AIM Astrocytes play a homeostatic role in the central nervous system and influence numerous aspects of neurophysiology via intracellular trafficking of vesicles. Intermediate filaments (IFs), also known as nanofilaments, regulate a number of cellular processes including organelle trafficking and adult hippocampal neurogenesis. We have recently demonstrated that the IF protein nestin, a marker of neural stem cells and immature and reactive astrocytes, is also expressed in some astrocytes in the unchallenged hippocampus and regulates neurogenesis through Notch signalling from astrocytes to neural stem cells, possibly via altered trafficking of vesicles containing the Notch ligand Jagged-1. METHODS We thus investigated whether nestin affects vesicle dynamics in astrocytes by examining single vesicle interactions with the plasmalemma and vesicle trafficking with high-resolution cell-attached membrane capacitance measurements and confocal microscopy. We used cell cultures of astrocytes from nestin-deficient (Nes-/- ) and wild-type (wt) mice, and fluorescent dextran and Fluo-2 to examine vesicle mobility and intracellular Ca2+ concentration respectively. RESULTS Nes-/- astrocytes exhibited altered sizes of vesicles undergoing full fission and transient fusion, altered vesicle fusion pore geometry and kinetics, decreased spontaneous vesicle mobility and altered ATP-evoked mobility. Purinergic stimulation evoked Ca2+ signalling that was slightly attenuated in Nes-/- astrocytes, which exhibited more oscillatory Ca2+ responses than wt astrocytes. CONCLUSION These results demonstrate at the single vesicle level that nestin regulates vesicle interactions with the plasmalemma and vesicle trafficking, indicating its potential role in astrocyte vesicle-based communication.

中文翻译：

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巢蛋白影响小鼠星形胶质细胞的融合孔动力学。

AIM星形胶质细胞在中枢神经系统中起着稳态作用，并通过细胞内小泡的运输影响神经生理学的许多方面。中间丝（IF），也称为纳米丝，调节许多细胞过程，包括细胞器运输和成年海马神经发生。我们最近证明，IF蛋白nestin是神经干细胞以及未成熟和反应性星形胶质细胞的标志物，它也在不受挑战的海马中的某些星形胶质细胞中表达，并通过从星形胶质细胞向神经干细胞的Notch信号传导调节神经发生，可能是通过改变含有Notch配体Jagged-1的囊泡。方法因此，我们通过高分辨率细胞附着膜电容测量和共聚焦显微镜检查了单囊泡与质膜和囊泡运输的相互作用，从而研究了巢蛋白是否影响星形胶质细胞的囊泡动力学。我们使用巢蛋白缺陷（Nes-/-）和野生型（wt）小鼠星形胶质细胞的细胞培养，以及荧光右旋糖酐和Fluo-2分别检测囊泡迁移率和细胞内Ca2 +浓度。结果Nes-/-星形胶质细胞的囊泡经历了完全裂变和瞬时融合，改变了囊泡的大小，改变了囊泡融合的孔的几何形状和动力学，降低了自发囊泡的活动性，并改变了ATP诱发的活动性。嘌呤能刺激引起Ca2 +信号传导，在Nes-/-星形胶质细胞中略微减弱，与野生型星形胶质细胞相比，其表现出更多的振荡性Ca2 +反应。结论这些结果表明，在单个囊泡水平上，巢蛋白调节囊泡与质膜和囊泡运输的相互作用，表明其在基于星形胶质细胞囊泡的交流中的潜在作用。