Journal of Cell Science (5)
B. van Deurs
Apical endocytosis of ricin in MDCK cells is regulated by the cyclooxygenase pathway
A. Llorente, B. van Deurs, O. Garred, P. Eker, K. Sandvig
Journal: Journal of Cell Science
J Cell Sci (2000) 113 (7): 1213–1221.
DOI: https://doi.org/10.1242/jcs.113.7.1213
Addition of arachidonic acid or stimulation of arachidonic acid production by secretory phospholipase A2 selectively upregulated apical endocytosis of ricin in MDCK cells without affecting basolateral endocytosis. Electron microscopic studies revealed that MDCK cells treated with secretory phospholipase A2 and incubated with horseradish peroxidase had an increased number of normal appearing peroxidase-labeled endosomes and no sign of membrane ruffling. Moreover, inhibition of basal arachidonic acid release, either by decreasing the cytosolic phospholipase A(2) activity or the diacylglycerol lipase activity, reduced the rate of apical endocytosis. Furthermore, indomethacin, an inhibitor of the cyclooxygenase pathway, counteracted the stimulation of endocytosis seen with both secretory phospholipase A2 and arachidonic acid, suggesting that formation of eicosanoids such as prostaglandins could be essential for the regulation. This idea was supported by the finding that prostaglandin E2, the predominant prostaglandin formed in kidney, also upregulated ricin uptake. The regulatory effect of the cyclooxygenase pathway on apical endocytosis of ricin was found to be independent of protein kinases A and C, which are known to selectively control apical clathrin-independent endocytosis in polarized cells.
Endocytic mechanisms responsible for uptake of GPI-linked diphtheria toxin receptor
G. Skretting, M.L. Torgersen, B. van Deurs, K. Sandvig
DOI: https://doi.org/10.1242/jcs.112.22.3899
We have here used diphtheria toxin as a tool to investigate the type of endocytosis used by a glycosylphosphatidylinositol-linked molecule, a glycosylphosphatidylinositol-linked version of the diphtheria toxin receptor that is able to mediate intoxication. The receptor is expressed in HeLa cells where clathrin-dependent endocytosis can be blocked by overexpression of mutant dynamin. Diphtheria toxin intoxicates cells by first binding to cell-surface receptors, then the toxin is endocytosed, and upon exposure to low endosomal pH, the toxin enters the cytosol where it inhibits protein synthesis. Inhibition of protein synthesis by the toxin can therefore be used to probe the entry of the glycosylphosphatidylinositol-linked receptor into an acidic compartment. Furthermore, degradation of the toxin can be used as an indicator of entry into the endosomal/lysosomal compartment. The data show that although expression of mutant dynamin inhibits intoxication mediated via the wild-type receptors, mutant dynamin does not affect intoxication or endocytosis and degradation of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor. Confocal microscopy demonstrated that diphtheria toxin is transported to vesicles containing EEA1, a marker for early endosomes. Biochemical and ultrastructural studies of the HeLa cells used reveal that they have very low levels of caveolin-1 and that they contain very few if any caveolae at the cell surface. Furthermore, the endocytic uptake of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor was not reduced by methyl-beta-cyclodextrin or by nystatin which both disrupt caveolar structure and functions. Thus, uptake of a glycosylphosphatidylinositol-linked protein, in this case the diphtheria toxin receptor, into the endosomal/lysosomal system can occur independently of both caveolae and clathrin-coated vesicles.
Expression of caveolin-1 and polarized formation of invaginated caveolae in Caco-2 and MDCK II cells
U. Vogel, K. Sandvig, B. van Deurs
J Cell Sci (1998) 111 (6): 825–832.
DOI: https://doi.org/10.1242/jcs.111.6.825
We have studied caveolin-1 expression and the frequency and distribution of typical invaginated caveolae as they are identified by electron microscopy in the polarized epithelial cell lines MDCK II and Caco-2. In wild-type MDCK II cells caveolin expression is high and more than 400 caveolae/mm filter were observed at the basolateral membrane. No caveolae were found at the apical surface. By contrast, wild-type Caco-2 cells do not express caveolin-1 and have extremely few, if any caveolae. Caco-2 cells were stably transfected with the gene for caveolin-1 in order to investigate if the formation of caveolae is polarized also in these cells. We have isolated Caco-2 clones expressing different levels of caveolin-1, where the level of expression varies from 10–100% of the endogenous level in MDCK II cells. Caveolin-1 expression in Caco-2 cells gives rise to a marked immunofluorescense labeling mainly at the lateral plasma membrane. By electron microscopy an increase from less than 4 caveolae/mm filter in wild-type Caco-2 cells to 21–76 caveolae/mm filter in Caco-2 clones transfected with caveolin-1 was revealed and these caveolae were exclusively localized to the basolateral membrane. Thus expression of heterologous caveolin-1 in Caco-2 cells leads to polarized formation of caveolae, but there is a lack of correlation between the amount of caveolin expressed in the cells and the number of caveolae, suggesting that factors in addition to caveolin are required for generation of caveolae.
Destabilization of plasma membrane structure by prevention of actin polymerization. Microtubule-dependent tubulation of the plasma membrane
B. van Deurs, F. von Bulow, F. Vilhardt, P.K. Holm, K. Sandvig
Electron microscopy of thick (0.2-1.0 micron) sections of cytochalasin D-treated cells fixed in the presence of Ruthenium red revealed an extensive, surface-connected tubular compartment in HEp-2 cells. The tubules measured 120–220 nm in diameter and at least up to 6 microns in length. Morphometric analysis showed that in control cells about 0.2% of the total plasma membrane area (defined as all Ruthenium red-labeled membrane) appeared as vesicular or tubular profiles beneath the cell surface. However, after 15–30 minutes of cytochalasin D incubation about 4% of the total plasma membrane area is tubulated, and after 60–105 minutes as much as about 15% of the total plasma membrane appears as tubules. Clathrin-coated pits and caveolae-like structures were occasionally associated with the tubular membrane. Moreover, immunogold labeling showed that the tubular membrane contained transferrin receptors at about the same density as the nontubulated plasma membrane. Examination of cells in which endosomes and lysosomes were labeled with horseradish peroxidase before or after exposure to cytochalasin D showed that these organelles remained spherical, and that no horseradish peroxidase was present in the tubules. Moreover, the surface to volume ratio remained constant with increasing time of cytochalasin D incubation. Accordingly, the surface-connected tubules were not derived from endocytic structures but were formed by invagination of the plasma membrane. The tubule formation is reversible. When microtubules are depolymerized by nocodazole or colchicine treatment before the cells are exposed to cytochalasine D, tubule formation is strongly inhibited. Hence, the cytochalasin D-induced plasma membrane tubulation depends on intact microtubules.
Coated pits with pinocytosis in Tetrahymena
J.R. Nilsson, B. van Deurs
J Cell Sci (1983) 63 (1): 209–222.
DOI: https://doi.org/10.1242/jcs.63.1.209
The possible role in pinocytosis of coated pits at the parasomal sacs of Tetrahymena has been studied using cationized ferritin (CF; pI = 8.5) as a marker of membrane and content. It is shown that CF binds evenly to the surface, including the coated pits, of Tetrahymena in an inorganic salt medium (to avoid formation of food vacuoles) at the normal growth temperature. Moreover, CF is internalized by coated vesicles (shown to be truly free by thin serial-section analysis) and delivered initially (1-5 min of incubation) to cisterna near the cell surface. Later (5-10 min) CF occurs also in autophagic vacuoles, formed as a result of starvation, and eventually (15-90 min) it is present in preformed (old) food vacuoles. These observations indicate that the coated pits at the parasomal sacs of Tetrahymena function in adsorptive pinocytosis in much the same manner as coated pits at the surface of mammalian cells.