Ranjan R, Bronk P, Zinsmaier K E
Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6074, USA.
J Neurosci. 1998 Feb 1;18(3):956-64. doi: 10.1523/JNEUROSCI.18-03-00956.1998.
The entire deletion of the cysteine string protein (CSP) gene causes a temperature-sensitive (ts) block of evoked neurotransmission in Drosophila. CSP has been found to interact in vitro with the clathrin-uncoating ATPase HSC70, suggesting a potential role of CSP in vesicle recycling. Using FM1-43 imaging, we analyzed whether the ts block of neurotransmission in csp mutants is caused by a defect in vesicle exocytosis or vesicle recycling. We determined that FM1-43-labeled synaptic boutons of csp mutant neuromuscular junctions fail to destain at 32 degrees C after K+ depolarization, and that FM1-43 dye uptake cannot be evoked by K+ stimulation at 32 degrees C. However, when we stimulated dye uptake independent of depolarization by using black widow spider venom (BWSV), we observed endocytotic uptake of FM1-43. This suggests that endocytosis exhibits no primary ts defect. In addition, we found no ts defect of vesicle recycling at 32 degrees C that would correlate with the ts block of neurotransmission. We also discovered that BWSV and the calcium ionophore calcimycin stimulate FM1-43 destaining and quantal release in csp mutants at 32 degrees C when depolarization fails to evoke any response. The wild-type-like, calcimycin-induced response in csp null mutants indicates that some aspect of the depolarization-dependent calcium signaling pathway must be impaired, either calcium entry, calcium action, or both. Collectively, our results indicate that the csp mutation affects calcium secretion coupling of evoked exocytosis but not vesicle recycling. This supports the hypothesis that CSP links synaptic vesicles to calcium secretion coupling.
半胱氨酸串珠蛋白（CSP）基因的完全缺失会导致果蝇中诱发神经传递出现温度敏感（ts）阻滞。已发现CSP在体外与网格蛋白脱衣被ATP酶HSC70相互作用，这表明CSP在囊泡循环中可能发挥作用。我们使用FM1-43成像分析了csp突变体中神经传递的ts阻滞是否由囊泡胞吐作用或囊泡循环缺陷引起。我们确定，csp突变体神经肌肉接头处经FM1-43标记的突触小体在32℃下K⁺去极化后无法褪色，并且在32℃下K⁺刺激无法诱发FM1-43染料摄取。然而，当我们使用黑寡妇蜘蛛毒液（BWSV）刺激与去极化无关的染料摄取时，我们观察到了FM1-43的内吞摄取。这表明内吞作用没有原发性ts缺陷。此外，我们发现在32℃下囊泡循环没有与神经传递的ts阻滞相关的ts缺陷。我们还发现，当去极化未能引发任何反应时，BWSV和钙离子载体钙霉素在32℃下可刺激csp突变体中的FM1-43褪色和量子释放。csp基因敲除突变体中类似野生型的钙霉素诱导反应表明，去极化依赖性钙信号通路的某些方面一定受到了损害，要么是钙内流、钙作用，要么两者皆有。总体而言，我们的结果表明，csp突变影响诱发胞吐作用中钙分泌的偶联，但不影响囊泡循环。这支持了CSP将突触囊泡与钙分泌偶联联系起来的假说。
