Actin sliding on reconstituted myosin filaments containing only one myosin heavy chain isoform.
We developed a technique to reconstitute myosin filaments containing only one myosin heavy chain (MyHC) isoform. Myosin was extracted from single skinned fibers of rabbit psoas muscle to ensure formation of filaments from only one MyHC isoform. Myosin filaments of up to about 20 microm in length were reconstituted by dialysing the extracted myosin against a buffer of slowly decreasing ionic strength. Length and diameter of the reconstituted myosin filaments were determined by electron microscopy. The reconstituted filaments were very heterogeneous in length, filament diameter was found to increase with length. The reconstituted myosin filaments were found to be functionaly bipolar like native thick filaments. Actin sliding towards the center of a reconstituted myosin filament occurred at 6.2 microm/s. Away from the center of these myosin filaments, i.e., in the unphysiological direction, actin-sliding velocity was found to be only 1.5 microm/s. We used these reconstituted myosin filaments to test whether ordered orientation and a more physiological environment for myosin molecules within reconstituted filaments can explain our previous finding that sliding velocity of actin filaments in in vitro motility assays with randomly attached myosin molecules extracted from single fibers is 4-8-fold slower than unloaded shortening velocity in muscle fibers even when experimental conditions and MyHC isoforms are identical (Thedinga E et al., (1999) J Muscle Res Cell Motil 20(8): 785-796).
Scholz
Tim
T
Medizinische Hochschule Hannover, Molekular- und Zellphysiologie, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
Brenner
Bernhard
B
D008841:Actin Cytoskeleton / Q000502:physiology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D007700:Kinetics; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D018995:Myosin Heavy Chains / Q000502:physiology* / Q000648:ultrastructure; D009218:Myosins / Q000302:isolation & purification / Q000502:physiology* / Q000648:ultrastructure; D010590:Phalloidine / Q000031:analogs & derivatives* / Q000737:chemistry; D011485:Protein Binding; D016658:Psoas Muscles / Q000737:chemistry; D011817:Rabbits; D012235:Rhodamines / Q000737:chemistry
D000911:Antibodies, Monoclonal; D012235:Rhodamines; D010590:Phalloidine; C041085:tetramethylrhodaminylphalloidine; D018995:Myosin Heavy Chains; D009218:Myosins; D016650:Fluorescein-5-isothiocyanate