Pharmacological attenuation of Paramecium fluid-phase endocytosis

Spectrophotometric quantification of fluid phase endocytosis in the presence of different pharmacological compounds was performed in the model unicellular eukaryote Paramecium. The kinetics of Lucifer Yellow Carbohydrazide (LY) uptake in cells exposed to forskolin and isoproterenol--known to stimulate phagocytosis in this cell--was analyzed. Reduction in both the rate of endocytosis and total accumulation of fluid phase marker was observed following the treatment. Forskolin diminished total LY accumulation by 11% and 21% after 5 min and 25 min of incubation, respectively, whereas the rate of uptake was lowered by 21% in comparison to control cells. The inhibitory effect ofisoproterenol was less pronounced than that of forskolin. The total accumulation of LY was decreased by 11% in 5 min as compared to the untreated cells and this effect was persistent upon further exposition to this reagent up to 25 min. To better understand these observations, the effect of inhibitors of PKA and cAMP phosphodiesterase on fluid phase uptake was tested. 3-isobutyl-1-methyl xanthine (IBMX) caused 12% decrease in LY accumulation after 5 min of incubation. In combination with isoproterenol or forskolin, IBMX enhanced their inhibitory effect on fluid endocytosis, which was lowered by 25% and 29%, respectively. The strongest inhibitory effect on fluid endocytosis was exerted by the 10 microM PKA inhibitor, which diminished endocytosis by 35% in 5 min. These results suggest that Paramecium fluid phase uptake may be regulated through activation of PKA, although the precise mechanism of this process has not yet been elucidated.     

Potential sites of PI-3 kinase function in the endocytic pathway revealed by the PI-3 kinase inhibitor, wortmannin

Previously we have shown that PDGF receptor mutants that do not bind PI- 3 kinase internalize after ligand binding, but fail to downregulate and degrade. To define further the role of PI-3 kinase in trafficking processes in mammalian cells, we have investigated the effects of a potent inhibitor of PI-3 kinase activity, wortmannin. At nanomolar concentrations, wortmannin inhibited both the transfer of PDGF receptors from peripheral compartments to juxtanuclear vesicles, and their subsequent degradation. In contrast, the delivery of soluble phase markers to lysosomes, assessed by the accumulation of Lucifer yellow (LY) in perinuclear vesicles after 120 min of incubation, was not blocked by wortmannin. Furthermore, wortmannin did not affect the rate of transferrin uptake, and caused only a small decrease in its rate of recycling. Thus, the effects of wortmannin on PDGFr trafficking are much more pronounced than its effects on other endocytic events. Unexpectedly, wortmannin also caused a striking effect on the morphology of endosomal compartments, marked by tubulation and enlargement of endosomes containing transferrin or LY. This effect was somewhat similar to that produced by brefeldin A, and was also blocked by pre-treatment of cells with aluminum fluoride (AlF4-). These results suggest two sites in the endocytic pathway where PI-3 kinase activity may be required: (a) to sort PDGF receptors from peripheral compartments to the lysosomal degradative pathway; and (b) to regulate the structure of endosomes containing lysosomally directed and recycling molecules. This latter function could be mediated through the activation of AlFt4-)-sensitive GTP-binding proteins downstream of PI-3 kinase.     

A prelysosomal compartment sequesters membrane-impermeant fluorescent dyes from the cytoplasmic matrix of J774 macrophages

After the membrane impermeant dye Lucifer Yellow is introduced into the cytoplasmic matrix of J774 cells, the dye is sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. In the present work we studied the intracellular transport of Lucifer Yellow in J774 macrophages and the nature of the cytoplasmic vacuoles into which this dye is sequestered. When the lysosomal system of J774 cells was prelabeled with a Texas red ovalbumin conjugate and Lucifer Yellow was then loaded into the cytoplasm of the cells by ATP-mediated permeabilization of the plasma membrane, the vacuoles that sequestered Lucifer Yellow 30 min later were distinct from the Texas red-stained lysosomes. After an additional 30 min Lucifer Yellow and Texas red colocalized in the same membrane bound compartments, indicating that the Lucifer Yellow had been delivered to lysosomes. We next prelabeled the plasma membrane of J774 cells with anti-macrophage antibody and Texas red protein A before Lucifer Yellow was loaded into the cells. The phase-lucent vacuoles that subsequently sequestered Lucifer Yellow also stained with Texas red, showing that they were part of the endocytic pathway. J774 cells were fractionated on percoll density gradients either 15 or 60 min after Lucifer Yellow was introduced into the cytoplasmic matrix of the cells. In cells fractionated after 15 min, Lucifer Yellow was contained within the fractions of light buoyant density that contain plasma membrane and endosomes; the dye later appeared in vesicles of higher density which contained lysosomes. Secretion of Lucifer Yellow from the cytoplasmic matrix of J774 cells is inhibited by the organic anion transport blocker probenecid. We found that probenecid also reversibly inhibited sequestration of dye, indicating that sequestration of dye within cytoplasmic vacuoles was also mediated by organic anion transporters. These studies show that the vacuoles that sequester Lucifer Yellow from the cytoplasmic matrix of J774 cells possess the attributes of endosomes. Thus, in addition to their role in sorting of membrane bound and soluble substances, macrophage endosomes may play a role in the accumulation and transport of molecules resident in the soluble cytoplasm.     

Internalization of lucifer yellow in Candida albicans by fluid phase endocytosis

Lucifer yellow (LY), an impermeable fluorescent dye used as a marker for fluid phase endocytosis, was internalized by Candida albicans. As observed by fluorescence microscopy, incubation of C. albicans with LY in potassium phosphate buffer (pH 6.0) and glucose (2%, w/v) resulted in localization of the dye inside vacuoles. Sodium azide and carbonyl cyanide m-chlorophenylhydrazone, which are inhibitors of energy metabolism, decreased the uptake of the dye. The optimum temperature for uptake was 30 degrees C; no internalization was observed at 0 degrees C. Quantification of cell-associated LY by fluorescence spectrometry showed an uptake linear with time and not saturable over a 400-fold range of concentration. Thus, C. albicans internalized LY into vacuoles by a nonsaturable and time-, temperature- and energy-dependent process consistent with fluid phase endocytosis. Both the yeast and mould phase of this dimorphic fungus endocytosed LY. Growth in complex medium appeared to be required to enable the cells to internalize LY. However, addition of peptone or yeast extract to the phosphate buffer/glucose assay medium interfered with LY uptake by causing an apparent increase of exocytosis. These studies provide the first evidence of fluid phase endocytosis in C. albicans and may explain how some large molecules, such as toxins and cationic proteins, enter C. albicans.     

Uptake of Lucifer Yellow by plant cells in the presence of endocytotic inhibitors

Summary Lucifer Yellow (LY), a membrane-impermeant anion, was able to enterArabidopsis thaliana cells. LY was taken up by fluid-phase endocytosis and a plasmalemmal anionic carrier mechanism. Both mechanisms were shown to be concentration-dependent. At 0.1 mg/ml, LY was mainly taken up via fluid-phase endocytosis and concentrated in vesicular-like structures. At a ten-fold higher concentration (1 mg/ml), a plasmalemmal anionic carrier system allowed LY uptake and its accumulation in the central vacuole by a vacuolar anionic transporter. Chloroquine, cytochalasin B, monensin, and phorbol-12-myristate-13-acetate (PMA) hindered LY endocytosis. Brefeldin A did not modify LY uptake. The probenecidsensitive carrier uptake machinery showed sensitivity to chloroquine and PMA. Therefore the probenecid-sensitive transport mechanism seems to be complex and involve both acidification of a compartment and protein kinase C activity.Abbreviations CH carbohydrazide - DMSO dimethylsulfoxide - LY Lucifer Yellow - MES 2-[N-morpholino]-ethanesulfonic acid - MS Murashige and Skoog's medium - PMA phorbol-12-myristate-13-acetate - NAA naphthalene acetic acid     

Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages

Lucifer Yellow CH (LY) is an excellent probe for fluid-phase pinocytosis. It accumulates within the macrophage vacuolar system, is not degraded, and is not toxic at concentrations of 6.0 mg/ml. Its uptake is inhibited at 0 degree C. Thioglycollate-elicited mouse peritoneal macrophages were found to exhibit curvilinear uptake kinetics of LY. Upon addition of LY to the medium, there was a brief period of very rapid cellular accumulation of the dye (1,400 ng of LY/mg protein per h at 1 mg/ml LY). This rate of accumulation most closely approximates the rate of fluid influx by pinocytosis. Within 60 min, the rate of LY accumulation slowed to a steady-state rate of 250 ng/mg protein per h which then continued for up to 18 h. Pulse-chase experiments revealed that the reduced rate of accumulation under steady-state conditions was due to efflux of LY. Only 20% of LY taken into the cells was retained; the remainder was released back into the medium. Efflux has two components, rapid and slow; each can be characterized kinetically as a first-order reaction. The kinetics are similar to those described by Besterman et al. (Besterman, J. M., J. A. Airhart, R. C. Woodworth, and R. B. Low, 1981, J. Cell Biol. 91:716-727) who interpret fluid-phase pinocytosis as involving at least two compartments, one small, rapidly turning over compartment and another apparently larger one which fills and empties slowly. To search for processes that control intracellular fluid traffic, we studied pinocytosis after treatment of macrophages with horseradish peroxidase (HRP) or with the tumor promoter phorbol myristate acetate (PMA). HRP, often used as a marker for fluid-phase pinocytosis, was observed to stimulate the rate of LY accumulation in macrophages. PMA caused an immediate four- to sevenfold increase in the rate of LY accumulation. Both HRP and PMA increased LY accumulation by stimulating influx and reducing the percentage of internalized fluid that is rapidly recycled. A greater proportion of endocytosed fluid passes into the slowly emptying compartment (presumed lysosomes). These experiments demonstrate that because of the considerable efflux by cells, measurement of marker accumulation inaccurately estimates the rate of fluid pinocytosis. Moreover, pinocytic flow of water and solutes through cytoplasm is subject to regulation at points beyond the formation of pinosomes.     

Endocytosis in cultured rat alveolar type II cells: effect of lysosomotropic weak bases on the processes.

We investigated the uptake of Lucifer yellow and surfactant complexed with gold (S-G) by isolated alveolar Type II cells. The fluid phase marker Lucifer yellow did not reach lamellar bodies (LB) even after prolonged incubation time, whereas S-G was internalized and found in LB. Treatment of Type II cells with lysosomotropic weak bases (NH4Cl and chloroquine) resulted in dilation of endosomes, lysosomes, and LB. The effect of these agents on LB resulted in disappearance of their lamellar organization, as detected by polarized light and electron microscopy. After incubation in lysosomotropic agent-free medium, endocytosis of Lucifer yellow and S-G in treated cells was mainly directed towards large vacuoles resembling either multivesicular bodies (MVB) or lysosomes. The possible relationship between LB, MVB, and lysosomes in freshly isolated as well as cultured alveolar Type II cells is discussed.     

Uptake of Lucifer yellow CH in leaves ofCommelina communis is mediated by endocytosis

Summary Lucifer yellow CH (LY) uptake into intact leaves ofCommelina communis has been studied with conventional fluorescence microscopy as well as confocal laser scanning microscopy. LY, a highly fluorescent tracer for apoplastic transport in plants and fluid phase endocytosis in animal cells, accumulates in the vacuole of leaf cells. However, considerable differences in the ability to take up LY were observed among the various cell types. Mesophyll cells take up large amounts of the dye whereas epidermal cells, including guard and subsidiary cells, showed no fluorescence in their vacuoles. An exception to this are trichome cells which show considerable accumulation of LY. When introduced into the cytoplasm of mesophyll protoplasts ofC. communis by means of a patch-clamp pipette, LY does not enter the vacuole. This supports the contention that exogenous LY can only gain access to the vacuole via endocytosis. Differences in the capacity for LY uptake may therefore reflect differences in endocytotic activity.Abbreviations CLSM Confocal laser scanning microscopy - DIC differential interference contrast - LY Lucifer yellow CH - PM plasma membrane     

PKCβ-dependent phosphorylation of the glycine transporter 1

The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the glycine transporter 1 (GlyT1) and the clearance rate for glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the years, it has been shown that PKC tightly regulates the activity of several neurotransmitter transporters. In the present work, by stably expressing three N-terminus GlyT1 isoforms in porcine aortic endothelial cells and assaying for [³²P]-orthophosphate metabolic labeling, we demonstrated that the isoforms GlyT1a, GlyT1b, and GlyT1c were constitutively phosphorylated, and that phosphorylation was dramatically enhanced, in a time dependent fashion, after PKC activation by phorbol ester. The phosphorylation was PKC-dependent, since pre-incubation of the cells with bisindolylmaleimide I, a selective PKC inhibitor, abolished the phorbol ester-induced phosphorylation. Blotting with specific anti-phospho-tyrosine antibodies did not yield any signal that could correspond to GlyT1 tyrosine phosphorylation, suggesting that the phosphorylation occurs at serine and/or threonine residues. In addition, a 23–40%-inhibition on V_max was obtained by incubation with phorbol ester without a significant change on the apparent Km value. Furthermore, pre-incubation of the cells with the selective PKCα/β inhibitor Gö6976 abolished the downregulation effect of phorbol ester on uptake and phosphorylation, whereas the selective PKCβ inhibitors (PKCβ inhibitor or LY333531) prevented the phosphorylation without affecting glycine uptake, defining a specific role of classical PKC on GlyT1 uptake and phosphorylation. Taken together, these data suggest that conventional PKCα/β regulates the uptake of glycine, whereas PKCβ is responsible for GlyT1 phosphorylation.     

The effect of K+/H+ antiporter nigericin on gap junction permeability

The K+/H+ antiporter nigericin inhibits the intercellular exchange of the fluorescent dye Lucifer Yellow between DM15-transformed fibroblasts derived from the Djungarian hamster. The efficacy of nigericin action was related to its concentration and time of incubation. The nigericin-induced uncoupling effect on gap junctions was reversible and was shown to be based on its ability to cause cystolic acidification. The effect of nigericin on dye-coupling in intact and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) pretreated cells did not differ, indicating that the uncoupling effect of H⁺ on gap junctions in DM15 cells was not mediated by the TPA-dependent isoform of protein kinase C.Abbreviations: BCECF, 2,7-bis-(2-carboxyethyl)-5(6)carboxyfluoresceine - BS, bovine serum - LY, Lucifer Yellow - pH_i, intercellular pH - PKC, protein kinase C - TPA, 12-0-tetradecanoylphorbol-13-acetate     

Organic anion transport in macrophage membrane vesicles

Cells of the J774 mouse macrophage-like cell line possess organic anion transporter that transport fluorescent dyes such as Lucifer Yellow out of the cytoplasmic matrix of the cells; the dye is both sequestered in endosomes and secreted into the extracellular medium. Lucifer Yellow that is sequestered within endosomes is subsequently delivered to the lysosomal compartment. In the present studies we demonstrated that probenecid inhibited removal of Lucifer Yellow from the soluble cytoplasm and sequestration into membrane bound organelles by quantitating Lucifer Yellow fluorescence in both soluble and membrane-associated fractions of J774 cells. In addition, we examined the uptake of Lucifer Yellow into isolated subcellular organelles derived from J774 cells. Lucifer Yellow transport in the organellar fraction of J774 cell homogenates was temperature- and pH-dependent and did not require ATP. Subcellular organelles from J774 cells were fractionated into endosome- and lysosome-enriched fractions by Percoll density gradient centrifugation. Lucifer Yellow was preferentially taken up by vesicles of the endosome-enriched fraction, and this transport was inhibited by probenecid. These studies provide direct evidence that probenecid inhibits Lucifer Yellow transport out of the cytoplasmic matrix and into cytoplasmic vacuoles in J774 cells and that organic anion transport in isolated organelles derived from J774 cells occurs preferentially in endosome, rather than in lysosome-enriched fractions; they suggest that Lucifer Yellow is carried across membranes via a secondary active transport process that requires proton symptom or hydroxyl anion antiport.     

PKCβ-dependent phosphorylation of the glycine transporter 1

The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the glycine transporter 1 (GlyT1) and the clearance rate for glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the years, it has been shown that PKC tightly regulates the activity of several neurotransmitter transporters. In the present work, by stably expressing three N-terminus GlyT1 isoforms in porcine aortic endothelial cells and assaying for [³²P]-orthophosphate metabolic labeling, we demonstrated that the isoforms GlyT1a, GlyT1b, and GlyT1c were constitutively phosphorylated, and that phosphorylation was dramatically enhanced, in a time dependent fashion, after PKC activation by phorbol ester. The phosphorylation was PKC-dependent, since pre-incubation of the cells with bisindolylmaleimide I, a selective PKC inhibitor, abolished the phorbol ester-induced phosphorylation. Blotting with specific anti-phospho-tyrosine antibodies did not yield any signal that could correspond to GlyT1 tyrosine phosphorylation, suggesting that the phosphorylation occurs at serine and/or threonine residues. In addition, a 23-40%-inhibition on V_max was obtained by incubation with phorbol ester without a significant change on the apparent Km value. Furthermore, pre-incubation of the cells with the selective PKCα/β inhibitor Gö6976 abolished the downregulation effect of phorbol ester on uptake and phosphorylation, whereas the selective PKCβ inhibitors (PKCβ inhibitor or LY333531) prevented the phosphorylation without affecting glycine uptake, defining a specific role of classical PKC on GlyT1 uptake and phosphorylation. Taken together, these data suggest that conventional PKCα/β regulates the uptake of glycine, whereas PKCβ is responsible for GlyT1 phosphorylation.     

Tubular lysosomes accompany stimulated pinocytosis in macrophages

A network of tubular lysosomes extends through the cytoplasm of J774.2 macrophages and phorbol ester-treated mouse peritoneal macrophages. The presence of this network is dependent upon the integrity of cytoplasmic microtubules and correlates with high cellular rates of accumulation of Lucifer Yellow (LY), a marker of fluid phase pinocytosis. We tested the hypothesis that the efficiency of LY transfer between the pinosomal and lysosomal compartments is increased in the presence of tubular lysosomes by asking how conditions that deplete the tubular lysosome network affect pinocytic accumulation of LY. Tubular lysosomes were disassembled in cells treated with microtubule-depolymerizing drugs or in cells that had phagocytosed latex beads. In unstimulated peritoneal macrophages, which normally contain few tubular lysosomes and which exhibit relatively inefficient transfer of pinocytosed LY to lysosomes, such treatments had little effect on pinocytosis. However, in J774 macrophages and phorbol ester-stimulated peritoneal macrophages, these treatments markedly reduced the efficiency of pinocytic accumulation of LY. We conclude that a basal level of solute accumulation via pinocytosis proceeds independently of the tubular lysosomes, and that an extended tubular lysosomal network contributes to the elevated rates of solute accumulation that accompany macrophage stimulation. Moreover, we suggest that the transformed mouse macrophage cell line J774 exhibits this stimulated pinocytosis constitutively.     

Rabies virus entry into cultured rat hippo campalneurons

Rabies virus entry into cultured hippocampal neurons was investigated by immunofluorescence and electron microscopy. Hippocampal neurons were susceptible to rabies virus infection and became filled with viral antigen 1 day after infection. Infection was inhibited by the lysosomotropic agents chloroquine and ammonium chloride. To study entry, neurons were adsorbed with rabies virus at 4°C and warmed to 37°C for short periods of time prior to fixation and localization of viral antigen by immunofluorescence microscopy. By 5 min at 37°C, viral antigen was localized to puncta in the cell body and dendrites and in synapses along dendrites. Little viral antigen was present in axons. Cells adsorbed with rabies virus were incubated with tracers for early endosomes. The endocytic tracers or markers Lucifer Yellow, transferrin receptor, dextran, and wheat germ agglutinin co-localized with rabies virus, indicating that rabies virus enters an endosome compartment shortly after uptake. Rabies virus also co-localized with LysoTracker Red, an acidotropic probe, indicating that some of the virus-containing endosomes are acidified. Rabies virus also co-localized with synapsin I, a synaptic vesicle marker, in nerve terminals but did not co-localize with lysosomal glycoprotein. By electron microscopy, after adsorption of virus and warming for 10 min, virus particles were present in coated pits, coated vesicles, and vacuolar membrane compartments in processes and axon terminals. It is concluded that rabies virus enters the somatodendritic domain and axon terminals of cultured hippocampal neurons by adsorptive endocytosis and is located in endosomes shortly after uptake.     

Hyperosmolarity inhibits galactosyl receptor-mediated but not fluid phase endocytosis in isolated rat hepatocytes

We have investigated the effects of hyperosmolarity induced by sucrose on the fluid phase endocytosis of the fluorescent dye lucifer yellow CH (LY) and the endocytosis of 125I-asialo-orosomucoid (ASOR) by the galactosyl receptor system in isolated rat hepatocytes. Continuous uptake of LY by cells at 37 degrees C is biphasic, occurs for 3-4 h, and then plateaus. Permeabilized cells or crude membranes do not bind LY at 4 or 37 degrees C. Intact cells also do not accumulate LY at 4 degrees C. The rate and extent of LY accumulation are concentration- and energy-dependent, and internalized LY is released from permeabilized cells. Efflux of internalized LY from washed cells is also biphasic and occurs with halftimes of approximately 38 and 82 min. LY is taken up into vesicles throughout the cytoplasm and the perinuclear region with a distribution pattern typical of the endocytic pathway. LY, therefore, behaves as a fluid phase marker in hepatocytes. LY has no effect on the uptake of 125I-ASOR at 37 degrees C. The rate of LY uptake by cells in suspension is not affected for at least 30 min by up to 0.2 M sucrose. The rate of endocytosis of 125I-ASOR, however, is progressively inhibited by increasing the osmolality of the medium with sucrose (greater than 98% with 0.2 M sucrose; Oka and Weigel (1988) J. Cell. Biochem. 36, 169-183). Hyperosmolarity completely inhibits endocytosis of 125I-ASOR by the galactosyl receptor, whereas fluid phase endocytosis of LY is unaffected. Cultured hepatocytes contained about 100 coated pits/mm of apical membrane length as assessed by transmission electron microscopy. In the presence of 0.4 M sucrose, only 17 coated pits/mm of membrane were observed, an 83% decrease. Only a few percent of the total cellular fluid phase uptake in hepatocytes is due to the coated pit endocytic pathway. We conclude that the fluid phase and receptor-mediated endocytic processes must operate via two separate pathways.     

Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein

Previously, we reported that fluid-phase endocytosis of native LDL by PMA-activated human monocytederived macrophages converted these macrophages into cholesterol-enriched foam cells (Kruth, H. S., Huang, W., Ishii, I., and Zhang, W. Y. (2002) J. Biol. Chem. 277, 34573-34580). Uptake of fluid by cells can occur either by micropinocytosis within vesicles (<0.1 microm diameter) or by macropinocytosis within vacuoles ( approximately 0.5-5.0 microm) named macropinosomes. The current investigation has identified macropinocytosis as the pathway for fluid-phase LDL endocytosis and determined signaling and cytoskeletal components involved in this LDL endocytosis. The phosphatidylinositol 3-kinase inhibitor, LY294002, which inhibits macropinocytosis but does not inhibit micropinocytosis, completely blocked PMA-activated macrophage uptake of fluid and LDL. Also, nystatin and filipin, inhibitors of micropinocytosis from lipid-raft plasma membrane domains, both failed to inhibit PMA-stimulated macrophage cholesterol accumulation. Time-lapse video phase-contrast microscopy and time-lapse digital confocal-fluorescence microscopy with fluorescent DiI-LDL showed that PMA-activated macrophages took up LDL in the fluid phase by macropinocytosis. Macropinocytosis of LDL depended on Rho GTPase signaling, actin, and microtubules. Bafilomycin A1, the vacuolar H+-ATPase inhibitor, inhibited degradation of LDL and caused accumulation of undegraded LDL within macropinosomes and multivesicular body endosomes. LDL in multivesicular body endosomes was concentrated >40-fold over its concentration in the culture medium consistent with macropinosome shrinkage by maturation into multivesicular body endosomes. Macropinocytosis of LDL taken up in the fluid phase without receptor-mediated binding of LDL is a novel endocytic pathway that generates macrophage foam cells. Macropinocytosis in macrophages and possibly other vascular cells is a new pathway to target for modulating foam cell formation in atherosclerosis.     

Selective modulation of the endocytic uptake of ricin and fluid phase markers without alteration in transferrin endocytosis

Cytochalasin D was found to reduce the endocytosis of ricin and the fluid phase markers [14C]sucrose and Lucifer Yellow in Vero cells without reducing the uptake of transferrin. The number of coated pits at the plasma membrane was not affected by the treatment. Cytochalasin D also reduced the endocytosis of ricin in cells where uptake of transferrin from coated pits was blocked by low cytosolic pH. Colchicine had a similar effect as cytochalasin D. Both drugs inhibited the exocytosis of ricin from the cells, and they reduced the rate by which ricin intoxicated the cells. Cytochalasin D had essentially no effect on the ability of the cells to bind transferrin, whereas colchicine reduced the binding to some extent. Epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) increased the endocytic uptake of ricin in A431 cells both under normal culture conditions and when the coated pit/coated vesicle pathway was blocked by acidification of the cytosol. In contrast, EGF and TPA had no stimulatory effect on the uptake of transferrin at normal cytoplasmic pH, and they did not abolish the ability of low cytoplasmic pH to inhibit endocytic uptake of transferrin. The results indicate that cytochalasin D and colchicine selectively inhibit endocytic uptake from non-clathrin-coated areas of the cell membrane whereas EGF and TPA stimulate it. The data support the view that there are different endocytic mechanisms, and they indicate that at least in some cell types the non-clathrin-coated endocytosis can be modulated.     

Low cytoplasmic pH inhibits endocytosis and transport from the trans- Golgi network to the cell surface

A fibroblast mutant cell line lacking the Na+/H+ antiporter was used to study the influence of low cytoplasmic pH on membrane transport in the endocytic and exocytic pathways. After being loaded with protons, the mutant cells were acidified at pH 6.2 to 6.8 for 20 min while the parent cells regulated their pH within 1 min. Cytoplasmic acidification did not affect the level of intracellular ATP or the number of clathrin-coated pits at the cell surface. However, cytosolic acidification below pH 6.8 blocked the uptake of two fluid phase markers, Lucifer Yellow and horseradish peroxidase, as well as the internalization and the recycling of transferrin. When the cytoplasmic pH was reversed to physiological values, both fluid phase endocytosis and receptor-mediated endocytosis resumed with identical kinetics. Low cytoplasmic pH also inhibited the rate of intracellular transport from the Golgi complex to the plasma membrane. This was shown in cells infected by the temperature-sensitive mutant ts 045 of the vesicular stomatitis virus (VSV) using as a marker of transport the mutated viral membrane glycoprotein (VSV-G protein). The VSV-G protein was accumulated in the trans-Golgi network (TGN) by an incubation at 19.5 degrees C and was transported to the cell surface upon shifting the temperature to 31 degrees C. This transport was arrested in acidified cells maintained at low cytosolic pH and resumed during the recovery phase of the cytosolic pH. Electron microscopy performed on epon and cryo-sections of mutant cells acidified below pH 6.8 showed that the VSV-G protein was present in the TGN. These results indicate that acidification of the cytosol to a pH less than 6.8 inhibits reversibly membrane transport in both endocytic and exocytic pathways. In all likelihood, the clathrin and nonclathrin coated vesicles that are involved in endo- and exocytosis cannot pinch off from the cell surface or from the TGN below this critical value of internal pH.     

Cell-cell interactions in the process of differentiation of thyroid epithelial cells into follicles: a study by microinjection and fluorescence microscopy on in vitro reconstituted thyroid follicles

Thyroid cells, cultured in the presence of thyroid stimulating hormone, reorganized within 36-48 hr into follicular structures, the in vitro reconstituted thyroid follicles or RTF. By microinjection of fluorescent probes either into the neoformed intrafollicular lumen (IL) or into cells forming the follicles, we have studied the development and some functional properties of cell-cell contacts involved in a) the formation of the thyroid follicular lumen and b) the communication between thyrocytes within the follicle. The probes were compounds of either low (Lucifer Yellow: LY) or high molecular weight (Dextran labeled with fluorescein: FITC-Dextran and Cascade Blue conjugated to bovine serum albumin: CB-BSA). LY microinjected into IL of 2-9-day-old RTF was seen to label circular spaces with a diameter ranging from 10 to 100 microns. The cells delimiting the IL remained unlabeled. The fluorescent dye remained concentrated in IL for up to 24 hr. FITC-Dextran or CB-BSA microinjected into IL behaved as LY; the probes were restrained into the lumen. A 2 hr incubation of RTF with iodide induced alterations of the structure of IL; an effect mediated by an organic form of actively trapped iodide. A 15-30 min incubation of RTF in a low CA2+ medium caused the opening of IL visualized by the progressive decrease of the fluorescence of probes preinjected into the lumenal space. The same but more rapid effect was obtained by microinjection of EGTA into the IL. The low Ca2(+)-dependent opening of IL was also demonstrated by the release into the medium of thyroglobulin present in IL. Microinjection of LY in a cell involved in the follicle structure led to the rapid labeling of the other cells forming the follicle but LY did not penetrate the IL. Unlike LY, the distribution of FITC-Dextran or CB-BSA injected into cells delimiting the lumen was restricted to the microinjected cells. Alterations of medium or intralumenal Ca2+ concentration which caused the opening of IL did not affect the cell-to-cell transfer of LY. By using fluorescent probe microinjection, we show that the in vitro thyrocyte histiotypic differentiation leads to the reconstitution of functional intercellular junctions: tight junctions insuring the tightness of the neoformed lumen and gap junctions mediating the cell-to-cell exchange of small molecules. The structure of the thyroid follicles appears to be under the control of both extracellular and intralumenal Ca2+ concentrations.     

Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes

At 4 degrees C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37 degrees C receptor-mediated endocytosis of transferrin occurred. Uptake at 37 degrees C exceeded binding at 4 degrees C by 2.5-fold and saturated after 20-30 min. During uptake at 37 degrees C, bound transferrin was internalized into a trypsin- resistant space. Trypsinization at 4 degrees C destroyed surface receptors, but with subsequent incubation at 37 degrees C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37 degrees C colloidal gold-transferrin (AuTf) clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37 degrees C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatase-negative compartments. AuTf, like transferrin, was released with subsequent incubation at 37 degrees C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.