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Myosin II dynamics and cortical flow during contractile ring formation in Dictyostelium cells.

Yumura S - J. Cell Biol. (2001)

Bottom Line: These results indicate that myosin II in the contractile ring performs dynamic turnover via its heavy chain phosphorylation.Because GFP-3ALA myosin II did not show the recovery, it served as a useful marker of myosin II movement, which enabled us to demonstrate cortical flow of myosin II toward the equator for the first time.Thus, cortical flow accompanies the dynamic exchange of myosin II during the formation of contractile rings.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan. yumura@po.cc.yamaguchi-u.ac.jp

ABSTRACT
Myosin II is a major component of a contractile ring. To examine if myosin II turns over in contractile rings, fluorescence of GFP-myosin II expressed in Dictyostelium cells was bleached locally by laser illumination, and the recovery was monitored. The fluorescence recovered with a half time of 7.01 +/- 2.62 s. This recovery was not caused by lateral movement of myosin II from the nonbleached area, but by an exchange with endoplasmic myosin II. Similar experiments were performed in cells expressing GFP-3ALA myosin II, of which three phosphorylatable threonine residues were replaced with alanine residues. In this case, recovery was not detected within a comparable time range. These results indicate that myosin II in the contractile ring performs dynamic turnover via its heavy chain phosphorylation. Because GFP-3ALA myosin II did not show the recovery, it served as a useful marker of myosin II movement, which enabled us to demonstrate cortical flow of myosin II toward the equator for the first time. Thus, cortical flow accompanies the dynamic exchange of myosin II during the formation of contractile rings.

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FRAP experiments of GFP–myosin II in the contractile ring. The rectangle area (arrow) of a contractile ring was photobleached. The number at the left corner of each image indicates time after photobleaching (in seconds). (A) A representative series of images of a dividing cell before and after photobleaching. (B) The time course of fluorescence recovery. The curve was generated by fitting. (C) The data of fluorescence density shown in B is plotted as logarithm as described in Materials and methods. Because the data points are scattered around a single line, this reaction is mainly composed of a single enzymatic reaction. The fluorescence recovered with a half time of 7.01 ± 2.62 (n = 16). Bar, 5 μm.
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fig1: FRAP experiments of GFP–myosin II in the contractile ring. The rectangle area (arrow) of a contractile ring was photobleached. The number at the left corner of each image indicates time after photobleaching (in seconds). (A) A representative series of images of a dividing cell before and after photobleaching. (B) The time course of fluorescence recovery. The curve was generated by fitting. (C) The data of fluorescence density shown in B is plotted as logarithm as described in Materials and methods. Because the data points are scattered around a single line, this reaction is mainly composed of a single enzymatic reaction. The fluorescence recovered with a half time of 7.01 ± 2.62 (n = 16). Bar, 5 μm.

Mentions: GFP-tagged myosin II was expressed extrachromosomally in MHC cells as described (Moores et al., 1996). This means that all MHCs in the cells were tagged with GFP. To assess if myosin II turns over in the contractile rings by the FRAP method, a small area of a contractile ring of cells expressing GFP-tagged myosin II was photobleached by scanning laser illumination (Fig. 1) . The fluorescence intensity of the bleached region shows rapid recovery after photobleaching (Fig. 1 B). A half time of recovery (t1/2) was 7.01 ± 2.62 (SD; n = 16) s. The time course of recovery showed a simple exponential rise to a maximum by curve fitting (Fig. 1, B and C), indicating that this process is mainly composed of a single enzymatic reaction.


Myosin II dynamics and cortical flow during contractile ring formation in Dictyostelium cells.

Yumura S - J. Cell Biol. (2001)

FRAP experiments of GFP–myosin II in the contractile ring. The rectangle area (arrow) of a contractile ring was photobleached. The number at the left corner of each image indicates time after photobleaching (in seconds). (A) A representative series of images of a dividing cell before and after photobleaching. (B) The time course of fluorescence recovery. The curve was generated by fitting. (C) The data of fluorescence density shown in B is plotted as logarithm as described in Materials and methods. Because the data points are scattered around a single line, this reaction is mainly composed of a single enzymatic reaction. The fluorescence recovered with a half time of 7.01 ± 2.62 (n = 16). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196877&req=5

fig1: FRAP experiments of GFP–myosin II in the contractile ring. The rectangle area (arrow) of a contractile ring was photobleached. The number at the left corner of each image indicates time after photobleaching (in seconds). (A) A representative series of images of a dividing cell before and after photobleaching. (B) The time course of fluorescence recovery. The curve was generated by fitting. (C) The data of fluorescence density shown in B is plotted as logarithm as described in Materials and methods. Because the data points are scattered around a single line, this reaction is mainly composed of a single enzymatic reaction. The fluorescence recovered with a half time of 7.01 ± 2.62 (n = 16). Bar, 5 μm.
Mentions: GFP-tagged myosin II was expressed extrachromosomally in MHC cells as described (Moores et al., 1996). This means that all MHCs in the cells were tagged with GFP. To assess if myosin II turns over in the contractile rings by the FRAP method, a small area of a contractile ring of cells expressing GFP-tagged myosin II was photobleached by scanning laser illumination (Fig. 1) . The fluorescence intensity of the bleached region shows rapid recovery after photobleaching (Fig. 1 B). A half time of recovery (t1/2) was 7.01 ± 2.62 (SD; n = 16) s. The time course of recovery showed a simple exponential rise to a maximum by curve fitting (Fig. 1, B and C), indicating that this process is mainly composed of a single enzymatic reaction.

Bottom Line: These results indicate that myosin II in the contractile ring performs dynamic turnover via its heavy chain phosphorylation.Because GFP-3ALA myosin II did not show the recovery, it served as a useful marker of myosin II movement, which enabled us to demonstrate cortical flow of myosin II toward the equator for the first time.Thus, cortical flow accompanies the dynamic exchange of myosin II during the formation of contractile rings.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan. yumura@po.cc.yamaguchi-u.ac.jp

ABSTRACT
Myosin II is a major component of a contractile ring. To examine if myosin II turns over in contractile rings, fluorescence of GFP-myosin II expressed in Dictyostelium cells was bleached locally by laser illumination, and the recovery was monitored. The fluorescence recovered with a half time of 7.01 +/- 2.62 s. This recovery was not caused by lateral movement of myosin II from the nonbleached area, but by an exchange with endoplasmic myosin II. Similar experiments were performed in cells expressing GFP-3ALA myosin II, of which three phosphorylatable threonine residues were replaced with alanine residues. In this case, recovery was not detected within a comparable time range. These results indicate that myosin II in the contractile ring performs dynamic turnover via its heavy chain phosphorylation. Because GFP-3ALA myosin II did not show the recovery, it served as a useful marker of myosin II movement, which enabled us to demonstrate cortical flow of myosin II toward the equator for the first time. Thus, cortical flow accompanies the dynamic exchange of myosin II during the formation of contractile rings.

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