Cargo and dynamin regulate clathrin-coated pit maturation.
Cargo and dynamin regulate clathrin-coated pit maturation.
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DOI：
10.1371/journal.pbio.1000057
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发表时间：
2009-03-17
期刊：
影响因子：
9.8
通讯作者：
Schmid SL
中科院分区：
文献类型：
--
作者：
Loerke D;Mettlen M;Yarar D;Jaqaman K;Jaqaman H;Danuser G;Schmid SL
全内反射荧光显微镜（TIR-FM）已成为研究网格蛋白介导的内吞作用的有力工具。然而，由于跟踪和量化其异质动态行为的困难，详细分析仅限于有限数量的选定的网格蛋白涂层坑（CCP）。为了确定网格蛋白包被囊泡形成过程中的中间体以及调节这些阶段进展的因素，我们使用粒子跟踪软件和统计方法来建立所有可见 CCP 轨迹的公正且完整的清单。我们确定了三种动态不同的 CCP 亚群：两种对应于中止中间体的短期亚群，以及一种寿命较长的生产性亚群。以一种依赖于 AP2 接头复合物的方式，增加货物浓度显着提高了生产性 CCP 的成熟效率，但对其寿命影响很小。相比之下，dynamin-2 GTPase 的小干扰 RNA (siRNA) 消耗和野生型或突变体 dynamin-1 的重新引入揭示了 dynamin 在控制流产中间体周转和 CCP 成熟速率中的作用。从这些数据中，我们推断存在内吞限制或检查点，对货物有反应并受动力调节。网格蛋白介导的内吞作用是真核细胞摄取分子的主要途径，并受到 GTP 酶动力的调节。衔接蛋白将网格蛋白招募到质膜上，网格蛋白包被的凹坑再次通过衔接子捕获跨膜货物分子。这些小凹内陷并夹断，形成网格蛋白包被的囊泡，将货物带入细胞。活细胞成像揭示了与质膜相关的网格蛋白包被的凹坑的动态行为中惊人的异质性，但这种异质性的本质及其功能含义尚不清楚。我们使用粒子跟踪软件建立了全内反射荧光显微镜可见的网格蛋白包被凹坑轨迹的无偏且完整的清单。通过统计分析，我们确定了三种动态不同的涂层坑亚群：两种对应于中止中间体的短寿命亚群，以及一种寿命较长的生产性亚群。每个亚群的比例及其寿命对分子扰动的独立响应。作为货物浓度、接头水平和动力突变体分析的系统调节的结果，我们假设存在内吞限制或检查点，其通过门控网格蛋白包被的凹坑的成熟来控制网格蛋白介导的内吞作用的速率。对活细胞图像的分析表明，网格蛋白介导的内吞作用是通过网格蛋白包被的凹坑的不同亚群进行的，并表明内吞作用检查点受动力调节并对货物负载做出反应。
Total internal reflection fluorescence microscopy (TIR-FM) has become a powerful tool for studying clathrin-mediated endocytosis. However, due to difficulties in tracking and quantifying their heterogeneous dynamic behavior, detailed analyses have been restricted to a limited number of selected clathrin-coated pits (CCPs). To identify intermediates in the formation of clathrin-coated vesicles and factors that regulate progression through these stages, we used particle-tracking software and statistical methods to establish an unbiased and complete inventory of all visible CCP trajectories. We identified three dynamically distinct CCP subpopulations: two short-lived subpopulations corresponding to aborted intermediates, and one longer-lived productive subpopulation. In a manner dependent on AP2 adaptor complexes, increasing cargo concentration significantly enhances the maturation efficiency of productive CCPs, but has only minor effects on their lifetimes. In contrast, small interfering RNA (siRNA) depletion of dynamin-2 GTPase and reintroduction of wild-type or mutant dynamin-1 revealed dynamin's role in controlling the turnover of abortive intermediates and the rate of CCP maturation. From these data, we infer the existence of an endocytic restriction or checkpoint, responsive to cargo and regulated by dynamin. Clathrin-mediated endocytosis is the major pathway for the uptake of molecules into eukaryotic cells and is regulated by the GTPase dynamin. Adaptor proteins recruit clathrin to the plasma membrane, where clathrin-coated pits capture transmembrane cargo molecules, again via adaptors. The pits invaginate and pinch off to form clathrin-coated vesicles that carry the cargo into the cell. Live cell imaging has revealed striking heterogeneity in the dynamic behavior of clathrin-coated pits associated with the plasma membrane, yet the nature of this heterogeneity and its functional implications are unknown. We used particle-tracking software to establish an unbiased and complete inventory of the trajectories of clathrin-coated pits visible by total internal reflection fluorescence microscopy. Through statistical analyses, we identified three dynamically distinct subpopulations of coated pits: two short-lived subpopulations corresponding to aborted intermediates, and one longer-lived productive subpopulation. The proportion of each subpopulation and their lifetimes respond independently to molecular perturbations. As a result of systematic modulation of cargo concentration, adaptor levels, and analysis of dynamin mutants, we postulate the existence of an endocytic restriction or checkpoint that governs the rate of clathrin-mediated endocytosis by gating the maturation of clathrin-coated pits. Analysis of live-cell images reveals that clathrin-mediated endocytosis proceeds via distinct subpopulations of clathrin-coated pits, and suggests that an endocytosis check-point is regulated by dynamin and responsive to cargo load.
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影响因子：
4.8
作者：
Keyel, PA;Watkins, SC;Traub, LM
通讯作者：
Traub, LM
影响因子：
11.8
作者：
Macia, Eric;Ehrlich, Marcelo;Kirchhausen, Tomas
通讯作者：
Kirchhausen, Tomas
DOI：
10.1083/jcb.150.5.1137
发表时间：
2000-09-04
期刊：
The Journal of cell biology
影响因子：
--
作者：
Sever S;Damke H;Schmid SL
通讯作者：
Schmid SL
影响因子：
8
作者：
Olivo-Marin, JC
通讯作者：
Olivo-Marin, JC
影响因子：
21.3
作者：
Merrifield, CJ;Feldman, ME;Almers, W
通讯作者：
Almers, W