Organelle movement in higher animal cells, especially in neurons, is known to be microtubule- based. Using axoplasm from squid giant axons the investigators have found that organelles exhibit, in addition to the well known microtubule- dependent motility, an equally prominent novel type of motility associated with an extensive actin filament network. This actin-associated movement is ATP-dependent, has myosin-like pharmacology, is unidirectional, and is independent of microtubules and microtubule-based motors. These findings show that actin-based organelle motility is driven by a myosin like motor and represent a major component of fast axonal organelle transport. Actin-based motility appears to operate in close association with microtubule-dependent motility to produce and regulate the movement of organelles. The goal of this proposal is to isolate this motor from squid giant axons and optic lobes and characterize its structure, function and regulation. The specific aims are as follows. The investigator will identify and isolate the myosin-like motor by subcellular fractionation of neural tissues from aquid (axoplasm and optic lobes). Experiments will be performed to obtain a purified preparation of active motor protein. After purification of the motor, the investigators will determine its molecular structure, enzymatic properties and ability to move organelles along actin filaments. Using an in vitro reconstituted system for actin-based organelle motility, the investigators will determine the mechanism by which the functional activities of the myosin-like motors are regulated. The investigators propose that calmodulin, via phosphorylation- dephosphorylation reactions, is involved in its regulation. The investigators will study the fine structure of the myosin-like motors and the organelle-motor-microfilament complexes using platinum shadowing, negative contrast and freeze-fracture electron microscopy.
NIH ExPorter
NIH ExPorter