Biomedical subjects

Bo van Deurs

Publications and source records attributed to Bo van Deurs.

18 recordsLinked to original sources

PubMed2006

EGF-induced activation of the EGF receptor does not trigger mobilization of caveolae.

Caveolae-dependent endocytosis has recently been proposed in the uptake of EGF receptor (EGFR) at high concentrations of ligand. Consistently, upon incubation of HEp2 and HeLa cells with methyl-beta-cyclodextrin, we observed a small inhibitory effect on endocytosis of ligated EGFR in HEp2 cells. However, immunoelectron microscopy showed the same relative amount of bound EGF localizing to caveolae on incubation with high and low concentrations of EGF, not supporting rapid recruitment of EGFR to caveolae. Live-cell microscopy furthermore demonstrated that incubating HEp2 cells with high concentrations of EGF did not increase the mobility of caveolae. By RNA-interference-mediated knockdown of clathrin heavy chain in HEp2 and HeLa cells, we found that endocytosis of EGFR was efficiently inhibited both at high and low concentrations of EGF. Our results show that caveolae are not involved in endocytosis of EGF-bound EGFR to any significant degree and that high concentrations of EGF do not further mobilize caveolae.

Caveolae↗

PubMed2005

Geldanamycin stimulates internalization of ErbB2 in a proteasome-dependent way.

The potent oncoprotein and receptor tyrosine kinase ErbB2 is remarkable because it resists efficient downregulation. However, ErbB2 can be downregulated by the HSP-90 inhibitor geldanamycin, but the underlying cellular mechanisms are uncertain. Apparently, delivery of ErbB2 to lysosomes, cleavage of the ErbB2 kinase domain and proteasomal activity are all processes that are involved. Using a non-invasive confocal microscopical assay allowing quantitative analysis of ErbB2 internalization in cell populations, we show that whereas ErbB2 is resistant to internalization in untreated SK-BR-3 cells, geldanamycin stimulates internalization and subsequent degradation in lysosomes. This process depends on proteasomal activity, which is a regulatory upstream event in ErbB2 internalization rather than the actual mechanism of degradation. ErbB2 can be internalized as a full-length protein, thus cleavage of the ErbB2 kinase domain is not a requirement for geldanamycin-stimulated internalization. Moreover, as shown by FRAP (fluorescence recovery after photobleaching) and electron microscopy, geldanamycin induces an increase in the amount of mobile ErbB2 and a redistribution of ErbB2 in the plasma membrane making the receptor accessible to endocytosis. Cells with most ErbB2 endocytosis also have the highest fraction of mobile ErbB2. It is concluded that geldanamycin stimulates internalization of full-length ErbB2 in a proteasome-dependent manner leading to lysosomal degradation.

Acetylcysteine↗

PubMed2005

Golgi vesiculation induced by cholesterol occurs by a dynamin- and cPLA2-dependent mechanism.

It was recently demonstrated that an increase in the cellular cholesterol level leads to vesiculation of the Golgi apparatus. This vesiculation affects the entire Golgi apparatus and is a reversible process. We have now started to elucidate the mechanism behind this cholesterol-induced vesiculation of the Golgi apparatus. Transient transfection of cells with dominant negative mutant constructs of dynamin 1 and 2 inhibited the vesiculation; expression of dynK44A in HeLa cells stably transfected with this construct had the same effect. However, the vesiculation seems to be independent of clathrin, as cholesterol-induced vesiculation still occurred following knock down of clathrin heavy chain in HeLa cells using RNA interference as well as in BHK cells where expression of antisense to clathrin heavy chain had been induced. Importantly, the cPLA2 inhibitor MAFP and the chelator BAPTA-AM that binds cytosolic Ca2+ inhibited the cholesterol-induced vesiculation, suggesting involvement of a cPLA2 that requires cytosolic Ca2+ for translocation to membranes. Furthermore, in response to an increased cellular cholesterol level, an EGFP-cPLA2 fusion protein translocated to the Golgi apparatus. Thus, our results demonstrate that the cholesterol-induced vesiculation of the Golgi apparatus is mediated by a cPLA2- and dynamin-dependent mechanism.

Animals↗

PubMed2005

Caveolae: stable membrane domains with a potential for internalization.

The role of caveolae in endocytosis is hotly debated. Here, we argue that most caveolae are stable microdomains at the cell surface. Only a small fraction of caveolae is constitutively internalized, leading to a quantitatively minor uptake of ligands and receptors. In addition, we suggest that a more pronounced downregulation of caveolae from the plasma membrane can occur, presumably stimulated by receptor cross-linking and clustering in caveolae. Finally, we propose that future studies dealing with internalization of caveolae should actually document such internalization and include kinetic data.

Caveolae↗

PubMed2004

The phagocyte NADPH oxidase depends on cholesterol-enriched membrane microdomains for assembly.

The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b558 complex, and cytosolic factors p47phox, p67phox and the small GTPase Rac, which translocate to the membrane to assemble the active complex following cell activation. We here show that insolubility of NADPH oxidase subunits in nonionic detergents TX-100, Brij-58, and Brij-98 is a consequence of inclusion into cholesterol-enriched membrane microdomains (lipid rafts). Thus, flavocytochrome b558, in a cholesterol-dependent manner, segregated to the bouyant low-density detergent-resistant membrane (DRM) fraction, and the cytosolic NADPH oxidase factors associated dynamically with low-density DRM. Further, superoxide production following cholesterol depletion was severely compromised in intact cells or in a cell-free reconstituted system, correlating with a reduced translocation of cytosolic phox subunits to the membrane. In analogy with the widely accepted role of lipid rafts as signaling platforms, our data indicate that cholesterol-enriched microdomains act to recruit and/or organize the cytosolic NADPH oxidase factors in the assembly of the active NADPH oxidase.

Animals↗

PubMed2004

Association with membrane protrusions makes ErbB2 an internalization-resistant receptor.

In contrast to the epidermal growth factor (EGF) receptor, ErbB2 is known to remain at the plasma membrane after ligand binding and dimerization. However, why ErbB2 is not efficiently down-regulated has remained elusive. Basically, two possibilities exist: ErbB2 is internalization resistant or it is efficiently recycled after internalization. By a combination of confocal microscopy, immunogold labeling electron microscopy, and biochemical techniques we show that ErbB2 is preferentially associated with membrane protrusions. Moreover, it is efficiently excluded from clathrin-coated pits and is not seen in transferrin receptor-containing endosomes. This pattern is not changed after binding of EGF, heregulin, or herceptin. The exclusion from coated pits is so pronounced that it cannot just be explained by lack of an internalization signal. Although ErbB2 is a raft-associated protein, the localization of ErbB2 to protrusions is not a result of raft binding. Also, an intact actin cytoskeleton is not required for keeping ErbB2 away from coated pits. However, after efficient cross-linking, ErbB2 is removed from protrusions to occur on the bulk membrane, in coated pits, and in endosomes. These data show that ErbB2 is a remarkably internalization-resistant receptor and suggest that the mechanism underlying the firm association of ErbB2 with protrusions also is the reason for this resistance.

Actins↗

PubMed2004

Sarcolemmal FAT/CD36 in human skeletal muscle colocalizes with caveolin-3 and is more abundant in type 1 than in type 2 fibers.

FAT/CD36 is a transmembrane protein that is thought to facilitate cellular long-chain fatty acid uptake. However, surprisingly little is known about the localization of FAT/CD36 in human skeletal muscle. By confocal immunofluorescence microscopy, we demonstrate high FAT/CD36 expression in endothelial cells and weaker but significant FAT/CD36 expression in sarcolemma in human skeletal muscle. No apparent intracellular staining was observed in the muscle cells. There are indications in the literature that caveolae may be involved in the uptake of fatty acids, possibly as regulators of FAT/CD36 or other fatty acid transporters. We show that in sarcolemma, FAT/CD36 colocalizes with the muscle-specific caveolae marker protein caveolin-3, suggesting that caveolae may regulate cellular fatty acid uptake by FAT/CD36. Furthermore, we provide evidence that FAT/CD36 expression is significantly higher in type 1 compared with type 2 fibers, whereas caveolin-3 expression is significantly higher in type 2 fibers than in type 1 fibers.

Adult↗

PubMed2004

Pathways followed by protein toxins into cells.

A number of protein toxins have an enzymatically active part, which is able to modify a cytosolic target. Some of these toxins, for instance ricin, Shiga toxin and cholera toxin, which we will focus on in this article, exert their effect on cells by first binding to the cell surface, then they are endocytosed, and subsequently they are transported retrogradely all the way to the ER before translocation of the enzymatically active part to the cytosol. Thus, studies of these toxins can provide information about pathways of intracellular transport. Retrograde transport to the Golgi and the ER seems to be dependent not only on different Rab and SNARE proteins, but also on cytosolic calcium, phosphatidylinositol 3-kinase and cholesterol. Comparison of the three toxins reveals differences indicating the presence of more than one pathway between early endosomes and the Golgi apparatus or, alternatively, that transport of different toxin-receptor complexes present in a certain subcompartment is differentially regulated.

Cholera Toxin↗

PubMed2004

Factor XII binding to endothelial cells depends on caveolae.

It is now generally accepted that factor XII (FXII) binds to cellular surfaces in the vascular system. One of the suggested receptors of this binding is the glycosylphosphatidylinositol-anchored urokinase-like plasminogen activator (u-PAR) harbored in caveolae/lipid rafts. However, binding of FXII to human umbilical vein endothelial cells (HUVEC) has never been shown to be localized to these specialized membrane structures. Using microscopical techniques, we here report that FXII binds to specific patches of the HUVEC plasma membrane with a high density of caveolae. Further investigations of FXII binding to caveolae were performed by sucrose density-gradient centrifugations. This showed that the majority of FXII, chemically cross-linked to HUVEC, could be identified in the same fractions of the gradient as caveolin-1, a marker of caveolae, while the majority of u-PAR was identified in noncaveolae lipid rafts. Accordingly, cholesterol-depleted cells were found to bind significantly reduced amounts of FXII. These observations, combined with the presence of a minority of u-PAR in caveolae concomitant with FXII binding, indicate that FXII binding to u-PAR may be secondary and depends upon the structural elements within caveolae. Thus, FXII binding to HUVEC depends on intact caveolae on the cellular surface.

Animals↗

PubMed2003

"Nonclassical" secretion of annexin A2 to the lumenal side of the enterocyte brush border membrane.

Annexin A2 is a member of the annexin family of Ca(2+)-dependent lipid binding proteins and believed to be engaged in membrane transport processes in a number of cell types. In small intestinal enterocytes, we localized annexin A2 to the brush border region, where it was found mainly on the lumenal side of the microvilli, showing an apical secretion by a "nonclassical" mechanism. In addition, annexin A2 was associated with surface-connected, deep apical tubules in the apical terminal web region and with an underlying pleiomorphic, tubulo-vesicular compartment (subapical compartment/multivesicular bodies). By subcellular fractionation, the 36 kDa full-length form of annexin A2 was approximately equally distributed between the Mg(2+)-precipitated fraction (containing intracellular and basolateral membranes) and the microvillar membrane fraction. In addition, a 33 kDa molecular form of annexin A2 was seen in the latter fraction that could be generated from the full-length annexin A2 by digestion with trypsin. Taken together, the results suggest that annexin A2 acts in exocytic apical membrane trafficking and is proteolytically cleaved in situ by pancreatic proteinases once it has become externalized to the lumenal side of the brush border membrane. On the basis of its well-known membrane fusogenic properties, we propose a model for the nonclassical membrane translocation of annexin A2.

Animals↗

PubMed2003

Induction of direct endosome to endoplasmic reticulum transport in Chinese hamster ovary (CHO) cells (LdlF) with a temperature-sensitive defect in epsilon-coatomer protein (epsilon-COP).

In the present study we demonstrate that ricin, apparently without passing through the Golgi apparatus, reaches the endoplasmic reticulum (ER) and intoxicates cells in which the Golgi apparatus has been vesiculated by depletion of epsilon-COP, a subunit of COPI. LdlF cells contain a temperature-sensitive mutation in epsilon-COP. At the nonpermissive temperature epsilon-COP is degraded, and the Golgi apparatus undergoes a morphological change. To study ricin transport in these cells we used ricin sulf-2, a modified ricin molecule containing glycosylation and sulfation sites. Measurements of the incorporation of radioactive mannose into ricin sulf-2 showed that ricin reached the ER in cells depleted of epsilon-COP. Importantly, by investigating the glycosylation of ricin sulf-2 that was modified with radioactive sulfate in the trans-Golgi network, it was demonstrated that transport of ricin to the ER via the Golgi apparatus was severely inhibited. Moreover, we found that ricin was able to intoxicate ldlF cells depleted of epsilon-COP in the presence of brefeldin A. In contrast, control cells were completely protected against ricin by brefeldin A. In conclusion, our results suggest that in ldlF cells depleted of epsilon-COP ricin might be transported to the ER by an induced brefeldin A-resistant pathway that circumvents the Golgi apparatus.

Animals↗

PubMed2003

Caveolae: anchored, multifunctional platforms in the lipid ocean.

The function of caveolae is hotly debated. It now seems clear that caveolae are stable membrane domains that are kept in place by the actin cytoskeleton. However, this stability can be perturbed, leading to caveolar internalization. Caveolae are important in the regulation of various signaling processes, such as nitric oxide activity, and in cholesterol efflux and cholesterol-ester uptake. Caveolin deficiency particularly affects the cardiovascular system and the lungs but, because the knockout mice are viable, none of the proposed functions appears to be essential. Rather than having a specific function, caveolae might be considered to be multifunctional organelles with a physiological role that varies depending on cell type and cellular needs.

Actin Cytoskeleton↗

PubMed2002

Transport of protein toxins into cells: pathways used by ricin, cholera toxin and Shiga toxin.

Ricin, cholera, and Shiga toxin belong to a family of protein toxins that enter the cytosol to exert their action. Since all three toxins are routed from the cell surface through the Golgi apparatus and to the endoplasmic reticulum (ER) before translocation to the cytosol, the toxins are used to study different endocytic pathways as well as the retrograde transport to the Golgi and the ER. The toxins can also be used as vectors to carry other proteins into the cells. Studies with protein toxins reveal that there are more pathways along the plasma membrane to ER route than originally believed.

Animals↗

PubMed2002

Membrane ruffling and macropinocytosis in A431 cells require cholesterol.

Cholesterol is important for the formation of caveolea and deeply invaginated clathrin-coated pits. We have now investigated whether formation of macropinosomes is dependent on the presence of cholesterol in the plasma membrane. Macropinocytosis in A431 cells was induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, a potent activator of protein kinase C (PKC). When cells were pretreated with methyl-beta-cyclodextrin to extract cholesterol, the phorbol ester was unable to induce the increased endocytosis of ricin otherwise seen, although PKC could still be activated. Electron microscopy revealed that extraction of cholesterol inhibited the formation of membrane ruffles and macropinosomes at the plasma membrane. Furthermore, cholesterol depletion inhibited the phorbol ester-induced reorganization of filamentous actin at the cell periphery, a prerequisite for the formation of membrane ruffles that close into macropinosomes. Under normal conditions the small GTPase Rac1 is activated by the phorbol ester and subsequently localized to the plasma membrane, where it induces the reorganization of actin filaments required for formation of membrane ruffles. Cholesterol depletion did not inhibit the activation of Rac1. However, confocal microscopy showed that extraction of cholesterol prevented the phorbol ester-stimulated localization of Rac1 to the plasma membrane. Thus, our results demonstrate that cholesterol is required for the membrane localization of activated Rac1, actin reorganization, membrane ruffling and macropinocytosis.

ADP-Ribosylation Factor 6↗

PubMed2002

Membrane traffic exploited by protein toxins.

A large number of protein toxins having enzymatically active A- and B-moieties that bind to cell surface receptors must be endocytosed before the A-moiety is translocated into the cytosol where it exerts its cytotoxic action. The accumulated information about the most well-studied toxins has provided a detailed picture of how they exploit the membrane trafficking systems of cells, and studies of toxin trafficking have revealed the existence of new pathways. The complexity of different endocytic mechanisms, as well as the multiple routes between endosomes and the Golgi apparatus and retrogradely to the endoplasmic reticulum (ER), are being unravelled by investigations of how toxins gain access to their targets. With increasing information about the internalization and intracellular trafficking of these opportunistic toxins, new avenues have been opened for their application in areas of medicine such as drug delivery and therapy.

Animals↗

PubMed2002

Sequestration of epidermal growth factor receptors in non-caveolar lipid rafts inhibits ligand binding.

Cholesterol depletion has been shown to increase mitogen-activated protein kinase activation in response to stimulation with epidermal growth factor (EGF) (Furuchi, T., and Anderson, R. G. W. (1998) J. Biol. Chem. 273, 21099-21104). However, the underlying mechanisms are unknown. We show that cholesterol depletion increases EGF binding, whereas cholesterol loading lowers EGF binding. Based on binding analyses, we demonstrate that the observed changes in EGF binding are caused by alterations in the number of EGF receptors available for ligand binding, whereas the affinity of the receptor for EGF remains unaltered. We also show by immunofluorescence that in unstimulated cells the EGF receptor is localized in non-caveolar lipid rafts containing the ganglioside GM1 and that patching of these rafts by cholera toxin B-chain causes co-patching of EGF receptors. Experiments with solubilization in different detergents at 4 degrees C show that the association of the EGF receptor with these rafts is sensitive to Triton X-100 extraction but insensitive to extraction with another non-ionic detergent, Brij 58. Furthermore, experiments with cholesterol-depleted cells show that the association is cholesterol-dependent. We propose that non-caveolar lipid rafts function as negative regulators of EGF receptor signaling by sequestering a fraction of the EGF receptors in a state inaccessible for ligand binding.

Cell Line↗

PubMed2002

Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking.

To investigate whether caveolae are involved in constitutive endocytic trafficking, we expressed N- and C- terminally green fluorescent protein (GFP)-tagged caveolin- 1 fusion proteins in HeLa, A431, and Madin-Darby canine kidney cells. The fusion proteins were shown by immunogold labeling to be sorted correctly to caveolae. By using confocal microscopy and photobleaching techniques, it was found that although intracellular structures labeled with GFP-tagged caveolin were dynamic, GFP-labeled caveolae were very immobile. However, after incubation with methyl- beta-cyclodextrin, distinct caveolae disappeared and the mobility of GFP-tagged caveolin in the plasma membrane increased. Treatment of cells with cytochalasin D caused lateral movement and aggregation of GFP-labeled caveolae. Therefore, both cholesterol and an intact actin cytoskeleton are required for the integrity of GFP-labeled caveolae. Moreover, stimulation with okadaic acid caused increased mobility and internalization of the labeled caveolae. Although the calculated mobile fraction (for t = infinity) of intracellular, GFP-tagged caveolin- associated structures was 70-90%, GFP-labeled caveolae in unstimulated cells had a mobile fraction of <20%, a value comparable to that previously reported for E-cadherin in junctional complexes. We therefore conclude that caveolae are not involved in constitutive endocytosis but represent a highly stable plasma membrane compartment anchored by the actin cytoskeleton.

Animals↗

PubMed2001

Pathways followed by ricin and Shiga toxin into cells.

The plant toxin ricin and the bacterial toxin Shiga toxin belong to a group of protein toxins that inhibit protein synthesis in cells enzymatically after entry into the cytosol. Ricin and Shiga toxin, which both have an enzymatically active moiety that inactivates ribosomes and a moiety that binds to cell surface receptors, enter the cytosol after binding to the cell surface, endocytosis by different mechanisms, and retrograde transport to the Golgi apparatus and the endoplasmic reticulum (ER). The toxins can be used to investigate the various transport steps involved, both the endocytic mechanisms as well as pathways for retrograde transport to the ER. Recent studies show that not only do several endocytic mechanisms exist in the same cell, but they are not equally sensitive to removal of cholesterol. New data have revealed that there is also more than one pathway leading from endosomes to the Golgi apparatus and retrogradely from the Golgi to the ER. Trafficking of protein toxins along these pathways will be discussed in the present article.

Animals↗