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The coexistence of different subtypes of voltage-dependent calcium channels (VDCC) within the same chromaffin cell (CC) and the marked interspecies variability in the proportion of VDCC subtypes that are present in the plasmalemma of the CCs raises the question on their roles in controlling different physiological functions. Particularly relevant seems to be the role of VDCCs in the regulation of the exocytotic neurotransmitter release process, and its tightly coupled membrane retrieval (endocytosis) process since both are Ca2+-dependent processes. This review is focused on the role of Ca2+ influx through L-type VDCC in the regulation of these two processes. It is currently accepted that the different VDCC subtypes (i.e., T, L, N, P/Q, R) contribute to exocytosis proportionally to their density of expression and gating properties. However, the pattern of stimulation defines a preferential role of the different subtypes of VDCC on exocytosis and endocytosis. Thus, L-type channels seem to control catecholamine release induced by prolonged stimuli while fast exocytosis in response to short square depolarizing pulses or action potentials is mediated by Ca2+ entering CCs through P/Q channels. The pattern of stimulation also influences the endocytotic process, and thus, electrophysiological data suggest the sustained Ca2+ entry through slow-inactivating L-type channels could be responsible for the activation of fast endocytosis.

Heavy metals are used in industrial applications, such as production of pesticides, batteries, alloys, and textile dyes. Excessive exposure may lead to specific disorders. The kidney is a target organ in heavy metal toxicity because of its ability to reabsorb and concentrate divalent metals. The extent of renal damage depends on the nature, the dose, and the time of exposure. In general, acute damage differs from chronic damage in its mechanism of toxicity. As a consequence, the clinical features and therapeutic approach are also different. Heavy metals in plasma exist in nondiffusible and ionized forms. The ionized form is toxic and produces acute toxicity; on the other hand the bound, inert form is conjugated with metallothionein and glutathione, which then are released into the blood by the liver and the kidney. These compounds subsequently are reabsorbed through an endocytotic process in segment S1 of the proximal tubule and can lead to chronic damage. Treatment regimens include chelation therapy, decontamination procedures, supportive care, and extracorporeal therapy. This chapter adds specific considerations for some of the most common metals.

Bioactive peptide LL-37/hCAP18, the only human member of the cathelicidin family, plays important roles in killing various pathogens, as well as in immune modulation. We demonstrate that LL-37 is internalized by human macrophages in a time-, dose-, temperature-, and peptide sequence–dependent endocytotic process. Both clathrin- and caveolae/lipid raft–mediated endocytosis pathways are involved in LL-37 internalization. We find that the P2X7 receptor (P2X7R) plays an important role in LL-37 internalization by human macrophages because significantly less internalized LL-37 was detected in macrophages pretreated with P2X7R antagonists or, more specifically, in differentiated THP-1 cells in which the P2X7R gene had been silenced. Furthermore, this P2X7R-mediated LL-37 internalization is primarily connected to the clathrin-mediated endocytosis pathway. In addition, our results demonstrate that internalized LL-37 traffics to endosomes and lysosomes and contributes to intracellular clearance of bacteria by human macrophages, coinciding with increased reactive oxygen species and lysosome formation. Finally, we show that human macrophages have the potential to import LL-37 released from activated human neutrophils. In conclusion, our study unveils a novel mechanism by which human macrophages internalize antimicrobial peptides to improve their intracellular pathogen clearance.

Under basal conditions the action potential firing rate of adrenal chromaffin cells is lower than 0.5 Hz. The maintenance of the secretory response at such frequencies requires a continuous replenishment of releasable vesicles. However, the mechanism that allows such vesicle replenishment remains unclear. Here, using membrane capacitance measurements on mouse chromaffin cells, we studied the mechanism of replenishment of a group of vesicles released by a single action potential-like stimulus (APls). The exocytosis triggered by APls (ETAP) represents a fraction (40%) of the immediately releasable pool, a group of vesicles highly coupled to voltage dependent calcium channels. ETAP was replenished with a time constant of 0.73 � 0.11 s, fast enough to maintain synchronous exocytosis at 0.2-0.5 Hz stimulation. Regarding the mechanism involved in rapid ETAP replenishment, we found that it depends on the ready releasable pool; indeed depletion of this vesicle pool significantly delays ETAP replenishment. On the other hand, ETAP replenishment also correlates with a dynamin-dependent fast endocytosis process (τ = 0.53 � 0.01 s). In this regard, disruption of dynamin function markedly inhibits the fast endocytosis and delays ETAP replenishment, but also significantly decreases the synchronous exocytosis during repetitive APls stimulation at low frequencies (0.2 and 0.5 Hz). Considering these findings, we propose a model in where both the transfer of vesicles from ready releasable pool and fast endocytosis allow rapid ETAP replenishment during low stimulation frequencies. Fil: Moya Diaz, José Abelino. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Alvarez, Yanina Daniela. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Montenegro, Mauricio Norman. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina Fil: Bayonés, Lucas. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina Fil: Belingheri, Ana Verónica. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: González-Jamett, Arlek M.. Universidad de Valparaiso; Chile Fil: Cárdenas, Ana M.. Universidad de Valparaiso; Chile Fil: Marengo, Fernando Diego. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina

International audience; The design, fabrication and test of an indium tin oxide (ITO) microdevice to investigate exocytotic behaviors of migratory dendritic cells (DCs) in confined three-dimensional environment were reported in this work. Indeed, immature DCs were able to migrate into micro-fabricated biocompatible polydimethylsiloxane (PDMS) channels that mimic their natural constrained environment of tissues for patrolling in search of danger associated antigens through an endocytotic process called macropinocytosis. In order to coordinate membrane trafficking and prevent cell volume increment, DCs will release part of their contents back to the extracellular medium while migrating. Through electrochemical measurements, we demonstrated that exocytotic events of migratory DCs could be monitored by our ITO microdevice. In addition, the transparency of ITO electrode should facilitate future combining assays of exocytosis with other fluorescence-based measurements of cell physiology.

We report that cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD) acutely applied to rat brain synaptosomes is accompanied by an immediate increase in transporter-mediated glutamate release and decrease in exocytotic release. To clarify the possible mechanisms underlying these phenomena, we investigated the influence of MbetaCD on synaptic vesicle acidification and exo/endocytotic process in nerve terminals. As shown by acridine orange fluorescence measurements, the application of MbetaCD to synaptosomes, as well as to isolated synaptic vesicles, led to the gradual leakage of the protons from the vesicles, whereas the application of MbetaCD complexed with cholesterol stimulated additional vesicle acidification and an increase in Ca2+-dependent exocytotic response. It was found that the treatment of nerve terminals with MbetaCD did not block Ca2+-triggered vesicle recycling. We suggest that cholesterol depletion of the plasma membrane with MbetaCD induces the removal of cholesterol from the membrane of synaptic vesicles resulting in immediate dissipation of synaptic vesicle proton gradient and redistribution of the neurotransmitter between the vesicular and cytosolic pools. The latter appears to be the main cause of a dramatic decrease in exocytotic and considerable increase in transporter-mediated release of L-[14C]glutamate.

Cortical granules are membrane-bound, electron dense cell inclusions known in oocytes of many animals, invertebrates [1-5] and vertebrates [6]. They are distributed in the peripheral ooplasm forming a monolayer beneath the plasma membrane [1,3,5]. These organelles are interpreted as Golgi complex productions [1,3,4,7,8]. In some metazoan (as crustacean), it is suggested that mitochondria take part in the origin of cortical granules [3]. Cortical granules contents differ among species (glycoproteins, mucopolysaccharide etc.) but their functions of preventing polyspermy remain similar [3,5,9,10] in the fertilization process. In planarians (Platyhelminthes, Tricladida), these inclusions are signalized for several species [4]. We present here an ultrastructural analysis of their genesis process in the marine gonochoristic triclad Sabussowia dioica where we demonstrate a novelty represented by the contribution of accessory cells in the formation of their peripheral content using an endocytotic process.

Protein delivery mediated by protein transduction domains (PTD) such as the HIV-1 TAT-PTD has emerged as a promising approach for neuroprotection. The objective of this study was to generate and evaluate the neuroprotective potential of TAT fusion proteins using constructs based on Bcl-2 anti-death family proteins. A TAT-Bcl-2 construct with the loop domain deleted (TAT-Bcl-2Δloop) was tested for its ability to transduce neuronal cells and to promote survival. The potential mechanism of TAT-mediated protein internalization in neural cells was also investigated. The purified TAT-Bcl-2Δloop binds to neural cell and rat brain mitochondria, and transduces cultured neural cell lines and primary cortical neurons when used at nm concentrations. Effective internalization of TAT-Bcl-2Δloop occurs at 37°C but not at 4°C, consistent with an endocytotic process. Both cell association and internalization require interaction of TAT-Bcl-2Δloop with cell surface heparan sulfate proteoglycans. TAT-mediated protein delivery in neuronal cells occurs through a lipid raft-dependent endocytotic process, inhibited by the cholesterol-sequestering agent nystatin. Transducible loop deleted Bcl-2 increases the survival of cortical neurons following trophic factor withdrawal and also rescues neural cell lines from staurosporine-induced death. These results support the concept of using protein transduction of Bcl-2 constructs for neuroprotection.

Crystal-like structures in vacuoles, precipitates in the cytoplasm and on the tonoplast membrane have been found to store remarkable amounts of Si in a number of higher plants. In most of the cases the final storage product is a SiO(2) gel. Accumulation inside the cells presumes a membrane and cytoplasm passage, driven by unknown transporters. Beside this uptake into the cytoplasm, Si-accumulating species possess a mechanism that does not involve a membrane and cytoplasm passage. Unusual small invaginations comprising the two membranes, plasmalemma and tonoplast, which enclose a small border of cytoplasm, were observed. The same cells contained vacuolar vesicles surrounded by two membranes, obviously derived from the invaginations. By energy-dispersive X-ray analysis and electron spectroscopic imaging, Si was shown in the invaginations and vacuolar vesicles. This novel endocytotic process allows the uptake of condensed, higher-molecular-weight Si compounds. In Zn hyperaccumulators, frequently SiO(2) precipitates were found in different cell compartments. Such plants showed the same invaginations and vacuolar vesicles, but Zn, colocalized with Si, was detected in these structures. Electron energy loss spectra confirmed the assumption that Zn-silicate is present in the vesicles. In the vacuoles the unstable Zn-silicate is degraded, forming SiO(2) precipitates, while the released Zn is bound to an unknown partner.

Hydrogel nanoparticles of cross-linked polyvinylpyrrolidone (PVP-NP) (35–50 nm in diameter) containing fluoresceinated dextran (FITC-Dx) were encapsulated in reconstituted Sendai viral envelopes containing only the fusion (F) protein (F-virosomes11Process for Producing a Targeted Gene (1997) US Patent 5, 683, 866).). Incubation of these loaded F-virosomes with human hepatoblastoma cells (HepG2) in culture resulted in membrane-fusion-mediated delivery of NPs to the cell cytoplasm, as inferred from the ability of cells to internalize FITC-Dx loaded PVP-NP (PVPf-NP) in the presence of azide (an inhibitor of the endocytotic process). Introduction of PVPf-NP into the HepG2 cells was assured by selective accumulation of FITC fluorescence in the cytosolic compartment. The structural integrity of the internalized PVPf-NP was also confirmed by fluorescence microscopy and ultracentrifugation analysis. The potential usefulness of PVP-NP-mediated cytosolic release of water soluble drugs both in vitro and in vivo has been established for the first time.

To investigate the cellular accumulation and intracellular localization of dimercaptosuccinate-coated iron oxide nanoparticles (D-IONPs) in oligodendroglial cells, we have synthesized IONPs that contain the fluorescent dye BODIPY (BP) in their coat (BP-D-IONPs) and have investigated the potential effects of the absence or presence of this dye on the particle uptake by oligodendroglial OLN-93 cells. Fluorescent BP-D-IONPs and non-fluorescent D-IONPs had similar hydrodynamic diameters and ζ-potentials of around 60 nm and -58 mV, respectively, and showed identical colloidal stability in physiological media with increasing particle size and positivation of the ζ-potential in presence of serum. After exposure of oligodendroglial OLN-93 cells to BP-D-IONPs or D-IONPs in the absence of serum, the specific cellular iron content increased strongly to around 1,800 nmol/mg. This strong iron accumulation was lowered for both types of IONPs by around 50 % on exposure of the cells at 4 °C and by around 90 % on incubation in presence of 10 % serum. The accumulation of both D-IONPs and BP-D-IONPs in the absence of serum was not affected by endocytosis inhibitors, whereas in the presence of serum inhibitors of clathrin-dependent endocytosis lowered the particle accumulation by around 50 %. These data demonstrate that oligodendroglial cells efficiently accumulate IONPs by an endocytotic process which is strongly affected by the temperature and the presence of serum and that BP-D-IONPs are a reliable tool to monitor by fluorescence microscopy the uptake and cellular fate of D-IONPs.

Calreticulin (CRT) is an endoplasmic reticulum chaperone protein that is involved in quality control process during protein folding and maturation. It plays an important role as a regulator of intracellular calcium homeostasis. Previously it has been shown that loss of CRT protein results in endoplasmic reticulum stress, increase in ubiquitin-proteasome activity, and resistance to apoptosis. Our preliminary studies illustrated that CRT deficient cells expresses significantly higher connexin 43 protein, however its function is significant suppressed. We showed that connexin 43 was accumulated within intracellular vesicles. Because connexin 43 function is dependent on its trafficking and localization, we hypothesized that loss of CRT function increases rate of endocytosis via caveolin dependent pathway. To test our hypothesis we measured the rate of uptake of fluorescently tagged-Wheat Germ Agglutinin, in wild type (wt) versus crt-/- mouse embryonic fibroblast cells (MEF). In crt-/- cells WGA was completely endocytosed within 30 minute incubation while in wt cells WGA was localized both on cell membrane and intracellular vesicles at 30 minute. The endocytotic process in crt -/- was significantly reduced by inhibiting proteasome activity with MG132. The caveolin-1 mRNA and protein levels were significantly increased in crt-/- cells compared to wt cells. However, there were no significant changes in clathrin protein level in wt versus crt-/- cells. Using Methyl-B-cylcodextrin to deplete cell cholesterol content, we demonstrated inhibition of endocytosis in crt-/- cells which confirms a major role for caveolin in endocytosis in these cells. Overall our data are the first to illustrate the inhibitory role of CRT in endocytotic pathways. Acknowledgment: This research was funded by Qatar National Research Fund (NPRP4-043-3-016).

Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) at synapses has been suggested to play an important role in the expression of synaptic plasticity. Both the regulated and the constitutive trafficking of synaptic AMPARs are thought to involve the insertion and removal of receptors by means of an exocytotic and endocytotic process, respectively. In contrast, N -methyl- d -aspartate (NMDA) receptors (NMDARs), which are colocalized with AMPARs at excitatory synapses, appear to be much less dynamic. Here, we present evidence supporting the idea that synaptic AMPARs turn over through a constitutive endocytotic process and that glutamate application greatly enhances this turnover of AMPARs. The glutamate-induced internalization of AMPARs requires a rise in postsynaptic Ca 2+ . The AMPAR internalization is mimicked by latrunculin A, a drug that selectively depolymerizes actin and is blocked by jasplakinolide, a drug which stabilizes actin filaments. The rate of endocytosis is not altered by glutamate application, whereas a clear enhancement is observed with insulin application. We propose a model in which the glutamate-induced dissociation of AMPARs from their anchor on the postsynaptic membrane involves actin depolymerization, which allows the released AMPARs to segregate from the NMDARs and diffuse to a presumably perisynaptic site, where they become available to an endocytotic machinery and are selectively internalized.

Although the possibility of targeting drugs to specific tissues and cells, as well as facilitating their uptake and cytoplasmic delivery has rendered liposomes a versatile drug carrier system with numerous potential applications in medicine, the molecular mechanisms of liposome-cell interactions are not understood well. Here we have reviewed the early and current concepts of liposome-cell interactions, including possible liposome receptors. Uptake of liposomes by cells can be modified by the lipid composition, particularly by the inclusion of steric stabilizers such as PEG-conjugated lipids. Such modifications also alter the circulation time and biodistribution of liposomes, which can thus be tailored for particular applications. The intracellular fate of encapsulated molecules can be modified by the use of pH-sensitive liposomes which can also be sterically stabilized. Cationic liposomes that can undergo lipid mixing with cellular membranes can deliver complexed DNA to cells, but most likely via an endocytotic process. Kinetic analysis of liposome-cell interactions can elucidate the numbers of liposome receptors of several types and the corresponding binding constants. It is likely that liposomes bind to different cell surface receptors on different cells, and that they utilize more than one type of receptor on a particular cell. The kinetic analysis also provides the rate constants of endocytosis and the percentages of liposomes that are bound or endocytosed.

This paper describes the uptake of Lucifer Yellow carbohydrazide and fluorescent dextrans labeled with fluorescein isothiocyanate or Sodium Green (molecular masses ranging from 522 to 2 × 106 Da) byDunaliella spp. halotolerant unicellular green algae isolated from salt pools in the Sinai peninsula. The fluorescent dyes were taken up into a set of vesicles around the nucleus and just above the chloroplast. It proved impossible to inhibit uptake of the fluorescent compounds in cells treated with a large variety of metabolic and other inhibitors. Cell labeling was complete within half a minute of addition of fluorescent compounds to the outside medium; efflux was equally rapid. The results are interpreted in terms of an endocytotic process whereby the outside medium, together with any substance dissolved in it, remains within vesicles enclosed within the cell body but cycles rapidly between the plasma membrane and the interior of the cell. The outside medium does not pass across the vesicular membrane, nor enters the cytosol.

Oncogene-driven adaptation of metabolism during tumorigenesis includes steps that stimulate the uptake of nutrients, especially glucose and glutamine, to sustain cell growth and proliferation. Macropinocytosis, a clathrin- and caveolin-independent endocytotic process that had previously been linked to the action of oncogenic Ras and Src, is now shown to contribute to amino acid uptake via enhanced delivery of extracellular proteins to lysosomes.

International audience; The aim of this contribution is to design, produce and characterize size-tuneable core-shell micelles from amphiphilic Tat-b-poly(trimethylene carbonate) (Tat-b-PTMC) molecular chimeras, and to explore their biological properties. Because the extensive characterization of nanomaterials is a pre-requisite to understand and rationalize their ensuing properties, we present a detailed description of Tat-b-PTMC micelles thanks to light scattering, AFM imaging and small angle neutron scattering analyses. In vitro, Tat-b-PTMC micelles were found to be rapidly and efficiently internalized by HeLa cells, with cellular uptake kinetics being mostly related to Tat peptide content and, to a lesser extent, to nanoparticle size. We also demonstrated that, after a first membrane-binding step, Tat-b-PTMC micelles were taken up by cells via an energy-dependent endocytotic process.

Uptake of 3,5,3'-triiodo-L-thyronine (T3) by isolated trout hepatocytes was characterized after 40 sec incubation of cells in a balanced salts medium containing [125]T3, and compared to L-thyroxine (T4) uptake (Riley and Eales, Gen. Comp. Endocrinol. 90, 31-42, 1993). T3 uptake resembled T4 uptake in several ways. There was a small (10%) diffusion component. The balance of the uptake was temperature- and energy-dependent and involved a protein carrier, but did not depend on the presence of Na+ in the medium or Na+ transport. Tyrosine and phenylalanine were ineffective competitors. Inhibition by colchicine and chloroquine indicated an endocytotic process. T3 uptake differed from T4 uptake in having a higher pH optimum (6-8) than T4 (5-6), and in having a much lower Kt (0.074 microM) than T4 (0.52 microM). T3 uptake was far more strongly inhibited than T4 uptake by TRIPROP (8% of control uptake), reverse T3 (9%), and 3,3'-diiodo-L-thyronine (9%). T4 inhibited T3 transport (Ki = 0.18 microM), but kinetic analyses indicated that the mutual inhibitions were noncompetitive, suggesting separate T3 and T4 binding sites. In conclusion, T3 uptake into isolated trout hepatocytes resembles that for T4 uptake in being an energy-dependent, carrier-mediated endocytotic process, but differs from T4 uptake in having a lower Kt, a higher pH optimum, and a greater sensitivity to inhibition by related iodothyronines. T3 and T4 uptakes may involve separate carrier systems, providing scope for individual control of T3 and T4 uptake by trout hepatocytes.

Insulin regulated aminopeptidase (IRAP) recognises "AT(4)-receptor" ligands like angiotensin IV (Ang IV) and peptidomimetics like AL-11. The metabolic stability and high affinity of [(3)H]AL-11 for catalytically active IRAP allowed its detection in Chinese hamster ovary (CHO-K1) cell membranes in the absence of chelators (Demaegdt et al., 2009). Here, we show that, contrary to [(3)H]Ang IV, [(3)H]AL-11 displays high affinity and specificity for IRAP in intact CHO-K1 cells as well. After binding to IRAP at the surface, [(3)H]AL-11 is effectively internalized by an endocytotic process. Unexpectedly, surface binding and internalization of [(3)H]AL-11 was not affected by pretreating the cells with Ang IV but declined with AL-11. In the latter case surface expression of IRAP even increased. After elimination of simpler explanations, it is proposed that metabolically stable "AT(4)-receptor" ligands undergo semi-continuous cycling between the cell surface and endosomal compartments. The in vivo efficacy of stable and unstable "AT(4)-receptor" ligands could therefore differ.

We investigated the distribution and fate of liposomes after their intravenous injection into a mouse. Liposomes were composed of dimyristoylphosphatidylcholine, cholesterol and dicetylphosphate (7:2:1, molar ratio) with or without lactosylceramide. They were characterized as small unilamellar vesicles, approximately 100 nm in diameter, using gel-exclusion chromatography on a Sephacryl S-1000, freeze-fracture electron microscope and dynamic light scattering method. Liposomes were very stable in serum as seen by the results of leakage of the entrapped marker and electrophoresis experiments. We demonstrated that liposomes were internalized by way of an endocytotic process via coated vesicles detected in the electron microscope. The increase in liver uptake of lactosylceramide-bearing liposomes was mostly accounted for by enhanced uptake in the parenchymal cells, while uptake by non-parenchymal cells was only slightly increased. This observation supported the notion that a galactose-specific receptor was involved in liver uptake of lactosylceramide liposomes. The lactosylceramide-bearing liposomes were preferentially recovered in the liver and were found to first be predominantly localized in the mitochondria-lysosomal fraction. They were then decomposed by lysosomal enzymes, and the hydrolyzed components were reincorporated into membrane phospholipids in the microsomal fraction. At the same time, a rapid and reversible exchange of phosphatidylcholine between microsomes and mitochondria was demonstrated.

Hepatitis B virus (HBV) is a major cause of liver disease. HBV primarily infects hepatocytes by a still poorly understood mechanism. After an endocytotic process, the nucleocapsids are released into the cytoplasm and the relaxed circular rcDNA genome is transported towards the nucleus where it is converted into covalently closed circular cccDNA. Replication of the viral genome occurs via an RNA pregenome (pgRNA) that binds to HBV polymerase (P). P initiates pgRNA encapsidation and reverse transcription inside the capsid. Matured, rcDNA containing nucleocapsids can re-deliver the RC-DNA to the nucleus, or be secreted via interaction with the envelope proteins as progeny virions.

In Saccharomyces cerevisiae, phosphate uptake is mainly dependent on the proton-coupled Pho84 permease under phosphate-limited growth conditions. Phosphate addition causes Pho84-mediated activation of the protein kinase A (PKA) pathway as well as rapid internalization and vacuolar breakdown of Pho84. We show that Pho84 undergoes phosphate-induced phosphorylation and subsequent ubiquitination on amino acids located in the large middle intracellular loop prior to endocytosis. The attachment of ubiquitin is dependent on the ubiquitin conjugating enzymes Ubc2 and Ubc4. In addition, we show that the Pho84 endocytotic process is delayed in strains with reduced PKA activity. Our results suggest that Pho84-mediated activation of the PKA pathway is responsible for its own downregulation by phosphorylation, ubiquination, internalization, and vacuolar breakdown.

Pathological calcifications are associated with many medical conditions including diabetes, breast cancer, and crystals-associated osteoarthritis. The deposition of calcium-containing crystals on cells induces detrimental cellular effects and speeds up the progression of associated diseases. We carried out the present study to test the hypotheses that calcium-containing crystals may stimulate the influx of other molecules existing in the extracellular fluid disturbing normal molecular signaling and that anti-calcification agent will inhibit such endocytotic process. We found that basic calcium phosphate (BCP) crystals greatly stimulated the endocytotic activity of cells by rendering the cells more permeable and that the anti-calcification agent phosphocitrate and several others inhibited the crystals-mediated endocytosis. This is the first study reporting that the endocytotic activity of cells is affected by BCP crystals and that such endocytotic activity can be inhibited by anti-calcification agents. Since calcium-containing crystals are associated with many human diseases and in many circumstances are associated with apoptotic bodies, extracellular and matrix vesicles where DNA fragments, small peptides, and minerals are released into extracellular space, the findings reported here are important for our understanding of the complex biological effects and the potential pathological role of calcium-containing crystals in crystals-associated diseases, and for the development of disease modifying agents as well.

The objective of this study was to clarify the renal uptake characteristics of oligonucleotides at a cellular level using LLC-PK1 renal epithelial cells derived from the proximal tubule. The association of [35S]-labeled 20-mer phosphodiester (PO) and phosphorothioate (PS) oligonucleotides with the monolayers of polarized LLC-PK1 cells cultured on polycarbonate filter was characterized after apical or basolateral application. The cellular association of PO and PS at both apical and basolateral membranes was time dependent and temperature dependent, and the apparent association amount of PS was larger than that of PO. The PO and PS association after apical application was saturable, with the apparent Km and Vmax values determined to be 5.4 microM and 0.14 nmol/mg protein for PO and 0.22 microM and 0.11 nmol/mg protein for PS, respectively. In contrast, almost linear kinetics were observed after basolateral application within a tested concentration range. The association was inhibited significantly by sodium azide and chloroquine, suggesting that an energy-dependent endocytotic process was involved. Internalization and subsequent transport to endosome and lysosome compartments of FITC-labeled oligonucleotides were shown by confocal laser scanning microscopy. The present study has demonstrated that both types of oligonucleotides are taken up by LLC-PK1 cells from both apical and basolateral surfaces probably via an endocytosis mechanism.

Various nonviral gene vectors, which simultaneously form polyplexes with DNA through electrostatic interaction, have been proposed so far. The mechanism of the polyplex-mediated gene transfer is thought to follow the general endocytotic process. Therefore, many researches have been focusing on each step during the intracellular trafficking to improve their efficiencies. For example, (1) interaction with plasma membrane, (2) internalization into the cells, (3) escape from the endosome, and (4) trafficking to nuclei have been widely studied. We have recently established some systems, with which introduced polyplexes were efficiently delivered into nuclei. Even in these systems, some non-viral carriers were found to lead to high gene transfection and the others could not. According to this finding, we focused on the last step following nuclear localization, that is, disassembly of the complexes and DNA recognition by the transcription factors.

The mechanism of surfactant protein (SP)-A-mediated lipid uptake by rat type II pneumocytes was investigated. In the absence of SP-A, freshly isolated type II pneumocytes actively take up very little if any liposomes. Most of the increase with time is independent of energy or temperature but is most likely due to spontaneous exchange of labeled lipids between liposomes and cell membranes. With 5 micrograms/ml SP-A, type II cells actively take up liposomes (244 pmol dipalmitoylphosphatidylcholine.h-1.10(6) cells-1). The effect of SP-A on uptake is temperature dependent and can be abolished by ATP depletion of the cells. Coincubation with an auto-anti-idiotypic antibody against the SP-A-binding protein BP55 on the cell membrane of type II pneumocytes inhibits SP-A-mediated lipid uptake by type II cells. With increasing amounts of extracellular SP-A present, increasing amounts of liposomes are taken up and directed toward a nondegrading compartment. We suggest that SP-A-mediated surfactant lipid uptake is a receptor-mediated endocytotic process involving BP55.

The digestive gland of Viviparus ater was studied using histochemical and ultrastructural methods. Only one cell type was observed in the tubule epithelium of the gland. The cells are involved in an endocytotic process mediated by clathrin-coated vesicles and in the intracellular digestion of food materials (thus they can be regarded as digestive cells). The different stages of digestion and exocytotic extrusion of residual bodies into the tubule lumen were shown by electron microscopy. Very few, small mucocytes are scattered among the digestive cells. Calcium concretions, glycogen-containing cells and endocrine cells are scattered in the area of connective tissue present among the digestive tubules.

The potential of reconstituted Sendai viral envelopes containing only the fusion protein (F-virosomes) was evaluated for a targeted cytosolic delivery of lysozyme to human hepatoblastoma cells (HepG2) in culture. 125I-Lysozyme loaded into F-virosomes was used to monitor its fusion-mediated transfer to the HepG2 cells. Using fusion assay based on the transfer of water soluble probe, we have demonstrated the existence of aqueous connection between F-virosomes and target cells. Target specificity of the F-virosomes was ensured by the strong interaction between terminal β-galactose moiety of F protein and the asialoglycoprotein receptor on the membrane of HepG2 cells. Incubation of the loaded F-virosomes with cells resulted in fusion-mediated injection, as inferred from the ability of cells to internalize lysozyme in the presence of azide (an inhibitor of the endocytotic process). Binding as well as fusion of the F-virosomes to HepG2 cells was solely mediated by the F protein. Introduction of 125I-lysozyme into the HepG2 cells was confirmed by selective accumulation of acid and anti-body-precipitable radioactivity in the cytosolic compartment. The structural integrity of the internalized lysozyme was also assessed. The potential usefulness of F-virosomes with defined specificities as biological carrier for both in vitro and in vivo cytosolic delivery of macromolecules and drugs has been established.

1. Serum lipoproteins play an important role in the in vivo transport of several porphyrinoid derivatives having a moderate or high degree of hydrophobicity. 2. There appears to exist a correlation between the extent of photosensitizer association with low-density lipoproteins (LDL) and the efficiency of tumour targeting by some classes of photosensitizers, such as differently sulphonated porphyrins and phthalocyanines, haematoporphyrin dialkylethers and unsubstituted phthalocyanines and naphthalocyanines. 3. In all cases, LDL-carried photosensitizers are preferentially released to malignant cells; hence, direct cell damage appears to be the major determinant of tumour damage consequent to photodynamic therapy. 4. Present evidence suggests that the LDL-associated photosensitizer is accumulated by tumour cells largely via a receptor-mediated endocytotic process. 5. Thus, the use of delivery systems for orientating a systemically injected photosensitizer towards lipoproteins has been explored; promising results have been obtained by incorporation of the dye into liposomal vesicles, oil emulsions or inclusion complexes, as well as by precomplexation of the dye with LDL. 6. Moreover, a suitable choice of the chemical constituents of the delivery system and the experimental conditions allows one to modulate the photosensitizer distribution among the different lipoproteins. 7. The occurrence of tumour-targeting strategies other than the LDL pathway is briefly discussed.

Publisher Summary This chapter describes electrical capacitance and the way it can be used to measure membrane surface area. To study exo- and endocytotic processes, it is necessary to have a parameter quantitatively related to its progress under different experimental conditions. Such information can be provided by a continuous measure of the membrane surface area as membrane is added or subtracted by the exo- or endocytotic process. Cell membrane elements such as channels, pumps, and exchangers may be directly studied through their electrophysiological effects on the membrane. Conversely, the equally important processes of exocytosis and endocytosis occur without producing clear electrical signals. The many ionic pathways of the membrane are protein molecules that reverse the membrane, and the proteins are thought to be held in a lipid matrix. The membrane can therefore be taken to be a sort of mosaic of insulating and conducting regions.

Enhanced video-fluorescence microscopy and microspectrofluorometry were used to characterize the internalization, distribution and retention of two photosensitizers, rose bengal — a xanthene dye — and disulphonated aluminium phthalocyanine in eukaryote Paramecium aurelia. Rose bengal, because of its anionic nature, cannot diffuse across the cell membrane and accumulates there preferentially. In a drug-free medium the membrane fluorescence disappears after a few minutes. Complexation of rose bengal with low density lipoproteins gives rise to a different fluorescence pattern, where, in addition to membrane localization and diffuse cytoplasmic fluorescence, highly fluorescent endosomes are observed, which persisted for at least 1 h after drug treatment. Disulphonated aluminium phthalocyanine, on the contrary, seems to be directly internalized through an endocytotic process leading to the appearance of fluorescent endosomes, exhibiting a long persistence, together with cytoplasmic diffuse fluorescence. The presence of low density lipoproteins does not modify the internalization of the drug significantly, because of the very low yield of the complexation reaction. The potential of rose bengal as a photosensitizer for photodynamic therapy is discussed.

The release of rhizobia from infection threads of mung bean (Vigna radiata) root nodules is an endocytotic process. The peribacteroid membrane surrounding the released bacteria is initially derived from the host plasma membrane which surrounds the infection thread and not from the nuclear envelope as previously reported by Prasad and De. Endoplasmic reticulum (ER) profiles and Golgi vesicles fuse with the infection thread cell wall and adjacent host plasma membrane. Although some ER profiles were continuous with the outer membrane of the nuclear envelope, no continuities of the nuclear envelope with the infection thread, the host plasma membrane, or the peribacteroid membrane were observed. Furthermore, no blebbing of the nuclear envelope was observed.

the covalent carbohydrate in the toxic lectin ricin has been modified by periodate treatment, and analysis of the products of this reaction indicates that only mannose residues are oxidized. HeLa cell toxicity, cell-surface binding, inhibition of cell-free protein synthesis, and other properties have been investigated as functions of the number of mannose residues modified. The data indicate an important role for mannose in ricin conformation and in the endocytotic process; however, mannose appears not to be essential for binding the toxin to cells.

A transmission electron microscopic study of nodules of Vicia faba has demonstrated that rhizobia are released from the infection threads by an endocytotic process. The rhizobia escape from unwalled regions of the infection thread or unwalled droplets of thread matrix and upon escape are surrounded by a peribacteroid membrane which is derived from the plasma membrane bounding the unwalled regions of thread matrix. Thus, the release of bacteria in V. faba nodules is essentially identical to that reported in other leguminous nodules and the peribacteroid membrane does not arise de novo as previously reported by other workers.

Plant protoplasts isolated from tomato fruit locule tissue take up 0·12 μ polystyrene latex spheres by an endocytotic process. Freeze-etching enables the process to be clearly visualized in the electron microscope and provides important data from face view membrane fractures. When protoplasts are glutaraldehyde fixed prior to mixing with latex, the spheres adhere to the plasmalemma and cause some localized membrane distortion. This shows that adhesive forces are sufficient to initiate the invagination process. Freeze-etch membrane fracture faces carry numerous granules, the density of which is lower in the invaginating region. This reduction is in accordance with the expected value if only that part of the membrane which comes in intimate contact with the sphere expands to surround it. There is no change in the granule densities in the surrounding membrane. In fractured membrane surface views distinct phases can be delimited, and their relative occurrence indicates that the second half of the invagination process (i.e. after the diameter of the sphere has reached the membrane plane) is some thirty times quicker than the first half. It is proposed this indicates operation of surface tension forces during the fast phase, and it may be that cellular energy is required only for the initial slow phase of membrane expansion.

A number of studies have suggested that during the synaptic transmission synaptic vesicles of the nerve terminal fuse with the presynaptic membrane and release their neurotransmitter content into the synaptic cleft [ 1 ] . After the physiological response has been elicited, the neurotransmitters are either inactivated by degradation or by reuptake [2], but little is known about the fate of the vesicular membrane material. Based on electron microscopy of frog neuromuscular synapses treated with horseradish peroxidase, Heuser and Reese [3] have postulated that the synaptic vesicle membrane was added to the nerve terminal surface during exocytosis and then retrieved by means of coated vesicles via an endocytotic process, distant from the synaptic cleft. The coated vesicles formed at that site, were then recycled into new synaptic vesicles by way of the intermediate cisternae. Jdrgensen and Mellerup [4] have shown that intracerebrally injected bovine serum albumin could be incorporated into a preparation of rat brain synaptic vesicles by an endocytotic process. A prerequisite for endocytotic models should be the presence of vesicular membrane material in the synaptic plasma membrane, and in order to detect this it is necessary to compare the composition of synaptic plasma membranes with that of the synaptic vesicles. In this investigation we have compared by means

During oocyte growth in the frog Discoglossus pictus two patterns of vitellogenesis are described. The first one consists of the transformation of multivesicular bodies into yolk platelets; the second is the result of a typical endocytotic process, as described in other species. The peculiarity in Discoglossus vitellogenesis consists of a regional difference of these features of vitellogenesis in vitellogenic oocytes: the multivesicular bodies transforming into yolk platelets are found only in the germinative area—the central portion of the animal half—whereas deep crypts with numerous endocytotic pits are found only in the vegetal half. The probable meaning of this regional difference in vitellogenic oocytes is discussed.

Native horseradish peroxidase (HRP) is known to pass rapidly through glomeruli when injected into rats. We have found that a conjugate of HRP with poly-lysine is readily endocytosed by glomerular epithelial cells (GEC). We have used this conjugate to study the GEC endocytotic process in male Wistar rats. The conjugate has an approximate molecular weight of 55-58,000, a pI of greater than 10.0, and almost the same secondary conformation as HRP; it does not increase urinary protein excretion significantly or alter the morphology of the renal glomeruli. After intravenous injection of the conjugate, it could be found in the GBM from 1 min to 4 h. At 1 min, it was evenly distributed on GEC foot processes and plasma membrane. GEC start to take up the conjugate from 1 min post-injection, by cellular membrane invagination. This reached a maximum at 4 h. Some of the endocytosed conjugate passed to lysosomes from the endosomal system. The amount of peroxidase demonstrable in the glomerular epithelial cells was considerably reduced by 24 h.

Publisher Summary This chapter discusses the oxidative metabolism of phagocytosing leukocytes. One difficulty in the study of phagocytotic metabolism is that the changes induced by particles are complicated by the endocytotic process, including the internalization of the plasma membrane. This can be avoided by using non-particulate reagents, which trigger metabolic changes characteristic of phagocytosis. The chapter highlights the uptake of oxygen and the release of superoxide anions by guinea pig polymorphonuclear leukocytes stimulated either by cytochalasin E or bacteria. In a study described in the chapter, the oxygen-uptake trace was obtained with a Clark-type oxygen electrode. The cytochalasin is a relatively weak stimulant of the oxygen uptake compared to bacteria, but the former caused the release of a larger amount of superoxide anions than the latter. The cytochalasins also stimulate the production of hydrogen peroxide, which can be measured continuously by the fluorescence increase resulting from the formation of a fluorescent product from homovanillic acid in the presence of horseradish peroxidase.

We have employed fluorescein and 125l-labeled elicitors of the defense response in soybeans to monitor the cellular distribution and movement of elicitors following their addition to a soybean cell suspension culture. Our results indicate that the macromolecular elicitors first bind to the cell surface and then internalize in a temperature- and energy-dependent endocytotic process. Within a few hours, virtually all of the elicitor is concentrated in the major vacuole or tonoplast of the cell. Nonspecific (control) proteins neither bound to the cell surface nor internalized in parallel assays.

The endocytotic process in cultured human RPE cells was observed after 1 min, 20 min, and 2 h incubation with cationized ferritin. Within 1 min the ferritin particles were seen to attach to the cell membrane, especially between microvilli. Uncoated and coated pits could be recognized on the cell membranes, and uncoated and coated endocytotic vesicles were found in the cytoplasm after 20 min of incubation. These vesicles were surrounded by abundant microfilaments and had no visible membranes. Loss of membrane may be an initial step in the process of developing into the irregular clumps of ferritin particles found inside the plasma membrane. With time, more irregular clumps of ferritin, smaller than the particles introduced during incubation, appeared just beneath the cell membrane. Lysosomes were adjacent to the clumps of ferritin particles associated with microtobules and finally degraded these particles. The phagolysosomes containing many particles were surrounded by many microtubules. Small ferritin particles surrounded but had not entered the rough endoplasmic reticulums, and no particles were seen either around or in the Golgi apparatus.

Dry-cleaving is introduced as a new technique for visualization of the cytoplasmic side of adherent membrane in cultured cells. Basically, cells are fixed and critical point-dried in situ and subsequently broken by means of adhesive tape. The plane of cleavage is dependent on the fixation scheme applied. The method has been used for the study of substrate-adherent membranes in primary cultured hepatocytes, in combination with a variety of scanning- and transmission electron microscopic (SEM and TEM) techniques. It is shown that a two-dimensional filamentous web is apposed to the entire hepatocytic plasma membrane. Particular patterns within the web can be recognized, among which a ‘spider-web’ pattern appears to be associated with the early stage of coated vesicle development. During successive stages of coated vesicle formation the coat appears to be connected to filaments, directly in its mid-stage and by means of radial spokes in its final stage. The significance of the spider-web pattern with respect to the endocytotic process and the interrelationship of coated vesicles and filamentous structures are discussed.

Incubation of primary cultures of rat hepatocytes with the local anesthetics, procaine or lidocaine, had little or no effect on insulin uptake or degradation but caused an inhibition of insulin-stimulated glycogenesis. While exposure of cultures to the amines, monodansylcadaverine or CH3NH2, resulted in significant dose-dependent decreases in glycogenesis, only monodansylcadaverine (an inhibitor of receptor clustering) decreased uptake whereas CH3NH2 (a lysosomotropic agent) caused increases in both insulin uptake and degradation. When cells were treated with agents which inhibit glycolysis (NaF, 2-deoxy-D-glucose) or oxidative metabolism (2,4-dinitrophenol, carbonyl cyanide m-chlorophenyl hydrazone, NaN3, antimycin A), pronounced inhibitions of each of the bioactivities studied (syntheses of glycogen, protein, lipid) were observed, but only the glycolytic inhibitors decreased insulin uptake. These results suggest that insulin is internalized by an endocytotic process involving receptor clustering and requiring metabolic energy derived from glycolysis. The post-receptor biosynthetic processes involved in the expression of the biological activities of insulin (syntheses of glycogen, protein, lipid) require energy produced by oxidative metabolism while the degradation of insulin is carried out by nonlysosomal mechanisms which are not energy-requiring.

Membrane transport of vitamin B12 (cyanocobalamin; Cbl) into mammalian cells is mediated by the serum protein transcobalamin II (TCII). In mouse leukaemia L1210 cells, TCII-Cbl binds to membrane receptors in a rapid, temperature-independent step and is internalized by a slow, temperature-dependent process. To delineate the location of receptors on these cells, we have constructed a visual probe by covalently coupling purified TCII-Cbl to submicrometre latex particles (minibeads). We report here that when L1210 cells are incubated with minibeads containing TCII-Cbl at 4 degrees C and examined by scanning electron microscopy (SEM), the particles are found attached predominantly to microvilli. Incubation of the cells at 37 degrees C results in the internalization of the minibeads. As visualized by transmission electron microscopy (TEM), this endocytotic process seems to occur in clathrin-coated pits and vesicles at the cell surface.

Cyanide-insensitive respiration of polymorphonuclear leukocytes is induced by the D or E type of cytochalasins, a family of antibiotics which inhibits various cellular motile functions including membrane movement(Nakagawara et al.,1974). The respiratory stimulation is accompanied by the release of superoxide anions and the activated hexose monophosphate oxidative pathway but without any formation of phagosomes(Nakagawara et al.,1976). The cytochalasins D and E are ideal reagents for studying phagocytotic metabolic changes, because they inhibit endocytotic process at the same time. We are going to show the properties of the burst reactions triggered with cytochalasin D or E and compare the reactions with those occur during the ingestion of bacteria.

The acute effect of a physiological concentration (1 mU/l) of thyrotropin (TSH) on the activity of four lysosomal enzymes in the thyroid follicular lining cell has been studied by quantitative cytochemical techniques. N-acetyl-beta-glucosaminidase (NAG) activity was increased by 14% after 10 min TSH stimulation and NAG and beta-galactosidase activities were increased by 24% and 25% respectively (P less than 0.05) after 20 min stimulation and by 40% and 45% (P less than 0.05) respectively after 30 min stimulation with TSH, indicating an early processing of these carbohydrate residues in thyroglobulin. Acid phosphatase activity, an acid hydrolase unrelated to the hydrolysis of thyroglobulin, was unchanged 30 min after TSH stimulation. Leucyl-beta-naphthylaminidase (LNA) activity changed biphasically with peak activities of 7 and 25 min possibly representing an early fusion of endocytotic vesicles and lysosomes and later the release process of the thyroid hormones. The changes in LNA activity and thus membrane permeability were not reflected in the other enzyme activities studied. This may indicate that the TSH regulation of lysosomal enzyme activities could be independent to the endocytotic process, which is known to involve fusion of lysosomes and endocytotic vesicles. In conclusion we have demonstrated for the first time with physiological concentrations of TSH a specific acute regulation of some lysosomal enzyme activities which may be involved in thyroglobulin processing. Further, these effects may be independent of the changes in lysosomal membrane permeability due to formation of secondary lysosomes.

Livers of rats were perfused with dextran solutions, containing no red cells. Changes in the dextran concentration due to water shifts between the intra- and extracellular space of the liver were recorded continuously by polarimetric measurement. Cytochalasin B causes an uptake of potassium and water, followed by a release of water and a spontaneously reversible release of potassium, while the liver weight increases. From these effects an uptake of dextran, probably by an endocytotic process, is concluded.

An endocytotic process in Xenopus eggs was studied by direct visualization of the fluorescent tracer lucifer yellow CH in 1-μm-thick sections. Eggs fixed 3 min after activation show small fluorescent vesicles in the cortex; those fixed after 30 min show mainly large vesicles. This process is related to membrane recycling after cortical granule exocytosis and can account for the observed decrease of the effective membrane capacity.

Goodchild , D. J. (Commonwealth Scientific and Industrial Research Organization, Canberra, Australia), and F. J. Bergersen . Electron microscopy of the infection and subsequent development of soybean nodule cells. J. Bacteriol. 92: 204–213. 1966—Electron microscopy of thin sections of the developing central tissue cells of young soybean root nodules has shown that infection is initiated by a few infection threads which penetrate cells of the young central tissue. Extension growth of the threads may be a result of pressure developed from the growth of the bacteria within the threads. Release of bacteria from a thread is preceded by the development on an infection thread of a bulge with a cellulose-free membrane-bounded extension; bacteria move from this into the host cells by an endocytotic process and remain enclosed in an infection vacuole which is bounded by a membrane of host-cell origin. Multiplication of the intracellular bacteria takes place within these vacuoles. Until the host cell becomes filled with bacteria, the vacuoles separate into discrete units at each division. Later, division of the bacteria occurs within each vacuole, thus leading to the mature structure of the central tissue cells in which several bacteria are enclosed within each membrane-bounded unit.