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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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cdc12p localizes to the cell division ring in wild-type cells. (A) Specificity of the anti-cdc12 affinity-purified antibody. 5 μg of  crude whole yeast cell extracts were analyzed by Western blotting using affinity-purified anti-cdc12 antibody. (Lane 1) Extract from  wild-type cells. (Lane 2) Extract from cells overexpressing cdc12p. Cells transformed with the plasmid pnmt-cdc12 (FC291) were shifted  to derepressing conditions (minimal media without thiamine) for 20 h at 30°C. Molecular weight markers (kD) are indicated. Serial dilutions showed that the pnmt-cdc12 extract contains 10–20-fold more cdc12p than the wild-type extracts (data not shown). (B–K) cdc12p localization in wild-type cells. Wild-type cells were grown in YE5S at 30°C in exponential phase, fixed, and processed for immunofluorescence using anti-cdc12 antibody and DAPI (see Materials and Methods for details). (B, D, F, H, and J) Anti-cdc12 staining. (C, E, G, I,  and K) DAPI staining. (B and C) A typical field of cells containing cells from different phases of the cell cycle. Note examples of ring  staining. (e) Cell in early mitosis in which the ring is beginning to form in an asymmetrical fashion. (i) Interphase with no specific cdc12  staining. (B, inset) Cross-section of a cdc12p ring. (D and E) Cell in early mitosis; (F and G) cell in early anaphase; (H and I) cell in midanaphase; (J and K) cell in postanaphase during ring closure. Patchy cytoplasmic staining is also seen in cells lacking cdc12p, and thus  probably represents background staining (data not shown). Bar, 10 μm.
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Figure 3: cdc12p localizes to the cell division ring in wild-type cells. (A) Specificity of the anti-cdc12 affinity-purified antibody. 5 μg of crude whole yeast cell extracts were analyzed by Western blotting using affinity-purified anti-cdc12 antibody. (Lane 1) Extract from wild-type cells. (Lane 2) Extract from cells overexpressing cdc12p. Cells transformed with the plasmid pnmt-cdc12 (FC291) were shifted to derepressing conditions (minimal media without thiamine) for 20 h at 30°C. Molecular weight markers (kD) are indicated. Serial dilutions showed that the pnmt-cdc12 extract contains 10–20-fold more cdc12p than the wild-type extracts (data not shown). (B–K) cdc12p localization in wild-type cells. Wild-type cells were grown in YE5S at 30°C in exponential phase, fixed, and processed for immunofluorescence using anti-cdc12 antibody and DAPI (see Materials and Methods for details). (B, D, F, H, and J) Anti-cdc12 staining. (C, E, G, I, and K) DAPI staining. (B and C) A typical field of cells containing cells from different phases of the cell cycle. Note examples of ring staining. (e) Cell in early mitosis in which the ring is beginning to form in an asymmetrical fashion. (i) Interphase with no specific cdc12 staining. (B, inset) Cross-section of a cdc12p ring. (D and E) Cell in early mitosis; (F and G) cell in early anaphase; (H and I) cell in midanaphase; (J and K) cell in postanaphase during ring closure. Patchy cytoplasmic staining is also seen in cells lacking cdc12p, and thus probably represents background staining (data not shown). Bar, 10 μm.

Mentions: To localize the cdc12 gene product, we raised polyclonal rabbit antibodies against a cdc12 FH1 domain protein fragment expressed in E. coli. The affinity-purified antibody recognized on an immunoblot a single band of ∼220 kD (Fig. 3 A), which is consistent with the size predicted from the nucleotide sequence. This 220-kD protein was confirmed to be cdc12p by showing that the level of this protein is increased ∼10–20-fold in cells containing a plasmid that overexpresses cdc12p from the thiamine-regulated nmt promoter (Fig. 3 A). Measurements of cdc12p levels both from synchronized cells and from cells arrested in G2 (cdc25 block) and G1 phases (cdc10 block) showed that cdc12p is expressed throughout the cell cycle with no large fluctuations in protein levels or mobility shifts in different phases of the cell cycle (data not shown).


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)

cdc12p localizes to the cell division ring in wild-type cells. (A) Specificity of the anti-cdc12 affinity-purified antibody. 5 μg of  crude whole yeast cell extracts were analyzed by Western blotting using affinity-purified anti-cdc12 antibody. (Lane 1) Extract from  wild-type cells. (Lane 2) Extract from cells overexpressing cdc12p. Cells transformed with the plasmid pnmt-cdc12 (FC291) were shifted  to derepressing conditions (minimal media without thiamine) for 20 h at 30°C. Molecular weight markers (kD) are indicated. Serial dilutions showed that the pnmt-cdc12 extract contains 10–20-fold more cdc12p than the wild-type extracts (data not shown). (B–K) cdc12p localization in wild-type cells. Wild-type cells were grown in YE5S at 30°C in exponential phase, fixed, and processed for immunofluorescence using anti-cdc12 antibody and DAPI (see Materials and Methods for details). (B, D, F, H, and J) Anti-cdc12 staining. (C, E, G, I,  and K) DAPI staining. (B and C) A typical field of cells containing cells from different phases of the cell cycle. Note examples of ring  staining. (e) Cell in early mitosis in which the ring is beginning to form in an asymmetrical fashion. (i) Interphase with no specific cdc12  staining. (B, inset) Cross-section of a cdc12p ring. (D and E) Cell in early mitosis; (F and G) cell in early anaphase; (H and I) cell in midanaphase; (J and K) cell in postanaphase during ring closure. Patchy cytoplasmic staining is also seen in cells lacking cdc12p, and thus  probably represents background staining (data not shown). Bar, 10 μm.
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Figure 3: cdc12p localizes to the cell division ring in wild-type cells. (A) Specificity of the anti-cdc12 affinity-purified antibody. 5 μg of crude whole yeast cell extracts were analyzed by Western blotting using affinity-purified anti-cdc12 antibody. (Lane 1) Extract from wild-type cells. (Lane 2) Extract from cells overexpressing cdc12p. Cells transformed with the plasmid pnmt-cdc12 (FC291) were shifted to derepressing conditions (minimal media without thiamine) for 20 h at 30°C. Molecular weight markers (kD) are indicated. Serial dilutions showed that the pnmt-cdc12 extract contains 10–20-fold more cdc12p than the wild-type extracts (data not shown). (B–K) cdc12p localization in wild-type cells. Wild-type cells were grown in YE5S at 30°C in exponential phase, fixed, and processed for immunofluorescence using anti-cdc12 antibody and DAPI (see Materials and Methods for details). (B, D, F, H, and J) Anti-cdc12 staining. (C, E, G, I, and K) DAPI staining. (B and C) A typical field of cells containing cells from different phases of the cell cycle. Note examples of ring staining. (e) Cell in early mitosis in which the ring is beginning to form in an asymmetrical fashion. (i) Interphase with no specific cdc12 staining. (B, inset) Cross-section of a cdc12p ring. (D and E) Cell in early mitosis; (F and G) cell in early anaphase; (H and I) cell in midanaphase; (J and K) cell in postanaphase during ring closure. Patchy cytoplasmic staining is also seen in cells lacking cdc12p, and thus probably represents background staining (data not shown). Bar, 10 μm.
Mentions: To localize the cdc12 gene product, we raised polyclonal rabbit antibodies against a cdc12 FH1 domain protein fragment expressed in E. coli. The affinity-purified antibody recognized on an immunoblot a single band of ∼220 kD (Fig. 3 A), which is consistent with the size predicted from the nucleotide sequence. This 220-kD protein was confirmed to be cdc12p by showing that the level of this protein is increased ∼10–20-fold in cells containing a plasmid that overexpresses cdc12p from the thiamine-regulated nmt promoter (Fig. 3 A). Measurements of cdc12p levels both from synchronized cells and from cells arrested in G2 (cdc25 block) and G1 phases (cdc10 block) showed that cdc12p is expressed throughout the cell cycle with no large fluctuations in protein levels or mobility shifts in different phases of the cell cycle (data not shown).

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