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K Sandvig

Publications and source records attributed to K Sandvig.

At least 91 records · Page 5Linked to original sources
PubMed1991

The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations.

The transfer of molecules from the cell surface to the early endosomes is mediated by preendosomal vesicles. These vesicles, which have pinched off completely from the plasma membrane but not yet fused with endosomes, form the earliest compartment along the endocytic route. Using a new assay to distinguish between free and cell surface connected vesicle profiles, we have characterized the preedosomal compartment ultrastructurally. Our basic experimental setup was labeling of the entire cell surface at 4 degrees C with Con A-gold, warming of the cells to 37 degrees C to allow endocytosis, followed by replacing incubation medium with fixative, all within either 30 or 60 s. Then the fixed cells were incubated with anti-Con A-HRP to distinguish truly free (gold labeled) endocytic vesicles from surface-connected structures. Finally, analysis of thin (20-30 nm) serial sections and quantification of vesicle diameters were carried out. Based on this approach it is shown that the preendosomal compartment comprises both clathrin-coated and non-coated endocytic vesicles with approximately the same frequency but with distinct diameter distributions, the average noncoated vesicle being smaller (95 nm) than the average coated one (110 nm). In parallel experiments, using an anti-transferrin receptor gold-conjugate as a specific marker for clathrin-dependent endocytosis it is also shown that uncoating of coated vesicles plays only a minor role for the total frequency of noncoated vesicles. Furthermore, after perturbation of clathrin-dependent endocytosis by potassium depletion where uptake of transferrin is blocked, noncoated endocytic vesicles with Con A-gold, but not coated vesicles, exist already after 30 and 60 s. Finally, it is shown that the existence of small, free vesicles in the short-time experiments cannot be ascribed to recycling from the early endosomes.

Cell Compartmentation↗
PubMed1991

Peptides fused to the amino-terminal end of diphtheria toxin are translocated to the cytosol.

Diphtheria toxin belongs to a group of toxic proteins that enter the cytosol of animal cells. We have here investigated the effect of NH2-terminal extensions of diphtheria toxin on its ability to become translocated to the cytosol. DNA fragments encoding peptides of 12-30 amino acids were fused by recombinant DNA technology to the 5'-end of the gene for a mutant toxin. The resulting DNA constructs were transcribed and translated in vitro. The translation products were bound to cells and then exposed to low pH to induce translocation across the cell membrane. Under these conditions all of the oligopeptides tested, including three viral peptides and the leader peptide of diphtheria toxin, were translocated to the cytosol along with the enzymatic part (A-fragment) of the toxin. Neither hydrophobic nor highly charged sequences blocked translocation. The results are compatible with a model in which the COOH-terminus of the A-fragment first crosses the membrane, whereas the NH2-terminal region follows behind. The possibility of using nontoxic variants of diphtheria toxin as vectors to introduce peptides into the cytosol to elicit MHC class I-restricted immune response and clonal expansion of the relevant CD8+ cytotoxic T lymphocytes is discussed.

Amino Acid Sequence↗
PubMed1991

Ricin transport in brefeldin A-treated cells: correlation between Golgi structure and toxic effect.

Whereas brefeldin A (BFA) protected a number of cell lines against the protein toxin ricin, two of the cell lines tested were not protected but rather sensitized to ricin by BFA. EM studies revealed that upon addition of BFA the Golgi stacks in cells which were protected against the toxin rapidly transformed into a characteristic tubulo-vesicular reticulum connected to the endoplasmic reticulum, and subcellular fractionation experiments showed that galactosyl transferase disappeared from the Golgi fractions where it was normally located. EM and subcellular fractionation also indicated that in contrast to the Golgi stacks, the trans-Golgi network (TGN) remained intact and that internalized ricin was still localized in the TGN both when BFA was added before and after the toxin. Thus, BFA does not prevent fusion of ricin-containing vesicles with the TGN, and unlike resident proteins in Golgi stacks, ricin is not transported back to ER upon treatment of cells with BFA. Two kidney epithelial cell lines, MDCK and PtK2, were not protected against ricin by BFA, and EM studies of MDCK cells revealed that BFA did not alter the morphology of the Golgi complex in these cells. Also, subcellular fractionation revealed that, in contrast to the other cell types tested, the localization of galactosyl transferase in the gradients was not affected by BFA treatment. The data show that there is a correlation between BFA-induced disassembly of the Golgi stacks and protection against ricin, and they demonstrate that the structural organization of the Golgi apparatus is affected by BFA to different extents in various cell lines.

Animals↗
PubMed1991

Bacterial protein toxins acting on intracellular targets.

A number of bacterial toxins act on targets located in the cytosol. Diphtheria toxin, Pseudomonas aeruginosa exotoxin A and shigella toxin inhibit protein synthesis by enzymatic inactivation of elongation factor 2 or the 60 S ribosomal subunit. These toxins enter the cells by receptor-mediated endocytosis, followed by translocation across the membranes of intracellular organelles. Also a number or toxins that are not cytocidal act on targets in the cytosol. A number of nontoxic bacterial proteins are able to modify enzymatically intracellular molecules. Some of these proteins could be considered for targeting to special cells followed by translocation to obtain defined physiological effects.

ADP Ribose Transferases↗
PubMed1990

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

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

Animals↗
PubMed1990

Role of Na(+)-H+ and Cl(-)-HCO3- antiports in the regulation of cytosolic pH near neutrality.

In Vero cells, Na(+)-H+ antiport as well as Na(+)-coupled and Na(+)-independent Cl(-)-HCO3- antiport are involved in regulation of cytosolic pH (pHi) after large (unphysiological) deviations from neutrality. In this paper we have studied to which extent each of the three antiports is involved in regulation of pHi after small deviations from neutrality expected to occur under physiological conditions. At physiological extracellular pH (pHo), inhibition of Na(+)-H+ exchange by amiloride did not alter pHi. At neutral and alkaline pHo, pHi was found to be lower in the presence of HCO3- than in its absence, whereas at acidic pHo, pHi was higher in the presence of HCO3- than in its nominal absence. Above pHi 6.5, the activity of the Na(+)-coupled Cl(-)-HCO3- antiport was higher than the Na(+)-H+ antiport. After a small reduction of pHi, the recovery of steady-state pHi was entirely dependent on Na(+)-coupled Cl(-)-HCO3- antiport, whereas after more pronounced acidification, also Na(+)-H+ exchange contributed to the acid extrusion. The Na(+)-independent Cl(-)-HCO3- antiport, which acts as an acidifying mechanism, was strongly activated at pHi greater than 7.1. The results indicate that at physiological pHo the steady-state pHi is largely determined by the activity of the two Cl(-)-HCO3- antiports, and they suggest that Na(+)-H+ exchange does not influence the resting pHi under these conditions.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

Translocation of diphtheria toxin to the cytosol and formation of cation selective channels.

A number of protein toxins act by translocating an enzymatically active polypeptide to the cytosol. The translocation process is best understood in the case of diphtheria toxin which binds to cell surface receptors, is then taken up by endocytosis and is subsequently translocated to the cytosol, where it inactivates elongation factor 2. The translocation of the enzymatically active part of the toxin can be induced at the level of the plasma membrane upon exposure to low pH of cells with surface-bound toxin. Receptor molecules appear to be involved in the translocation process, which also requires an inward directed H(+)-gradient and permeant anions. Cation-selective channels are formed in the membrane upon toxin entry. The B-fragment alone is much more efficient in inducing channels than the whole toxin. The current model of the translocation process is discussed.

Biological Transport↗
PubMed1990

Endocytosis, intracellular transport and transcytosis of the toxic protein ricin by a polarized epithelium.

The toxic plant protein ricin binds to both the apical and basolateral surface domains of MDCK (strain I) cells grown on polycarbonate filters. Endocytosis of 125I-labeled ricin was not only higher from the basolateral than from the apical surface--an observation which can be explained by the higher surface area of the basolateral surface--but it also appeared to be more efficient when measured as a percentage of total cell-associated ricin. Monovalent ricin-horseradish peroxidase (Ri-HRP), which is known to behave like native ricin with respect to intracellular transport, also binds to, and is taken up from, both the apical and the basolateral surfaces. Initially, after 10 to 15 min, molecules taken up from the two surface domains at 37 degrees C are present in two separate (basolateral and apical) early endosomal populations. This can also be obtained by incubating for 60 min at 18 degrees C. However, after 30 to 60 min at 37 degrees C, most internalized ligand is found in apical lysosomes, regardless from which surface endocytosis took place. Experiments with endocytosis of cationized ferritin from the apical pole and HRP or Ri-HRP from the basolateral pole showed that intermixing in apical lysosomes (or prelysosomes) of molecules taken up from the two poles occurs. Bidirectional transcytosis involving coated pits of both 125I-labeled ricin and Ri-HRP was demonstrated and was found to be most efficient (as measured in per cent of endocytosed toxin) from the apical pole. Transcytosis was strongly reduced at 18 degrees C, and no transepithelial transport of ricin could be measured at 4 degrees C. Transcytosed ricin was intact and could intoxicate new cells. Finally, delivery of ricin internalized from both the apical and the basolateral surface to the apically localized trans-Golgi network occurred at 37 degrees C but not at 18 degrees C, and ricin inhibited protein synthesis largely with the same kinetics following uptake from the two poles. Incubation at 18 degrees C strongly inhibited the toxic effect of ricin. These data show that ricin can intoxicate epithelia from both sides and also penetrate tight epithelial barriers in intact form.

Animals↗
PubMed1989

Inhibition of chloride/bicarbonate antiports in monkey kidney cells (Vero) by non-steroidal anti-inflammatory drugs.

Two chloride/bicarbonate antiport mechanisms are involved in the regulation of cytosolic pH (pHi) in Vero cells, namely Na+-dependent chloride/bicarbonate antiport to normalize pHi after acidification of the cytosol, and Na+-independent Cl-/HCO3- exchange to regulate pHi back to normal after alkalinization of the cytosol. We have tested the effects of the non-steroidal anti-inflammatory drugs acetylsalicylic acid (aspirin), salicylic acid, indomethacin and piroxicam on chloride/bicarbonate exchange and on chloride self exchange in Vero cells. All these drugs were found to inhibit both the Na+-independent and the Na+-linked chloride/bicarbonate antiport in a dose dependent manner. The Na+-independent chloride/bicarbonate antiport was inhibited by lower doses of the drugs than the Na+-linked antiport. The ability of the drugs to inhibit chloride self exchange did not vary much with varying external pH, indicating that the inhibitory effect is due to the anionic form of the drugs. Inhibition occurred immediately upon addition of the drugs, and it was rapidly reversible, indicating that the inhibitory effect is due to direct interaction of the drugs with chloride/bicarbonate antiport, and not to inhibition of prostaglandin synthesis. The relevance of our findings to the clinical effects of the drugs is discussed.

Animals↗
PubMed1989

Translocation of diphtheria toxin A-fragment to the cytosol. Role of the site of interfragment cleavage.

Diphtheria toxin contains a trypsin-sensitive region with 3 closely spaced arginines in the sequence (Asn189, Arg190, Val191, Arg192, Arg193, Ser194). Cleavage of the toxin to yield A- and B-fragments ("nicking") appears to occur in a stochastic manner after either of these arginine residues. Isoelectric focusing of A-fragment prepared in vitro showed four bands of varying intensity with pI between 4.5 and 5.0, three of which could be accounted for by the three different cleavage sites. Exposure of cells with surface-bound toxin to pH less than 5.3 induces translocation of A-fragment to a position where it is shielded from external Pronase, presumably in the cytosol. A-fragment translocated in this manner had the same pI as the most acidic A-fragments, indicating that only A-fragments lacking both Arg192 and Arg193 are translocation-competent. This was confirmed by amino acid sequencing. Treatment of A-fragment with carboxypeptidase B eliminated the two bands with the highest pI while there was a concomitant increase in the bands corresponding to the two most acidic A-fragments. Such treatment of nicked diphtheria toxin increased the amount of translocated A-fragment and the ability of toxin to form cation-selective pores in the cell membrane. The site of trypsin cleavage therefore appears to be one of the factors limiting toxin entry to the cytosol.

Animals↗
PubMed1989

Formation of active diphtheria toxin in vitro based on ligated fragments of cloned mutant genes.

An intact gene coding for wild type diphtheria toxin was reconstituted in vitro from fragments of cloned inactive diphtheria toxin mutants with defects in different parts of the gene. The reconstituted DNA template was amplified using the Taq DNA polymerase chain reaction, providing a virtually unlimited supply. The toxin was expressed in vitro by transcription from a T3 RNA polymerase promoter, followed by translation of the mRNA in a rabbit reticulocyte lysate system. Diphtheria toxin synthesised in this manner was shown to be as toxic as natural toxin and behaved like natural toxin in every way tested. This can serve as a general approach for study of toxic proteins, opening up the possibility of in vitro gene manipulation. Reconstitution of the wild type gene from fragments followed by polymerase chain reaction amplification bypasses the hazards of cloning protein toxins.

Animals↗
PubMed1989

Role of anions in low pH-induced translocation of diphtheria toxin.

Previous work has shown that when Vero cells with surface-bound diphtheria toxin are exposed to low pH, toxin entry across the plasma membrane is induced and that this entry involves two steps, insertion of the B-fragment of the toxin into the membrane and translocation of the enzymatically active A-fragment to the cytosol. Here we have studied the role of permeant anions in this process. It was found that when the B-fragment was inserted into the membrane, part of it, a 25-kDa polypeptide, was shielded from externally added Pronase. This insertion did not require permeant anions. The translocation of the A-fragment was monitored by measuring either its ability to inhibit protein synthesis in the cells or the appearance of radioactively labeled 21-kDa fragment after treatment of the cells with externally applied Pronase. The translocation of the A-fragment was dependent on the presence of permeant anions in the medium. However, when the cells were depleted of Cl- by incubation in Cl- free buffer at high pH, translocation of the A-fragment did not require permeant anions in the medium. The possibility that translocation of the A-fragment is inhibited by an outward directed chloride gradient rather than by the absence of chloride is discussed.

Animals↗
PubMed1989

Membrane interactions of diphtheria toxin analyzed using in vitro synthesized mutants.

We have developed a system to study the interactions of diphtheria toxin with the cell surface using non-toxic mutant proteins synthesized in vitro. Proteins obtained by N-terminal deletions containing the whole B fragment bound strongly to cells. Deletions extending into the B fragment did not yield an autonomous binding domain. Loss of only the N-terminal 3 kd of the B fragment significantly impaired the ability to recognize the receptor. This, together with previous reports that the C-terminal end of the B fragment is required for binding, suggests that both ends of the B fragment are necessary for receptor recognition. Receptor bound diphtheria toxin undergoes a conformational change at pH less than 5.3 that results in translocation of the A fragment to the cytosol and the appearance of a B fragment-derived 25 kd polypeptide (P25) resistant to externally applied protease. Only the B fragment was required for generation of P25. N-terminal deletions of 130 amino acids or more resulted in proteins that gave rise to P25 at higher pH than full length toxin. Furthermore, a second protease-inaccessible polypeptide of 18 kd (P18) was observed.

Animals↗
PubMed1989

Permeabilization of the plasma membrane by deletion mutants of diphtheria toxin.

Diphtheria toxin B-fragment binds to cell-surface receptors and facilitates translocation of the enzymatically active A-fragment to the cytosol. In this process the B-fragment inserts into the plasma membrane and induces formation of cation-selective channels. We examined the ability of a number of diphtheria toxin-derived molecules translated in vitro to permeabilize cells. Two proteins consisting of the whole B-fragment and small parts of the A-fragment, and one protein comprising most of the B-fragment alone, were more efficient than full-length toxin in permeabilizing the plasma membrane to monovalent cations. Two shorter B-fragment-derived proteins, with 3 and 10 kd N-terminal deletions, permeabilized the cells to sulfate and sucrose in addition to monovalent cations. The relationship between channel formation and toxin translocation is discussed.

Animals↗
PubMed1989

Internalized ricin and the plasma membrane glycoprotein MAM-6 colocalize in the trans-Golgi network of T47D human breast carcinoma cells.

We have analyzed the intracellular transport of endocytosed ricin in the human breast carcinoma cell line T47D. Cells were incubated with ricin (10 micrograms/ml) for 1 h at 37 degrees C. Marked reduction in the protein synthesis did not take place until the end of this period. To detect ricin immunocytochemically, a rabbit anti-ricin serum was used. Gel electrophoresis followed by immunoblotting revealed that the antiserum reacted specifically with ricin and detected both the ricin A-chain and the ricin B-chain. Immunofluorescence experiments showed endocytosed ricin in endosomal and lysosomal vacuoles throughout the cytoplasm, as well as in a typical perinuclear position corresponding to the Golgi region. Using the monoclonal mouse antibody 115D8 directed toward the high-molecular-weight membrane glycoprotein MAM-6 of human breast epithelial cells, we similarly obtained a swarms of small vesicles throughout the cytoplasm. To further analyze the apparent colocalization of ricin and MAM-6 in the perinuclear Golgi region, immunogold cytochemistry on ultracryosections was performed. MAM-6 was detected mainly in Golgi stacks and associated trans-Golgi network (TGN) profiles, in 0.1 to 0.2-micron secretory vesicles, and on the cell surface. Ricin was detected on the cell surface, in endosomes and lysosomes, and also in the TGN. Furthermore, by using immunogold double labeling, internalized ricin was found to colocalize with MAM-6 in the TGN.

Antibodies, Monoclonal↗