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-coat ed 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 SP-LMM hinge, and th e other at the C terminus of the myosin rod. These monoclonal antibodi es were used to provide increasing flexibility in the mode of attachme nt. Fast skeletal muscle myosin monomers were bound to the surfaces th rough the specific interaction with these monoclonal antibodiess and t he 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 cour se of several hours. Attachment of myosin through the anti-S2 and anti -LMM monoclonal antibodies yielded a maximum velocity of 10 mu m/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower vel ocity of 4-5 mu m/s. Each antibody showed a characteristic minimum myo sin density below which sliding movement was no longer supported and a n exponential dependence of actin filament velocity on myosin surface density below V-max. Maximum sliding velocity was achieved over a rang e of myosin surface densities. Thus, the specific mode of attachment c an influence the characteristic velocity of actin filament movement an d 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.