Synaptic vesicle generation from activity-dependent bulk endosomes requires calcium and calcineurin.
Synaptic vesicle generation from activity-dependent bulk endosomes requires calcium and calcineurin.
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DOI：
10.1523/jneurosci.4697-12.2013
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发表时间：
2013-02-20
期刊：
影响因子：
--
通讯作者：
Cousin MA
中科院分区：
文献类型：
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作者：
Cheung G;Cousin MA
活动依赖性大量内吞（ADBE）是高频刺激中枢神经末梢时突触囊泡（SV）内吞的主要方式。ADBE直接从质膜产生内体，这意味着由于从细胞外空间的液相摄取，高浓度的钙将存在于其内部。利用形态学和荧光测定来追踪在原代神经元培养物中从本体内体产生SV。这一过程是功能解耦SV胞吐和质膜检索事件后，干预SV融合和内吞完成。无论是细胞内（BAPTA-AM）或内体（Rhod-葡聚糖）钙螯合抑制SV生成从散装内体，表明钙流出这个区室是这个过程的关键。V型ATP酶拮抗剂巴弗洛霉素A1也阻止了SV从大量内体的产生，表明内体酸化可能是钙外流所必需的。最后，钙依赖性蛋白磷酸酶钙调磷酸酶的药理学抑制阻断了内体SV的产生，将其鉴定为该过程中的关键下游效应物。这些结果揭示了细胞外钙的液相摄取及其随后在SV生命周期中的流出的新的关键作用。
Activity-dependent bulk endocytosis (ADBE) is the dominant mode of synaptic vesicle (SV) endocytosis during high frequency stimulation in central nerve terminals. ADBE generates endosomes direct from the plasma membrane, meaning that high concentrations of calcium will be present in their interior due to fluid phase uptake from the extracellular space. Morphological and fluorescent assays were utilised to track the generation of SVs from bulk endosomes in primary neuronal culture. This process was functionally uncoupled from both SV exocytosis and plasma membrane retrieval events by intervening only after SV fusion and endocytosis were completed. Either intracellular (BAPTA-AM) or intra-endosomal (Rhod-dextran) calcium chelation inhibited SV generation from bulk endosomes, indicating calcium efflux from this compartment is critical for this process. The V-type ATPase antagonist bafilomycin A1 also arrested SV generation from bulk endosomes indicating endosomal acidification may be required for calcium efflux. Finally pharmacological inhibition of the calcium-dependent protein phosphatase calcineurin blocked endosomal SV generation, identifying it as a key downstream effector in this process. These results reveal a novel and key role for the fluid phase uptake of extracellular calcium and its subsequent efflux in the SV lifecycle.