Limits...
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.

Show MeSH

Related in: MedlinePlus

Mid2p is regulated by SCF-dependent proteolysis. (A and B) In vivo ubiquitination assays. The cut2–myc mts3-1 (KGY1923) (lane 1), cut2–myc mts3-1 lid1-6 (KGY1948) (lane 2), cut2–myc mts3-1 skp1-A4 (KGY4050) (lane 3), mts3-1 (KGY574) (lane 5), mid2–myc (KGY2432) (lane 6), mid2–myc mts3-1 (KGY3687) (lane 7), mid2–myc mts3-1 lid1-6 (KGY1977) (lane 8), and mid2–myc mts3-1 skp1-A4 (KGY1978) (lane 9) strains containing pREP1-His6–ubiquitin (Ub) and the mid2-myc mts3-1 strain (KGY3687) without vector (lane 4) were grown at 25°C for 22 h in the absence of thiamine to induce His6–Ub expression and then shifted to 36°C for 4 h. Ub-conjugated proteins were isolated and immunoblotted for either Myc (A) or Ub (B). (C) Denatured protein lysates from wild-type (KGY246), mid2–HA(KGY2843), mid2–HA mts3-1(KGY3307), pop1Δ mid2–HA (KGY3702), pop2Δ mid2–HA (KGY3699), and mid2–HA pop1Δ pop2Δ (KGY3700) strains were immunoblotted with anti-HA (12CA5) to detect Mid2p–HA and anti-PSTAIR to detect Cdc2p.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172762&req=5

fig3: Mid2p is regulated by SCF-dependent proteolysis. (A and B) In vivo ubiquitination assays. The cut2–myc mts3-1 (KGY1923) (lane 1), cut2–myc mts3-1 lid1-6 (KGY1948) (lane 2), cut2–myc mts3-1 skp1-A4 (KGY4050) (lane 3), mts3-1 (KGY574) (lane 5), mid2–myc (KGY2432) (lane 6), mid2–myc mts3-1 (KGY3687) (lane 7), mid2–myc mts3-1 lid1-6 (KGY1977) (lane 8), and mid2–myc mts3-1 skp1-A4 (KGY1978) (lane 9) strains containing pREP1-His6–ubiquitin (Ub) and the mid2-myc mts3-1 strain (KGY3687) without vector (lane 4) were grown at 25°C for 22 h in the absence of thiamine to induce His6–Ub expression and then shifted to 36°C for 4 h. Ub-conjugated proteins were isolated and immunoblotted for either Myc (A) or Ub (B). (C) Denatured protein lysates from wild-type (KGY246), mid2–HA(KGY2843), mid2–HA mts3-1(KGY3307), pop1Δ mid2–HA (KGY3702), pop2Δ mid2–HA (KGY3699), and mid2–HA pop1Δ pop2Δ (KGY3700) strains were immunoblotted with anti-HA (12CA5) to detect Mid2p–HA and anti-PSTAIR to detect Cdc2p.

Mentions: Because of the dynamic changes in Mid2p–HA levels during the cell cycle (Fig. 2 C), we tested if Mid2p was targeted for destruction by ubiquitin-mediated proteolysis. First, we examined whether Mid2p was ubiquitinated in vivo. In an mts3-1 mutant strain, multi-ubiquitinated conjugates accumulate at the restrictive temperature due to the abrogation of proteosome function (Gordon et al., 1996). Therefore, polyubiquitinated Mid2p–Myc would be expected to accrue in an mts3-1 strain, but only if the relevant E3 ubiquitin ligase was active. S. pombe securin, Cut2p, was used as a positive control for a ubiquitin-conjugated protein (Berry et al., 1999). Both Cut2p–Myc and Mid2p–Myc were readily detected as multi-ubiquitinated proteins in isolates of ubiquitin-conjugated proteins from an mts3-1 strain overproducing tagged ubiquitin (Fig. 3 A, lanes 1 and 7).


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 is regulated by SCF-dependent proteolysis. (A and B) In vivo ubiquitination assays. The cut2–myc mts3-1 (KGY1923) (lane 1), cut2–myc mts3-1 lid1-6 (KGY1948) (lane 2), cut2–myc mts3-1 skp1-A4 (KGY4050) (lane 3), mts3-1 (KGY574) (lane 5), mid2–myc (KGY2432) (lane 6), mid2–myc mts3-1 (KGY3687) (lane 7), mid2–myc mts3-1 lid1-6 (KGY1977) (lane 8), and mid2–myc mts3-1 skp1-A4 (KGY1978) (lane 9) strains containing pREP1-His6–ubiquitin (Ub) and the mid2-myc mts3-1 strain (KGY3687) without vector (lane 4) were grown at 25°C for 22 h in the absence of thiamine to induce His6–Ub expression and then shifted to 36°C for 4 h. Ub-conjugated proteins were isolated and immunoblotted for either Myc (A) or Ub (B). (C) Denatured protein lysates from wild-type (KGY246), mid2–HA(KGY2843), mid2–HA mts3-1(KGY3307), pop1Δ mid2–HA (KGY3702), pop2Δ mid2–HA (KGY3699), and mid2–HA pop1Δ pop2Δ (KGY3700) strains were immunoblotted with anti-HA (12CA5) to detect Mid2p–HA and anti-PSTAIR to detect Cdc2p.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172762&req=5

fig3: Mid2p is regulated by SCF-dependent proteolysis. (A and B) In vivo ubiquitination assays. The cut2–myc mts3-1 (KGY1923) (lane 1), cut2–myc mts3-1 lid1-6 (KGY1948) (lane 2), cut2–myc mts3-1 skp1-A4 (KGY4050) (lane 3), mts3-1 (KGY574) (lane 5), mid2–myc (KGY2432) (lane 6), mid2–myc mts3-1 (KGY3687) (lane 7), mid2–myc mts3-1 lid1-6 (KGY1977) (lane 8), and mid2–myc mts3-1 skp1-A4 (KGY1978) (lane 9) strains containing pREP1-His6–ubiquitin (Ub) and the mid2-myc mts3-1 strain (KGY3687) without vector (lane 4) were grown at 25°C for 22 h in the absence of thiamine to induce His6–Ub expression and then shifted to 36°C for 4 h. Ub-conjugated proteins were isolated and immunoblotted for either Myc (A) or Ub (B). (C) Denatured protein lysates from wild-type (KGY246), mid2–HA(KGY2843), mid2–HA mts3-1(KGY3307), pop1Δ mid2–HA (KGY3702), pop2Δ mid2–HA (KGY3699), and mid2–HA pop1Δ pop2Δ (KGY3700) strains were immunoblotted with anti-HA (12CA5) to detect Mid2p–HA and anti-PSTAIR to detect Cdc2p.
Mentions: Because of the dynamic changes in Mid2p–HA levels during the cell cycle (Fig. 2 C), we tested if Mid2p was targeted for destruction by ubiquitin-mediated proteolysis. First, we examined whether Mid2p was ubiquitinated in vivo. In an mts3-1 mutant strain, multi-ubiquitinated conjugates accumulate at the restrictive temperature due to the abrogation of proteosome function (Gordon et al., 1996). Therefore, polyubiquitinated Mid2p–Myc would be expected to accrue in an mts3-1 strain, but only if the relevant E3 ubiquitin ligase was active. S. pombe securin, Cut2p, was used as a positive control for a ubiquitin-conjugated protein (Berry et al., 1999). Both Cut2p–Myc and Mid2p–Myc were readily detected as multi-ubiquitinated proteins in isolates of ubiquitin-conjugated proteins from an mts3-1 strain overproducing tagged ubiquitin (Fig. 3 A, lanes 1 and 7).

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