 mutant cells generally exhibit a cdc12p spot with a single cdc12p strand extending in a random direction. Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested. Finally, we found that cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

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Localization of  cdc12p in different actin ring  mutants during mitosis. (A)  cdc12-299 (FC127); (B) cdc3313 (FC114); (C) cdc8-346  (FC131); (D) cdc15-287  (FC55); (E and F) cdc4-377  (FC361). Mutant cells were  grown in YE5S at permissive  temperature (25°C), and then  shifted to restrictive temperature (35.5°C) for 4 h. Cells  were fixed and processed for  immunofluorescence with  anti-cdc12 antibody, anti-tubulin antibody or anti-actin antibody, and DAPI. On the basis of spindle and nuclear  morphology, cells pictured  are in anaphase, the cell cycle  period of maximum cdc12p  ring staining in wild-type cells.  (A–C) Cells are in mitosis in  which two nuclei are dividing  into four and thus exhibit  double spindles; (E–F) cells  exhibit single spindles. (A–C)  Mutants exhibit no specific  cdc12p staining; the cytoplasmic staining is nonspecific background staining. (E and F) Note the medial spot of cdc12p (arrow) in the  cdc4 mutant, which localizes to the origin of an actin aster (F, middle panel, labeled a). Bar, 10 μm.
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Figure 6: Localization of cdc12p in different actin ring mutants during mitosis. (A) cdc12-299 (FC127); (B) cdc3313 (FC114); (C) cdc8-346 (FC131); (D) cdc15-287 (FC55); (E and F) cdc4-377 (FC361). Mutant cells were grown in YE5S at permissive temperature (25°C), and then shifted to restrictive temperature (35.5°C) for 4 h. Cells were fixed and processed for immunofluorescence with anti-cdc12 antibody, anti-tubulin antibody or anti-actin antibody, and DAPI. On the basis of spindle and nuclear morphology, cells pictured are in anaphase, the cell cycle period of maximum cdc12p ring staining in wild-type cells. (A–C) Cells are in mitosis in which two nuclei are dividing into four and thus exhibit double spindles; (E–F) cells exhibit single spindles. (A–C) Mutants exhibit no specific cdc12p staining; the cytoplasmic staining is nonspecific background staining. (E and F) Note the medial spot of cdc12p (arrow) in the cdc4 mutant, which localizes to the origin of an actin aster (F, middle panel, labeled a). Bar, 10 μm.

Mentions: Next, we examined expression and localization of cdc12p in the different actin ring mutants. First, immunoblot analysis showed that expression of cdc12p was not altered in cdc3, cdc4, cdc8, cdc15, or mid1 mutants (data not shown). Second, we examined cdc12p localization in different mutant backgrounds. cdc3-313, cdc8-346, and cdc15-287 mutants generally showed no specific cdc12p staining during mitosis, much like a cdc12-299 mutant (Fig. 6, A–D, left panels). In addition, cdc15 mutant cells that overexpressed cdc12p did not exhibit any cdc12p spot or ring staining in either interphase or mitotic cells (data not shown). Thus, cdc3 (profilin homologue), cdc8 (tropomyosin homologue), and cdc15 genes are necessary for cdc12p localization at the ring, and at least cdc15 is also required for cdc12p spot formation.


cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin.

Chang F, Drubin D, Nurse P - J. Cell Biol. (1997)

Localization of  cdc12p in different actin ring  mutants during mitosis. (A)  cdc12-299 (FC127); (B) cdc3313 (FC114); (C) cdc8-346  (FC131); (D) cdc15-287  (FC55); (E and F) cdc4-377  (FC361). Mutant cells were  grown in YE5S at permissive  temperature (25°C), and then  shifted to restrictive temperature (35.5°C) for 4 h. Cells  were fixed and processed for  immunofluorescence with  anti-cdc12 antibody, anti-tubulin antibody or anti-actin antibody, and DAPI. On the basis of spindle and nuclear  morphology, cells pictured  are in anaphase, the cell cycle  period of maximum cdc12p  ring staining in wild-type cells.  (A–C) Cells are in mitosis in  which two nuclei are dividing  into four and thus exhibit  double spindles; (E–F) cells  exhibit single spindles. (A–C)  Mutants exhibit no specific  cdc12p staining; the cytoplasmic staining is nonspecific background staining. (E and F) Note the medial spot of cdc12p (arrow) in the  cdc4 mutant, which localizes to the origin of an actin aster (F, middle panel, labeled a). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 6: Localization of cdc12p in different actin ring mutants during mitosis. (A) cdc12-299 (FC127); (B) cdc3313 (FC114); (C) cdc8-346 (FC131); (D) cdc15-287 (FC55); (E and F) cdc4-377 (FC361). Mutant cells were grown in YE5S at permissive temperature (25°C), and then shifted to restrictive temperature (35.5°C) for 4 h. Cells were fixed and processed for immunofluorescence with anti-cdc12 antibody, anti-tubulin antibody or anti-actin antibody, and DAPI. On the basis of spindle and nuclear morphology, cells pictured are in anaphase, the cell cycle period of maximum cdc12p ring staining in wild-type cells. (A–C) Cells are in mitosis in which two nuclei are dividing into four and thus exhibit double spindles; (E–F) cells exhibit single spindles. (A–C) Mutants exhibit no specific cdc12p staining; the cytoplasmic staining is nonspecific background staining. (E and F) Note the medial spot of cdc12p (arrow) in the cdc4 mutant, which localizes to the origin of an actin aster (F, middle panel, labeled a). Bar, 10 μm.
Mentions: Next, we examined expression and localization of cdc12p in the different actin ring mutants. First, immunoblot analysis showed that expression of cdc12p was not altered in cdc3, cdc4, cdc8, cdc15, or mid1 mutants (data not shown). Second, we examined cdc12p localization in different mutant backgrounds. cdc3-313, cdc8-346, and cdc15-287 mutants generally showed no specific cdc12p staining during mitosis, much like a cdc12-299 mutant (Fig. 6, A–D, left panels). In addition, cdc15 mutant cells that overexpressed cdc12p did not exhibit any cdc12p spot or ring staining in either interphase or mitotic cells (data not shown). Thus, cdc3 (profilin homologue), cdc8 (tropomyosin homologue), and cdc15 genes are necessary for cdc12p localization at the ring, and at least cdc15 is also required for cdc12p spot formation.

Bottom Line: Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes.Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested.Finally, we found that cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

View Article: PubMed Central - PubMed

Affiliation: Imperial Cancer Research Fund, London, United Kingdom. fc99@columbia.edu

ABSTRACT
As in many other eukaryotic cells, cell division in fission yeast depends on the assembly of an actin ring that circumscribes the middle of the cell. Schizosaccharomyces pombe cdc12 is an essential gene necessary for actin ring assembly and septum formation. Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes. Using indirect immunofluorescence, we show that cdc12p is located in the cell division ring and not in other actin structures. When overexpressed, cdc12p is located at a medial spot in interphase that anticipates the future ring site. cdc12p localization is altered in actin ring mutants. cdc8 (tropomyosin homologue), cdc3 (profilin homologue), and cdc15 mutants exhibit no specific cdc12p staining during mitosis. cdc4 mutant cells exhibit a medial cortical cdc12p spot in place of a ring. mid1 mutant cells generally exhibit a cdc12p spot with a single cdc12p strand extending in a random direction. Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested. Finally, we found that cdc12 and cdc3 mutants show a synthetic-lethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.

Show MeSH
Related in: MedlinePlus