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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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Phenotype of cdc12+ and cdc12::ura4 deletion cells. (A  and B). Wild-type cells stained with rhodamine-phalloidin, showing normal interphase (A) and mitotic (B) actin patterns. (C–L)  cdc12::ura4 cells stained with rhodamine-phallodin (C, E, G, and  I), DAPI (D, F, H, and J), or calcofluor (K and L). Diploid ura4/ ura4 leu1/leu1 ade6-M16/ade6-M22 cdc12+/cdc12::ura4 cells were  sporulated, and the spores were germinated in media containing  no uracil, so only cdc12::ura4 spores could germinate. Spores  were grown for 15 h at 30°C, fixed, and stained. (C–F) cdc12::ura4  cells in interphase, showing normal interphase polarized actin  distribution at the cell tips. Cell in D has two nuclei, and cell in F  has proceeded through another generation and contains four nuclei. (G–J) cdc12::ura4 mitotic cells with actin dots instead of an  actin ring. Cell in G is focused on the middle of the cell, while the  cell in I is focused on the cell surface. Nuclear patterns in H and I  are characteristic of cells in mitosis (see Fig. 6 A for comparison).  Calcofluor-stained cells show no septum cell wall deposition even  after multiple nuclear divisions. The rounded bulge that stains  with Calcofluor (K and L) in cdc12::ura4 cells represents the site  of the original spore wall. Bar, 10 μm.
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Figure 2: Phenotype of cdc12+ and cdc12::ura4 deletion cells. (A and B). Wild-type cells stained with rhodamine-phalloidin, showing normal interphase (A) and mitotic (B) actin patterns. (C–L) cdc12::ura4 cells stained with rhodamine-phallodin (C, E, G, and I), DAPI (D, F, H, and J), or calcofluor (K and L). Diploid ura4/ ura4 leu1/leu1 ade6-M16/ade6-M22 cdc12+/cdc12::ura4 cells were sporulated, and the spores were germinated in media containing no uracil, so only cdc12::ura4 spores could germinate. Spores were grown for 15 h at 30°C, fixed, and stained. (C–F) cdc12::ura4 cells in interphase, showing normal interphase polarized actin distribution at the cell tips. Cell in D has two nuclei, and cell in F has proceeded through another generation and contains four nuclei. (G–J) cdc12::ura4 mitotic cells with actin dots instead of an actin ring. Cell in G is focused on the middle of the cell, while the cell in I is focused on the cell surface. Nuclear patterns in H and I are characteristic of cells in mitosis (see Fig. 6 A for comparison). Calcofluor-stained cells show no septum cell wall deposition even after multiple nuclear divisions. The rounded bulge that stains with Calcofluor (K and L) in cdc12::ura4 cells represents the site of the original spore wall. Bar, 10 μm.

Mentions: Germinated cdc12::ura4 spores were examined for effects on actin and septum organization. Wild-type cells form cortical actin patches at the ends of cells during interphase and an actin ring during mitosis (Fig. 2, A and B; Marks and Hyams, 1985). cdc12::ura4 cells had normal interphase cortical actin patches (Fig. 2, C and E), but only delocalized actin patches instead of an actin ring during mitosis (Fig. 2, G and I). In some mitotic cells, actin dots were concentrated in the zones where the actin ring would have formed (Fig. 2 I). cdc12::ura4 cells became multinucleate (Fig. 2), indicating that nuclear division occurred in the absence of cytokinesis. This phenotype is identical to that seen in temperature-sensitive cdc12 alleles at restrictive temperature (Chang et al., 1996; Marks et al., 1987; Nurse et al., 1976). Thus, cdc12 is required for actin ring assembly during mitosis, but is not required for actin organization during interphase.


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)

Phenotype of cdc12+ and cdc12::ura4 deletion cells. (A  and B). Wild-type cells stained with rhodamine-phalloidin, showing normal interphase (A) and mitotic (B) actin patterns. (C–L)  cdc12::ura4 cells stained with rhodamine-phallodin (C, E, G, and  I), DAPI (D, F, H, and J), or calcofluor (K and L). Diploid ura4/ ura4 leu1/leu1 ade6-M16/ade6-M22 cdc12+/cdc12::ura4 cells were  sporulated, and the spores were germinated in media containing  no uracil, so only cdc12::ura4 spores could germinate. Spores  were grown for 15 h at 30°C, fixed, and stained. (C–F) cdc12::ura4  cells in interphase, showing normal interphase polarized actin  distribution at the cell tips. Cell in D has two nuclei, and cell in F  has proceeded through another generation and contains four nuclei. (G–J) cdc12::ura4 mitotic cells with actin dots instead of an  actin ring. Cell in G is focused on the middle of the cell, while the  cell in I is focused on the cell surface. Nuclear patterns in H and I  are characteristic of cells in mitosis (see Fig. 6 A for comparison).  Calcofluor-stained cells show no septum cell wall deposition even  after multiple nuclear divisions. The rounded bulge that stains  with Calcofluor (K and L) in cdc12::ura4 cells represents the site  of the original spore wall. Bar, 10 μm.
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Figure 2: Phenotype of cdc12+ and cdc12::ura4 deletion cells. (A and B). Wild-type cells stained with rhodamine-phalloidin, showing normal interphase (A) and mitotic (B) actin patterns. (C–L) cdc12::ura4 cells stained with rhodamine-phallodin (C, E, G, and I), DAPI (D, F, H, and J), or calcofluor (K and L). Diploid ura4/ ura4 leu1/leu1 ade6-M16/ade6-M22 cdc12+/cdc12::ura4 cells were sporulated, and the spores were germinated in media containing no uracil, so only cdc12::ura4 spores could germinate. Spores were grown for 15 h at 30°C, fixed, and stained. (C–F) cdc12::ura4 cells in interphase, showing normal interphase polarized actin distribution at the cell tips. Cell in D has two nuclei, and cell in F has proceeded through another generation and contains four nuclei. (G–J) cdc12::ura4 mitotic cells with actin dots instead of an actin ring. Cell in G is focused on the middle of the cell, while the cell in I is focused on the cell surface. Nuclear patterns in H and I are characteristic of cells in mitosis (see Fig. 6 A for comparison). Calcofluor-stained cells show no septum cell wall deposition even after multiple nuclear divisions. The rounded bulge that stains with Calcofluor (K and L) in cdc12::ura4 cells represents the site of the original spore wall. Bar, 10 μm.
Mentions: Germinated cdc12::ura4 spores were examined for effects on actin and septum organization. Wild-type cells form cortical actin patches at the ends of cells during interphase and an actin ring during mitosis (Fig. 2, A and B; Marks and Hyams, 1985). cdc12::ura4 cells had normal interphase cortical actin patches (Fig. 2, C and E), but only delocalized actin patches instead of an actin ring during mitosis (Fig. 2, G and I). In some mitotic cells, actin dots were concentrated in the zones where the actin ring would have formed (Fig. 2 I). cdc12::ura4 cells became multinucleate (Fig. 2), indicating that nuclear division occurred in the absence of cytokinesis. This phenotype is identical to that seen in temperature-sensitive cdc12 alleles at restrictive temperature (Chang et al., 1996; Marks et al., 1987; Nurse et al., 1976). Thus, cdc12 is required for actin ring assembly during mitosis, but is not required for actin organization during interphase.

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