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Assembly of normal actomyosin rings in the absence of Mid1p and cortical nodes in fission yeast.

Huang Y, Yan H, Balasubramanian MK - J. Cell Biol. (2008)

Bottom Line: In this study, we test this model in cells that are unable to assemble cortical nodes.Although the cortical nodes play a role in the timing of ring assembly, we find that they are dispensable for the assembly of orthogonal actomyosin rings.Thus, a mechanism that is independent of cortical nodes is sufficient for the assembly of normal actomyosin rings.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604.

ABSTRACT
Cytokinesis in many eukaryotes depends on the function of an actomyosin contractile ring. The mechanisms regulating assembly and positioning of this ring are not fully understood. The fission yeast Schizosaccharomyces pombe divides using an actomyosin ring and is an attractive organism for the study of cytokinesis. Recent studies in S. pombe (Wu, J.Q., V. Sirotkin, D.R. Kovar, M. Lord, C.C. Beltzner, J.R. Kuhn, and T.D. Pollard. 2006. J. Cell Biol. 174:391-402; Vavylonis, D., J.Q. Wu, S. Hao, B. O'Shaughnessy, and T.D. Pollard. 2008. Science. 319:97-100) have suggested that the assembly of the actomyosin ring is initiated from a series of cortical nodes containing several components of this ring. These studies have proposed that actomyosin interactions bring together the cortical nodes to form a compacted ring structure. In this study, we test this model in cells that are unable to assemble cortical nodes. Although the cortical nodes play a role in the timing of ring assembly, we find that they are dispensable for the assembly of orthogonal actomyosin rings. Thus, a mechanism that is independent of cortical nodes is sufficient for the assembly of normal actomyosin rings.

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Membrane-associated nodes of Rlc1p, Cdc15p, and Cdc12p are not detected in cells lacking Mid1p. (A) cdc25-22 and cdc25-22 mid1Δ cells expressing Rlc1p-GFP, Cdc15p-GFP, or Cdc12p-GFP were arrested at the G2/M boundary by incubation at the restrictive temperature of 36°C. Cells were shifted to the permissive temperature of 24°C, and images were captured 30–45 min after a shift down. In some instances, as indicated, LatA was added to the culture to facilitate visualization of the cortical nodes of Cdc15p and Cdc12p. (B and C) cdc25-22 cells expressing Rlc1p-mCherry and Cdc15p-GFP (B) or Rlc1p-mCherry and Cdc12p-GFP (C) were cultured as described in A and treated with LatA, and images were captured. Bars, 5 μm.
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fig1: Membrane-associated nodes of Rlc1p, Cdc15p, and Cdc12p are not detected in cells lacking Mid1p. (A) cdc25-22 and cdc25-22 mid1Δ cells expressing Rlc1p-GFP, Cdc15p-GFP, or Cdc12p-GFP were arrested at the G2/M boundary by incubation at the restrictive temperature of 36°C. Cells were shifted to the permissive temperature of 24°C, and images were captured 30–45 min after a shift down. In some instances, as indicated, LatA was added to the culture to facilitate visualization of the cortical nodes of Cdc15p and Cdc12p. (B and C) cdc25-22 cells expressing Rlc1p-mCherry and Cdc15p-GFP (B) or Rlc1p-mCherry and Cdc12p-GFP (C) were cultured as described in A and treated with LatA, and images were captured. Bars, 5 μm.

Mentions: In S. pombe, Wu et al. (2006) have shown that Mid1p assembles first into nodes and recruits other proteins (including type II myosin and its light chain Rlc1p, formin Cdc12p, FCH-BAR protein Cdc15p, and IQGAP-Rng2p) into nodes (Motegi et al., 2000, 2004; Paoletti and Chang, 2000). Fig. 1 A shows examples of Rlc1p-3GFP nodes in wild-type cells and their absence in mid1Δ cells, which is consistent with experiments of Wu et al. (2006). Furthermore, as shown by Wu et al. (2006), we have found that Cdc15p organizes into a few nodelike structures (15 ± 7 nodes/cell; n = 17; Fig. 1 A) and that the number and intensity of these structures is increased (45 ± 18 nodes/cell; n = 22; Fig. 1 A) upon treatment with latrunculin A (LatA), which prevents actin polymerization (Ayscough et al., 1997). We have also been able to observe colocalization of Cdc15p-GFP nodes with Rlc1p-mCherry nodes in LatA-treated cells (Fig. 1 B). As shown by Wu et al. (2006), we have found that organization of Cdc15p into nodes in untreated and LatA-treated cells depended on Mid1p function. Although we have been able to detect Rlc1p and Cdc15p in cortical nodes, we were unable to detect the formin Cdc12p in cortical nodes in unperturbed cells, which is consistent with studies by Chang (1999) and Yonetani et al. (2008). Nevertheless, as described by Wu et al. (2006), we did detect Cdc12p nodes upon LatA treatment (Fig. 1 A). Again, organization of Cdc12p nodes in LatA-treated cells depended on Mid1p function. Approximately 51% of Cdc12p nodes (n = 270) generated by LatA treatment colocalized with Rlc1p nodes (Fig. 1 C). We have also found that Rlc1p nodes are not detected in mutants defective in the fission yeast Polo kinase Plo1p (unpublished data), which is a known regulator of fission yeast Mid1p (Bahler et al., 1998). Collectively, these studies established that some actomyosin ring components (such as Rlc1p and Cdc15p) are detected in nodes in unperturbed cells, whereas others (such as Cdc12p) are detected in nodes only upon LatA treatment. Furthermore, our experiments as well as other studies (Motegi et al., 2004; Wu et al., 2006) have established that the organization of cortical nodes depends on Mid1p function.


Assembly of normal actomyosin rings in the absence of Mid1p and cortical nodes in fission yeast.

Huang Y, Yan H, Balasubramanian MK - J. Cell Biol. (2008)

Membrane-associated nodes of Rlc1p, Cdc15p, and Cdc12p are not detected in cells lacking Mid1p. (A) cdc25-22 and cdc25-22 mid1Δ cells expressing Rlc1p-GFP, Cdc15p-GFP, or Cdc12p-GFP were arrested at the G2/M boundary by incubation at the restrictive temperature of 36°C. Cells were shifted to the permissive temperature of 24°C, and images were captured 30–45 min after a shift down. In some instances, as indicated, LatA was added to the culture to facilitate visualization of the cortical nodes of Cdc15p and Cdc12p. (B and C) cdc25-22 cells expressing Rlc1p-mCherry and Cdc15p-GFP (B) or Rlc1p-mCherry and Cdc12p-GFP (C) were cultured as described in A and treated with LatA, and images were captured. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2600752&req=5

fig1: Membrane-associated nodes of Rlc1p, Cdc15p, and Cdc12p are not detected in cells lacking Mid1p. (A) cdc25-22 and cdc25-22 mid1Δ cells expressing Rlc1p-GFP, Cdc15p-GFP, or Cdc12p-GFP were arrested at the G2/M boundary by incubation at the restrictive temperature of 36°C. Cells were shifted to the permissive temperature of 24°C, and images were captured 30–45 min after a shift down. In some instances, as indicated, LatA was added to the culture to facilitate visualization of the cortical nodes of Cdc15p and Cdc12p. (B and C) cdc25-22 cells expressing Rlc1p-mCherry and Cdc15p-GFP (B) or Rlc1p-mCherry and Cdc12p-GFP (C) were cultured as described in A and treated with LatA, and images were captured. Bars, 5 μm.
Mentions: In S. pombe, Wu et al. (2006) have shown that Mid1p assembles first into nodes and recruits other proteins (including type II myosin and its light chain Rlc1p, formin Cdc12p, FCH-BAR protein Cdc15p, and IQGAP-Rng2p) into nodes (Motegi et al., 2000, 2004; Paoletti and Chang, 2000). Fig. 1 A shows examples of Rlc1p-3GFP nodes in wild-type cells and their absence in mid1Δ cells, which is consistent with experiments of Wu et al. (2006). Furthermore, as shown by Wu et al. (2006), we have found that Cdc15p organizes into a few nodelike structures (15 ± 7 nodes/cell; n = 17; Fig. 1 A) and that the number and intensity of these structures is increased (45 ± 18 nodes/cell; n = 22; Fig. 1 A) upon treatment with latrunculin A (LatA), which prevents actin polymerization (Ayscough et al., 1997). We have also been able to observe colocalization of Cdc15p-GFP nodes with Rlc1p-mCherry nodes in LatA-treated cells (Fig. 1 B). As shown by Wu et al. (2006), we have found that organization of Cdc15p into nodes in untreated and LatA-treated cells depended on Mid1p function. Although we have been able to detect Rlc1p and Cdc15p in cortical nodes, we were unable to detect the formin Cdc12p in cortical nodes in unperturbed cells, which is consistent with studies by Chang (1999) and Yonetani et al. (2008). Nevertheless, as described by Wu et al. (2006), we did detect Cdc12p nodes upon LatA treatment (Fig. 1 A). Again, organization of Cdc12p nodes in LatA-treated cells depended on Mid1p function. Approximately 51% of Cdc12p nodes (n = 270) generated by LatA treatment colocalized with Rlc1p nodes (Fig. 1 C). We have also found that Rlc1p nodes are not detected in mutants defective in the fission yeast Polo kinase Plo1p (unpublished data), which is a known regulator of fission yeast Mid1p (Bahler et al., 1998). Collectively, these studies established that some actomyosin ring components (such as Rlc1p and Cdc15p) are detected in nodes in unperturbed cells, whereas others (such as Cdc12p) are detected in nodes only upon LatA treatment. Furthermore, our experiments as well as other studies (Motegi et al., 2004; Wu et al., 2006) have established that the organization of cortical nodes depends on Mid1p function.

Bottom Line: In this study, we test this model in cells that are unable to assemble cortical nodes.Although the cortical nodes play a role in the timing of ring assembly, we find that they are dispensable for the assembly of orthogonal actomyosin rings.Thus, a mechanism that is independent of cortical nodes is sufficient for the assembly of normal actomyosin rings.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604.

ABSTRACT
Cytokinesis in many eukaryotes depends on the function of an actomyosin contractile ring. The mechanisms regulating assembly and positioning of this ring are not fully understood. The fission yeast Schizosaccharomyces pombe divides using an actomyosin ring and is an attractive organism for the study of cytokinesis. Recent studies in S. pombe (Wu, J.Q., V. Sirotkin, D.R. Kovar, M. Lord, C.C. Beltzner, J.R. Kuhn, and T.D. Pollard. 2006. J. Cell Biol. 174:391-402; Vavylonis, D., J.Q. Wu, S. Hao, B. O'Shaughnessy, and T.D. Pollard. 2008. Science. 319:97-100) have suggested that the assembly of the actomyosin ring is initiated from a series of cortical nodes containing several components of this ring. These studies have proposed that actomyosin interactions bring together the cortical nodes to form a compacted ring structure. In this study, we test this model in cells that are unable to assemble cortical nodes. Although the cortical nodes play a role in the timing of ring assembly, we find that they are dispensable for the assembly of orthogonal actomyosin rings. Thus, a mechanism that is independent of cortical nodes is sufficient for the assembly of normal actomyosin rings.

Show MeSH
Related in: MedlinePlus