Isolation and Characterization of Endocytic Vesicles

Mah, Vivian

Abstract

All living cells are capable to some extent of uptaking molecules from their environment by carrier-mediated or controlled diffusion mechanisms as well as by endocytosis. The process of endocytosis includes phagocytosis, fluid-phase pinocytosis, and receptor-mediated endocytosis. Plasma membrane has been known to internalize specifically hormones, proteins, iron ions, cholesterol, etc. though binding of specific surface receptors. Although the process of endocytosis is widespread and may be responsible for various physiological roles, little is known about its causative mechanism. Recent studies have shown that the endocytosed contents are first accumulated in the prelysosomal compartment, reprocessed, and eventually delivered to secondary lysosomes. The digestion products of lysosomes are either released from the cells or exported to the cytoplasm. The mechanism of endocytic transfer is unknown. The difficulty in illucidating the molecular transfer mechanism is largely due to the fact that classical techniques of subcellular fractionation have not been effective in the isolation of endocytic vesicles (endosomes) for biochemical analysis. This project proposes a new approach to isolate endosomes by magnetic separation chromatography. In this procedure, a ligand (asialoglycoprotein) with a marker enzyme (horseradish peroxidase) is coupled to micromagnetic beads (Biomag) prior to in vitro internalization by the adult white rat liver cells. The resulting endosomes are then separated magnetically from all other organelles and the fluid contents after cell disruption. The marker enzyme provides a convenient tracer for the endosomes. By adjusting the time of ligand internalization and pulse-chase intervals, a variety of endosome populations can be harvested. Biochemical characterization of these endosome populations would shed light on the molecular mechanism of endocytosis. The outcome of this research would contribute to the understanding of the dynamic behavior of the plasma membrane and may lead to the realization of the ultimate goal of providing an effective way for the plasma membrane to deliver specific therapeutic molecules to needed cells.