Reversible Inhibition of Endocytosis in Cultured Neurons from the Drosophila Temperature-Sensitive Mutant Shibire

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Abstract
The Drosophila mutant, shibirets1 (shits1), is paralyzed at restrictive temperatures (> 29oC) by a reversible block in synaptic transmission. Heat pulses deplete synaptic vesicles in nerve terminals and inhibit endocytic internalization of plasma membrane in garland cells and oocytes. In dissociated cultures of larval central nervous system (CNS), a temperature-sensitive defect is also expressed in shits1 neurons: at 30oC, growth cone formation is retarded and neurite outgrowth is arrested. We now report that we have examined constitutive endocytosis in Drosophila CNS culture and have demonstrated directly an endocytic defect in shits1 neurons. At the permissive temperature, 20-22oC, both shits1and wild-type neurons actively endocytosed fluorescein-labelled dextran (40KD, 5%) or horseradish peroxidase (HRP, 1%). Within 5 min, HRP was seen in vesicles, cup-shaped bodies, tubules and multivesicular bodies in neurites and cell bodies. In contrast, endocytosis was inhibited in cultures derived from the temperature-sensitive paralytic shi by a 15 min heat pulse (30oC). Even after 30 min of HRP exposure at 30oC, HRP-containing membranes were absent from almost all shits1 neurites; a minority of cell bodies had a few HRP-containing vesicles. The temperature-dependent block in endocytosis was readily reversed at 20oC. Interestingly, the block was overcome by high concentration of external cations: shits1 neurons in culture actively took up HRP in numerous vesicles at 30oC if 18mM Ca2+ or Mg2 was added to the medium. Our results support the notion that membrane recycling plays a critical role in regulating neurite outgrowth. This study also provides baseline information for further mutational analysis of the mechanism underlying the membrane cycling process in cultured neurons.