This project determines the structure of neuronal and glial cytoplasm, particularly as it pertains to axoplasmic transport, and the organization of 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 organelle movements along microtubules. Microtubules isolated from the axoplasm of the squid giant axon continue to support movements of organelles for many hours. However, actin filaments in other cells support similar movements of organelles. A protein translocator responsible for these organelle movements has been characterized, a 600 KD protein with 110 KD and 60-65 KD doublet peptides. This protein induces beads to move along purified microtubules in the presence of ATP. When a monoclonal antibody column (directed towards the 110 KD subunit) is used to purify this translocator it has the same peptide components and supports similar movements. Based on its size, pharmacological properties, and asymetrical shape seen by molecular shadowing, this translocator protein belongs to a new class of motility protein, which we call kinesin. Kinesin appears to be of general significance in cellular motility. The organelle movements, induced by kinesin are always directed towards the plus ends of microtubules, a direction corresponding to anterograde axonal transport. However, brain extracts stripped of kinesin with monoclonal antibody induce bead movement in the retrograde direction. Our current efforts are concentrated on purifying the translocator for retrograde movements and on determining whether organelles selectively bind translocators in order to explain how they are selected for anterograde or retrograde transport.
| Galbraith, Catherine G; Yamada, Kenneth M; Galbraith, James A (2007) Polymerizing actin fibers position integrins primed to probe for adhesion sites. Science 315:992-5 |
| Colina, Claudia; Rosenthal, Joshua J C; DeGiorgis, Joseph A et al. (2007) Structural basis of Na(+)/K(+)-ATPase adaptation to marine environments. Nat Struct Mol Biol 14:427-31 |
| Satpute-Krishnan, Prasanna; DeGiorgis, Joseph A; Conley, Michael P et al. (2006) A peptide zipcode sufficient for anterograde transport within amyloid precursor protein. Proc Natl Acad Sci U S A 103:16532-7 |
| DeGiorgis, Joseph A; Reese, Thomas S; Bearer, Elaine L (2002) Association of a nonmuscle myosin II with axoplasmic organelles. Mol Biol Cell 13:1046-57 |
| Galbraith, J A; Reese, T S; Schlief, M L et al. (1999) Slow transport of unpolymerized tubulin and polymerized neurofilament in the squid giant axon. Proc Natl Acad Sci U S A 96:11589-94 |
