|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1998 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1997 : ¥1,800,000 (Direct Cost : ¥1,800,000)
1) Growth cones are considered as the precursor of presynaptic terminals. To elucidate the minimal requirement of exocytotic molecular machinery, we examined the characteristics of exocytosis in growth cones using alpha-latrotoxin (alpha-LTX). The major biochemical difference between the adult presynptic terminal (SPS) and the isolated growth cone (IGC) was that rab3A was converted to GTP-form in the former, but remained GDP-form in the latter. We found that rabphilin was localized in much smaller amounts in IGC than in SPS.Following a supply of rabphilin, the IGC obtained as highly alpha-LTX-sensitivity as SPS, and the GDP-GTP conversion attached to rab3A on the GCV.In native IGCs, the GCVs had the SNAREs but riot NSF and alpha- SNAP ; while in the rabphilin-supplied IGC, the GCVs recruited NSF and alpha-SNAP, and the SNARE-NSF-SNAP complex was formed on the GCVs. Our results suggest that rab3A cycling develops later than the SNARE by accumulation of rabphilin, and that it is involved in determining the efficiency of exocytosis forced by alpha-LTX.
2) Although growth cones responds to various modulators of neurite outgrowth, the signal-transducing mechanisms for these modulators in growth cones are unclear. I examined L-type VSCC-dependent signaling p-athways. In intact IGCs, Bay K 8644 (BK) induced much more rapid elevation of [Ca^<2+>] than that in SPS.Ca^<2+>-dependent phosphorylation of GAP-43 and MAROKS protein by protein kinase C (PKC) was enhanced in the JGC by BK, resulting in the release of these proteins from the membrane, which is consistent with our recent report. In addition, the Ca^<2+>-dependent degradation of brain spectrin by calpain was also enhanced by BK or GABA, consequently inducing the release of alpha-actinin from the membrane skeleton of growth cones. The activities of PKC and calpain were not inhibited by inhibitors of the other.