Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence for rapid turnover and compartmentation.

Abstract

The uptake and fate of pinocytosed fluid were investigated in monolayers of pulmonary alveolar macrophages and fetal lung fibroblasts using the fluid-phase marker, [14 C]sucrose . Initial experiments revealed that cellular accumulation of chromatographically repurified [14 C]sucrose was not linear with incubation time . Deviation from linearity was shown to be due to constant exocytosis of accumulating marker . Chromatographic analysis revealed that the cells were unable to metabolize sucrose and were releasing it intact by a process that was temperature-sensitive but not dependent on extracellular calcium and magnesium . A detailed analysis of the kinetics of exocytosis was undertaken by preloading cells with [ 1 'C] sucrose for various lengths of time and then monitoring the appearance of radioactivity into isotope-free medium . Results indicated that modeling the process of fluidphase pinocytosis and subsequent exocytosis required at least two intracellular compartments in series, one compartment being of small size and turning over very rapidly (t 1/2 = 5 min in macrophages, 6-8 min in fibroblasts) and the other compartment being apparently larger in size and turning over very slowly (t 1/2 = 180 min in macrophages, 430-620 min in fibroblasts) . Computer-simulation based on this model confirmed that the kinetics of efflux faithfully reflected the kinetics of influx and that the rate of efflux completely accounted for the deviation from linearity of accumulation kinetics . Moreover, the sizes of the compartments and magnitude of the intercom partment fluxes were such that the majority of fluid internalized in pinocytic vesicles was rapidly returned to the extracellular space via exocytosis . This result provides direct experimental evidence for a process previously thought necessary based solely on morphological and theoretical considerations . Furthermore, the turnover of pinocytosed fluid was so dynamic that accumulation deviated from linearity even within the first few minutes of incubation . We were able to show that the kinetics of exocytosis allowed calculation of the actual pinocytic rate, a rate that was nearly 50% greater than the apparent initial rate obtained from the slope of the uptake curve over the first 10 min .Pinocytosis may represent the afferent arm of a homeostatic mechanism for recycling the plasmalemma . The foundation for this hypothesis was laid by the work of Steinman et al . (37) . Their stereological analysis of pinocytosis in macrophages and L-cells revealed that the volume and surface area of incoming pinocytic vesicles was ten times greater than that which the secondary lysosomal compartment was shown to accommodate and, therefore, they postulated : (a) that interiorized pinocytic vesicle fluid must rapidly egress from the vacuolar system and (b) that pinocytic vesicle membrane is recycled back to the cell surface . Recently, the latter prediction has received experimental support both in mammalian (25, 34) and nonmammalian (6, 42) cells . These studies provided evide...

A multicompartmental model of fluid-phase endocytosis in rabbit liver parenchymal cells

“…Analysis of the kinetics of exocytosis of fluid-phase markers has previously suggested release from at least two compartments. A compartment with slow turnover similar to our compartment 3 was demonstrated by Besterman et al [23] in macrophages and fibroblasts. Entry of a fluid-phase marker into a slowly turning-over compartment has also been demonstrated in cultured rat hepatocytes [6].…”

“…In the present model, the rate constant for recycling of tracer from early endosomes was predicted to be 0.08 min-1, corresponding to a transit time of 12 min or a ti of 9 min, which is within the range of values published 2previously (t1 between 1 and 20 min) [6,[23][24][25][26] Vol. 262 compartment 3 to the medium was estimated to be 67 min.…”

“…Previous studies on isolated hepatocytes, macrophages, fibroblasts, L cells and adipocytes have shown that a marker internalized by fluid-phase endocytosis first enters a compartment that rapidly exchanges its contents with the extracellular medium [1,23,24]. In the present model, the rate constant for recycling of tracer from early endosomes was predicted to be 0.08 min-1, corresponding to a transit time of 12 min or a ti of 9 min, which is within the range of values published 2previously (t1 between 1 and 20 min) [6,[23][24][25][26] Vol.…”

Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process.

“…While membrane components are efficiently recycled back to the cell surface, acid-released ligands (t~2M and LDL) and bulk fluid (e.g., HRP) are lysosomally directed, albeit with lower efficiencies (Adams et al, 1982;Besterman et al, 1982;Greenspan and St. Clair, 1984;Yamashiro et al, 1989). Retained acid-released ligands and fluid phase molecules are then delivered to the late endosomes and lysosomes at the same rate (Ward et al, 1989), via a maturation process (Dunn and Maxfield, 1992;Stoorvogel et al, 1991).…”

RAB-10 Is Required for Endocytic Recycling in theCaenorhabditis elegansIntestine

“…Although it is sometimes difficult to image fluid phase markers in recycling endosomes in normal cells, perhaps because cargo is diluted within thin membrane tubules, the recycling of endocytosed fluid has been amply demonstrated in cultured mammalian cells (Tooze and Hollinshead, 1991;Apodaca et al, 1994b;Barroso and Sztul, 1994). At least 50% of internalized fluid recycles back into the culture medium, presumably in the same transport carriers as recycling receptors (Besterman et al, 1981;Bomsel et al, 1989;Gagescu et al, 2000). When recycling is blocked by pharmacological means in either MDCK cells (Apodaca et al, 1994a) or HepG2 cells (van Weert et al, 2000) gigantic endosomal structures similar to those described in C. elegans rme-1 mutant intestinal cells are formed.…”