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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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Phosphorylation-dependent release of myosin II from Triton ghosts. After 1 mM ATP was added to Triton ghosts, released myosin II from Triton ghosts was quantitated. Wild-type myosin II was readily released (○), but most of 3ALA myosin II (•) was not. The bars indicate phosphorylation of released heavy chains of wild-type myosin II. Each plot was an average of two experiments. 20% of 3ALA myosin II was released. This release was due to a partial disruption of Triton ghosts during contraction (Yumura, 1991).
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fig8: Phosphorylation-dependent release of myosin II from Triton ghosts. After 1 mM ATP was added to Triton ghosts, released myosin II from Triton ghosts was quantitated. Wild-type myosin II was readily released (○), but most of 3ALA myosin II (•) was not. The bars indicate phosphorylation of released heavy chains of wild-type myosin II. Each plot was an average of two experiments. 20% of 3ALA myosin II was released. This release was due to a partial disruption of Triton ghosts during contraction (Yumura, 1991).

Mentions: To gain further support for the hypothesis that the translocation of myosin II from the cortex to the endoplasm requires heavy chain phosphorylation, Triton X-100 cytoskeleton assays were performed. Triton X-100–insoluble cytoskeletons (Triton ghosts) contract upon the addition of 1 mM ATP (Yumura, 1991). At this time, almost all of myosin II associated with the cytoskeletons was released into the supernatant fraction (Fig. 8 , ○). In accordance with the release, MHCs were phosphorylated (Fig. 8, bars). On the contrary, most of 3ALA myosin II was not released (Fig. 8, •), although Triton ghosts of 3ALA cells could contract upon the addition of ATP. Similar experiments were performed by Egelhoff et al. (1993), in which ATP was added to the cells together with Triton X-100. In this case, it was unclear which fraction, the supernatant or Triton ghosts, contained the kinase activity. The present experiments indicate that the heavy chain kinase responsible for the release of myosin II was associated with Triton ghosts. Since the Triton ghosts are mainly composed of cortical cytoskeletons, the release of myosin II from the Triton ghosts represents its translocation toward the endoplasm (Yumura, 1991). Similar results were obtained from the previous experiments using membrane–cytoskeleton complexes, in which myosin II is released from them by the treatment with ATPγS as substrate for phosphorylation, followed by the addition of ITP as an energy source for myosin motor activity (Yumura and Kitanishi-Yumura, 1992, 1993). Thus, these in vitro experiments also support the model that the translocation of myosin II from the cortex to the endoplasm requires the phosphorylation of heavy chains.


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

Yumura S - J. Cell Biol. (2001)

Phosphorylation-dependent release of myosin II from Triton ghosts. After 1 mM ATP was added to Triton ghosts, released myosin II from Triton ghosts was quantitated. Wild-type myosin II was readily released (○), but most of 3ALA myosin II (•) was not. The bars indicate phosphorylation of released heavy chains of wild-type myosin II. Each plot was an average of two experiments. 20% of 3ALA myosin II was released. This release was due to a partial disruption of Triton ghosts during contraction (Yumura, 1991).
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Phosphorylation-dependent release of myosin II from Triton ghosts. After 1 mM ATP was added to Triton ghosts, released myosin II from Triton ghosts was quantitated. Wild-type myosin II was readily released (○), but most of 3ALA myosin II (•) was not. The bars indicate phosphorylation of released heavy chains of wild-type myosin II. Each plot was an average of two experiments. 20% of 3ALA myosin II was released. This release was due to a partial disruption of Triton ghosts during contraction (Yumura, 1991).
Mentions: To gain further support for the hypothesis that the translocation of myosin II from the cortex to the endoplasm requires heavy chain phosphorylation, Triton X-100 cytoskeleton assays were performed. Triton X-100–insoluble cytoskeletons (Triton ghosts) contract upon the addition of 1 mM ATP (Yumura, 1991). At this time, almost all of myosin II associated with the cytoskeletons was released into the supernatant fraction (Fig. 8 , ○). In accordance with the release, MHCs were phosphorylated (Fig. 8, bars). On the contrary, most of 3ALA myosin II was not released (Fig. 8, •), although Triton ghosts of 3ALA cells could contract upon the addition of ATP. Similar experiments were performed by Egelhoff et al. (1993), in which ATP was added to the cells together with Triton X-100. In this case, it was unclear which fraction, the supernatant or Triton ghosts, contained the kinase activity. The present experiments indicate that the heavy chain kinase responsible for the release of myosin II was associated with Triton ghosts. Since the Triton ghosts are mainly composed of cortical cytoskeletons, the release of myosin II from the Triton ghosts represents its translocation toward the endoplasm (Yumura, 1991). Similar results were obtained from the previous experiments using membrane–cytoskeleton complexes, in which myosin II is released from them by the treatment with ATPγS as substrate for phosphorylation, followed by the addition of ITP as an energy source for myosin motor activity (Yumura and Kitanishi-Yumura, 1992, 1993). Thus, these in vitro experiments also support the model that the translocation of myosin II from the cortex to the endoplasm requires the phosphorylation of heavy chains.

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