Regulation of Macropinocytosis by p21-activated Kinase-1

The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5′-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.

Ultrastructural alterations of choroid plexuses of lateral ventricles of rats (Rattus norvegicus) submitted to experimental chronic alcoholism

Adult male rats (Wistar lineage) were alcoholized with sugar cane liquor diluted at 30(0) GL during 300 days and sacrificed every 60 days in 5 stages. Samples of choroid plexuses of lateral ventricles were collected and examined at transmission electronic microscope to detect possible ultrastructural alterations and to raise possible pathological correlations. Gradual changes were observed in these animals during all the experiment: dilatation and enlargement of cisternae of Golgi complex, dilatation of RER, presence of digestive vacuoles and a large amount of pinocytic vesicles as well as vesicles with electronlucent content throughout cytoplasm, as well as an enlargement of intercellular space between basolateral interdigitation of the cells and of the connective tissue. The changes observed in the epithelium and connective tissue of choroid plexuses specially in 240 and 300 days of treatment are presumably due to a disturbance in hydroelectrolitic homeostasis, contributing to several morpho-functional disturbs of central nervous system. No changes were observed in the control group animals.

v-Src induces constitutive macropinocytosis in rat fibroblasts

The role of v-Src as regulator of fluid-phase pinocytosis was investigated in Rat-1 cells expressing a stable (Rat-1/BB16) or a thermosensitive (Rat-1/tsLA29) v-Src protein. In the second cell line, this protein is inactive when cells are cultured at 40 degrees C but recovers its tyrosine kinase activity upon transfer to 34 degrees C, resulting into a transformed phenotype. The rate of fluid-phase pinocytosis of the tracer horseradish peroxidase was 2-fold higher in v-Src-transformed fibroblasts (Rat-1/BB16, Rat-1/tsLA29 cultured at 34 degrees C) as compared to non-transformed cells (Rat-1, Rat-1/tsLA29 kept at 40 degrees C). In contrast, receptor-mediated endocytosis of transferrin was poorly affected, suggesting that structures distinct from clathrin-coated pits are involved in pinocytosis stimulation. By light and electron microscopy, transformed cells frequently contained large peroxidase-labeled pinocytic vesicles located near to membrane ruffles, demonstrating that stimulation of pinocytosis corresponds to induction of constitutive macropinocytosis. Stimulation of pinocytosis occurred more than 8 hours after transfer to the permissive temperature, whereas transfer to the non-permissive temperature partially reversed the stimulation within 2 hours. Protein synthesis inhibition for 6 hours abrogated pinocytosis stimulation in transformed cells, indicating that constitutive macropinocytosis induced by v-Src depends on continuous synthesis of a short-lived regulatory machinery.

A Clinical, Ultrastructural and Immunochemical Study of Dupuytren’s Disease

Aponeurotic tissue from seven normal subjects and from apparently unaffected branches, nodules and cords of 16 Dupuytren’s patients were compared. Control tissue was characterized by polymorphous cells, showing cytoplasmic microfilament bundles, numerous pinocytic vesicles, basement membrane-like structures, and a thick coat of interwoven filaments, and by type I- and III-positive heterogeneous collagen fibrils, fibronectin, vitronectin, decorin and proteoglycans. The clinically normal branches consisted of fibroblast-like cells, small type III-highly positive collagen fibrils, fibronectin and proteoglycans. Nodules and fibrotic cords contained fibroblast-like cells, type I and III collagen, fibronectin and proteoglycans. Myofibroblast-like cells in only five out of 16 patients were present. There was no relation between clinical stage and structural alterations; the whole aponeurosis always seemed to be involved; cord retraction would seem to depend on the interactions among fibroblast-like cells and matrix components and among matrix macro-molecules themselves.

Evidence for a Vacuolar-Type Proton ATPase in Entamoeba histolytica

Entamoeba histolytica Entamoeba histolytica is a primitive eukaryote that lacks mitochondria, Golgi and a well-developed endoplasmic reticulum. Close to half of the cell volume is occupied by pinocytic vesicles, which are in continuous turnover with the plasma membrane and perform functions that in higher eukaryotic cells are taken over by lysosomes. Similar to the latter, the amebal vesicles are acidified. We report here that bafilomycin AI, a specific inhibitor of vacuolar-type (V-) ATPases, suppressed this acidification at submicromolar concentrations; concom itantly, it inhibited pinocytosis. These results strongly suggest the presence of a V-ATPase in pinocytic vesicles of E. histolytica, and thereby support the notion that the V-ATPases in the organelles of higher eukaryotes are derived from an archaic plasma membrane-bound form.

Characterization of early compartments in fluid phase pinocytosis: a cell fractionation study

Flotation through a 5.6% Percoll gradient of pinosomes from Chinese hamster ovary (CHO) cells labelled during a 10 min internalization period with horseradish peroxidase (HRP), a solute, revealed two pinosomal populations, the expected low-buoyancy population and an unexpected buoyant population. The buoyant pinosomes that sedimented similarly to plasma membrane were not an artifact of HRP trapping during homogenization or of cell surface-adherent HRP. No trapping or cell surface adherence of HRP could be detected by biochemical or cytochemical assays, even after internalization periods as short as 15 s to 1 min. With short uptake times, the buoyant pinosome population was the major HRP positive vesicle population, suggesting a precursor-to-product relationship between the two populations. In pulse-chase experiments, the buoyant pinosome population was shown to be highly exocytic and the precursor to later pinosomes. By electron-microscope cytochemistry, rapidly labelled, HRP positive pinosomes (15 s to 1 min uptake) were typically smooth vesicles with a median diameter of approximately equal to 0.30 micron and a size range from approximately equal to 0.10 micron to greater than 1.0 micron in diameter. We suggest that these rapidly labelled structures are a very early stage in the intracellular processing of pinocytic vesicles.

Osmotically induced microinjection of ricin bypasses a ricin internalization defect in a Chinese hamster ovary mutant cell line

By osmotic lysis of pinocytic vesicles we were able to inject ricin or ricin A chain directly into the cytosol of Chinese hamster ovary cells. The lag time of 1 to 2 h before the onset of the inhibition of protein synthesis by ricin in intact cells was reduced to 15 to 30 min by this method. Preincubation of cells with a low concentration of nigericin, which was shown earlier to enhance the cytotoxicity of ricin, had no effect under this condition. Direct transfer of either intact ricin or the ricin A subunit by osmotic lysis of pinocytic vesicles into the cytosol of the ricin-resistant CHO mutant cell line 4-10 rendered the mutant 4-10 cells as sensitive to ricin as the CHO pro wild-type cells. Both the lag time and the rate of inhibition of protein synthesis in the wild-type and mutant cell lines after the introduction of ricin by osmotic lysis of pinocytic vesicles were the same. These results indicate that injection of ricin into the cytosol by osmotic lysis of pinosomes bypasses the internalization defect in the mutant cell line.

Osmotically induced microinjection of ricin bypasses a ricin internalization defect in a Chinese hamster ovary mutant cell line.

By osmotic lysis of pinocytic vesicles we were able to inject ricin or ricin A chain directly into the cytosol of Chinese hamster ovary cells. The lag time of 1 to 2 h before the onset of the inhibition of protein synthesis by ricin in intact cells was reduced to 15 to 30 min by this method. Preincubation of cells with a low concentration of nigericin, which was shown earlier to enhance the cytotoxicity of ricin, had no effect under this condition. Direct transfer of either intact ricin or the ricin A subunit by osmotic lysis of pinocytic vesicles into the cytosol of the ricin-resistant CHO mutant cell line 4-10 rendered the mutant 4-10 cells as sensitive to ricin as the CHO pro wild-type cells. Both the lag time and the rate of inhibition of protein synthesis in the wild-type and mutant cell lines after the introduction of ricin by osmotic lysis of pinocytic vesicles were the same. These results indicate that injection of ricin into the cytosol by osmotic lysis of pinosomes bypasses the internalization defect in the mutant cell line.