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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–3ALA myosin II in the contractile ring. (A) The circle area (arrow) of a contractile ring was photobleached. (B) Changes of fluorescence intensity in the bleached region. This is a representative figure of observed seven different cells. Note that the fluorescence did not recover. The number at the left corner of each image indicates time after photobleaching (in seconds). The fluorescence of the outside limb of the bleached region increased slightly. A leak from the laser illumination for photobleaching increased the fluorescence of GFP. GFP gene of GFP–3ALA myosin II was the original one from a jellyfish. On the other hand, the GFP gene of GFP-WT myosin II had a S65T mutation and the fluorescence intensity of this S65T GFP did not increase by weak illumination or a leak (Rizzuto et al., 1996). Bar, 5 μm.
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fig6: FRAP experiments of GFP–3ALA myosin II in the contractile ring. (A) The circle area (arrow) of a contractile ring was photobleached. (B) Changes of fluorescence intensity in the bleached region. This is a representative figure of observed seven different cells. Note that the fluorescence did not recover. The number at the left corner of each image indicates time after photobleaching (in seconds). The fluorescence of the outside limb of the bleached region increased slightly. A leak from the laser illumination for photobleaching increased the fluorescence of GFP. GFP gene of GFP–3ALA myosin II was the original one from a jellyfish. On the other hand, the GFP gene of GFP-WT myosin II had a S65T mutation and the fluorescence intensity of this S65T GFP did not increase by weak illumination or a leak (Rizzuto et al., 1996). Bar, 5 μm.

Mentions: FRAP experiments were performed in the cleavage furrow of cells expressing GFP–3ALA myosin II (Fig. 6) . Interestingly, fluorescence of the bleached region did not recover. In a complementary FLIP assay, the fluorescence of all areas except two small cortical areas was bleached in interphase GFP–3ALA myosin II cells (Fig. 7) . The fluorescence of nonbleached areas neither decreased nor diffused into the endoplasm. As observed in wild-type cells, there were not any changes in the distance between the two areas. These observations strongly suggest that the heavy chain phosphorylation is responsible for the turnover of cortical myosin II.


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

Yumura S - J. Cell Biol. (2001)

FRAP experiments of GFP–3ALA myosin II in the contractile ring. (A) The circle area (arrow) of a contractile ring was photobleached. (B) Changes of fluorescence intensity in the bleached region. This is a representative figure of observed seven different cells. Note that the fluorescence did not recover. The number at the left corner of each image indicates time after photobleaching (in seconds). The fluorescence of the outside limb of the bleached region increased slightly. A leak from the laser illumination for photobleaching increased the fluorescence of GFP. GFP gene of GFP–3ALA myosin II was the original one from a jellyfish. On the other hand, the GFP gene of GFP-WT myosin II had a S65T mutation and the fluorescence intensity of this S65T GFP did not increase by weak illumination or a leak (Rizzuto et al., 1996). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: FRAP experiments of GFP–3ALA myosin II in the contractile ring. (A) The circle area (arrow) of a contractile ring was photobleached. (B) Changes of fluorescence intensity in the bleached region. This is a representative figure of observed seven different cells. Note that the fluorescence did not recover. The number at the left corner of each image indicates time after photobleaching (in seconds). The fluorescence of the outside limb of the bleached region increased slightly. A leak from the laser illumination for photobleaching increased the fluorescence of GFP. GFP gene of GFP–3ALA myosin II was the original one from a jellyfish. On the other hand, the GFP gene of GFP-WT myosin II had a S65T mutation and the fluorescence intensity of this S65T GFP did not increase by weak illumination or a leak (Rizzuto et al., 1996). Bar, 5 μm.
Mentions: FRAP experiments were performed in the cleavage furrow of cells expressing GFP–3ALA myosin II (Fig. 6) . Interestingly, fluorescence of the bleached region did not recover. In a complementary FLIP assay, the fluorescence of all areas except two small cortical areas was bleached in interphase GFP–3ALA myosin II cells (Fig. 7) . The fluorescence of nonbleached areas neither decreased nor diffused into the endoplasm. As observed in wild-type cells, there were not any changes in the distance between the two areas. These observations strongly suggest that the heavy chain phosphorylation is responsible for the turnover of cortical myosin II.

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