Single particle tracking (SPT) is often the rate-limiting step in live cell imaging studies of sub-cellular dynamics. Here we present a tracking algorithm that addresses the principal challenges of SPT, namely high particle density, particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting. The algorithm first links particles between consecutive frames and then links the resulting track segments into complete trajectories. Both steps are formulated as global combinatorial optimization problems whose solution identifies the overall most likely set of particle trajectories throughout the movie. Using this approach, we show that the GTPase dynamin differentially affects the kinetics of long and short-lived endocytic structures, and that the motion of CD36 receptors along cytoskeleton-mediated linear tracks increases their aggregation probability. Both applications indicate the requirement for robust and complete tracking of dense particle fields to dissect the mechanisms of receptor organization at the level of the plasma membrane.

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.

The adaptor AP2 is required for initiation of clathrin-mediated endocytosis. Kadlecova et al. delineate the functional hierarchy of AP2 interactions with phosphatidylinositol lipids and cargo and their relationship to distinct steps in clathrin-coated pit nucleation and maturation in living cells.

Clathrin-coated pit size and dynamic behavior varies with low density lipoprotein receptor (LDLR) expression levels in a manner dependent on the LDLR-specific adaptors, Dab2 and ARH.

Diverse cargo molecules (i.e., receptors and ligand/receptor complexes) are taken into the cell by clathrin-mediated endocytosis (CME) utilizing a core machinery consisting of cargo-specific adaptors, clathrin and the GTPase dynamin. Numerous endocytic accessory proteins are also required, but their differential roles and functional hierarchy during CME are not yet understood. Here, we used a combination of quantitative live-cell imaging by total internal reflection fluorescence microscopy (TIR-FM), and decomposition of the lifetime distributions of clathrin-coated pits (CCPs) to measure independent aspects of CCP dynamics, including the turnover of abortive and productive CCP species and their relative contributions. Capitalizing on the sensitivity of this assay, we have examined the effects of specific siRNAmediated depletion of endocytic accessory proteins on CME progression. Of the 12 endocytic accessory proteins examined, we observed seven qualitatively different phenotypes upon protein depletion. From this data we derive a temporal hierarchy of protein function during early steps of CME. Our results support the idea that a subset of accessory proteins, which mediate coat assembly, membrane curvature, and cargo selection, can provide input into an endocytic restriction point/checkpoint mechanism that monitors CCP maturation. INTRODUCTIONClathrin-mediated endocytosis (CME) proceeds through the sequential stages of 1) clathrin nucleation at the membrane, 2) clathrin-coated pit (CCP) maturation and invagination, 3) CCP scission, and 4) vesicle uncoating (Conner and Schmid, 2003). The core components of the CME machinery, namely clathrin, the tetrameric adaptor protein AP-2, and the large GTPase dynamin, are assisted by numerous endocytic accessory proteins throughout all stages of CCP maturation (Schmid and McMahon, 2007). Although the function of the core components of the CME machinery is increasingly well understood, the exact function of most other endocytic accessory proteins remains unknown. Among these factors, some such as CALM, epsin1, and SNX9 recognize specific subclasses of cargo molecules (Howard et al., 1999;Harel et al., 2008;Kazazic et al., 2009). Others, such as intersectin, Eps15, SNX9, and endophilin have multiple protein-interaction domains (e.g., Eps15-homolgy [EH] or Src-homology 3 [SH3] domains) and are thought to have scaffolding functions (Miliaras and Wendland, 2004;Ungewickell and Hinrichsen, 2007). Endophilin, SNX9, and epsin have lipid curvature sensing and/or generating domains (e.g., BAR or ENTH domains) and are thought to regulate CME by introducing local biochemical and physical changes into the lipid bilayer, i.e., membrane curvature (Ford et al., 2002;Itoh and De Camilli, 2006). Finally, Hip1R, SNX9, and intersectin are thought to link CME to the actin cytoskeleton and actin dynamics (McPherson, 2002;Le Clainche et al., 2007;Yarar et al., 2007). How these diverse and partially redundant components of the endocytic machinery are integrated and function to create a robust mech...