Motility assays using myosin attached to surfaces through specific binding to monoclonal antibodies.
We have analyzed the dependence of actin filament movement on the mode of myosin attachment to surfaces. Monoclonal antibodies 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 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. These monoclonal antibodies were used to provide increasing flexibility in the mode of attachment. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these monoclonal antibodies and the sliding movement of fluorescently labeled actin filaments analyzed by video microscopy. Each of these antibodies produced stable, myosin-coated surfaces that supported uniform movement of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM monoclonal antibodies yielded a maximum velocity of 10 microns/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower velocity of 4-5 microns/s. Each antibody showed a characteristic minimum myosin density below which sliding movement was no longer supported and an exponential dependence of actin filament velocity on myosin surface density below Vmax. Maximum sliding velocity was achieved over a range of myosin surface densities. Thus, the specific mode of attachment can influence the characteristic velocity of actin filament movement and the surface density needed to support movement. These data are being used to analyze the dynamics of sliding filament assays and evaluate estimates of the average number of motor molecules per unit length of actin required to support movement.
Winkelmann
D A
DA
Department of Pathology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08540, USA.
Bourdieu
L
L
Kinose
F
F
Libchaber
A
A
D000199:Actins / Q000737:chemistry / Q000502:physiology; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D066298:In Vitro Techniques; D007700:Kinetics; D009068:Movement / Q000502:physiology; D009218:Myosins / Q000737:chemistry* / Q000276:immunology / Q000502:physiology; D013499:Surface Properties
D000199:Actins; D000911:Antibodies, Monoclonal; D009218:Myosins