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

摘要

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 tissue extracts, In this initial study, silica beads coated with an antibody that specifically recognizes the tail of myosin-V were used to immunoadsorb 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 motility using high resolution video DIC microscopy. The addition of brush border spectrin to the motility chamber enabled the growth of stable actin filament tracks that were similar to 4-fold longer than filaments grown in the absence of this actin crosslinking protein, The velocity of myosin-V immunoadsorbed from brain extracts was similar to that observed for purified myosin-V that was antibody-linked to beads or assessed 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 motor-containing head domain of myosin-V, Myosin-V motility was most robust in the absence of Ca2+. Interestingly, skeletal muscle tropomyosin and brush border spectrin had no detectable effect on myosin-Ti mechanochemistry. Myosin-V containing beads were also occasionally observed migrating directly on acrosomal processes in the absence of exogenously added actin, The use of laser tweezers to trap and position particles on the elongate actin filament tracks should expedite mechanochemical analysis of novel immunoadsorbed myosins that resist isolation using preparative techniques. Moreover, this assay should facilitate studies of myosin-dependent organelle/vesicle motility. [References: 49]