Biomedical subjects

J Davoust

Publications and source records attributed to J Davoust.

At least 55 records · Page 3Linked to original sources

PubMed1995

Role of B cell receptor Ig alpha and Ig beta subunits in MHC class II-restricted antigen presentation.

The ability of the B cell antigen receptors (BCRs) to enhance MHC class II-restricted antigen presentation was ascribed to mig-associated Ig alpha/Ig beta heterodimers. The relative role of Ig alpha and Ig beta subunits in antigen presentation was investigated by fusing their cytoplasmic tails to the extracellular and transmembrane domains of Fc receptors. Ig alpha and Ig beta chimera mediate antigen internalization and increase the efficiency of antigen presentation, but they drive antigens to different endosomal compartments. Furthermore, antigens internalized by either chimera are degraded and presented with different kinetics. The cytoplasmic tail of Ig alpha targets antigen towards a major population of newly synthesized MHC class II located in class II-rich compartments. In contrast, Ig beta targets antigen towards a minor population of recycling MHC class II molecules, located in transferrin receptor-containing endosomes. Altogether, our data indicate that the composition of BCR could be therefore an important way to modulate the immune response.

Animals↗

PubMed1994

Evidence for protein tyrosine kinase involvement in ligand-induced TCR/CD3 internalization and surface redistribution.

Triggering of the TCR/CD3 complex can lead to its internalization and modulation from the cell surface. In the present study, we address the question of the dependence of internalization on protein tyrosine kinase (PTK) activation. With use of an activating anti-clonotypic (anti-Ti) mAb on a CTL clone, we have shown that the PTK inhibitors genistein and tyrphostin 25 delayed anti-Ti-induced internalization, but did not affect fluid phase protein uptake or transferrin receptor cycling. Confocal microscopy with use of fluorescent anti-Ti mAb revealed that the inhibition of TCR internalization corresponded to the induction of large patches that were localized in cell membrane areas depleted of polymerized actin, the formation of which was dependent on the combined action of the anti-Ti mAb and the PTK inhibitors. In contrast to the effect of these PTK inhibitors, depletion of Src-like PTKs by T cell pretreatment with herbimycin A led to an increased rate of anti-Ti-induced internalization. Internalization induced by the monovalent Fab fraction of anti-Ti mAb was similarly affected by the PTK inhibitors, although the extent of induced internalization was less by approximately one-half. An analysis of substrates phosphorylated in kinase assays on TCR/CD3 immunoprecipitates of the CTL, which were activated by anti-Ti mAb in both the absence and presence of genistein, identified protein bands in which phosphorylation or association with CD3 was inhibited in the presence of genistein. Thus, a genistein-sensitive PTK activity seems to control ligand-induced TCR/CD3 complex redistribution and internalization.

Actins↗

PubMed1994

Restricted distribution of connexin40, a gap junctional protein, in mammalian heart.

Connexin40 (Cx40) is a member of the connexin family of gap junction proteins. Its mRNA, abundant in lung, is also present in mammalian heart, although in lower amount. Rabbit antipeptide antibodies directed to the COOH terminus (residues 335 to 356) of rat Cx40 were characterized to investigate the distribution of Cx40 in rat and guinea pig cardiac tissues. The affinity-purified antibodies detect specifically a major protein (M(r), 40,000) in immunoblots of total extracts from rat lung and rat and guinea pig heart. In sections of guinea pig atrial tissue treated for immunofluorescence, a strong labeling associated with myocytes was seen with a distribution consistent with that of intercalated disks. The results of immunoelectron microscopy carried out with guinea pig atrial tissue showed that epitopes recognized by these antibodies were exclusively associated with gap junctions. These results, added to those of control experiments, demonstrate that antibodies 335-356 are specific for Cx40. Double-labeling experiments carried out with lung sections using anti-factor VIII and anti-Cx40 antibodies suggest that Cx40 is expressed in blood vessel endothelial cells. In guinea pig and rat heart sections, investigated using both immunofluorescence and immunoperoxidase techniques, a signal was also found to be associated with vascular walls. In guinea pig heart, only atrial myocytes are Cx40-positive. No labeling was detected in ventricular myocytes, including those of the His bundle and the bundle branches, which otherwise do express connexin43 (Cx43). In rat heart Cx40-expressing myocytes are localized in branches, and the Purkinje fibers. Cx43 is not detected either in the His bundle or in the proximal parts of the bundle branches, and consequently, Cx40 is the first connexin demonstrated in this region of the rat conduction system. Cx40 was not detected in the working ventricular myocytes. Double-labeling experiments carried out with hen anti-Cx43 antibodies and rabbit anti-Cx40 antibodies demonstrated that, in tissues expressing both Cx43 and Cx40, these two connexins were localized in the same immunoreactive sites. A few sites, however, appear to contain only one or the other of these two connexins.

Amino Acid Sequence↗

PubMed1994

GPI membrane anchor is determinant in intracellular accumulation of apical plasma membrane proteins in the non-polarized human colon cancer cell line HT-29 18.

We have compared the intracellular localization of plasma membrane proteins anchored either with a transmembrane segment or with a glycosylphosphatidylinositol moiety to estimate the effects of membrane anchor on protein segregation in the non-polarized form of the human colon cancer cell line HT-29 18. We have monitored two endogenous proteins: the carcinoembryonic antigen, a glycosylphosphatidylinositol protein and the transmembrane protein dipeptidyl peptidase IV, and two transfected proteins: the glycosylphosphatidylinositol protein Thy-1 and an engineered transmembrane form of Thy-1. Using immunocytochemistry on ultra-thin cryosections and confocal microscopy, we detected a carcinoembryonic antigen-rich vesicular compartment, excluding classical pre-lysosomal and lysosomal markers such as mannose 6-phosphate receptor, lamp-1 and cathepsin D. This compartment, where carcinoembryonic antigen accumulated, excluded the transmembrane protein dipeptidyl peptidase IV and was reduced during the polarization of the cells. Moreover, the glycosylphosphatidylinositol form of Thy-1 also accumulated in the carcinoembryonic antigen-rich compartment whereas the transmembrane form of Thy-1 was excluded. We proposed that, in the non-polarized HT-29 18 cells, accumulation of glycosylphosphatidylinositol proteins independently of transmembrane proteins reveals different intracellular fates for proteins according to their anchor in the plasma membrane.

Carcinoembryonic Antigen↗

PubMed1994

Programmed cell death in Dictyostelium.

Programmed cell death (PCD) of Dictyostelium discoideum cells was triggered precisely and studied quantitatively in an in vitro system involving differentiation without morphogenesis. In temporal succession after the triggering of differentiation, PCD included first an irreversible step leading to the inability to regrow at 8 hours. At 12 hours, massive vacuolisation was best evidenced by confocal microscopy, and prominent cytoplasmic condensation and focal chromatin condensation could be observed by electron microscopy. Membrane permeabilization occurred only very late (at 40-60 hours) as judged by propidium iodide staining. No early DNA fragmentation could be detected by standard or pulsed field gel electrophoresis. These traits exhibit some similarity to those of previously described non-apoptotic and apoptotic PCD, suggesting the hypothesis of a single core molecular mechanism of PCD emerging in evolution before the postulated multiple emergences of multicellularity. A single core mechanism would underly phenotypic variations of PCD resulting in various cells from differences in enzymatic equipment and mechanical constraints. A prediction is that some of the molecules involved in the core PCD mechanism of even phylogenetically very distant organisms, e.g. Dictyostelium and vertebrates, should be related.

Animals↗

PubMed1993

The invariant chain induces compact forms of class II molecules localized in late endosomal compartments.

The invariant chain (Ii) binds to newly synthesized major histocompatibility complex (MHC) class II molecules and is targeted to an acidic compartment where it is degraded. To evaluate its role on the conformation and the subcellular distribution of murine MHC class II molecules we have established stable L cell transfectants expressing class II IAk heterodimers alone or in conjunction with p31 and p41 Ii chains. In these cells, class II molecules were present under three forms: alpha beta heterodimers bearing high mannose carbohydrate moieties, and fully glycosylated alpha beta heterodimers that are sensitive or resistant to sodium dodecyl sulfate dissociation at 20 degrees C. The latter class II molecules called compact heterodimers, were here highly induced in Ii-positive cells. Using in situ iodination of endosomal compartments, class II heterodimers were detected in late endosomal compartments essentially as compact forms in Ii-positive cells, and as non-compact forms in Ii-negative cells. Using confocal microscopy, IAk molecules were located in compartments distinct from early endosomes labeled with transferrin, but partially coincident with vesicles containing fluid-phase markers, and highly coincident with compartments containing large amounts of cathepsins B, D, H, and L in Ii-positive and Ii-negative cells. At the ultrastructural level, class II molecules were mostly present in multivesicular bodies, even without Ii expression. But Ii chains were needed to induce an efficient presentation of the hen egg lysozyme antigen and were sufficient to promote a major conformational change of the late endosomal, and/or lysosomal resident, class II molecules. Ii molecules are presumably playing a chaperoning function favoring the association of peptides with class II molecules in endosomal compartments.

Antigen Presentation↗

PubMed1993

Major histocompatibility complex class II-restricted presentation of secreted and endoplasmic reticulum resident antigens requires the invariant chains and is sensitive to lysosomotropic agents.

We have tested the involvement of the invariant chains (Ii) p31 and p41 in the presentation of peptides derived from hen egg lysozyme (HEL) constructs targeted to different intracellular compartments within transfected fibroblasts. The endogenous HEL constructs were either present in the cytosol (HELc), secreted (HELs), or linked to the mammalian (KDEL C-terminal sequence that causes retention of HEL in the endoplasmic reticulum (ER)/pre-Golgi recycling compartment (HELr). Using Ii-negative antigen-presenting cells, the presentation of HELr to a HEL 46-61 specific T cell hybridoma was far less efficient than the presentation of the HELs. High levels of Ii expression enhanced drastically the presentation of the HEL 46-61 determinant derived from both HELr and HELs. HELr and HELs presentation was fully sensitive to lysosomotropic agents such as chloroquine, indicating that the formation of complexes between major histocompatibility complex (MHC) class II molecules and determinants derived from endogenous antigens entering the secretory pathway is taking place in an acidic compartment. The degradation and dissociation of Ii might be a prerequisite for the efficient presentation of endogenously derived determinants by MHC class II molecules, as for the presentation of most exogenous antigens. All our results are compatible with the notion that endogenous molecules being translocated into the lumen of the ER could be presented by class II molecules through a processing pathway involving an acidic compartment in which Ii chains dissociate from class II molecules.

Animals↗

PubMed1993

Structural compartmentalization of MHC class II signaling function.

Major histocompatibility complex (MHC) class II molecules are critical restricting elements in the generation of thymus-dependent immune responses. Recent studies indicate that in addition to providing a composite epitope for recognition by T-cell antigen receptors, MHC class II molecules function in signal transduction through interaction with other cellular proteins. Mutational analyses indicate that structural information necessary for these functions is compartmentalized in different aspects of the molecular complex. Here, William Wade and colleagues review the structural basis of this MHC class II function as defined in the I-A alpha and -beta chains.

Amino Acid Sequence↗

PubMed1993

Microtubule organization during the cell cycle of the primitive eukaryote dinoflagellate Crypthecodinium cohnii.

The complete microtubular system of the dinoflagellate Crypthecodinium cohnii Biecheler is described, as seen by confocal laser scanning fluorescence microscopy and labelling with anti-beta-tubulin antibody. This technique allowed us to observe the organization of the subcortical and internal cytoskeletons and the mitotic microtubular system, and their changes during the cell cycle. These observations are compared with those made in cryosections by light microscopy and in fast-freeze-fixed, cryosubstituted cells by electron microscopy. We show the organization of the cortical microtubules, and in particular of the thick microtubular bundles arranged as a three-pronged fork from which they seem to emanate. This fork emerges from a peculiar cytoplasmic zone at the pole of the cell and is in contact with the region of the kinetosomes, at the cingulum. During the G1 phase, only a single, radial microtubular bundle (a "desmose") is observable in the inner part of the cytoplasm. One of its ends is near the flagellar bases and the other end is close to the nucleus in the centrosome region. During the S phase, the flagella drop off, the cell encysts and the kinetosomes duplicate. In mitosis, the cortical microtubules and the intracytoplasmic microtubular bundles do not depolymerize. The microtubular fork, desmose and centrosome double and migrate, while the divided kinetosomes stay in the same place. Later, the centrosomes organize the extranuclear spindle, which is connected to the kinetosome region by the microtubular desmose. The convergent end of the three-pronged fork seems to be in contact with the centrosome region. In early and mid-prophase, thick microtubular bundles pass through the nucleus in cytoplasmic channels and converge towards the two poles. Asters were never seen at the spindle poles. The channels and microtubular bundles in the spindle double in number during late prophase and lengthen in early anaphase. The spindle bundles diverge in late anaphase, extend to very near the plasma membrane and depolymerize during telophase. The cleavage furrow in which tubulin and actin are characterized appears in anaphase, formed by invagination of plasma membrane in the kinetosome region. The structure and rearrangements of the Crypthecodinium cohnii microtubular system are compared with those of other dinoflagellates and protists and of higher eukaryotes.

Animals↗

PubMed1992

Tyrosine-containing motif that transduces cell activation signals also determines internalization and antigen presentation via type III receptors for IgG.

Type III receptors for IgG (Fc gamma RII; ref. 1), high-affinity IgE receptors (Fc epsilon RI; ref. 2), as well as the T- and B-cell antigen receptors, consist of multiple components with specialized ligand-binding and signal transduction functions. Fc gamma RII alpha (ligand-binding) and gamma (signal-transducing) subunits are expressed in macrophages, a cell type involved in the uptake of antigen, its processing and the presentation of the resulting peptides to major histocompatibility complex class II-restricted T lymphocytes. Here we show that murine Fc gamma RIII, transfected into Fc gamma R-negative antigen-presenting B-lymphoma cells, mediate rapid ligand internalization and strongly increase the efficiency of antigen presentation when antigen is complexed to IgG. Efficient internalization and antigen presentation via Fc gamma RIII did not require the cytoplasmic domain of the ligand-binding alpha-chain, but did require the gamma-subunit. Using chimaeric molecules, we show that gamma-chain contains a signal for receptor internalization and that the mutation of either of the two tyrosine residues present in its cytoplasmic domain prevents efficient internalization and antigen presentation of immune complexes. Thus, associated chains and their tyrosine-containing motif are not exclusively involved in cell activation, but also determine multimeric receptor internalization.

Amino Acid Sequence↗

PubMed1992

Apical to basolateral surface area ratio and polarity of MDCK cells grown on different supports.

We have established conditions under which Madin-Darby canine kidney cells develop a well-polarized monolayer on polycarbonate filters and on transparent filters. These filters have biochemical and mechanical advantages over the nitrocellulose filters which have been widely used. Transepithelial resistance was established 10 h after plating and stabilized after 24 h. The distribution of protein antigens was followed by surface immunofluorescence and quantitated by a surface immunoassay that we developed. Uvomorulin was localized to the lateral membrane, with low amounts detectable on the basal membrane. The 58-kDa antigen was distributed over the entire basolateral domain, including cell processes extending into the filter pores. This distribution was confirmed by immunogold labeling of frozen sections. The 114-kDa antigen was found to be present at similar surface densities on both the apical and the basolateral domain. The support used for growth had profound effects on the cell morphology. A morphometric analysis of the plasma membrane of both strains of the cell line showed an increase in the number and size of the microvilli, and a smoother basal membrane as compared to published data on nitrocellulose filters. The apical to basolateral surface area ratio was therefore modified.

Animals↗

PubMed1991

Overproduction and secretion of beta 2-microglobulin by a rat thymic epithelial cell line that expresses MHC class I heavy chain.

Major histocompatibility complex (MHC) class I antigens are constituted of dimers consisting of a peripheral light chain, beta 2-microglobulin (beta 2m) and a transmembrane heavy chain whose cell surface expression depends on its assembly with beta 2m. In contrast, soluble beta 2m can be secreted in the absence of heavy chain expression. The presence of beta 2m in medium conditioned by a rat thymic epithelial cell line, IT45-R1 (IT45) prompted us to investigate whether beta 2m could be secreted by cells that express MHC class I antigens. IT45 cells produce three to five times more beta 2m in the culture supernatant than another rat thymic epithelial cell line, IT26-R21 (IT26). The IT45 cell line exported beta 2m through a constitutive pathway of secretion, as indicated by the kinetics of production and localization of intracellular beta 2m. Although cells from the IT45 cell line expressed a much higher amount of beta 2m as compared to IT26 and NBT II cells (a rat bladder epithelial cell line), all three of these cell lines expressed the same amount of membrane and intracellular MHC class I heavy chain. These data are thus consistent with a constitutive secretion of beta 2m dependent upon an overexpression of MHC class I light chain as compared to the heavy chain. The amount of beta 2m mRNA and the ratio of beta 2m versus MHC class I heavy chain transcripts were higher in IT45 than in IT26 cells, indicating that overexpression of beta 2m in IT45 cells could be due to an enhanced level of beta 2m mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Apical and basal Forssman antigen in MDCK II cells: a morphological and quantitative study.

We have studied the surface distribution of a glycosphingolipid (the Forssman antigen) in MDCK II and CCL39 cells. The Forssman antigen is mobile on the surface of both these cell lines. Its surface distribution is homogenous on non-polarized cells. Under conditions where MDCK II cells are well polarized, the Forssman antigen is present in equal amounts on the apical membrane and on the basal membrane and its processes. Very little Forssman antigen can be detected on the lateral membrane. The nature of the mechanism excluding the Forssman antigen from the lateral domain remains to be determined. This surface distribution is established within hours after plating and was observed with cells grown on different types of filters. The surface density of the Forssman antigen on the apical and on the basal domain has been estimated. No involvement of the basal Forssman antigen in cell attachment could be demonstrated. However, the apical Forssman antigen appears to be essential to the establishment of the cells in culture.

Animals↗

PubMed1990

Mouse B lymphocyte specific endocytosis and recycling of MHC class II molecules.

In B lymphocytes, the processing of exogenous proteins and the subsequent binding of antigenic peptides to class II molecules encoded by the major histocompatibility complex (MHC) occurs most likely within endocytic compartments. To examine the endocytic transport of MHC class II molecules, we used (i) surface iodination followed by internalization, pronase treatment and immunoprecipitation, (ii) in situ iodination of endosomal compartments, and (iii) confocal microscopy to visualize the fate of fluorescence coupled Fab fragments. In murine I-Ak, I-Ek positive B lymphoma cells, cell surface MHC class II molecules are partially protected from pronase digestion after 15 min at 37 degrees C and recycled back to the cell surface within the next 30 min. The fluorescence coupled Fab fragments are delivered to juxtanuclear endocytic compartments in 15 min. In contrast to the murine B cells, L fibroblasts transfected with either I-A alpha beta k or I-E alpha k beta k,d fail to internalize their surface class II molecules. A fraction of class II molecules, however, is still present in endosomal compartments as detected after in situ iodination in L fibroblasts. We conclude that the recipient L fibroblasts lack one or several factors needed for the transport of MHC class II molecules from the cell surface to the endosomes. We suggest that in murine B lymphoma cells, antigenic peptides can gain access to a pool of recycling class II molecules whereas in L cells they meet newly synthesized class II molecules targeted to the endosomal compartments.

Animals↗

PubMed1990

Confocal microscopy as a tool to reveal the tridimensional organization of intracellular lumens and intercellular cysts in a human colon adenocarcinoma cell line.

Adenocarcinoma cells often form intracellular lumens and intercellular cysts. In order to study the structural relationships between these lumens and the apical domain of normal enterocytes, we have applied electron microscopy and confocal microscopy to a cloned cell line derived from the human colon adenocarcinoma cell line LoVo which express a high number of intracellular lumens and intercellular cysts. Microvilli reminiscent of those detected in the brush border of small intestinal cells are formed in the two types of compartments. By immunofluorescence, we found that a 135 kDa membrane glycoprotein characterized by a monoclonal Ab and normally associated with the brush-border of enterocytes is expressed at the surface of the intracellular lumens and intercellular cysts present in the adenocarcinoma cells. Comparison of fluorescence and reflection contrast micrographs obtained by confocal microscopy demonstrate the presence of spherical intracellular lumens in the juxtanuclear region of single cells, and of more complex shaped intercellular cysts located within clusters of cells. The later cells form junctional complexes limiting an apical plasma membrane domain in contact with the intercellular cyst. It is suggested that the intracellular lumens may represent the abortive form of an apical plasma membrane due to the lack of components required to establish epithelial cell contacts. As opposed to conventional fluorescence microscopy, confocal microscopy allows rapid inspection of the tridimensional organization of intracellular lumens and intercellular cysts even when they are located in cell multilayers.

Adenocarcinoma↗

PubMed1990

A signaling role for the cytoplasmic segment of the CD8 alpha chain detected under limiting stimulatory conditions.

To test for the functional importance of the cytoplasmic segment of the CD8 molecule, a mouse T-cell hybridoma expressing a T-cell receptor specific for the class I major histocompatibility complex product H-2Kb was transfected with a set of CD8 alpha-chain (Ly-2) and/or beta-chain (Ly-3) genes encoding polypeptides with carboxyl-terminal truncations or substitutions. When challenged with Kb-positive splenocytes, transfectants expressing Ly-2 homodimers that lacked cytoplasmic tails responded nearly as effectively as wild-type Ly-2 transfectants. However in marked contrast to the wild-type Ly-2 transfectants, tailless Ly-2 transfectants were greatly impaired in their ability to respond to Kb-transfected L cells. Coexpression of the Ly-3 gene did not restore this impaired response. The unique functional property of the Ly-2 alpha cytoplasmic segment was further supported by the analysis of a chimeric Ly-3 subunit in which the cytoplasmic segment was replaced by the one from the Ly-2 alpha subunit. When associated with a soluble Ly-2 subunit lacking a transmembrane segment, the chimeric Ly-3 was indeed sufficient to restore the response to Kb-transfected L cells. Since the lateral mobility of the tailless Ly-2 molecules on the cell surface was nearly identical to that of the wild-type Ly-2 molecules, their partially impaired function may indicate that they have lost their cis-acting signaling properties but retained their ability to bind class I products of the major histocompatibility complex.

Amino Acid Sequence↗

PubMed1990

Microtubules are stabilized in confluent epithelial cells but not in fibroblasts.

Rhodamine-tagged tubulin was microinjected into epithelial cells (MDCK) and fibroblasts (Vero) to characterize the dynamic properties of labeled microtubules in sparse and confluent cells. Fringe pattern fluorescence photobleaching revealed two components with distinct dynamic properties. About one-third of the injected tubulin diffused rapidly in the cytoplasm with a diffusion coefficient of 1.3-1.6 x 10(-8) cm2/s. This pool of soluble cytoplasmic tubulin was increased to greater than 80% when cells were treated with nocodazole, or reduced to approximately 20% upon treatment of cells with taxol. Fluorescence recovery of the remaining two-thirds of labeled tubulin occurred with an average half-time (t1/2) of 9-11 min. This pool corresponds to labeled tubulin associated with microtubules, since it was sensitive to treatment of cells with nocodazole and since taxol increased its average t1/2 to greater than 22 min. Movement of photobleached microtubules in the cytoplasm with rates of several micrometers per minute was shown using very small interfringe distances. A significant change in the dynamic properties of microtubules occurred when MDCK cells reached confluency. On a cell average, microtubule half-life was increased about twofold to approximately 16 min. In fact, two populations of cells were detected with respect to their microtubule turnover rates, one with a t1/2 of approximately 9 min and one with a t1/2 of greater than 25 min. Correspondingly, the rate of incorporation of microinjected tubulin into interphase microtubules was reduced about twofold in confluent MDCK cells. In contrast to the MDCK cells, no difference in microtubule dynamics was observed in sparse and confluent populations of Vero fibroblasts, where the average microtubule half-life was approximately 10 min. Thus, microtubules are significantly stabilized in epithelial but not fibroblastic cells grown to confluency.

Animals↗

PubMed1989

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

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

Animals↗