Hypertonic sucrose inhibition of endocytic transport suggests multiple early endocytic compartments

Abstract

Incubation of animal cells with hypertonic sucrose and polyethylene glycol (PEG) 1,000 renders endosomes sensitive in situ to hypotonic shock (Okada and Rechsteiner, 1982). We found that: 1) in vitro endosomes were osmotically insensitive; and 2) hypertonic sucrose inhibited transport from very early endosomes to lysosomes. Endocytic vesicles were labeled by incubating Chinese hamster ovary (CHO) cells for 1-10 min at 37 degrees C with horseradish peroxidase (HRP) and/or fluorescein isothiocyanate-conjugated dextran (FITC-dextran). Cell fractions prepared in 0.25 M sucrose were hypotonically shocked by dilution with 5 mM Na phosphate buffer, pH 6.7, to a final sucrose concentration of 0.05 M. After hypotonic shock, endocytized HRP and FITC-dextran pelleted with membrane while lysosomal hydrolases did not. The HRP activity in the pellet was latent, suggesting that endosomes were resistant to osmotic shock. Uptake in the presence of hypertonic sucrose had little effect on the subsequent osmotic sensitivity of the endosomes. Uptake in the presence of hypertonic sucrose and PEG 1,000 rendered endosomes fragile to cell homogenization. Unexpectedly, the inclusion of hypertonic sucrose in the uptake and chase media inhibited the appearance of HRP in lysosomes. HRP internalized during a 10-min uptake appeared as if it were present in two physically distinct compartments, one accessible to transport inhibition by exogenous sucrose ("very early" endosomes) and the other not ("early" endosomes). After a brief uptake (1-3 min), postincubation of CHO cells in 0.25 M sucrose-containing media completely blocked transport of internalized HRP to lysosomes. This blockage could be partially relieved by cointernalization of invertase with HRP. These results suggest that transport between multiple early endosome populations is sensitive to intraorganellar osmotic conditions.

RNA interference by osmotic lysis of pinosomes: liposome-independent transfection of siRNAs into mammalian cells

“…In our experiments, that would correspond to an uptake of 4 10 5 siRNA molecules per cell every 15 min during a single round of treatment. Consistent with previous reports (4), we confirmed that the osmotic shock leading to the lysis of the pinosomes within the cell is required to obtain the release of the contents of the pinocytotis vesicles (Figure 2, E and F).…”

“…Once cells have taken up the siRNAs in pinocytotic vesicles, the vesicles are lysed by a shock in hypotonic medium that results in the release of the compound into the cytosol. The pinocytotic vesicles do not fuse with lysosomes (4) and therefore store their cargo until osmotic lysis is forced. The pinocytosis technique, already established in the early 1980s by Okada and Rechsteiner (3), has been mainly used for the delivery of proteins or fluorescent dyes into the cells.…”

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

“…Also, using a cholesterol depletor, methyl-b-cyclodextrin, they observed a partial blockade PKC-induced functional downregulation, despite changes in DAT internalization equivalent to that seen with PMA treatments. Further support for physiologic trafficking-independent regulation of DAT after PKC activation has been obtained from studies of brain synaptosomes, where functional downregulation has been observed after phorbol ester treatment in the presence of high sucrose, which blocks endocytosis (Park et al, 1988). The Vaughan laboratory more recently provided evidence that the trafficking-independent effects of PKC may involve regulation of palmitoylation of DAT.…”

Defective Proximal Tubule Lysosomal Acidification by Bence Jones Proteins

“…PLC/late endosome is usually a large pleomorphic structure that, after separation on Percoll gradients, has a light buoyant density. In contrast, lysosomes are simple-secretion granule-like vesicles that are significantly denser [33]. Although there is no record of any molecule being present in lysosomes that is absent in the PLC/late endosomes, there are a number of proteins found in the latter, often in very high concentrations, that are not detected in lysosomes, such as cation-independent MPR [18].…”

“…Two distinct compartments have been identified in the terminal stages of the endocytic pathway: the proximal pre-lysosomal compartment (PLC or late endosome) and the more distal lysosomes [33, 34, 35]. These two compartments are structurally and functionally distinct and are supposed to be in dynamic equilibrium.…”