Reconstitution of clathrin-independent endocytosis at the apical domain of permeabilized MDCK II cells: requirement for a Rho-family GTPase.

This paper studies the endocytosis of ricin at the apical pole of polarized MDCK II cells after permeabilization of the cells basolaterally with streptolysin O. Ricin endocytosis after the addition of cytosol with an ATP-regenerating system was 2-3-fold higher than after the addition of a transport medium. A similar increase in ricin endocytosis was obtained by reconstitution of dialyzed cytosol with the nonhydrolyzable GTP analog, GTP gamma S, in the presence of an ATP-regenerating system. The nonhydrolyzable GDP analog, GDP beta S, did not increase ricin uptake. In contrast to the data obtained with ricin, GTP gamma S was found to inhibit apical transferrin uptake in MDCK II cells transfected with the human transferrin receptor, and the data thus imply that GTP gamma S supports clathrin-independent endocytosis. Electron microscopy (EM) demonstrated that free endocytic vesicles were formed from the apical pole of permeabilized MDCK II cells in the presence of GTP gamma S and that both a ricin-HRP conjugate, HRP, and cationized gold were endocytosed. Ricin endocytosis in the presence of intact cytosol, as well as GTP gamma S-stimulated ricin uptake, was inhibited by Clostridium botulinum C3 transferase, an enzyme found to inactivate Rho proteins. The data demonstrate that apical clathrin-independent endocytosis functions in the presence of GTP gamma S, and suggest that one or more of the small GTP binding proteins of the Rho family is involved in regulation of the apical clathrin-independent endocytosis in MDCK II cells.

Apical macropinocytosis in polarized MDCK cells: regulation by N-ethylmaleimide-sensitive proteins.

In cells tested so far endocytosis seems to be dependent on N-ethylmaleimide (NEM)-sensitive proteins, and treatment with NEM results in a complete block of endocytosis. We here demonstrate that treatment of polarized MDCK I cells with NEM strongly increased endocytosis of ricin and horseradish peroxidase at the apical side, and electron microscopy revealed NEM-induced formation of large macropinosomes at the apical pole. The NEM-stimulated apical endocytosis seemed to involve phosphatidylinositol-3 kinase, protein kinase C and phospholipase D and it was dependent on ATP. Moreover, in contrast to endocytosis in nonpolarized cells ricin endocytosis at the basolateral side continued in the presence of NEM whereas endocytosis of transferrin was blocked. Furthermore, recycling of ricin endocytosed in the absence of NEM was not inhibited on either side upon addition of NEM demonstrating the existence of a NEM-resistant fusion machinery. The results suggest that the fusogenic property of both the apical and the basolateral plasma membrane of MDCK cells differs from that typically observed in cells unable to polarize.

Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles.

The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-beta-cyclodextrin (MbetaCD) to selectively extract cholesterol from the plasma membrane. MbetaCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MbetaCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MbetaCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MbetaCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.

Photochemical internalization: a novel technology for delivery of macromolecules into cytosol.

The therapeutic usefulness of macromolecules, such as in gene therapy, is often limited by an inefficient transfer of the macromolecule to the cytosol and a lack of tissue-specific targeting. The possibility of photochemically releasing macromolecules from endosomes and lysosomes into the cytosol was examined. Endocytosed macromolecules and photosensitizer were exposed to light and intracellular localization and the expression of macomolecules in the cytosol was analyzed. This novel technology, named photochemical internalization (PCI), was found to efficiently deliver type I ribosome-inactivating proteins, horseradish peroxidase, a p21ras-derived peptide, and a plasmid encoding green fluorescent protein into cytosol in a light-dependent manner. The results presented here show that PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that PCI may have a variety of useful applications for site-specific drug delivery, e.g., in gene therapy, vaccination, and cancer treatment.

Liposome-bound Zn (II)-phthalocyanine. Mechanisms for cellular uptake and photosensitization.

In the present study, cellular uptake of a liposomal formulation of ZnPc (CGP 55847) has been studied in human cervix carcinoma cells of the line NHIK 3025. The cellular uptake of ZnPc is found to be completed after 4-8 h of incubation. The maximum level of ZnPc in the cells after incubation with 1 microgram/ml ZnPc in E2a medium containing 3% serum is 60 ng/mg protein. The cellular uptake is attenuated by the presence of serum and at low temperature of the incubation medium, but the activation energy (30 kJ/mol) and fluorescence microscopic analysis of cells incubated with ZnPc at 0 degree C indicate that ZnPc is taken up into cells by a diffusion-mediated pathway. Measurements of subcellular marker enzymes have been performed immediately after light exposure of ZnPc-treated cells. The mitochondrial marker enzyme (cytochrome c oxidase) and the marker enzyme for the Golgi apparatus (UDP galactosyl transferase), but not those for lysosomes (beta-N-acetyl-D-glucosaminidase) and endoplasmic reticulum (NADPH cytochrome c reductase), are inactivated upon photodynamic treatment. These results indicate that ZnPc is mainly located in the Golgi apparatus and the mitochondria of NHIK 3025 cells. In contrast, photoactivated Photofrin is found to reduce the activity of UDP galactosyl transferase, but not that of NADPH cytochrome c reductase. The tetraphenylporphine TPPS2a and light reduce the activity of NADPH cytochrome c reductase, without influencing the activity of UDP galactosyl transferase. TPPS4 and light do not attenuate the activities of UDP galactosyl transferase and NADPH cytochrome c reductase.