IN-VITRO MOTILITY OF IMMUNOADSORBED BRAIN MYOSIN-V USING A LIMULUS ACROSOMAL PROCESS AND OPTICAL TWEEZER-BASED ASSAY

To facilitate functional studies of novel myosins, we have developed a strategy for characterizing the mechanochemical properties of motors isolated by immunoadsorption directly from small amounts of crude tiss ue extracts, In this initial study, silica beads coated with an antibo dy that specifically recognizes the tail of myosin-V were used to immu noadsorb this motor protein from brain extracts, The myosin-containing beads were then positioned with optical tweezers onto actin filaments nucleated from Limulus sperm acrosomal processes and observed for mot ility using high resolution video DIC microscopy. The addition of brus h border spectrin to the motility chamber enabled the growth of stable actin filament tracks that were similar to 4-fold longer than filamen ts grown in the absence of this actin crosslinking protein, The veloci ty of myosin-V immunoadsorbed from brain extracts was similar to that observed for purified myosin-V that was antibody-linked to beads or as sessed using the sliding actin filament assay, Motile beads containing myosin-V immunoadsorbed from brain extracts bound poorly to nucleated actin filaments and were incapable of linear migrations following the addition of a different antibody that specifically recognizes the mot or-containing head domain of myosin-V, Myosin-V motility was most robu st in the absence of Ca2+. Interestingly, skeletal muscle tropomyosin and brush border spectrin had no detectable effect on myosin-Ti mechan ochemistry. Myosin-V containing beads were also occasionally observed migrating directly on acrosomal processes in the absence of exogenousl y added actin, The use of laser tweezers to trap and position particle s on the elongate actin filament tracks should expedite mechanochemica l analysis of novel immunoadsorbed myosins that resist isolation using preparative techniques. Moreover, this assay should facilitate studie s of myosin-dependent organelle/vesicle motility.