(134c) Towards Quantitative Understanding of Mass Transfer in the Endocytic Pathway

The endocytic pathway is central to a number of critical cellular functions such as uptake and trafficking of nutrients and drug or gene carriers. Cargo internalized by endocytosis is encapsulated in endosomes and eventually transferred to lysosomes where it is further processed. Mass transfer from endosomes to lysosomes is thus a primary function of the endocytic pathway. Transfer of mass from endosomes to lysosomes relies on two distinct, tightly regulated processes?mobility of endosomes and lysosomes on microtubules and exchange of material between endosome-lysosome and lysosome-lysosome through clustering and fusion. Although biochemical pathways that regulate these processes have been well studied, the interplay between transport and vesicle interactions and their impact on mass transfer in the endocytic pathway are not quantitatively understood.

To study mass transfer in the endocytic pathway, we studied the motion of endosomes and lysosomes in human skin fibroblasts using video microscopy. We also studied clustering of endocytic vesicles (endosomes and lysosomes) in real time and exchange of mass between endocytic vesicles at several time points. Vesicle transport properties were manipulated by chemical and/or genetic means and the effects of these changes on the rate of inter-vesicle mass transfer was examined. The results showed that mass transfer in the endocytic pathway depends on a delicate balance between transport on microtubules and vesicle clustering. Transport on microtubules was quantified in terms of fundamental parameters including speed, direction, run length, and pauses whereas vesicle clustering was quantified in terms of cluster size, rate of fusion, and rate of disaggregation. Excessive aggregation or reduced aggregation significantly reduced the efficiency of mass transfer from endosomes to lysosomes. The results showed that there exists an optimal combination of transport and aggregation parameters that leads to optimal mass transfer. Our studies provide a quantitative, spatial and mechanistic picture of the endocytic pathway?from single vesicle to whole cell level. This knowledge has important implications in understanding the intracellular transport of drug and gene carriers as well as pathology of diseases associated with malfunctioning of the endocytic pathway.