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A structural model for phosphorylation control of Dictyostelium myosin II thick filament assembly.

Liang W, Warrick HM, Spudich JA - J. Cell Biol. (1999)

Bottom Line: Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II- phenotypes.These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments.The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307, USA.

ABSTRACT
Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II- phenotypes. Here we report a suppressor screen of Dictyostelium myosin II- cells containing 3 x Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control. These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments. The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

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Representative rotary shadowed myosin II molecules in high ionic strength (200 mM KCl). (A) Upper panel: myosin II molecule in the straight conformation. Middle panel: myosin II molecule in a bent conformation. Lower panel: an extreme case of the bent conformation, where the COOH terminus of the tail folds back tightly to make the molecule look shorter. Bar, 0.1 μm. (B) Position of bend in EM images of 3×Asp myosin II monomers. The majority of 3×Asp myosin IIs bend at 1,200 Å, approximately two-thirds length of the tail from the head–neck junction. A second region at ∼1000 Å was also observed. n = 210.
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Figure 4: Representative rotary shadowed myosin II molecules in high ionic strength (200 mM KCl). (A) Upper panel: myosin II molecule in the straight conformation. Middle panel: myosin II molecule in a bent conformation. Lower panel: an extreme case of the bent conformation, where the COOH terminus of the tail folds back tightly to make the molecule look shorter. Bar, 0.1 μm. (B) Position of bend in EM images of 3×Asp myosin II monomers. The majority of 3×Asp myosin IIs bend at 1,200 Å, approximately two-thirds length of the tail from the head–neck junction. A second region at ∼1000 Å was also observed. n = 210.

Mentions: Dictyostelium 3×Asp myosin II molecules were monomeric at high ionic strength. Rotary shadowed 3×Asp myosin II exhibited primarily two conformations under this condition: straight and bent monomers (Fig. 4 A). Various forms of the bent monomers were observed. In 20% of the bent monomer images, the COOH terminus of the tail folded back tightly and resulted in an apparently shorter tail (Fig. 4 A, lower panel). 77% of the 3×Asp myosin II molecules were found to be in the bent conformation (n = 400). On the other hand, only 23% of the wild-type myosin II molecules were found to be bent (n = 280). The percentage of the bent wild-type molecules is consistent with the previous finding that freshly purified wild-type myosin IIs are 20–30% phosphorylated in the heavy chain (Kuczmarski and Spudich 1980).


A structural model for phosphorylation control of Dictyostelium myosin II thick filament assembly.

Liang W, Warrick HM, Spudich JA - J. Cell Biol. (1999)

Representative rotary shadowed myosin II molecules in high ionic strength (200 mM KCl). (A) Upper panel: myosin II molecule in the straight conformation. Middle panel: myosin II molecule in a bent conformation. Lower panel: an extreme case of the bent conformation, where the COOH terminus of the tail folds back tightly to make the molecule look shorter. Bar, 0.1 μm. (B) Position of bend in EM images of 3×Asp myosin II monomers. The majority of 3×Asp myosin IIs bend at 1,200 Å, approximately two-thirds length of the tail from the head–neck junction. A second region at ∼1000 Å was also observed. n = 210.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169343&req=5

Figure 4: Representative rotary shadowed myosin II molecules in high ionic strength (200 mM KCl). (A) Upper panel: myosin II molecule in the straight conformation. Middle panel: myosin II molecule in a bent conformation. Lower panel: an extreme case of the bent conformation, where the COOH terminus of the tail folds back tightly to make the molecule look shorter. Bar, 0.1 μm. (B) Position of bend in EM images of 3×Asp myosin II monomers. The majority of 3×Asp myosin IIs bend at 1,200 Å, approximately two-thirds length of the tail from the head–neck junction. A second region at ∼1000 Å was also observed. n = 210.
Mentions: Dictyostelium 3×Asp myosin II molecules were monomeric at high ionic strength. Rotary shadowed 3×Asp myosin II exhibited primarily two conformations under this condition: straight and bent monomers (Fig. 4 A). Various forms of the bent monomers were observed. In 20% of the bent monomer images, the COOH terminus of the tail folded back tightly and resulted in an apparently shorter tail (Fig. 4 A, lower panel). 77% of the 3×Asp myosin II molecules were found to be in the bent conformation (n = 400). On the other hand, only 23% of the wild-type myosin II molecules were found to be bent (n = 280). The percentage of the bent wild-type molecules is consistent with the previous finding that freshly purified wild-type myosin IIs are 20–30% phosphorylated in the heavy chain (Kuczmarski and Spudich 1980).

Bottom Line: Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II- phenotypes.These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments.The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307, USA.

ABSTRACT
Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II- phenotypes. Here we report a suppressor screen of Dictyostelium myosin II- cells containing 3 x Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control. These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments. The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

Show MeSH
Related in: MedlinePlus