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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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Cortical flow of 3ALA myosin II. (A) The equatorial region that would be constricted later was photobleached. (B) The profiles of the relative fluorescence intensity within boxed areas in the images of A were shown. The gray lines were drawn to pass each of two peaks at 0:00 and 236:22 s. The fluorescence within the boxed areas gradually moved toward the equator after photobleaching. This is a representative figure of observed 12 different cells. The movie version of this experiment can be seen at http://www.jcb.org/cgi/content/full/200011013/DC1. Bar, 5 μm.
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fig9: Cortical flow of 3ALA myosin II. (A) The equatorial region that would be constricted later was photobleached. (B) The profiles of the relative fluorescence intensity within boxed areas in the images of A were shown. The gray lines were drawn to pass each of two peaks at 0:00 and 236:22 s. The fluorescence within the boxed areas gradually moved toward the equator after photobleaching. This is a representative figure of observed 12 different cells. The movie version of this experiment can be seen at http://www.jcb.org/cgi/content/full/200011013/DC1. Bar, 5 μm.

Mentions: The fluorescence recovery of GFP-WT myosin II cells was too rapid to trace the bleached region even if myosin II filaments moved along the cortex. The property of nonrecovery of GFP–3ALA myosin II cells after photobleaching made it possible to mark the cortical myosin II and to observe it for a longer period. When parts of a cleavage region in early telophase were bleached, the unbleached cortical fluorescence flanking the furrow moved toward the equator (Fig. 9) . Because 3ALA myosin II was in filamentous form in the cortex as observed in Fig. 5, it is concluded that myosin II filaments move toward the equator along the cortex. This is the first direct evidence that cortical myosin II flows toward the equator in Dictyostelium. The rate of the flow of individual filaments might not be uniform because slight increase in fluorescence in the central part of the bleached region occurred preceding the flow of major fluorescence toward the equator. However, they still moved toward the equator as a mass.


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

Yumura S - J. Cell Biol. (2001)

Cortical flow of 3ALA myosin II. (A) The equatorial region that would be constricted later was photobleached. (B) The profiles of the relative fluorescence intensity within boxed areas in the images of A were shown. The gray lines were drawn to pass each of two peaks at 0:00 and 236:22 s. The fluorescence within the boxed areas gradually moved toward the equator after photobleaching. This is a representative figure of observed 12 different cells. The movie version of this experiment can be seen at http://www.jcb.org/cgi/content/full/200011013/DC1. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Cortical flow of 3ALA myosin II. (A) The equatorial region that would be constricted later was photobleached. (B) The profiles of the relative fluorescence intensity within boxed areas in the images of A were shown. The gray lines were drawn to pass each of two peaks at 0:00 and 236:22 s. The fluorescence within the boxed areas gradually moved toward the equator after photobleaching. This is a representative figure of observed 12 different cells. The movie version of this experiment can be seen at http://www.jcb.org/cgi/content/full/200011013/DC1. Bar, 5 μm.
Mentions: The fluorescence recovery of GFP-WT myosin II cells was too rapid to trace the bleached region even if myosin II filaments moved along the cortex. The property of nonrecovery of GFP–3ALA myosin II cells after photobleaching made it possible to mark the cortical myosin II and to observe it for a longer period. When parts of a cleavage region in early telophase were bleached, the unbleached cortical fluorescence flanking the furrow moved toward the equator (Fig. 9) . Because 3ALA myosin II was in filamentous form in the cortex as observed in Fig. 5, it is concluded that myosin II filaments move toward the equator along the cortex. This is the first direct evidence that cortical myosin II flows toward the equator in Dictyostelium. The rate of the flow of individual filaments might not be uniform because slight increase in fluorescence in the central part of the bleached region occurred preceding the flow of major fluorescence toward the equator. However, they still moved toward the equator as a mass.

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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