Immunocytochemical study of endocytotic structures accumulated in HeLa cells transformed with a temperature-sensitive mutant of dynamin.

Dynamin is a 100-kD GTPase, which is required for clathrin-mediated endocytosis. Recent studies have revealed that dynamin is closely involved in clathrin-coated vesicle formation. In this study we investigated the ultrastructure of endocytotic structures accumulated in HeLa cells that were transformed with a temperature-sensitive (ts) mutant of dynamin to clarify which step was blocked in dynts cells. Endocytosis of transferrin receptors was restricted at the level of surface-connected membrane structures. Tubular and vesicular membrane invaginations were accumulated in the cells' peripheral regions, suggesting that the endocytosis was blocked just before the pinching-off steps in coated vesicle formation. The "collared" tubes, which were reported to be localized in nerve terminals in shibirets1 flies and GTPgammaS-treated synaptosomes, were not observed in the dynts cells even at nonpermissive temperature. The distribution pattern of dynamin in deeply invaginated coated pits in dynts cells was similar to that in dynwt cells but not to that in dynK44A cells, which are other endocytosis-defective mutant cells. These morphological data suggest that dynts blocked the pinching-off steps in clathrin-coated vesicle formation, which may be caused by a different mechanism from that of dynK44A cells.     

Control mechanisms governing the infectivity of Chlamydia trachomatis for HeLa cells: mechanisms of endocytosis

The mechanism by which Chlamydia trachomatis is endocytosed by host cells is unclear. Studies of the kinetics of chlamydial attachment and uptake in the susceptible HeLa 229 cell line showed that chlamydial endocytosis was rapid and saturable but limited by the slow rate of chlamydial attachment. To overcome this limitation and to investigate the mechanism of endocytosis, chlamydiae were centrifuged onto the host cell surface in the cold to promote attachment. Endocytosis of the adherent chlamydiae was initiated synchronously by rapid warming to 36 degrees C. Electron micrographs of chlamydial uptake 5 min after onset showed that chlamydial ingestion involves movement of the host cell membrane, leading to interiorization in tight, endocytic vacuoles which were not clathrin coated. Chlamydial ingestion was not inhibited by monodansylcadaverine or amantadine, inhibitors of receptor-mediated endocytosis and chlamydiae failed to displace [3H]sucrose from micropinocytic vesicles. Chlamydial endocytosis was markedly inhibited by cytochalasin D, an inhibitor of host cell microfilament function, and by vincristine or vinblastine, inhibitors of host cell microtubules. Hyperimmune rabbit antibody prevented the ingestion of adherent chlamydiae, suggesting that endocytosis requires the circumferential binding of chlamydial and host cell surface ligands. These findings were incompatible with the suggestion that chlamydiae enter cells by taking advantage of the classic mechanism of receptor-mediated endocytosis into clathrin-coated vesicles, used by the host cell for the internalization of beta-lipoprotein and other macromolecules, but were consistent with the hypothesis that chlamydiae enter cells by a microfilament-dependent zipper mechanism.     

Evidence that globular Golgi clusters in mitotic HeLa cells are clustered tubular endosomes.

By conventional electron microscopy we observed in mitotic HeLa cells the structures termed Golgi clusters by Lucocq et al. (J. Cell Biol. 104, 865-874 (1987)) and interpreted by them as clusters of vesicular remnants of the Golgi apparatus. Golgi clusters consist of tubular and vesicular profiles about 50 nm in diameter, sometimes associated with larger 250 nm vesicles. When cultures of HeLa cells were incubated for 60 min or 120 min with medium containing high specific activity horseradish peroxidase (HRP) at 10 mg/ml we found that the membrane-bound compartments in the Golgi clusters in mitotic cells contained heavy deposits of HRP reaction product. Neither interphase nor mitotic HeLa cells contain an endogenous peroxidase activity. We concluded that Golgi clusters are an endocytic compartment and confirmed this by showing that Golgi clusters could be labeled with two other endocytic tracers--bovine serum albumin conjugated to colloidal gold and transferrin conjugated to HRP. When cultures were incubated with HRP for only 15 min most of the Golgi clusters in the mitotic cells were either unlabeled or consisted of a mixture of HRP-labeled and unlabeled profiles. Since during mitosis endocytosis is inhibited this was the expected result. When interphase HeLa cells were incubated with Brefeldin A (BFA), the Golgi apparatus disassembled and immunofluorescence microscopy showed that 1,4 beta galactosyltransferase had relocated to the endoplasmic reticulum. When cells in the presence of BFA and lacking the Golgi apparatus were allowed to endocytose HRP and then entered mitosis, typical HRP-labeled Golgi clusters were seen in the mitotic cells. It is therefore highly unlikely that these structures contain membrane derived from the Golgi cisternae that are sensitive to BFA, including in HeLa cells those containing galactosyltransferase. Finally, we found that interphase HeLa cells incubated with okadaic acid contain structures that are morphologically indistinguishable from Golgi clusters but can be labeled by endocytic tracer. Taken together, this evidence indicates that most, if not all, of the membrane-bound compartments in Golgi clusters are tubular early endosomes.     

Redundant and Distinct Functions for Dynamin-1 and Dynamin-2 Isoforms

A role for dynamin in clathrin-mediated endocytosis is now well established. However, mammals express three closely related, tissue-specific dynamin isoforms, each with multiple splice variants. Thus, an important question is whether these isoforms and splice variants function in vesicle formation from distinct intracellular organelles. There are conflicting data as to a role for dynamin-2 in vesicle budding from the TGN. To resolve this issue, we compared the effects of overexpression of dominant-negative mutants of dynamin-1 (the neuronal isoform) and dynamin-2 (the ubiquitously expressed isoform) on endocytic and biosynthetic membrane trafficking in HeLa cells and polarized MDCK cells. Both dyn1(K44A) and dyn2(K44A) were potent inhibitors of receptor-mediated endocytosis; however neither mutant directly affected other membrane trafficking events, including transport mediated by four distinct classes of vesicles budding from the TGN. Dyn2(K44A) more potently inhibited receptor-mediated endocytosis than dyn1(K44A) in HeLa cells and at the basolateral surface of MDCK cells. In contrast, dyn1(K44A) more potently inhibited endocytosis at the apical surface of MDCK cells. The two dynamin isoforms have redundant functions in endocytic vesicle formation, but can be targeted to and function differentially at subdomains of the plasma membrane.     

Endocytosis at 0° C, 5° C,and 10° C in trophotaenial absorptive cells of goodeid embryos (Teleostei)

Summary The absorptive epithelium of the trophotaeniae of goodeid embryos is involved in the micropinocytotic uptake of protein macromolecules from the ovarian embryotrophe. Incubations of viable Xenoophorus captivus embryos in vitro with horseradish peroxidase (HRP) and/or cationized ferritin (CF) allows the tracing of the fluid-phase and receptor-mediated pathways, respectively. Effects of lowered temperature on both these endocytotic mechanisms have been investigated. At 10° C, trophotaenial absorptive cells (TACs) have a strong capacity to ingest marker proteins from double tracer media. Surface-bound ligands (CF) and solutes (HRP), taken up in primary pinocytic vesicles, are rapidly channelled to the endosomal compartment. Part of the ingested CF is segregated into dense apical tubules and small vesicles indicating that membrane recycling and transcytosis continue at 10° C. Adsorptive endocytosis of CF at 5° C proceeds at a decreased rate. After incubation periods of 30 min and 1 h, tracer molecules can be found in vesicular, tubular and vacuolar compartments of the apical endocytic zone. At 0° C, no uptake of ligand worth mentioning could be ascertained. Fluid-phase endocytosis, on the other hand, is observable at this temperature. Enzyme reaction product accumulates in flattened vacuoles rather than typical voluminous endosomes. After prolonged exposure to HRP, the epithelial junctional complex becomes leaky and the marker protein penetrates the intercellular space and the lateral lamellar membrane invaginations of TACs.Supported by the Deutsche Forschungsgemeinschaft     

Small-cell Lung Cancer (Human): Potentiation of Endocytic Membrane Activity by Voltage-gated Na<Superscript>+</Superscript> Channel Expression in Vitro

The possible functional role of voltage-gated Na⁺ channel (VGSC) expression in controlling endocytic membrane activity in human small-cell lung cancer (SCLC) cell lines (H69, H209, H510) was studied using uptake of horseradish peroxidase (HRP). The normal human airway epithelial (16HBE14o) cell line was used in a comparative approach. Uptake of HRP was vesicular, strongly temperature-sensitive and suppressed by cytoskeletal poisons (cytochalasin D and colchicine), consistent with endocytosis. Compared with the normal cells, HRP uptake into SCLC cells was kinetically more efficient, resulting in more than four-fold higher uptake under optimized conditions. Importantly, HRP uptake into SCLC cells was inhibited significantly by the specific VGSC blocker tetrodotoxin, as well as lidocaine and phenytoin. These effects were dose-dependent. None of these drugs had any effect on the uptake into the 16HBE14o cells. Uptake of HRP into SCLC cells was reduced by ∼66% in Na⁺-free medium and was partially (∼30%) dependent on extracellular Ca²⁺. The possibility that the endocytic activity in the H510 SCLC cells involved an endogenous cholinergic system was investigated by testing the effects of carbachol (a cholinergic receptor agonist) and eserine (an inhibitor of acetylcholinesterase). Both drugs inhibited HRP uptake, thereby suggesting that basal cholinergic activity occurred. It is concluded that VGSC upregulation could enhance metastatic cell behavior in SCLC by enhancing endocytic membrane activity.     

Tubular early endosomal networks in AtT20 and other cells

Using horseradish peroxidase (HRP) as a fluid-phase endocytic tracer, we observed through the electron microscope numerous tubular endosomes with a diameter of 30-50 nm and lengths of greater than 2 microns in thick sections (0.2-0.5 microns) of AtT20 cells. These tubular endosomes are multibranching and form local networks but not a single reticulum throughout the cytoplasm. They are sometimes in continuity with vesicular endosomal structures but have not been observed in continuity with AtT20 cell late endosomes. Tubular endosomal networks are not uniformly distributed throughout the cytoplasm, but are particularly abundant in growth cones, in patches below the plasma membrane of the cell body, and surrounding the centrioles and microtubule organizing center (MTOC). Tubular endosomes at all these locations receive HRP within the first 5 min of endocytosis but approximately 30 min of endocytosis are required to load the tubular endosomal networks with HRP so that their full extent can be visualized in the electron microscope. After 10 min of endocytosis, complete unloading occurs within 30 min of chase, but between 30 and 60 min are required to chase out all the tracer from the tubular endosomes loaded to steady state during 60 min endocytosis of 10 mg/ml HRP. In interphase cells, neither the loading nor unloading of tubular endosomes depends on microtubules but in cells blocked in mitosis by depolymerization of the mitotic spindle with nocodazole, HRP does not chase out of tubular endosomes. The thread-like shape of tubular endosomes is not dependent on microtubules. Furthermore, HRP is delivered to AtT20 tubular endosomes at 20 degrees C. All these properties indicate that AtT20 cell tubular endosomes are an early endocytic compartment distinct from late endosomes. Tubular endosomes like those in AtT20 cells have been seen in cells of the following lines: PC12, HeLa, Hep2, Vero, MDCK I and II, CCL64, RK13, and NRK; they are particularly abundant in the first three lines. In contrast, tubular endosomes are sparse in 3T3 and BHK21 cells. The tubular endosomes we have observed appear to be identical to the endosomal reticulum observed in the living Hep2 cells by Hopkins, C. R., A. Gibson, H. Shipman, and K. Miller. 1990.     

Increased endocytosis and formation of multivesicular bodies in phorbol ester-stimulated human monoblastic U-937 cells

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is known to arrest mitotic activity and induce macrophage differentiation in the U-937 monoblastic cell line. The acute effect of TPA on ultrastructural morphology and endocytic activity of U-937 cells was studied. TPA induced within 15 min a marked enlargement of multivesicular bodies (MVBs), comprising both volume and number of inclusion vesicles (other organelles appeared unchanged). At this stage the MBVs frequently showed tubular cytoplasmic extensions. Inclusion vesicles accumulated in MBVs with prolonged incubation (60 min). Horseradish peroxidase (HRP) and cationized ferritin (CF) added to the medium were routed preferentially to MVBs in TPA-stimulated cells. In contrast to MVBs of unstimulated cells many of the TPA-induced MVBs showed a positive cytochemical reaction to acid phosphatase. The MVBs in cells incubated with ionomycin, a calcium ionophore, did not differ from those of unstimulated cells. Cellular uptake of 125I-HRP was increased five times the control values already after 5 min of TPA stimulation. The uptake increased further with prolonged incubation (60 min), but at a slower rate. Together these indicate a TPA-induced transfer by endocytosis of portions of the plasma membrane to the lysosomal system via MVBs. Consideration of MVBs as part of the receptor-mediated endocytic pathway suggests that this effect of TPA might involve down-regulation of cell-surface receptors. The possibility of MVBs as a proton-sequestrating compartment, responsible for the cytoplasmic alkalinization previously reported for TPA-stimulated U-937 monoblastic cells, is discussed.     

The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits

The lymphocyte glycoprotein CD4 is constitutively internalized and recycled in nonlymphoid cells, but is excluded from the endocytic pathway in lymphocytic cells (Pelchen-Matthews, A., J. E. Armes, G. Griffiths, and M. Marsh. 1991. J. Exp. Med. 173: 575-587). Inhibition of CD4 endocytosis is dependent on CD4 expressing an intact cytoplasmic domain and is only observed in cells where CD4 can interact with the protein tyrosine kinase p56lck, a member of the src gene family. We have expressed p56lck, p60c-src, or chimeras of the two proteins in CD4-transfected NIH-3T3 or HeLa cells. Immunoprecipitation of CD4 and in vitro kinase assays showed that p56lck and the lck/src chimera, which contains the NH2 terminus of p56lck, can associate with CD4. In contrast, p60c-src and the src/lck chimera, which has the NH2 terminus of p60c-src, do not associate with CD4. Endocytosis assays using radioiodinated anti-CD4 monoclonal antibodies demonstrated that coexpression of CD4 with p56lck, but not with p60c-src, inhibited CD4 endocytosis, and that the extent of the inhibition depended directly on the relative levels of CD4 and p56lck expressed. The uptake of mutant CD4 molecules which cannot interact with p56lck was not affected. Measurement of the fluid-phase endocytosis of HRP or the internalization of transferrin indicated that the effect of p56lck was specific for CD4, and did not extend to other receptor-mediated or fluid-phase endocytic processes. Immunogold labeling of CD4 at the cell surface and observation by electron microscopy demonstrated directly that p56lck inhibits CD4 endocytosis by preventing its entry into coated pits.     

Vasopressin stimulates endocytosis in kidney collecting duct principal cells

In target epithelia, a vasopressin-induced water permeability increase is accompanied by the appearance of intramembranous particle (IMP) clusters, probably representing water-permeable patches, in the apical plasma membrane of responding cells. In the collecting duct principal cell, we have previously shown that these clusters are located in clathrin-coated pits. To determine whether vasopressin induces the endocytic uptake of these membrane domains in principal cells, we have examined the uptake of horseradish peroxidase (HRP) by principal cells of normal rats, vasopressin-deficient Brattleboro rats, and vasopressin-treated Brattleboro rats, following intravenous injection of HRP. By quantitative electron microscopy, principal cells of Brattleboro homozygous rats were found to take up much less HRP into cytoplasmic vesicles than normal rats, and HRP uptake was increased to normal levels in vasopressin-treated Brattleboro rats. Many invaginating coated pits at the cell surface were loaded with HRP reaction product, indicating their participation in the observed endocytosis of HRP. We conclude that vasopressin stimulates endocytosis in collecting duct principal cells. Since we have already shown that IMP clusters are found in coated pits at the cell surface, the endocytic removal of these putative water-permeable patches from the apical membrane seems to occur via a clathrin-mediated mechanism in this tissue.     

Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses

Enveloped viruses often enter cells via endocytosis; however, specific endocytic trafficking pathway(s) for many viruses have not been determined. Here we demonstrate, through the use of dominant-negative Rab5 and Rab7, that influenza virus (Influenza A/WSN/33 (H1N1) and A/X-31 (H3N2)) requires both early and late endosomes for entry and subsequent infection in HeLa cells. Time-course experiments, monitoring viral ribonucleoprotein colocalization with endosomal markers, indicated that influenza exhibits a conventional endocytic uptake pattern – reaching early endosomes after approximately 10 min, and late endosomes after 40 min. Detection with conformation-specific hemagglutinin antibodies indicated that hemagglutinin did not reach a fusion-competent form until the virus had trafficked beyond early endosomes. We also examined two other enveloped viruses that are also pH-dependent for entry – Semliki Forest virus and vesicular stomatitis virus. In contrast to influenza virus, infection with both Semliki Forest virus and vesicular stomatitis virus was inhibited only by the expression of dominant negative Rab5 and not by dominant negative Rab7, indicating an independence of late endosome function for infection by these viruses. As a whole, these data provide a definitive characterization of influenza virus endocytic trafficking and show differential requirements for endocytic trafficking between pH-dependent enveloped viruses .     

A quantitative analysis of the endocytic pathway in baby hamster kidney cells

A morphological analysis of the compartments of the endocytic pathway in baby hamster kidney (BHK) cells has been made using the fluid-phase marker horseradish peroxidase (HRP). The endocytic structures labeled after increasing times of endocytosis have been identified and their volume and surface densities measured. In the first 2 min of HRP uptake the volume density of the labeled structures increased rapidly and thereafter remained constant for the next 13-18 min. This plateau represents the volume density of endosome organelles and accounts for 0.65% of the cytoplasmic volume (or 6.8 microns 3 per cell). The labeled structures consist of tubular-cisternal elements which are frequently observed in continuity with 300-400 nm vesicles. After 15-20 min of internalization the volume density of HRP-labeled structures again increased rapidly and reached a second plateau between 30 and 60 min of labeling. This second increase corresponded to detectable levels of HRP reaching later, acid phosphatase (AcPase)-reactive compartments. These structures, comprising the prelysosomes and lysosomes, were mostly vesicular and collectively accounted for 3.5% of the cytoplasmic volume (or 37 microns 3 per cell). The absolute peripheral surface areas of the two classes of organelles (endosomes and prelysosomes/lysosomes) were estimated to be 430 and 370 microns 2 per cell, respectively. The volume of fluid internalized in the first 2 min of uptake was five- to sevenfold less than the volume of the compartment labeled in this time. To account for these results we propose that, after uptake from the cell surface, HRP is delivered to, and diluted in, endosomes that are preexisting organelles initially devoid of the marker. With increasing times of endocytosis the concentration of HRP in the early endosomes increases, as more of the marker enters this compartment. An elevation in HRP concentration in endosomes during the early time points was shown directly using anti- HRP antibodies and colloidal gold on cryosections. The stereological values given in the present study, in combination with earlier studies, provide a minimum estimate for both the total surface area of membranes and the rate of membrane synthesis in a BHK cell.     

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.     

Uptake of endocytic markers by rice cells: variations related to the growth phase

Endocytosis is now considered a basic cellular process common to plant cells. Although both non-specific and receptor-mediated endocytosis appear to take place in plant cells, the physiological role of the latter remains unclear. We have investigated the endocytic process in rice cell suspensions using two biotinylated proteins, peroxidase and bovine serum albumin (bHRP and bBSA), as markers. First, we show that markers are internalized by rice cells and appear in intracellular membranes. The uptake of the two markers is temperature dependent, saturable with time and markers dose and it is competed by free biotin. Thus, it shows the properties of a receptor-mediated process. We also show that uptake of markers is strongly influenced by growth phase as optimal uptake occurs during the lag phase, but the initiation of the exponential growth phase decreases uptake drastically. Arrest of the cell cycle by starvation of either a nutrient (phosphate) or a growth regulator (2,4-dichlorophenoxyacetic acid), both components of the culture medium, does not modify the rate of bBSA uptake. Subsequent readdition of these components results in growth recovery and a dramatic decrease in bBSA uptake. On the other hand, nocodazole treatment, a method to arrest the cell cycle by microtubule depolymerization, inhibited bBSA uptake. The possible causes for this arrest of endocytosis are discussed.     

Endocytosis, intracellular transport, and turnover of anionic and cationic proteins in cultured mouse peritoneal macrophages

It was previously shown that cultured mouse peritoneal macrophages ingest anionic derivatives of horseradish peroxidase (HRP) and ferritin by fluid-phase endocytosis and accumulate them in lysosomes. Cationic derivatives were taken up by adsorptive endocytosis and transported to lysosomes but subsequently appeared also in stacked cisternae, tubules, and vesicles of the Golgi complex. In the present investigation, the effect of molecular net charge on the rate of cellular inactivation of a protein was studied. The results demonstrate that anionized HRP was inactivated at a higher initial rate than cationized HRP. This is in agreement with the finding that the cationic protein partly escaped from the digestive compartment of the cells, that means the lysosomes. The effects of phorbol myristate acetate (PMA)--a diterpene ester and a tumor promoter--and monensin--a carboxylic ionophore and a perturbant of the Golgi complex--on fluid-phase endocytosis of HRP and intracellular transport of cationized ferritin (CF) were also studied. PMA stimulated fluid-phase endocytosis of HRP but did not interfere with transport of CF to the Golgi complex. Contrarily, monensin inhibited uptake of HRP and almost totally blocked transport of CF to the Golgi complex. The findings support the idea that membrane and content of endocytic vesicles are treated separately. The content is emptied into lysosomes where macromolecular material normally is degraded. The membrane becomes part of the lysosomal envelope in connection with endocytic vesicle-lysosome fusion. Subsequently, membrane patches are detached from the lysosomes and reutilized. This is at least partly mediated via the Golgi complex and particularly its tubular and vesicular parts. Since the cationic tracers do not bind to the membrane in a stable way, it is not possible to extend the conclusions to individual membrane constituents.     

Reversible pinocytosis of horseradish peroxidase in lymphoid cells

A detailed study of fluid phase endocytosis of horseradish peroxidase (HRP) in rat lymph node cells (LNC) is presented in this paper. Preliminary experiments have shown that HRP was internalized by non-receptor-mediated endocytosis and interacted minimally or not at all with plasma membrane of LNC, and can then be considered as a true fluid phase marker for these cells. Kinetics of uptake of HRP was found not to be linear with incubation time at 37 degrees C and deviation from linearity can be attributed to constant exocytosis of HRP. The kinetics of exocytosis cannot be described by a single exponential process. Rather, a minimum of two exponentials is required to account for exocytosis. This suggests that at least two intracellular compartments are involved in this process. The first turns over very rapidly with a t 1/2 release of about 3 min and is saturated after 10 min of exposure with HRP. The second, which turns over very slowly, is characterized by a t 1/2 release of about 500 min and accounts for the intracellular accumulation of HRP. Similar biphasic kinetics of exocytosis were observed with unfractionated LNC, with T lymphocyte-enriched LNC and with lymphocytes purified according to their density. This suggests that most, if not all, LNC are able to release HRP and that each cell type is endowed with the two intracellular compartments. Kinetics of uptake of HRP in these two compartments indicated that they are probably filled by two endocytic pathways, at least partially independent. Taken together, these results seem to indicate that a rapid membrane recycling occurs in lymphocytes. Furthermore, the weak base ammonium chloride and the carboxylic ionophore monensin were shown in our study to inhibit fluid phase endocytosis of HRP. The inhibition was time-dependent and required a preincubation of the cells with the drugs to be observed. Our results suggest that a perturbation of the vesicular traffic or a sequestration of membranes involved in HRP uptake is induced by these drugs. Under these conditions the release of cell-associated HRP was also reduced and to the same extent as the inhibition of uptake. Distribution of HRP between the two compartments and the t 1/2 release of HRP from either compartment were not perturbed. Taken together these results seem to indicate that exocytosis is not specifically affected by these drugs. Inhibition of uptake in drug-treated cells could result from a general decrease of membrane recycling or to the formation of smaller pinocytic vesicles with a different surface to volume ratio.     

CD36 is not involved in scavenger receptor-mediated endocytic uptake of glycolaldehyde- and methylglyoxal-modified proteins by liver endothelial cells

Circulating proteins modified by advanced glycation end-products (AGE) are mainly taken up by liver endothelial cells (LECs) via scavenger receptor-mediated endocytosis. Endocytic uptake of chemically modified proteins by macrophages and macrophage-derived cells is mediated by class A scavenger receptor (SR-A) and CD36. In a previous study using SR-A knockout mice, we demonstrated that SR-A is not involved in endocytic uptake of AGE proteins by LECs [Matsumoto et al. (2000) Biochem. J. 352, 233-240]. The present study was conducted to determine the contribution of CD36 to this process. Glycolaldehyde-modified BSA (GA-BSA) and methylglyoxal-modified BSA (MG-BSA) were used as AGE proteins. 125I-GA-BSA and 125I-MG-BSA underwent endocytic degradation by these cells at 37 degrees C, and this process was inhibited by several ligands for the scavenger receptors. However, this endocytic uptake of 125I-GA-BSA by LECs was not inhibited by a neutralizing anti-CD36 antibody. Similarly, hepatic uptake of (111)In-GA-BSA after its intravenous injection was not significantly attenuated by co-administration of the anti-CD36 antibody. These results clarify that CD36 does not play a significant role in elimination of GA-BSA and MG-BSA from the circulation, suggesting that the receptor involved in endocytic uptake of circulating AGE proteins by LEC is not SR-A or CD36.     

Endocytosis is regulated by protein kinase A, but not protein kinase C in a secretory epithelial cell line.

Endocytosis in the chloride secreting epithelial cell line T84 was monitored by uptake of the fluid-phase markers FITC-dextran and horseradish peroxidase (HRP). Uptake of marker was inhibited by incubation of cells at 4 degrees C, consistent with an endocytic uptake. Although activation of the cAMP-dependent second messenger pathway has been shown to stimulate exocytosis in this cell line, it caused a 63% reduction in endocytosis as measured by uptake of fluid-phase markers. In contrast, the presence of the protein kinase C activator phorbol-myristate acetate (PMA) caused no significant reduction in the level of endocytosis compared to control, nor did it reverse the inhibitory effect of PKA activation. The data thus suggest that endocytosis in T84 cells is regulated through activation of protein kinase A, but not through activation of protein kinase C.     

Effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of Hep 2 cells by picornaviruses

Potassium depletion after a brief exposure of the cells to hypotonic medium was used to inhibit endocytosis from coated pits in Hep 2 cells. After such treatment the endocytic uptake of transferrin was arrested, and electron microscopy revealed that virtually no coated pits were present at the cell surface, while smooth (uncoated) pits were abundant. Under the same conditions the cells were strongly protected against poliovirus, while the cytopathogenic effect of human rhinovirus type 2, HRV 2, was increased. The cytopathogenic effect of encephalomyocarditis (EMC) virus was only slightly affected. Potassium depletion without hypotonic shock reduced the endocytic uptake of transferrin 2-3-fold and the number of coated pits at the cell surface about 3-fold. Furthermore, the cells were not protected against poliovirus after such treatment. The data indicate that the productive uptake of poliovirus occurs by receptor-mediated endocytosis from coated pits, while the productive uptake of the other two picornaviruses may occur by another endocytic pathway. In order to efficiently arrest endocytosis from coated pits in these cells, hypotonic shock seems to be a critical component of the potassium depletion protocol.     

Studies on endocytic mechanisms of the Menkes copper-translocating P-type ATPase (ATP7A; MNK) – Endocytosis of the Menkes protein

The human X-linked recessive copper deficiency disorder, Menkes disease, is caused by mutations in the ATP7A (MNK) gene, which encodes a transmembrane copper-transporting P-type ATPase (MNK). The MNK protein is localised to the Golgi apparatus and relocalises to the plasma membrane when copper levels are elevated. Previous studies have identified a C-terminal di-leucine endocytic motif (L1487L1488) in MNK, thought to direct it into the clathrin-mediated endocytic pathway. To determine whether MNK is internalised via clathrin-dependent endocytosis, this pathway was blocked in MNK-overexpressing HeLa cells by the transient expression of dominant negative dynamin and Eps15 mutants. MNK internalisation was not inhibited in such cells. MNK internalisation was inhibited in cells treated with hypertonic sucrose that not only blocked clathrin-mediated endocytosis but also fluid-phase endocytosis. These studies, together with earlier studies on the requirement for L1487L1488, suggest that MNK can utilise both clathrin-dependent and clathrin-independent endocytosis in HeLa cells.