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An anillin homologue, Mid2p, acts during fission yeast cytokinesis to organize the septin ring and promote cell separation.

Tasto JJ, Morrell JL, Gould KL - J. Cell Biol. (2003)

Bottom Line: Simanis. 1996.Mid2p colocalizes with septins, and mid2 Delta cells display disorganized, diffuse septin rings and a cell separation defect similar to septin deletion strains. mid2 gene expression and protein levels fluctuate during the cell cycle in a sep1- and Skp1/Cdc53/F-box (SCF)-dependent manner, respectively, implying that Mid2p activity must be carefully regulated.Overproduction of Mid2p depolarizes cell growth and affects the organization of both the septin and actin cytoskeletons.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

ABSTRACT
Anillin is a conserved protein required for cell division (Field, C.M., and B.M. Alberts. 1995. J. Cell Biol. 131:165-178; Oegema, K., M.S. Savoian, T.J. Mitchison, and C.M. Field. 2000. J. Cell Biol. 150:539-552). One fission yeast homologue of anillin, Mid1p, is necessary for the proper placement of the division site within the cell (Chang, F., A. Woollard, and P. Nurse. 1996. J. Cell Sci. 109(Pt 1):131-142; Sohrmann, M., C. Fankhauser, C. Brodbeck, and V. Simanis. 1996. Genes Dev. 10:2707-2719). Here, we identify and characterize a second fission yeast anillin homologue, Mid2p, which is not orthologous with Mid1p. Mid2p localizes as a single ring in the middle of the cell after anaphase in a septin- and actin-dependent manner and splits into two rings during septation. Mid2p colocalizes with septins, and mid2 Delta cells display disorganized, diffuse septin rings and a cell separation defect similar to septin deletion strains. mid2 gene expression and protein levels fluctuate during the cell cycle in a sep1- and Skp1/Cdc53/F-box (SCF)-dependent manner, respectively, implying that Mid2p activity must be carefully regulated. Overproduction of Mid2p depolarizes cell growth and affects the organization of both the septin and actin cytoskeletons. In the presence of a nondegradable Mid2p fragment, the septin ring is stabilized and cell cycle progression is delayed. These results suggest that Mid2p influences septin ring organization at the site of cell division and its turnover might normally be required to permit septin ring disassembly.

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Mid2p functional domains. (A) Diagram of full-length Mid2p depicting the PEST and PH domains. The fine vertical lines indicate the positions of amino acids 148, 336, and 569. (B) The ability of HA– or GFP–Mid2 fusion proteins produced from the nmt41 promoter to rescue the mid2Δ (KGY3135) phenotype, their localization pattern in KGY3135, and the overproduction phenotype of untagged constructs when expressed from the full-strength nmt1 promoter in wild-type cells (KGY246). MR, medial ring; CP, cytoplasmic; PM, plasma membrane; N, nuclear; O.P., overproduction. (C) Wild-type cells expressing the indicated fragments were lysed under denaturing conditions, and the lysates were subjected to an anti-HA immunoprecipitation and then immunoblotted. An asterisk indicates the expected size of a fusion protein. Bands not present in vector control lane are degradation products. (D) Image of live mid2Δ cells expressing GFP–Mid2p(336–704) under control of the derepressed nmt41 promoter. Bar, 5 μm. (E and F) A cdc25-22 mid2Δ strain (KGY4062) containing either (E) pREP41HA–Mid2p(1–704) or (F) pREP41HA–Mid2p(336–704) was grown for 22 h in the absence of thiamine at 25°C, shifted to 36°C for 4 h, and then released to 25°C in the presence of excess thiamine while time points were collected every 20 min for immunoblot analysis. HA–Mid2p and Cdc2p levels were determined by immunoblotting with 12CA5 and PSTAIR, respectively. Mitotic progression was determined by assaying for Cdc13p fluctuations by immunoblotting.
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fig7: Mid2p functional domains. (A) Diagram of full-length Mid2p depicting the PEST and PH domains. The fine vertical lines indicate the positions of amino acids 148, 336, and 569. (B) The ability of HA– or GFP–Mid2 fusion proteins produced from the nmt41 promoter to rescue the mid2Δ (KGY3135) phenotype, their localization pattern in KGY3135, and the overproduction phenotype of untagged constructs when expressed from the full-strength nmt1 promoter in wild-type cells (KGY246). MR, medial ring; CP, cytoplasmic; PM, plasma membrane; N, nuclear; O.P., overproduction. (C) Wild-type cells expressing the indicated fragments were lysed under denaturing conditions, and the lysates were subjected to an anti-HA immunoprecipitation and then immunoblotted. An asterisk indicates the expected size of a fusion protein. Bands not present in vector control lane are degradation products. (D) Image of live mid2Δ cells expressing GFP–Mid2p(336–704) under control of the derepressed nmt41 promoter. Bar, 5 μm. (E and F) A cdc25-22 mid2Δ strain (KGY4062) containing either (E) pREP41HA–Mid2p(1–704) or (F) pREP41HA–Mid2p(336–704) was grown for 22 h in the absence of thiamine at 25°C, shifted to 36°C for 4 h, and then released to 25°C in the presence of excess thiamine while time points were collected every 20 min for immunoblot analysis. HA–Mid2p and Cdc2p levels were determined by immunoblotting with 12CA5 and PSTAIR, respectively. Mitotic progression was determined by assaying for Cdc13p fluctuations by immunoblotting.

Mentions: To determine which regions of the protein dictated Mid2p localization and were important for its function, a series of mid2 constructs were generated and tested for their ability to rescue the mid2Δ cell separation defect, their localization pattern, and any overexpression phenotype (Fig. 7 B). Immunoblot analysis of anti-HA immunoprecipitations from denatured lysates confirmed that these fragments were all produced (Fig. 7 C).


An anillin homologue, Mid2p, acts during fission yeast cytokinesis to organize the septin ring and promote cell separation.

Tasto JJ, Morrell JL, Gould KL - J. Cell Biol. (2003)

Mid2p functional domains. (A) Diagram of full-length Mid2p depicting the PEST and PH domains. The fine vertical lines indicate the positions of amino acids 148, 336, and 569. (B) The ability of HA– or GFP–Mid2 fusion proteins produced from the nmt41 promoter to rescue the mid2Δ (KGY3135) phenotype, their localization pattern in KGY3135, and the overproduction phenotype of untagged constructs when expressed from the full-strength nmt1 promoter in wild-type cells (KGY246). MR, medial ring; CP, cytoplasmic; PM, plasma membrane; N, nuclear; O.P., overproduction. (C) Wild-type cells expressing the indicated fragments were lysed under denaturing conditions, and the lysates were subjected to an anti-HA immunoprecipitation and then immunoblotted. An asterisk indicates the expected size of a fusion protein. Bands not present in vector control lane are degradation products. (D) Image of live mid2Δ cells expressing GFP–Mid2p(336–704) under control of the derepressed nmt41 promoter. Bar, 5 μm. (E and F) A cdc25-22 mid2Δ strain (KGY4062) containing either (E) pREP41HA–Mid2p(1–704) or (F) pREP41HA–Mid2p(336–704) was grown for 22 h in the absence of thiamine at 25°C, shifted to 36°C for 4 h, and then released to 25°C in the presence of excess thiamine while time points were collected every 20 min for immunoblot analysis. HA–Mid2p and Cdc2p levels were determined by immunoblotting with 12CA5 and PSTAIR, respectively. Mitotic progression was determined by assaying for Cdc13p fluctuations by immunoblotting.
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fig7: Mid2p functional domains. (A) Diagram of full-length Mid2p depicting the PEST and PH domains. The fine vertical lines indicate the positions of amino acids 148, 336, and 569. (B) The ability of HA– or GFP–Mid2 fusion proteins produced from the nmt41 promoter to rescue the mid2Δ (KGY3135) phenotype, their localization pattern in KGY3135, and the overproduction phenotype of untagged constructs when expressed from the full-strength nmt1 promoter in wild-type cells (KGY246). MR, medial ring; CP, cytoplasmic; PM, plasma membrane; N, nuclear; O.P., overproduction. (C) Wild-type cells expressing the indicated fragments were lysed under denaturing conditions, and the lysates were subjected to an anti-HA immunoprecipitation and then immunoblotted. An asterisk indicates the expected size of a fusion protein. Bands not present in vector control lane are degradation products. (D) Image of live mid2Δ cells expressing GFP–Mid2p(336–704) under control of the derepressed nmt41 promoter. Bar, 5 μm. (E and F) A cdc25-22 mid2Δ strain (KGY4062) containing either (E) pREP41HA–Mid2p(1–704) or (F) pREP41HA–Mid2p(336–704) was grown for 22 h in the absence of thiamine at 25°C, shifted to 36°C for 4 h, and then released to 25°C in the presence of excess thiamine while time points were collected every 20 min for immunoblot analysis. HA–Mid2p and Cdc2p levels were determined by immunoblotting with 12CA5 and PSTAIR, respectively. Mitotic progression was determined by assaying for Cdc13p fluctuations by immunoblotting.
Mentions: To determine which regions of the protein dictated Mid2p localization and were important for its function, a series of mid2 constructs were generated and tested for their ability to rescue the mid2Δ cell separation defect, their localization pattern, and any overexpression phenotype (Fig. 7 B). Immunoblot analysis of anti-HA immunoprecipitations from denatured lysates confirmed that these fragments were all produced (Fig. 7 C).

Bottom Line: Simanis. 1996.Mid2p colocalizes with septins, and mid2 Delta cells display disorganized, diffuse septin rings and a cell separation defect similar to septin deletion strains. mid2 gene expression and protein levels fluctuate during the cell cycle in a sep1- and Skp1/Cdc53/F-box (SCF)-dependent manner, respectively, implying that Mid2p activity must be carefully regulated.Overproduction of Mid2p depolarizes cell growth and affects the organization of both the septin and actin cytoskeletons.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

ABSTRACT
Anillin is a conserved protein required for cell division (Field, C.M., and B.M. Alberts. 1995. J. Cell Biol. 131:165-178; Oegema, K., M.S. Savoian, T.J. Mitchison, and C.M. Field. 2000. J. Cell Biol. 150:539-552). One fission yeast homologue of anillin, Mid1p, is necessary for the proper placement of the division site within the cell (Chang, F., A. Woollard, and P. Nurse. 1996. J. Cell Sci. 109(Pt 1):131-142; Sohrmann, M., C. Fankhauser, C. Brodbeck, and V. Simanis. 1996. Genes Dev. 10:2707-2719). Here, we identify and characterize a second fission yeast anillin homologue, Mid2p, which is not orthologous with Mid1p. Mid2p localizes as a single ring in the middle of the cell after anaphase in a septin- and actin-dependent manner and splits into two rings during septation. Mid2p colocalizes with septins, and mid2 Delta cells display disorganized, diffuse septin rings and a cell separation defect similar to septin deletion strains. mid2 gene expression and protein levels fluctuate during the cell cycle in a sep1- and Skp1/Cdc53/F-box (SCF)-dependent manner, respectively, implying that Mid2p activity must be carefully regulated. Overproduction of Mid2p depolarizes cell growth and affects the organization of both the septin and actin cytoskeletons. In the presence of a nondegradable Mid2p fragment, the septin ring is stabilized and cell cycle progression is delayed. These results suggest that Mid2p influences septin ring organization at the site of cell division and its turnover might normally be required to permit septin ring disassembly.

Show MeSH
Related in: MedlinePlus