Novel mode of cooperative binding between myosin and Mg2+ -actin filaments in the presence of low concentrations of ATP.
Cooperative interaction between myosin and actin filaments has been detected by a number of different methods, and has been suggested to have some role in force generation by the actomyosin motor. In this study, we observed the binding of myosin to actin filaments directly using fluorescence microscopy to analyze the mechanism of the cooperative interaction in more detail. For this purpose, we prepared fluorescently labeled heavy meromyosin (HMM) of rabbit skeletal muscle myosin and Dictyostelium myosin II. Both types of HMMs formed fluorescent clusters along actin filaments when added at substoichiometric amounts. Quantitative analysis of the fluorescence intensity of the HMM clusters revealed that there are two distinct types of cooperative binding. The stronger form was observed along Ca(2+)-actin filaments with substoichiometric amounts of bound phalloidin, in which the density of HMM molecules in the clusters was comparable to full decoration. The novel, weaker form was observed along Mg(2+)-actin filaments with and without stoichiometric amounts of phalloidin. HMM density in the clusters of the weaker form was several-fold lower than full decoration. The weak cooperative binding required sub-micromolar ATP, and did not occur in the absence of nucleotides or in the presence of ADP and ADP-Vi. The G680V mutant of Dictyostelium HMM, which over-occupies the ADP-Pi bound state in the presence of actin filaments and ATP, also formed clusters along Mg(2+)-actin filaments, suggesting that the weak cooperative binding of HMM to actin filaments occurs or initiates at an intermediate state of the actomyosin-ADP-Pi complex other than that attained by adding ADP-Vi.
Tokuraku
Kiyotaka
K
Department of Chemical Science and Engineering, Miyakonojo National College of Technology, 473-1 Yoshio-cho, Miyakonojo-shi, Miyazaki 885-8567, Japan. tokuraku@miyakonojo-nct.ac.jp
Kurogi
Rika
R
Toya
Ryo
R
Uyeda
Taro Q P
TQ
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004023:Dictyostelium; D008274:Magnesium / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D010590:Phalloidine / Q000378:metabolism; D011817:Rabbits
D015879:Myosin Subfragments; D010590:Phalloidine; D000255:Adenosine Triphosphate; D009218:Myosins; D008274:Magnesium