ESCRT-0 marks an APPL1-independent transit route for EGFR between the cell surface and the EEA1-positive early endosome.

Endosomal sorting complexes required for transport (ESCRT)-0 sorts ubiquitylated EGFR within the early endosome so that the receptor can be incorporated into intralumenal vesicles. An important question is whether ESCRT-0 acts solely upon EGFR that has already entered the vacuolar early endosome (characterised by the presence of EEA1) or engages EGFR within earlier compartments. Here, we employ a suite of software to determine the localisation of ESCRT-0 at subpixel resolution and to perform particle-based colocalisation analysis with other endocytic markers. We demonstrate that although some of the ESCRT-0 subunit Hrs (also known as HGS) colocalises with the vacuolar early endosome marker EEA1, most localises to a population of peripheral EEA1-negative endosomes that act as intermediates in transporting EGFR from the cell surface to more central early endosomes. The peripheral Hrs-labelled endosomes are distinct from APPL1-containing endosomes, but co-label with the novel endocytic adaptor SNX15. In contrast to ESCRT-0, ESCRT-I is recruited to EGF-containing endosomes at later times as they move to more a central position, whereas ESCRT-III is also recruited more gradually. RNA silencing experiments show that both ESCRT-0 and ESCRT-I are important for the transit of EGF to EEA1 endosomes.

Modes of correlated angular motion in live cells across three distinct time scales.

Particle tracking experiments with high speed digital microscopy yield the positions and trajectories of lipid droplets inside living cells. Angular correlation analysis shows that the lipid droplets have uncorrelated motion at short time scales (τ < 1 ms) followed by anti-persistent motion for lag times in the range of 1 ⩽ τ ⩽ 10 ms. The angular correlation at longer time scales, τ > 10 ms, becomes persistent, indicating directed movement. The motion at all time scales is associated with the lipid droplets being tethered to and driven along the microtubule network. The point at which the angular correlation changes from anti-persistent to persistent motion corresponds to the cross over between sub-diffusive and super diffusive motion, as observed by mean square displacement analysis. Correct analysis of the angular correlations of the detector noise is found to be crucial in modelling the observed phenomena.

First-passage-probability analysis of active transport in live cells.

The first-passage-probability can be used as an unbiased method for determining the phases of motion of individual organelles within live cells. Using high speed microscopy, we observe individual lipid droplet tracks and analyze the motor protein driven motion. At short passage lengths (<10(-2)µm), a log-normal distribution in the first-passage-probability as a function of time is observed, which switches to a Gaussian distribution at longer passages due to the running motion of the motor proteins. The mean first-passage times () as a function of the passage length (L), averaged over a number of runs for a single lipid droplet, follow a power law distribution ~L(α), α>2, at short times due to a passive subdiffusive process. This changes to another power law at long times where 1<α<2, corresponding to sub-ballistic superdiffusive motion, an active process. Subdiffusive passive mean square displacements are observed as a function of time, ~t(ß), where 0<ß<1 at short times again crossing over to an active sub-ballistic superdiffusive result 1<ß<2 at longer times. Consecutive runs of the lipid droplets add additional independent Gaussian peaks to a cumulative first-passage-probability distribution indicating that the speeds of sequential phases of motion are independent and biochemically well regulated. As a result we propose a model for motor driven lipid droplets that exhibits a sequential run behavior with occasional pauses.