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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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Photobleaching of a whole contractile ring. The fluorescence of a whole contractile ring region was bleached, but recovery was still observed. Note that the fluorescence intensity in the area except the contractile ring decreased after photobleaching. The number at the left corner of each image indicates time after photobleaching (in seconds). This is a representative figure of observed eight different cells. Bar, 5 μm.
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fig3: Photobleaching of a whole contractile ring. The fluorescence of a whole contractile ring region was bleached, but recovery was still observed. Note that the fluorescence intensity in the area except the contractile ring decreased after photobleaching. The number at the left corner of each image indicates time after photobleaching (in seconds). This is a representative figure of observed eight different cells. Bar, 5 μm.

Mentions: To examine from where the myosin II recovered in the bleached region came, two rectangular regions parallel to the cleavage axis were bleached, as shown in Fig. 2 . If the fluorescence intensity of the middle region between these two bleached regions decreases in accordance with the fluorescence recovery in the bleached regions, then it will suggest that myosin II moves along the cortex rapidly. Otherwise, the recovered myosin II must be derived from the endoplasm to be exchanged with the existing myosin II. The fluorescence intensity of the middle region did not show any decrease (Fig. 2 B). Furthermore, the fluorescence intensity of any region within the bleached areas increased at the same rate. If the fluorescence intensity of the limb region of bleached areas increased faster than the inner region, it could indicate that myosin II slid along the cortex. Therefore, the recovered myosin II did not slide from a neighbor cortex but came from the endoplasm. The fluorescence recovery was observed even after photobleaching of a whole contractile ring (Fig. 3) , further supporting this possibility.


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

Yumura S - J. Cell Biol. (2001)

Photobleaching of a whole contractile ring. The fluorescence of a whole contractile ring region was bleached, but recovery was still observed. Note that the fluorescence intensity in the area except the contractile ring decreased after photobleaching. The number at the left corner of each image indicates time after photobleaching (in seconds). This is a representative figure of observed eight different cells. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Photobleaching of a whole contractile ring. The fluorescence of a whole contractile ring region was bleached, but recovery was still observed. Note that the fluorescence intensity in the area except the contractile ring decreased after photobleaching. The number at the left corner of each image indicates time after photobleaching (in seconds). This is a representative figure of observed eight different cells. Bar, 5 μm.
Mentions: To examine from where the myosin II recovered in the bleached region came, two rectangular regions parallel to the cleavage axis were bleached, as shown in Fig. 2 . If the fluorescence intensity of the middle region between these two bleached regions decreases in accordance with the fluorescence recovery in the bleached regions, then it will suggest that myosin II moves along the cortex rapidly. Otherwise, the recovered myosin II must be derived from the endoplasm to be exchanged with the existing myosin II. The fluorescence intensity of the middle region did not show any decrease (Fig. 2 B). Furthermore, the fluorescence intensity of any region within the bleached areas increased at the same rate. If the fluorescence intensity of the limb region of bleached areas increased faster than the inner region, it could indicate that myosin II slid along the cortex. Therefore, the recovered myosin II did not slide from a neighbor cortex but came from the endoplasm. The fluorescence recovery was observed even after photobleaching of a whole contractile ring (Fig. 3) , further supporting this possibility.

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.

Show MeSH