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It has been suggested that the membrane recycling process in the Drosophila mutant shibirets1 (shits1) is reversibly blocked at restrictive temperature (greater than 29 degrees C) in various cell types, including neurons, as a result of enhanced vesicle fusion and disrupted membrane pinch-off (Poodry and Edgar, 1979; Koenig et al., 1983; Kosaka and Ikeda, 1983a, b). We analyzed the neuronal development of shits1 cells at the single-cell level by using dissociated larval CNS culture. Compared to normal cultures, there was a substantial reduction in neuronal adhesion to the substratum and a retardation of growth cone formation in shits1 cultures at 30 degrees C. Time-lapse studies showed that neurite initiation and elongation in shits1 cultures were suppressed at the restrictive temperature. Differentiated shits1 neurons exhibited accelerated reabsorption and retarded arborization of neurites at 30 degrees C. The above processes were reversible since normal outgrowth was restored when the temperature was lowered. These findings provide a new line of evidence that growth cone activity determines neurite initiation, elongation, and branching. Furthermore, if the primary defect of the shits1 mutation indeed resides in a membrane recycling mechanism common to different cell types, our results would also lend strong support to the notion that membrane recycling is crucial in the regulation of neuronal membrane adhesion and growth cone activity.

Reversible blockage of neurite development and growth cone formation in neuronal cultures of a temperature-sensitive mutant of Drosophila