Flexibility of myosin attachment to surfaces influences F-actin motion.
We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachment to surfaces. Monoclonal antibodies (mAbs) that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain (LC2) located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. This method of attachment provides a means of controlling the flexibility and density of myosin on the surface. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these mAbs, and the sliding movement of fluorescently labeled actin filaments was analyzed by video microscopy. Each of these antibodies produced stable myosin-coated surfaces that supported uniform motion of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM mAbs yielded significantly higher velocities (10 microns/s at 30 degrees C) than attachment through anti-LC2 (4-5 microns/s at 30 degrees C). For each antibody, we observed a characteristic value of the myosin density for the onset of F-actin motion and a second critical density for velocity saturation. The specific mode of attachment influences the velocity of actin filaments and the characteristic surface density needed to support movement.
Winkelmann
D A
DA
Department of Pathology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Bourdieu
L
L
Ott
A
A
Kinose
F
F
Libchaber
A
A
D000199:Actins / Q000737:chemistry* / Q000502:physiology; D000327:Adsorption; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002645:Chickens; D003101:Collodion; D000939:Epitopes / Q000032:analysis; D005898:Glass; D007150:Immunohistochemistry; D007700:Kinetics; D008961:Models, Structural; D009068:Movement; D018482:Muscle, Skeletal / Q000502:physiology*; D009218:Myosins / Q000737:chemistry* / Q000502:physiology; D013816:Thermodynamics; D014741:Video Recording
D000199:Actins; D000911:Antibodies, Monoclonal; D000939:Epitopes; D003101:Collodion; D009218:Myosins