Endocytic adaptor molecules reveal an endosomal population of clathrin by total internal reflection fluorescence microscopy
Endocytic adaptor molecules reveal an endosomal population of clathrin by total internal reflection fluorescence microscopy
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DOI：
10.1074/jbc.m312717200
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发表时间：
2004-03-26
影响因子：
4.8
通讯作者：
Traub, LM
中科院分区：
文献类型：
--
作者：
Keyel, PA;Watkins, SC;Traub, LM
大多数真核生物利用单个网格蛋白池在不同的细胞内位置组装网格蛋白包被的运输囊泡。涂层组装是一个循环过程。可溶性网格蛋白通过隔室特异性衔接子和/或辅助蛋白募集到膜表面。然后相邻的三聚体聚集在一起，组装成一个多面体晶格，逐渐内陷，从膜表面出芽，包裹着一个新生的运输囊泡，很快就没有了涂层。使用全内反射荧光显微镜跟踪网格蛋白动力学接近细胞表面，我们发现，大多数标记的网格蛋白结构是相对静态的，垂直进出的倏逝场，但很少横向运动。一小部分在微米距离上表现出快速的横向和定向运动。衔接蛋白，包括AP-2，ARH和Dab 2的α亚基也是相对静止的，几乎没有横向运动。一个荧光标记的AP-2 β 2亚基，纳入AP-2和AP-1衔接子复合物，表现出两种类型的行为。这表明，高度能动的网格蛋白斑点可能是不同的质膜相关的网格蛋白结构。当内吞的货物分子，如转铁蛋白或低密度脂蛋白，进入细胞后，他们表现出更多的横向运动比网格蛋白，并逐渐集中在核周区域，与经典的内体运输一致。重要的是，网格蛋白与内化的转铁蛋白部分共定位，但随着结构纵向移动而发散。因此，高度能动的网格蛋白结构明显不同于质膜，伴随转铁蛋白，并含有AP-1，揭示了网格蛋白结构的内体群体。
Most eukaryotes utilize a single pool of clathrin to assemble clathrin-coated transport vesicles at different intracellular locations. Coat assembly is a cyclical process. Soluble clathrin triskelia are recruited to the membrane surface by compartment-specific adaptor and/or accessory proteins. Adjacent triskelia then pack together to assemble a polyhedral lattice that progressively invaginates, budding off the membrane surface encasing a nascent transport vesicle that is quickly uncoated. Using total internal reflection fluorescence microscopy to follow clathrin dynamics close to the cell surface, we find that the majority of labeled clathrin structures are relatively static, moving vertically in and out of the evanescent field but with little lateral motion. A small minority shows rapid lateral and directed movement over micrometer distances. Adaptor proteins, including the alpha subunit of AP-2, ARH, and Dab2 are also relatively static and exhibit virtually no lateral movement. A fluorescently labeled AP-2 beta2 subunit, incorporated into both AP-2 and AP-1 adaptor complexes, exhibits both types of behavior. This suggests that the highly motile clathrin puncta may be distinct from plasma membrane-associated clathrin structures. When endocytosed cargo molecules, such as transferrin or low density lipoprotein, are followed into cells, they exhibit even more lateral motion than clathrin, and gradually concentrate in the perinuclear region, consistent with classical endosomal trafficking. Importantly, clathrin partially colocalizes with internalized transferrin, but diverges as the structures move longitudinally. Thus, highly motile clathrin structures are apparently distinct from the plasma membrane, accompany transferrin, and contain AP-1, revealing an endosomal population of clathrin structures.