Phosphatidylinositol-(4,5)-bisphosphate regulates clathrin-coated pit initiation, stabilization, and size.
Phosphatidylinositol-(4,5)-bisphosphate regulates clathrin-coated pit initiation, stabilization, and size.
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DOI：
10.1091/mbc.e11-04-0362
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发表时间：
2011-07-15
影响因子：
3.3
通讯作者：
Schmid SL
中科院分区：
文献类型：
--
作者：
Antonescu CN;Aguet F;Danuser G;Schmid SL
磷脂酰肌醇-（4，5）-二磷酸（PIP 2）是网格蛋白介导的胞吞作用（CME）中蛋白质的主要脂质结合伴侣。全内反射荧光显微镜结合计算机图像分析显示，在散装质膜和网格蛋白涂层的坑内的局部营业额的PIP 2合成的平衡控制多个不同的，但只有部分重叠的CME阶段。网格蛋白介导的内吞作用（CME）是哺乳动物细胞内化的主要机制。CME通过募集衔接子和网格蛋白以形成网格蛋白包被的凹坑（CCP）而启动。近一半的新生CCP流产，而其他的稳定未知的机制，并进行进一步成熟之前捏断形成网格蛋白包被囊泡（CCV）。磷脂酰肌醇-（4，5）-二磷酸（PIP 2）是内吞蛋白的主要脂质结合伴侣，是CCP组装所必需的，但目前对其在CCV形成中的作用知之甚少。使用小干扰RNA（siRNA）敲低和过表达，我们分析了操纵PIP 2的合成和营业额对CME的影响，通过定量全内反射荧光显微镜和计算分析。磷脂酰肌醇-4-磷酸-5-激酶不能在CCP中检测到，但在启动和控制CCP生长的速率和程度中起作用。相反，5′-肌醇磷酸酶synaptojanin 1定位于CCP并控制早期稳定和成熟效率。总之，这些结果表明，平衡的PIP 2合成的主体质膜和其局部周转内CCP控制多个阶段的CCV形成。
Phosphatidylinositol-(4,5)-bisphosphate (PIP2) is the main lipid binding partner of proteins involved in clathrin-mediated endocytosis (CME). Total internal reflection fluorescence microscopy coupled to computational image analysis revealed that the balance of PIP2 synthesis in the bulk plasma membrane and its local turnover within clathrin-coated pits control multiple distinct yet only partly overlapping stages of CME. Clathrin-mediated endocytosis (CME) is the major mechanism for internalization in mammalian cells. CME initiates by recruitment of adaptors and clathrin to form clathrin-coated pits (CCPs). Nearly half of nascent CCPs abort, whereas others are stabilized by unknown mechanisms and undergo further maturation before pinching off to form clathrin-coated vesicles (CCVs). Phosphatidylinositol-(4,5)-bisphosphate (PIP2), the main lipid binding partner of endocytic proteins, is required for CCP assembly, but little is currently known about its contribution(s) to later events in CCV formation. Using small interfering RNA (siRNA) knockdown and overexpression, we have analyzed the effects of manipulating PIP2 synthesis and turnover on CME by quantitative total internal reflection fluorescence microscopy and computational analysis. Phosphatidylinositol-4-phosphate-5-kinase cannot be detected within CCPs but functions in initiation and controls the rate and extent of CCP growth. In contrast, the 5′-inositol phosphatase synaptojanin 1 localizes to CCPs and controls early stabilization and maturation efficiency. Together these results suggest that the balance of PIP2 synthesis in the bulk plasma membrane and its local turnover within CCPs control multiple stages of CCV formation.