This project determines the structure of neuronal and glial cytoplasm, particularly as it pertains to axoplasmic transport, and the organization of the cytoplasm. Cultured myocytes, grown on grids, frozen, freeze-substituted, and examined directly at high voltages in an electronmicroscope have a cytoplasmic ground substance consisting of fine filaments instead of a microtubular meshwork, and distinct cytoplasmic domains characterized by different types of organelle movements. Filaments are isolated from the axoplasm of the squid giant axon along which organelles continue to move for many hours. Filaments previously observed with the video microscope and then examined in the electronmicroscope turn out to be single microtubules. However, similar filaments in certain cells are made of actin, so organelle movements may be actin based in some locations and microtubule based in others. Progress has been made towards characterizing the translocators for these organelle movements. Treatment of latex beads with a crude extract of squid brain induces the beads to move along microtubules. A 700 KD protein with 110 KD and 60-65 KD doublet peptides which was then purified moves beads along microtubules. A monoclonal antibody column (directed towards the 110 Kd subunit) was also used to purify this translocater. Based on its size and pharmacological properties this translocation is neither a dynein nor a myosin, so we have defined a new class of motility protein which we call kinesin. Kinesin appears to be of general significance in cellular motility. However, organelle movement induced by kinesin only translocates in a direction corresponding to anterograde axonal transport. However, brain extracts stripped of kinesin with antibody induce bead movement in the retrograde direction. Our current efforts are concentrated on purifying the retrograde direction. Our current efforts are concentrated on purifying retrograde translocator and determining whether it is selectively bound to some organelles.
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