Particle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Since manually detecting and following large numbers of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized, for the first time, an open competition, in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to important practical conclusions for users and developers.

SUMMARY The GTPase dynamin, a key player in endocytic membrane fission, interacts with numerous proteins that regulate actin dynamics and generate/sense membrane curvature. To determine the functional relationship between these proteins and dynamin, we have analyzed endocytic intermediates that accumulate in cells that lack dynamin (derived from dynamin 1 and 2 double conditional knockout mice). In these cells, actin nucleating proteins, actin and BAR domain proteins accumulate at the base of arrested endocytic clathrin-coated pits where they support the growth of dynamic long tubular necks. These results, which we show reflect the sequence of events in wildtype cells, demonstrate a concerted action of these proteins prior to, and independent of, dynamin, and emphasize similarities between clathrin-mediated endocytosis in yeast and higher eukaryotes. Our data also demonstrate that the relationship between dynamin and actin is intimately connected to dynamin’s endocytic role and that dynamin terminates a powerful actin- and BAR protein-dependent tubulating activity.

Endophilin is a membrane binding protein with curvature-generating/sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin, and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles but not of clathrin-coated pits at synapses. The absence of endophilin impairs, but does not abolish synaptic transmission and results in perinatal lethality, while partial endophilin absence causes severe neurological defects, including epilepsy, and neurodegeneration. Our data supports a model in which endophilin recruitment to coated pit necks, due to its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.

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