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.

Protons dictate the charge and structure of macromolecules and are used as energy currency by eukaryotic cells. The unique function of individual organelles therefore depends on the establishment and stringent maintenance of a distinct pH. This, in turn, requires a means to sense the prevailing pH and to respond to deviations from the norm with effective mechanisms to transport, produce or consume proton equivalents. A dynamic, finely tuned balance between proton-extruding and proton-importing processes underlies pH homeostasis not only in the cytosol, but in other cellular compartments as well.

Electrostatic interactions with negatively charged membranes contribute to the subcellular targeting of proteins with polybasic clusters or cationic domains. Although the anionic phospholipid phosphatidylserine is comparatively abundant, its contribution to the surface charge of individual cellular membranes is unknown, partly because of the lack of reagents to analyze its distribution in intact cells. We developed a biosensor to study the subcellular distribution of phosphatidylserine and found that it binds the cytosolic leaflets of the plasma membrane, as well as endosomes and lysosomes. The negative charge associated with the presence of phosphatidylserine directed proteins with moderately positive charge to the endocytic pathway. More strongly cationic proteins, normally associated with the plasma membrane, relocalized to endocytic compartments when the plasma membrane surface charge decreased on calcium influx.