Cholesterol and F-actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane
Cholesterol and F-actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane
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DOI：
10.1113/jphysiol.2013.265447
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发表时间：
2014-02-15
影响因子：
5.5
通讯作者：
Charlton, Milton P.
中科院分区：
文献类型：
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作者：
Dason, Jeffrey S.;Smith, Alex J.;Charlton, Milton P.
关键点从突触前质膜上捕获的突触囊泡中提取胆固醇会导致突触囊泡蛋白在胞吐作用后分散，囊泡胆固醇既调节突触前磷脂酰肌醇（4（5）─突触囊泡循环过程中的二磷酸水平和肌动蛋白分布。肌动蛋白聚合的抑制导致蛋白质从被困的突触囊泡中分散出来，并损害突触囊泡的循环。囊泡胆固醇和肌动蛋白在突触囊泡的再循环过程中，突触囊泡的脂质和蛋白质共同将突触囊泡蛋白质限制在突触前质膜上，因此，膜或突触囊泡脂质的改变可能通过影响突触囊泡蛋白质的再循环而影响突触进行持续的胞吐和胞吞的能力，突触囊泡（SV）及其蛋白质必须被再循环以维持突触传递。我们测试的假设，SV胆固醇是需要适当的SV蛋白在活突触前末梢回收过程中的排序。我们使用的可逆块内吞作用的果蝇温度敏感的发动蛋白突变体shibire-ts 1陷阱exocytosed SV蛋白，然后研究这些蛋白质的分布在突触前质膜的共聚焦显微镜实验治疗的效果。SV蛋白synaptotagmin，vglut和csp在对照实验中SV捕获后聚集，但分散在用胆固醇螯合剂甲基-β-环糊精处理以提取SV胆固醇的样品中。SV捕获后，突触前末梢中存在磷脂酰肌醇（4，5）-二磷酸（PIP 2）的积累，并且在SV胆固醇提取后这种积累减少。PIP 2积累减少与突触前末梢中肌动蛋白的破坏积累有关。与囊泡胆固醇提取类似，在SV蛋白被捕获在质膜上后，latrunculinA对肌动蛋白的破坏导致SV蛋白的分散，并由于内吞作用受损而阻止突触传递的恢复。我们的研究结果表明，囊泡胆固醇是所需的exocytosed SV蛋白在突触前质膜的聚集，并与涉及调节PIP 2积累和局部肌动蛋白聚合的胆固醇的机制是一致的。因此，膜或SV脂质的改变可能会影响突触的能力，通过损害SV蛋白质的再循环进行持续的突触传递。
Key pointsExtraction of cholesterol from synaptic vesicles trapped on the presynaptic plasma membrane causes synaptic vesicle proteins to disperse after exocytosis.Vesicular cholesterol regulates both presynaptic phosphatidylinositol (4,5)-bisphosphate levels and actin distribution during synaptic vesicle recycling.Inhibition of actin polymerization results in the dispersal of proteins from trapped synaptic vesicles and impairs synaptic vesicle recycling.Vesicular cholesterol and actin together confine synaptic vesicle proteins on the presynaptic plasma membrane during synaptic vesicle recycling.Alteration of membrane or synaptic vesicle lipids might therefore affect the ability of synapses to undergo sustained exocytosis and endocytosis by compromising the recycling of synaptic vesicle proteins.Synaptic vesicles (SVs) and their proteins must be recycled for sustained synaptic transmission. We tested the hypothesis that SV cholesterol is required for proper sorting of SV proteins during recycling in live presynaptic terminals. We used the reversible block of endocytosis in the Drosophila temperature-sensitive dynamin mutant shibire-ts1 to trap exocytosed SV proteins, and then examined the effect of experimental treatments on the distribution of these proteins within the presynaptic plasma membrane by confocal microscopy. SV proteins synaptotagmin, vglut and csp were clustered following SV trapping in control experiments but dispersed in samples treated with the cholesterol chelator methyl-beta-cyclodextrin to extract SV cholesterol. There was accumulation of phosphatidylinositol (4,5)-bisphosphate (PIP2) in presynaptic terminals following SV trapping and this was reduced following SV cholesterol extraction. Reduced PIP2 accumulation was associated with disrupted accumulation of actin in presynaptic terminals. Similar to vesicular cholesterol extraction, disruption of actin by latrunculinA after SV proteins had been trapped on the plasma membrane resulted in the dispersal of SV proteins and prevented recovery of synaptic transmission due to impaired endocytosis following relief of the endocytic block. Our results demonstrate that vesicular cholesterol is required for aggregation of exocytosed SV proteins in the presynaptic plasma membrane and are consistent with a mechanism involving regulation of PIP2 accumulation and local actin polymerization by cholesterol. Thus, alteration of membrane or SV lipids may affect the ability of synapses to undergo sustained synaptic transmission by compromising the recycling of SV proteins.