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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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3ALA myosin II filaments localized at the cortex. GFP–3ALA myosin II cells were fixed and observed by a confocal laser microscope. Serial optical sections from the bottom (A) to the top (F). Thickness of each section was ∼0.3 μm, and the interval was 0.3 μm. Myosin II filaments were observed only along the cortex. The large fluorescent spot near the center, which was an aggregate of filaments, was frequently observed in 3ALA myosin II cells (Egelhoff et al., 1993). Bar, 5 μm.
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fig5: 3ALA myosin II filaments localized at the cortex. GFP–3ALA myosin II cells were fixed and observed by a confocal laser microscope. Serial optical sections from the bottom (A) to the top (F). Thickness of each section was ∼0.3 μm, and the interval was 0.3 μm. Myosin II filaments were observed only along the cortex. The large fluorescent spot near the center, which was an aggregate of filaments, was frequently observed in 3ALA myosin II cells (Egelhoff et al., 1993). Bar, 5 μm.

Mentions: It was suggested that the localization of myosin II to the cortex requires the dephosphorylation of heavy chains because 3ASP mutant myosin II, carrying aspartate residues in place of phosphorylatable threonine residues, does not localize to the cortex but disperses in the endoplasm (Egelhoff et al., 1993). Conversely, 3ALA mutant myosin II localizes only to the cortex (Yumura and Uyeda, 1997a). Confocal microscopy of cells expressing the GFP-version of this mutant myosin confirmed its cortical localization by serial optical sectioning (Fig. 5) .


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

Yumura S - J. Cell Biol. (2001)

3ALA myosin II filaments localized at the cortex. GFP–3ALA myosin II cells were fixed and observed by a confocal laser microscope. Serial optical sections from the bottom (A) to the top (F). Thickness of each section was ∼0.3 μm, and the interval was 0.3 μm. Myosin II filaments were observed only along the cortex. The large fluorescent spot near the center, which was an aggregate of filaments, was frequently observed in 3ALA myosin II cells (Egelhoff et al., 1993). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: 3ALA myosin II filaments localized at the cortex. GFP–3ALA myosin II cells were fixed and observed by a confocal laser microscope. Serial optical sections from the bottom (A) to the top (F). Thickness of each section was ∼0.3 μm, and the interval was 0.3 μm. Myosin II filaments were observed only along the cortex. The large fluorescent spot near the center, which was an aggregate of filaments, was frequently observed in 3ALA myosin II cells (Egelhoff et al., 1993). Bar, 5 μm.
Mentions: It was suggested that the localization of myosin II to the cortex requires the dephosphorylation of heavy chains because 3ASP mutant myosin II, carrying aspartate residues in place of phosphorylatable threonine residues, does not localize to the cortex but disperses in the endoplasm (Egelhoff et al., 1993). Conversely, 3ALA mutant myosin II localizes only to the cortex (Yumura and Uyeda, 1997a). Confocal microscopy of cells expressing the GFP-version of this mutant myosin confirmed its cortical localization by serial optical sectioning (Fig. 5) .

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
Related in: MedlinePlus