Auxilin-1對PC12細胞的胞吞胞吐作用之調控

Abstract

神經細胞透過胞吐、胞吞作用在突觸末端將神經傳導物質釋放、回收以維持細胞膜的恆定。其中一種胞吞作用是透過clathrin調控，當clathrin包覆的小泡回收至細胞後，Hsc70與auxilin共同作用使得clathrin離開小泡回收再使用，小泡也會進入lysosome回收，或者直接經由endosome回到胞吐機轉路徑。Auxilin-1是特定表現在神經細胞中的亞型，具有與PIP2結合的PTEN-like domain、與AP-2、dynamin、clathrin等胞吞作用相關蛋白結合的central domain，以及與Hsc70結合的J domain。本實驗室先前使用yeast-two-hybrid screening搜尋在大鼠cDNA library中，可與NCS-1作用的蛋白；發現NCS-1會和auxilin的C端部分結合（胺基酸序列444至970）。已知NCS-1能調控神經傳導物質的釋放，而auxilin則參與在胞吞作用機轉中，為進一步瞭解兩者的交互作用，對調控胞吞胞吐機制的調控，我們將人類auxili-1的C端基因片段（胺基酸序列465至970，C-half）與ECFP做成融合蛋白，表現在PC12細胞中，並以電生理實驗，探討auxilin分佈位置及對胞吞胞吐作用的影響。在PC12細胞中，auxilin-C half均勻分佈在細胞中，即使胞吐作用旺盛，也未觀察到聚集在細胞膜附近，這可能與缺乏PTEN domain有關。然而和位在細胞膜的NCS-1共同過量表現在細胞時，auxilin-1 C-half也會出現在細胞膜上，顯示NCS-1與auxilin-1 C-half有交互作用。而auxilin-1 C-half大量表現，會使內生性的clathrin無法聚集在細胞膜上，顯示auxilin-1會影響clathrin的分佈。電生理的測量發現，auxilin-1 C-half的表現，會使胞吐作用減少，而無法與Hsc70結合的auxilin-1 C-half H934Q突變型則沒有影響。這些結果顯示auxilin可能透過與NCS-1、clathrin及Hsc70的交互作用，調控小泡循環，進而影響神經傳導物質的分泌。

Neurons release neurotransmitter by exocytosis and recycle the fused vesicles by endocytosis to maintain the membrane homeostasis. Clathrin-mediated endocytosis is one of the major pathways to form an invaginated membrane patch; after fission from the plasma membrane, the clathrin coat would be destabilized and dissociated from the vesicle by Hsc70 and auxilin. These vesicles will fuse with lysosome or be recycled for exocytosis. Auxilin-1 is a neuron-specific isoform and has three domains: an N-terminal PTEN-domain for PIP2 binding, a central domain for AP-2, dynamin, and clathrin interactions, and a C-terminal J domain for Hsc70 interaction. Our previous studies have shown that a rat cDNA clone covers the C-half (a. a. 444 ~ 970) of auxilin-1 was identified by yeast-two-hybrid screening using neuronal calcium sensor-1 (NCS-1) as the bait. NCS-1 has been shown to modulate the neurotransmitter release and auxilin is involved in clathrin-mediated endocytosis pathway; therefore, we are interested in characterizing how the interaction between NCS-1 and auxilin modulates exo-endocytosis. The auxilin-1 C-terminal fragment (a.a. 465 ~ 970, auxilin-1 C-half) was fused with the ECFP and subcloned to a mammalian expression vectors. The cloned was expressed in PC12 and the exo-endocytosis was investigated by electrophysiological approaches. In NGF-treated PC12 cell, auxilin-1 C-half was mainly distributed in the cytosol and not changed by depolarization treatment. However, when co-expressed with NCS-1, the auxilin-1 C-half was localized at the plasma membrane. In addition, endogenous clathrin could not aggregate at the plasma membrane in cells overexpressing the auxilin-1 C-half. When cells were perforated patched and the membrane capacitance was monitored, the evoked exocytosis was inhibited by auxilin-1 C-half, but not the inactive mutant. These results suggest that NCS-1 may interact with auxilin to guide the localization of clathrin to the plasma membrane for vesicle recycling and modulates the exo-endocytosis.