Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages
Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages
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DOI：
10.1073/pnas.0609795104
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发表时间：
2006-12-19
影响因子：
11.1
通讯作者：
Toomre, Derek
中科院分区：
文献类型：
--
作者：
Perera, Rushika M.;Zoncu, Roberto;Toomre, Derek
磷脂酰肌醇被认为在网格蛋白包被纹孔（CCP）动力学中起重要作用。生物化学和结构研究表明磷脂酰肌醇（4，5）二磷酸[PI（4，5）P-2]与内吞网格蛋白衔接子直接相互作用，而使用无细胞系统或完整细胞的功能研究表明PI（4，5）P2合成和去磷酸化在网格蛋白包被和去包被中的重要性。此外，参与PI（4，5）P2代谢的激酶和磷酸酶的遗传操作导致突触囊泡再循环和其他形式的网格蛋白依赖性内吞的主要缺陷。然而，这些酶在CCP的实时成像研究尚未进行。我们已经使用全内反射荧光显微镜（TIRFM）可视化的时空招聘synaptojanin 1（SJ1），聚磷酸肌醇磷酸酶，及其结合伙伴嗜铬蛋白的CCP。引人注目的是，我们观察到两个主要的SJ1剪接变异体的CCP的差异时间招聘。145-kDa的同种型，在大脑中表达的主要同种型，被迅速招募作为一个"爆发"，与嗜热蛋白，在CCP形成的后期阶段。相反，非神经元普遍表达的170 kDa的SJ1亚型存在于CCP形成的所有阶段。这些结果提高了动态磷酸肌醇代谢可能发生在CCP的整个生命周期的可能性。
Phosphoinositides are thought to play an important role in clathrin-coated pit (CCP) dynamics. Biochemical and structural studies have shown a direct interaction of phosphatidylinositol (4,5)bisphosphate [PI(4,5)P-2] with endocytic clathrin adaptors, whereas functional studies using cell-free systems or intact cells have demonstrated the importance of PI(4,5)P2 synthesis and dephosphorylation in clathrin coating and uncoating, respectively. Furthermore, genetic manipulations of kinases and phosphatases involved in PI(4,5)P2 metabolism result in major defects in synaptic vesicle recycling and other forms of clathrin-dependent endocytosis. However, live imaging studies of these enzymes at CCPs have not been conducted. We have used multicolor total internal reflection fluorescence microscopy (TIRFM) to visualize the spatial-temporal recruitment of synaptojanin 1 (SJ1), a polyphosphoinositide phosphatase, and its binding partner enclophilin to CCPs. Strikingly, we observed differential temporal recruitment of the two major SJ1 splice variants to CCPs. The 145-kDa isoform, the predominant isoform expressed in the brain, was rapidly recruited as a "burst," together with enclophilin, at a late stage of CCP formation. In contrast, the nonneuronal ubiquitously expressed 170-kDa isoform of SJ1 was present at all stages of CCP formation. These results raise the possibility that dynamic phosphoinositide metabolism may occur throughout the lifetime of a CCP.