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.

SUMMARY Numerous endocytic accessory proteins (EAPs) mediate assembly and maturation of clathrin-coated pits (CCPs) into cargo-containing vesicles. Analysis of EAP function through bulk measurement of cargo uptake has been hampered due to potential redundancy among EAPs and, as we show here, the plasticity and resilience of clathrin-mediated endocytosis (CME). Instead, EAP function is best studied by uncovering the correlation between variations in EAP association to individual CCPs and the resulting variations in maturation. However, most EAPs bind to CCPs in low numbers, making the measurement of EAP association via fused fluorescent reporters highly susceptible to detection errors. Here, we present a framework for unbiased measurement of EAP recruitment to CCPs and their direct effects on CCP dynamics. We identify dynamin and the EAP-binding α-adaptin appendage domain of the AP2 adaptor as switches in a regulated, multistep maturation process and provide direct evidence for a molecular checkpoint in CME.

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 (CME) is the major endocytic pathway in mammalian cells. It is responsible for the uptake of transmembrane receptors and transporters, for remodeling plasma membrane composition in response to environmental changes, and for regulating cell surface signaling. CME occurs via the assembly and maturation of clathrin-coated pits that concentrate cargo as they invaginate and pinch off to form clathrin-coated vesicles. In addition to the major coat proteins, clathrin triskelia and adaptor protein complexes, CME requires a myriad of endocytic accessory proteins and phosphatidylinositol lipids. CME is regulated at multiple steps-initiation, cargo selection, maturation, and fission-and is monitored by an endocytic checkpoint that induces disassembly of defective pits. Regulation occurs via posttranslational modifications, allosteric conformational changes, and isoform and splice-variant differences among components of the CME machinery, including the GTPase dynamin. This review summarizes recent findings on the regulation of CME and the evolution of this complex process.

The STIM1-ORAI1 pathway of store-operated Ca2+ entry is an essential component of cellular Ca2+ signaling1. STIM1 senses depletion of intracellular Ca2+ stores in response to physiological stimuli, and relocalises within the endoplasmic reticulum (ER) to plasma membrane (PM)-apposed junctions, where it recruits and gates open plasma membrane ORAI1 Ca2+ channels. Here we used a genome-wide RNAi screen to identify filamentous septin proteins as critical regulators of store-operated Ca2+ entry. Septin filaments and phosphatidylinositol 4,5-bisphosphate (PIP2) rearrange locally at ER-PM junctions prior to and during formation of STIM1-ORAI1 clusters, facilitating STIM1 targeting to these junctions and promoting the stable recruitment of ORAI1. Septin rearrangement at junctions is required for PIP2 reorganisation and efficient STIM1-ORAI1 communication. Septins are known to demarcate specialized membrane regions such as dendritic spines, the yeast bud, and the primary cilium, and to serve as membrane diffusion barriers and/or signaling hubs in cellular processes including vesicle trafficking, cell polarity, and cytokinesis2–4. Our data show that septins also organise the highly localised plasma membrane domains important in STIM1-ORAI1 signaling, and indicate that septins may organise membrane microdomains relevant to other signaling processes.