MICROTUBULE DYNAMICS IN A CYTOSOLIC EXTRACT OF FETAL-RAT BRAIN

Brain microtubule dynamics were studied by video-enhanced differential interference contrast microscopy in a cytosolic extract from fetal ra t brain, prepared under conditions designed to produce minimal alterat ions in microtubule stability. With urchin sperm axoneme fragments as nucleation seeds, the extract was shown to support cellular-like micro tubule dynamics. Microtubules elongated from one end of the axonemes, and did not spontaneously self-assemble in the absence of axonemes. Th e following microtubule kinetic parameters were measured in the extrac t: velocity of elongation (8.1 mm/min), velocity of rapid shortening ( 5.8 mm/min), catastrophe frequency (0.17 min(-1)), and rescue frequenc y (1 min(-1)). These parameters were in close agreement with reported values for growth cones of living neurons. Microtubule properties in t he fetal brain extract were shown to be affected by agents with known effects on the cytoskeleton. pp60(c-src), a tyrosine kinase important in cell adhesion molecule-dependent axon growth, caused small increase s in the frequency of microtubule catastrophe (0.31 min(-1)) and rescu e (2 min(-1)) without changing the velocities of elongation or rapid-s hortening. Although pp60(c-src) phosphorylated purified porcine brain tubulin in vitro, it did not elicit significant changes in its polymer ization properties, suggesting that other cytoskeletal proteins in the brain extract are involved in modulating microtubule dynamics. The cy tosolic extract of fetal rat brain provides a useful system for studyi ng regulation of microtubule assembly in neuronal growth cones.