Sequential appearance of cytoskeletal components during the early stages of neurite outgrowth from cerebellar granule cells in vitro.

Cerebellar granule cells derived from one-week-old rat pups have been placed into culture to examine the expression of microfilament, microtubular and neurofilament proteins in developing neurites up to 2 days in vitro. Immunofluorescence and rhodamine-phalloidin studies have shown that the first cytoskeletal element to appear in the developing neurite was the microfilament network which was present in cell processes and expansions from at least 8 minutes in vitro. Rhodamine-phalloidin fluorescence was maintained in the developing neurite over 2 days and was enriched in some growth cones. At between 30 and 60 min in culture microtubules (reacting with anti alpha-tubulin antibodies) appeared in the neurites but did not ramify throughout the growth cone at this or later time points. The expression of tyrosylated alpha-tubulin in microtubules of the developing neurite appeared to precede that of acetylated alpha-tubulin. Only one of the neurofilament subunits, the 200 kDa subunit, could be positively identified in granule cell processes in these cultures. The developmental pattern of cytoskeletal protein expression in granule cell neurites in vitro may reflect a process of stabilisation of the growing neurite behind an active, motile growth cone.