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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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Cell cycle regulation of Mid2p. (A) Localization of Mid2p–GFP in live cells (KGY2422) (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211126/DC1). (B) mid2–GFP cells were fixed with ethanol and stained with DAPI to visualize DNA. At least 70 cells from each cell cycle stage (as determined by microtubule staining) were counted to determine the percentage of cells, indicated below the images, with a medial Mid2p–GFP signal. Representative images of each stage are shown. (C and E) mid2–HA (KGY2843) and wild-type (KGY246) cells were grown to mid-log phase, and cultures synchronized in G2 were obtained by centrifugal elutriation. Cell samples were collected at the indicated times after synchronization and processed for protein (C) or RNA (E). (C) Cell cycle progression was monitored by determining septation index and Cdc13p levels. Mid2p–HA, Cdc13p, and Cdc2p (which served as a loading control in all of our immunoblots) were detected by immunoblotting with 12CA5, anti-Cdc13p, and anti-PSTAIR antibodies, respectively. (D) Mid2p–HA (KGY2843) was immunoprecipitated with 12CA5 antibodies and incubated with λ-phosphatase or mock treated. (E) Northern analysis of mid2 transcripts from asynchronous cultures of mid2Δ (KGY3135), mid2–HA (KGY2843), and mid2–myc (KGY2432) strains and a synchronized culture of wild-type cells (KGY246). Cell synchrony was monitored by septation index. his3 mRNA served as a loading control. Bars, 5 μm.
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fig2: Cell cycle regulation of Mid2p. (A) Localization of Mid2p–GFP in live cells (KGY2422) (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211126/DC1). (B) mid2–GFP cells were fixed with ethanol and stained with DAPI to visualize DNA. At least 70 cells from each cell cycle stage (as determined by microtubule staining) were counted to determine the percentage of cells, indicated below the images, with a medial Mid2p–GFP signal. Representative images of each stage are shown. (C and E) mid2–HA (KGY2843) and wild-type (KGY246) cells were grown to mid-log phase, and cultures synchronized in G2 were obtained by centrifugal elutriation. Cell samples were collected at the indicated times after synchronization and processed for protein (C) or RNA (E). (C) Cell cycle progression was monitored by determining septation index and Cdc13p levels. Mid2p–HA, Cdc13p, and Cdc2p (which served as a loading control in all of our immunoblots) were detected by immunoblotting with 12CA5, anti-Cdc13p, and anti-PSTAIR antibodies, respectively. (D) Mid2p–HA (KGY2843) was immunoprecipitated with 12CA5 antibodies and incubated with λ-phosphatase or mock treated. (E) Northern analysis of mid2 transcripts from asynchronous cultures of mid2Δ (KGY3135), mid2–HA (KGY2843), and mid2–myc (KGY2432) strains and a synchronized culture of wild-type cells (KGY246). Cell synchrony was monitored by septation index. his3 mRNA served as a loading control. Bars, 5 μm.

Mentions: To better understand the role of Mid2p in cell separation, its subcellular localization was examined. A carboxy-terminal Mid2p–GFP fusion protein was constructed by homologous recombination at the endogenous locus of mid2+ in order to maintain its physiological expression level. Mid2p–GFP localized to the medial region in 17% of the cells in an asynchronous population (Fig. 2 A). Using time-lapse microscopy, Mid2p–GFP was observed to initially form a single ring, split into two rings as the septum formed, and then disappear as cells separated (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211126/DC1). Mid2p–GFP was stable to a variety of fixation procedures, thereby permitting it to be visualized in fixed cells also stained with DAPI and antibodies to tubulin. In this case, Mid2p was only detected at the ring in late anaphase cells that lacked spindles (Fig. 2 B; unpublished data).


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)

Cell cycle regulation of Mid2p. (A) Localization of Mid2p–GFP in live cells (KGY2422) (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211126/DC1). (B) mid2–GFP cells were fixed with ethanol and stained with DAPI to visualize DNA. At least 70 cells from each cell cycle stage (as determined by microtubule staining) were counted to determine the percentage of cells, indicated below the images, with a medial Mid2p–GFP signal. Representative images of each stage are shown. (C and E) mid2–HA (KGY2843) and wild-type (KGY246) cells were grown to mid-log phase, and cultures synchronized in G2 were obtained by centrifugal elutriation. Cell samples were collected at the indicated times after synchronization and processed for protein (C) or RNA (E). (C) Cell cycle progression was monitored by determining septation index and Cdc13p levels. Mid2p–HA, Cdc13p, and Cdc2p (which served as a loading control in all of our immunoblots) were detected by immunoblotting with 12CA5, anti-Cdc13p, and anti-PSTAIR antibodies, respectively. (D) Mid2p–HA (KGY2843) was immunoprecipitated with 12CA5 antibodies and incubated with λ-phosphatase or mock treated. (E) Northern analysis of mid2 transcripts from asynchronous cultures of mid2Δ (KGY3135), mid2–HA (KGY2843), and mid2–myc (KGY2432) strains and a synchronized culture of wild-type cells (KGY246). Cell synchrony was monitored by septation index. his3 mRNA served as a loading control. Bars, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172762&req=5

fig2: Cell cycle regulation of Mid2p. (A) Localization of Mid2p–GFP in live cells (KGY2422) (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211126/DC1). (B) mid2–GFP cells were fixed with ethanol and stained with DAPI to visualize DNA. At least 70 cells from each cell cycle stage (as determined by microtubule staining) were counted to determine the percentage of cells, indicated below the images, with a medial Mid2p–GFP signal. Representative images of each stage are shown. (C and E) mid2–HA (KGY2843) and wild-type (KGY246) cells were grown to mid-log phase, and cultures synchronized in G2 were obtained by centrifugal elutriation. Cell samples were collected at the indicated times after synchronization and processed for protein (C) or RNA (E). (C) Cell cycle progression was monitored by determining septation index and Cdc13p levels. Mid2p–HA, Cdc13p, and Cdc2p (which served as a loading control in all of our immunoblots) were detected by immunoblotting with 12CA5, anti-Cdc13p, and anti-PSTAIR antibodies, respectively. (D) Mid2p–HA (KGY2843) was immunoprecipitated with 12CA5 antibodies and incubated with λ-phosphatase or mock treated. (E) Northern analysis of mid2 transcripts from asynchronous cultures of mid2Δ (KGY3135), mid2–HA (KGY2843), and mid2–myc (KGY2432) strains and a synchronized culture of wild-type cells (KGY246). Cell synchrony was monitored by septation index. his3 mRNA served as a loading control. Bars, 5 μm.
Mentions: To better understand the role of Mid2p in cell separation, its subcellular localization was examined. A carboxy-terminal Mid2p–GFP fusion protein was constructed by homologous recombination at the endogenous locus of mid2+ in order to maintain its physiological expression level. Mid2p–GFP localized to the medial region in 17% of the cells in an asynchronous population (Fig. 2 A). Using time-lapse microscopy, Mid2p–GFP was observed to initially form a single ring, split into two rings as the septum formed, and then disappear as cells separated (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211126/DC1). Mid2p–GFP was stable to a variety of fixation procedures, thereby permitting it to be visualized in fixed cells also stained with DAPI and antibodies to tubulin. In this case, Mid2p was only detected at the ring in late anaphase cells that lacked spindles (Fig. 2 B; unpublished data).

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