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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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Mid1p and cortical nodes are important for orthogonal ring assembly in early mitosis. (A) mid1-18 cells expressing Rlc1p-GFP were arrested in metaphase by overproduction of the mitotic checkpoint protein Mad2p expressed under control of the thiamine-repressible nmt1 promoter. Cells were grown in the absence of thiamine for 18 h at 24°C, shifted to 36°C for 3.5 h, and fixed and stained with Tat1 antibodies (tubulin), Cdc4p antibodies (rings), and DAPI (nuclei). (B) Quantitation of orthogonal rings and misoriented rings in metaphase-arrested mid1-18 cells. (C) Schematic representation of imaging experiments in D and E. (D) Time-lapse imaging of myosin II ring/cable assembly in metaphase-arrested mid1-18 cells. 0 min refers to the time when the imaging was initiated (2.5 h after the shift to 36°C). (E) Time-lapse imaging of unraveling of a preformed medial myosin II ring in a metaphase-arrested mid1-18 cell upon shift to the restrictive temperature (red arrows). Bars, 5 μm.
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fig4: Mid1p and cortical nodes are important for orthogonal ring assembly in early mitosis. (A) mid1-18 cells expressing Rlc1p-GFP were arrested in metaphase by overproduction of the mitotic checkpoint protein Mad2p expressed under control of the thiamine-repressible nmt1 promoter. Cells were grown in the absence of thiamine for 18 h at 24°C, shifted to 36°C for 3.5 h, and fixed and stained with Tat1 antibodies (tubulin), Cdc4p antibodies (rings), and DAPI (nuclei). (B) Quantitation of orthogonal rings and misoriented rings in metaphase-arrested mid1-18 cells. (C) Schematic representation of imaging experiments in D and E. (D) Time-lapse imaging of myosin II ring/cable assembly in metaphase-arrested mid1-18 cells. 0 min refers to the time when the imaging was initiated (2.5 h after the shift to 36°C). (E) Time-lapse imaging of unraveling of a preformed medial myosin II ring in a metaphase-arrested mid1-18 cell upon shift to the restrictive temperature (red arrows). Bars, 5 μm.

Mentions: To independently test whether Mid1p was important for ring assembly in early mitotic cells, we arrested mid1-18 cells expressing Rlc1p-GFP at metaphase by overproduction of the mitotic checkpoint protein Mad2p (He et al., 1997). We performed three different experiments. First, we induced the expression of Mad2p, shifted cells to the restrictive temperature for mid1-18, and fixed and stained these cells with antibodies against Cdc4p, tubulin, and DAPI. In this experiment, we found that >85% of metaphase-arrested mid1-18 cells contained nonorthogonal myosin II cables (Fig. 4, A and B). Second, we imaged the process of myosin II ring assembly in metaphase-arrested mid1-18 cells (Fig. 4, C and D). We found that myosin II cables and bundles assembled efficiently in metaphase-arrested mid1-18 cells. However, these myosin II cables failed to become organized into rings even upon incubation for nearly 90 min (Fig. 4 D). Finally, we induced the expression of Mad2p, allowed myosin II ring assembly at the permissive temperature for mid1-18, and subsequently shifted cells to the restrictive temperature (Fig. 4, C and E). We found that shift of metaphase-arrested mid1-18 cells with a preexisting myosin II ring to the restrictive temperature led to unraveling and disassembly of this ring (Fig. 4 E, red arrows). These observations suggested that Mid1p and possibly the nodes were important for organization of actomyosin rings in early mitosis and that Mid1p function might be continuously required for the medial maintenance of the actomyosin ring until septation. The fact that metaphase-arrested unseptated mid1-18 cells failed to organize orthogonal rings ruled out the possibility that prevention of septation in cps1-191 mid1− mutants was solely responsible for actomyosin ring organization. Furthermore, this result suggested that some aspect of regulation occurring after metaphase is responsible for the organization of orthogonal rings in cps1-191 mid1− cells.


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)

Mid1p and cortical nodes are important for orthogonal ring assembly in early mitosis. (A) mid1-18 cells expressing Rlc1p-GFP were arrested in metaphase by overproduction of the mitotic checkpoint protein Mad2p expressed under control of the thiamine-repressible nmt1 promoter. Cells were grown in the absence of thiamine for 18 h at 24°C, shifted to 36°C for 3.5 h, and fixed and stained with Tat1 antibodies (tubulin), Cdc4p antibodies (rings), and DAPI (nuclei). (B) Quantitation of orthogonal rings and misoriented rings in metaphase-arrested mid1-18 cells. (C) Schematic representation of imaging experiments in D and E. (D) Time-lapse imaging of myosin II ring/cable assembly in metaphase-arrested mid1-18 cells. 0 min refers to the time when the imaging was initiated (2.5 h after the shift to 36°C). (E) Time-lapse imaging of unraveling of a preformed medial myosin II ring in a metaphase-arrested mid1-18 cell upon shift to the restrictive temperature (red arrows). Bars, 5 μm.
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Related In: Results  -  Collection

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fig4: Mid1p and cortical nodes are important for orthogonal ring assembly in early mitosis. (A) mid1-18 cells expressing Rlc1p-GFP were arrested in metaphase by overproduction of the mitotic checkpoint protein Mad2p expressed under control of the thiamine-repressible nmt1 promoter. Cells were grown in the absence of thiamine for 18 h at 24°C, shifted to 36°C for 3.5 h, and fixed and stained with Tat1 antibodies (tubulin), Cdc4p antibodies (rings), and DAPI (nuclei). (B) Quantitation of orthogonal rings and misoriented rings in metaphase-arrested mid1-18 cells. (C) Schematic representation of imaging experiments in D and E. (D) Time-lapse imaging of myosin II ring/cable assembly in metaphase-arrested mid1-18 cells. 0 min refers to the time when the imaging was initiated (2.5 h after the shift to 36°C). (E) Time-lapse imaging of unraveling of a preformed medial myosin II ring in a metaphase-arrested mid1-18 cell upon shift to the restrictive temperature (red arrows). Bars, 5 μm.
Mentions: To independently test whether Mid1p was important for ring assembly in early mitotic cells, we arrested mid1-18 cells expressing Rlc1p-GFP at metaphase by overproduction of the mitotic checkpoint protein Mad2p (He et al., 1997). We performed three different experiments. First, we induced the expression of Mad2p, shifted cells to the restrictive temperature for mid1-18, and fixed and stained these cells with antibodies against Cdc4p, tubulin, and DAPI. In this experiment, we found that >85% of metaphase-arrested mid1-18 cells contained nonorthogonal myosin II cables (Fig. 4, A and B). Second, we imaged the process of myosin II ring assembly in metaphase-arrested mid1-18 cells (Fig. 4, C and D). We found that myosin II cables and bundles assembled efficiently in metaphase-arrested mid1-18 cells. However, these myosin II cables failed to become organized into rings even upon incubation for nearly 90 min (Fig. 4 D). Finally, we induced the expression of Mad2p, allowed myosin II ring assembly at the permissive temperature for mid1-18, and subsequently shifted cells to the restrictive temperature (Fig. 4, C and E). We found that shift of metaphase-arrested mid1-18 cells with a preexisting myosin II ring to the restrictive temperature led to unraveling and disassembly of this ring (Fig. 4 E, red arrows). These observations suggested that Mid1p and possibly the nodes were important for organization of actomyosin rings in early mitosis and that Mid1p function might be continuously required for the medial maintenance of the actomyosin ring until septation. The fact that metaphase-arrested unseptated mid1-18 cells failed to organize orthogonal rings ruled out the possibility that prevention of septation in cps1-191 mid1− mutants was solely responsible for actomyosin ring organization. Furthermore, this result suggested that some aspect of regulation occurring after metaphase is responsible for the organization of orthogonal rings in cps1-191 mid1− cells.

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