Marxen M, Volknandt W, Zimmermann H
Biozentrum der J.W. Goethe-Universität, AK Neurochemie, Frankfurt am Main, Germany.
Neuroscience. 1999;94(3):985-96. doi: 10.1016/s0306-4522(99)00351-6.
It is now well established that the membrane of synaptic vesicles is recycled following exocytosis. However, little is known concerning the identity of the primary or secondary endocytic structures and their molecular composition. Using cultured rat cerebellar granule cells we combined uptake of horseradish peroxidase as a fluid phase marker and immunogold labeling for a variety of presynaptic proteins to assess the molecular identity of the stimulation-induced endocytic compartments. Short periods (5 or 30 s) of stimulation with 50 mM KCl were followed by periods of recovery for up to 30 min. Stimulation resulted in the formation of horseradish-peroxidase-filled vacuoles in the axonal varicosities as the apparent primary endocytic compartment. Horseradish peroxidase-filled synaptic vesicles were formed when stimulated cells were allowed to recover in horseradish peroxidase-free culture medium. Horseradish peroxidase-filled vacuoles as wells as vesicles contained the synaptic vesicle membrane proteins VAMP II, synaptotagmin, SV2, and synaptophysin, the vesicle-associated proteins rab 3A and synapsin I, and in addition SNAP-25. No incorporation of vesicle proteins into the plasma membrane was observed. Horseradish peroxidase-filled vesicles and vacuoles generated on incubation of unstimulated granule cells with horseradish peroxidase for prolonged periods of time were equally immunolabeled. Renewed stimulation of prestimulated granule cells with either 100 mM KCl or 30 microM Ca2+ ionophore A23187 resulted in a reduction of horseradish peroxidase-filled vacuoles suggesting that the vacuolar membrane compartment was exocytosis-competent. Our results suggest that varicosities of cultured cerebellar granule cells possess a fast stimulation-induced pathway for recycling the entire synaptic vesicle membrane compartment. The primary endocytic compartment represents not a synaptic vesicle but a somewhat larger vesicle protein-containing vacuolar entity from which smaller vesicles of identical protein composition may be regenerated. Endocytic vacuoles and synaptic vesicles share membrane and membrane-associated proteins and presumably also major functional properties.
现已充分证实，突触小泡膜在胞吐作用后会被循环利用。然而，关于初级或次级内吞结构的身份及其分子组成却知之甚少。我们利用培养的大鼠小脑颗粒细胞，将辣根过氧化物酶作为液相标记物的摄取与针对多种突触前蛋白的免疫金标记相结合，以评估刺激诱导的内吞区室的分子身份。用50 mM KCl进行短时间（5或30秒）刺激，随后恢复长达30分钟。刺激导致轴突膨体中形成充满辣根过氧化物酶的液泡，这似乎是初级内吞区室。当刺激后的细胞在不含辣根过氧化物酶的培养基中恢复时，会形成充满辣根过氧化物酶的突触小泡。充满辣根过氧化物酶的液泡以及小泡含有突触小泡膜蛋白VAMP II、突触结合蛋白、SV2和突触素，囊泡相关蛋白rab 3A和突触蛋白I，此外还有SNAP - 25。未观察到囊泡蛋白掺入质膜。长时间用辣根过氧化物酶孵育未刺激的颗粒细胞所产生的充满辣根过氧化物酶的小泡和液泡具有相同的免疫标记。用100 mM KCl或30 microM钙离子载体A23187再次刺激预刺激的颗粒细胞，会导致充满辣根过氧化物酶的液泡减少，这表明液泡膜区室具有胞吐能力。我们的结果表明，培养的小脑颗粒细胞的膨体拥有一条快速的刺激诱导途径，用于循环整个突触小泡膜区室。初级内吞区室并非代表突触小泡，而是一个稍大的含囊泡蛋白的液泡实体，从中可以再生出蛋白质组成相同的较小囊泡。内吞液泡和突触小泡共享膜和膜相关蛋白，并且大概也具有主要的功能特性。
