Analysis of intracellular translocation of choline acetyltransferase by bioimaging

胆碱乙酰转移酶(ChAT)是乙酰胆碱的合成酶，在多种动物中涉及多种ChAT基因的异构体。由于这些异构体大多是非编码剪接变体，因此只有68 kDa的同源ChAT蛋白在体内产生。最近的证据表明，存在一种编码ChAT基因剪接变体的蛋白质，它缺乏大鼠ChAT基因的外显子6-9。编码的蛋白被命名为外周型ChAT(PChAT)，因为经Western印迹和免疫组织化学证实，它在外周神经系统中优先表达。然而，pChAT的功能意义尚不清楚。为了获得这个问题的线索，我们使用绿色荧光蛋白(GFP)在活体人类胚胎肾细胞(HEK293细胞)和PC-12细胞中检测了pChAT和众所周知的普通型ChAT(CChAT)在细胞内转运方面的可能差异。激光共聚焦扫描显微镜显示，pChAT-GFP在胞浆中可检测到，而cChAT-GFP在胞浆和胞核中均可检测到。经核输出途径抑制剂软霉素B处理后，pChAT-GFP在胞浆和胞核均可检测到，表明pChAT可以转位到胞核。相反，软霉素B处理似乎不影响核内cChAT-GFP的含量。与蛋白激酶C抑制剂孵育后，pChAT-GFP在细胞核内的积聚增加，但cChAT-GFP在细胞核内的积聚增加。这些结果清楚地表明，pChAT和cChAT在细胞内转运方面存在差异，这可能反映了pChAT和cChAT在生理作用上的不同。

Choline acetyltransferase(ChAT), the synthesizing enzyme for acetylcholine, has been implicated to involve multiple isoforms of ChAT mRNA in several animals. Since these isoforms are mostly non-coding splice variants, only a homologous ChAT protein of about 68 kDa has been shown to be produced in vivo. Recent evidence indicates the existence of a protein coding splice variant of ChAT mRNA, which lacks exons 6-9 of the rat ChAT gene. The encoded protein was designated ChAT of a peripheral type(pChAT), because of its preferential expression in the peripheral nervous system as confirmed by Western blot and immunohistochemistry. However, functional significance of pChAT is unknown. To obtain a clue to this question, we examined a possible difference in intracellular trafficking between pChAT and the well-known ChAT of the common type(cChAT) using green fluorescent protein(GFP) in living human embryonic kidney cells (HEK293 cells) and PC-12 cells. Confocal laser scanning microscopy revealed that pChAT-GFP was detectable in the cytoplasm but not in the nucleus, whereas cChAT-GFP was found in both cytoplasm and nucleus. Following treatment with leptomycin B, a nuclear export pathway inhibitor, pChAT-GFP became detectable in both cytoplasm and nucleus, indicating that pChAT can be translocated to the nucleus. In contrast, the leptomycin B treatment did not seem to affect the content of intranuclear cChAT-GFP. After incubation with protein kinase C inhibitors, enhanced accumulation of pChAT-GFP but not cChAT-GFP occurred in the nucleus. These results clearly indicate that pChAT varies from cChAT in intracellular transportation, probably reflecting the difference in physiological roles between pChAT and cChAT.