Membrane flow during pinocytosis. A stereologic analysis.

Abstract

HRP has been used as a cytochemical marker for a stereologic analysis of pinocytic vesicles and secondary lysosomes in cultivated macrophages and L cells. Evidence is presented that the diaminobenzidine technique (a) detects all vacuoles containing enzyme and (b) distinguishes between incoming pinocytic vesicles and those which have fused with pre-existing lysosomes to form secondary lysosomes.The HRP reactive pinocytic vesicle space fills completely within 5 min after exposure to enzyme, while the secondary lysosome compartment is saturated in 45-60 min. The size distribution of sectioned (profile) vacuole diameters was measured at equilibrium and converted to actual (spherical) dimensions using a technique modified from Dr. S. D. Wicksell.The most important findings in this study have to do with the rate at which pinocytosed fluid and surface membrane move into the cell and on their subsequent fate. Each minute macrophages form at least 125 pinocytic vesicles having a fractional vol of 0.43% of the cell's volume and a fractional area of 3.1% of the cell's surface area. The fractional volume and surface area influx rates for L cells were 0.05% and 0.8% per minute respectively. Macrophages and L cells thus interiorize the equivalent of their cell surface area every 33 and 125 min.During a 3-h period, the size of the secondary lysosome compartment remains constant and represents 2.5% of the cell volume and 18% of the surface area. Each hour, therefore, the volume and surface area of incoming vesicles is 10 times greater than the dimensions of the secondary lysosomes in both macrophages and L cells. This implies a rapid reduction in vesicle size during the formation of the secondary lysosome and the egress of pinocytosed fluid from the vacuole and the cell. In addition, we postulate that membrane components of the vacuole are subsequently recycled back to the cell surface.Pinocytosis involves the formation of a membranebound vesicle from the cell surface, trapping within it fluid and solutes from the extracellular environment. In most cells, these incoming pinocytic vesicles fuse with acid-hydrolase-containing granules or lysosomes. These events have been studied largely in relation to macromolecular and small particulate markers occupying the content of the incoming vesicle (e.g., 9, 15, 40, 41). In these instances, interiorized materials undergo digestion and/or sequestration within the lysosomal apparatus. Very little else is known about pinocytic vesicles. We lack quantitative information on their surface areas and volumes, especially in relation to

Role of Dynactin in Endocytic Traffic: Effects of Dynamitin Overexpression and Colocalization with CLIP-170

“…Regardless of the mechanism for endosome relocalization, our results suggest that cells overexpressing dynamitin have arrived at a new steadystate condition in which endosome movement has stopped. Under normal conditions, overall membrane flux is kept in balance so that export parallels import (Steinman et al, 1976). The bidirectional movement of individual organelles (e.g., lipid droplets) is also held in balance, because motility in both directions is altered in parallel when cells are subjected to physiological (Hamm-Alvarez et al, 1993) or genetic manipulation (Welte et al, 1998).…”

Dendritic cells: Understanding immunogenicity

“…Instead, the macrophage internalized HRP continually over long periods, and the bulk of the antigen was catabolized down to the level of amino acids. During these studies, we discovered that endocytosis required the rapid recycling of the internalized vesicle membrane [9,10]. As revealed by Brown and Goldstein [11], the recycling of endocytic receptors allows all cells to continually capture adsorbed proteins for efficient degradation in lysosomes.…”

Morphometric analysis of volumes and surface areas in membrane compartments during endocytosis in Acanthamoeba.

“…A model, taking into consideration these and other data on Acanthamoeba, is proposed to account for the observed membrane shifts . The data suggest that the vacuolar (digestive) system of Acanthamoeba is central to cellular control of endocytosis and membrane recycling.Evidence from several different kinds of studies suggests that the bulk of cell surface membrane in free-living endocytic cells is in a reversible equilibrium with cytoplasmic vacuolar system (4,6,12,(15)(16)(17)(18). The rate of cell surface turnover in these cells is very high, on the order of minutes (4,17,18) .…”