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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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Upon activation of the SIN, Mid1p and cortical nodes are not required for orthogonal ring assembly. (A) nda3-KM311 mid1Δ cells expressing Rlc1p-GFP and Sid4p-GFP were cultured at 18°C for 6 h and imaged by fluorescence microscopy. The SPBs are marked with pink asterisks. (B) Quantitation of orthogonal rings and misoriented rings in nda3-KM311 mid1Δ cells. (C) Prometaphase-arrested nda3-KM311 mid1Δ cells were fixed and stained with Alexa Fluor 488 phalloidin and DAPI to visualize the F-actin cables and rings and the nuclei. (D) Upon SIN activation, orthogonal actomyosin rings assemble in the absence of Mid1p and cortical nodes. Cells of the indicated genotypes expressing Rlc1p-GFP and Uch2p-GFP were arrested in interphase by treatment with 12 mM HU for 6 h at 24°C and shifted to 36°C to inactivate Cdc16p function in the presence of HU, and images were captured. Bars, 5 μm.
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fig5: Upon activation of the SIN, Mid1p and cortical nodes are not required for orthogonal ring assembly. (A) nda3-KM311 mid1Δ cells expressing Rlc1p-GFP and Sid4p-GFP were cultured at 18°C for 6 h and imaged by fluorescence microscopy. The SPBs are marked with pink asterisks. (B) Quantitation of orthogonal rings and misoriented rings in nda3-KM311 mid1Δ cells. (C) Prometaphase-arrested nda3-KM311 mid1Δ cells were fixed and stained with Alexa Fluor 488 phalloidin and DAPI to visualize the F-actin cables and rings and the nuclei. (D) Upon SIN activation, orthogonal actomyosin rings assemble in the absence of Mid1p and cortical nodes. Cells of the indicated genotypes expressing Rlc1p-GFP and Uch2p-GFP were arrested in interphase by treatment with 12 mM HU for 6 h at 24°C and shifted to 36°C to inactivate Cdc16p function in the presence of HU, and images were captured. Bars, 5 μm.

Mentions: To further assess whether Mid1p and the cortical nodes were important for ring assembly in metaphase, we arrested nda3-KM311 and nda3-KM311 mid1Δ cells at prometaphase by a shift to 18°C. nda3-KM311 is a cold-sensitive mutant allele of the sole gene encoding the fission yeast β-tubulin (Hiraoka et al., 1984). nda3-KM311 cells arrest with an actomyosin ring (Chang et al., 1996). Interestingly, we found that nearly 59% of nda3-KM311 mid1Δ cells contained misoriented rings, whereas the rest contained orthogonal rings (Fig. 5, A–C; Rlc1p and Sid4p, an SPB component [Chang and Gould, 2000], are shown in A, whereas F-actin is shown in C). In contrast, rings in the nda3-KM311 cells were always orthogonal (unpublished data). These results furthered the notion that the function of Mid1p and cortical nodes was important for organization of normal actomyosin rings in early mitosis. The slightly higher proportion of orthogonal rings observed in nda3 mid1Δ cells might be caused by the prolonged block of nda3-KM311 cells at the restrictive temperature.


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)

Upon activation of the SIN, Mid1p and cortical nodes are not required for orthogonal ring assembly. (A) nda3-KM311 mid1Δ cells expressing Rlc1p-GFP and Sid4p-GFP were cultured at 18°C for 6 h and imaged by fluorescence microscopy. The SPBs are marked with pink asterisks. (B) Quantitation of orthogonal rings and misoriented rings in nda3-KM311 mid1Δ cells. (C) Prometaphase-arrested nda3-KM311 mid1Δ cells were fixed and stained with Alexa Fluor 488 phalloidin and DAPI to visualize the F-actin cables and rings and the nuclei. (D) Upon SIN activation, orthogonal actomyosin rings assemble in the absence of Mid1p and cortical nodes. Cells of the indicated genotypes expressing Rlc1p-GFP and Uch2p-GFP were arrested in interphase by treatment with 12 mM HU for 6 h at 24°C and shifted to 36°C to inactivate Cdc16p function in the presence of HU, 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

fig5: Upon activation of the SIN, Mid1p and cortical nodes are not required for orthogonal ring assembly. (A) nda3-KM311 mid1Δ cells expressing Rlc1p-GFP and Sid4p-GFP were cultured at 18°C for 6 h and imaged by fluorescence microscopy. The SPBs are marked with pink asterisks. (B) Quantitation of orthogonal rings and misoriented rings in nda3-KM311 mid1Δ cells. (C) Prometaphase-arrested nda3-KM311 mid1Δ cells were fixed and stained with Alexa Fluor 488 phalloidin and DAPI to visualize the F-actin cables and rings and the nuclei. (D) Upon SIN activation, orthogonal actomyosin rings assemble in the absence of Mid1p and cortical nodes. Cells of the indicated genotypes expressing Rlc1p-GFP and Uch2p-GFP were arrested in interphase by treatment with 12 mM HU for 6 h at 24°C and shifted to 36°C to inactivate Cdc16p function in the presence of HU, and images were captured. Bars, 5 μm.
Mentions: To further assess whether Mid1p and the cortical nodes were important for ring assembly in metaphase, we arrested nda3-KM311 and nda3-KM311 mid1Δ cells at prometaphase by a shift to 18°C. nda3-KM311 is a cold-sensitive mutant allele of the sole gene encoding the fission yeast β-tubulin (Hiraoka et al., 1984). nda3-KM311 cells arrest with an actomyosin ring (Chang et al., 1996). Interestingly, we found that nearly 59% of nda3-KM311 mid1Δ cells contained misoriented rings, whereas the rest contained orthogonal rings (Fig. 5, A–C; Rlc1p and Sid4p, an SPB component [Chang and Gould, 2000], are shown in A, whereas F-actin is shown in C). In contrast, rings in the nda3-KM311 cells were always orthogonal (unpublished data). These results furthered the notion that the function of Mid1p and cortical nodes was important for organization of normal actomyosin rings in early mitosis. The slightly higher proportion of orthogonal rings observed in nda3 mid1Δ cells might be caused by the prolonged block of nda3-KM311 cells at the restrictive temperature.

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