Membrane traffic and the cellular uptake of cholera toxin

Lencer, Wayne I.; Hirst, Timothy R.; Holmes, Randall K.

doi:10.1016/s0167-4889(99)00070-1

Cited by 229 publications

(198 citation statements)

References 152 publications

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“…These results demonstrate that the internalization of the fluorescent GM 1 analog was perturbed by CtxB binding, and raise the possibility that internalization of endogenous GM 1 might also be affected by the bound toxin in some cell types. We speculate that this perturbation occurs because CtxB binding (five molecules of GM 1 bound per molecule of CtxB; Lencer et al, 1999) disrupts the normal interactions of GM 1 with neighboring lipids, proteins, and cholesterol that are required for clathrin-independent internalization.…”

Section: Ctxb (But Not Laccer) Internalization Mechanism Varies With mentioning

confidence: 99%

Selective Caveolin-1–dependent Endocytosis of Glycosphingolipids

Singh

Puri

Valiyaveettil

et al. 2003

MBoC

234

249

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We studied the endocytosis of fluorescent glycosphingolipid (GSL) analogs in various cell types using pathway-specific inhibitors and colocalization studies with endocytic markers and DsRed caveolin-1 (cav-1). Based on inhibitor studies, all GSLs tested were internalized predominantly (Ͼ80%) by a clathrin-independent, caveolar-related mechanism, regardless of cell type. In addition, fluorescent lactosylceramide (LacCer) colocalized with DsRed-cav-1 in vesicular structures upon endocytosis in rat fibroblasts. The internalization mechanism for GSLs was unaffected by varying the carbohydrate headgroup or sphingosine backbone chain length; however, a fluorescent phosphatidylcholine analog was not internalized via caveolae, suggesting that the GSL ceramide core may be important for caveolar uptake. Internalization of fluorescent LacCer was reduced 80 -90% in cell types with low cav-1, but was dramatically stimulated by cav-1 overexpression. However, even in cells with low levels of cav-1, residual LacCer internalization was clathrin independent. In contrast, cholera toxin B subunit (CtxB), which binds endogenous GM 1 , was internalized via clathrin-independent endocytosis in cells with high cav-1 expression, whereas significant clathrin-dependent uptake occurred in cells with low cav-1. Fluorescent GM 1 , normally internalized by clathrin-independent endocytosis in HeLa cells with low cav-1, was induced to partially internalize via the clathrin pathway in the presence of CtxB. These results suggest that GSL analogs are selectively internalized via a caveolar-related mechanism in most cell types, whereas CtxB may undergo "pathway switching" when cav-1 levels are low.

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Section: Ctxb (But Not Laccer) Internalization Mechanism Varies With mentioning

confidence: 99%

Selective Caveolin-1–dependent Endocytosis of Glycosphingolipids

Singh

Puri

Valiyaveettil

et al. 2003

MBoC

234

249

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“…A Rentsendorj et al mediated cell entry, which is known to mediate internalization and retrograde trafficking of cholera toxin 28 and SV40, 29,30 and facilitate toxin arrival to the Golgi. We found that indeed PB partially overlaps with the caveolae marker, caveolin-1, during uptake (Figure 7e and f).…”

Section: Penton Base Traffickingmentioning

confidence: 99%

Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer

et al. 2006

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The adenovirus (Ad) penton base protein facilitates viral infection by binding cell surface integrins, triggering receptormediated endocytosis and mediating endosomal penetration. Given these multiple functions, recombinant penton base proteins have been utilized as non-viral vehicles for gene transfer by our lab and others. Although we have previously demonstrated that penton base-derived vectors undergo integrin-specific binding and cell entry, less than desirable levels of gene expression have led us to re-evaluate the recombinant penton base as an agent for gene delivery. To do so, we have examined here the intracellular trafficking of an Ad serotype 5 (Ad5) recombinant penton base protein (PB). Here, we not only observed that PB utilizes a similar, typical trafficking pathway of whole Ad, but also found that PB entered HeLa cells through pathways not yet identified as contributing to cell entry by the whole virus. We show by high-resolution confocal microscopy and biochemical methods that binding to a v -integrins is a requirement for cell entry, but that early internalization stages did not substantially pass through clathrin-positive and early endosomal compartments. Moreover, a subpopulation of internalized protein localized with caveolin-positive compartments and Golgi markers, suggesting that a certain percentage of proteins pass through nonclathrin-mediated pathways. Similar to the virus, trafficking toward the nucleus was affected by disruption of microtubules and dynein. The majority of penton base molecules avoided the lysosome while facilitating early vesicle release of low molecular weight dextran molecules. In further support of a vesicle escape capacity, a subpopulation of internalized penton base appeared to enter the nucleus, as observed by high-resolution confocal microscopy and cell fractionation. As a confirmation of these findings, we demonstrate that a recombinant penton base facilitated cytosolic entry of an siRNA molecule as observed by RNA interference of a marker gene. Based on our findings here, we suggest that whereas soluble penton base proteins may enter cells through clathrinand non-clathrin-mediated pathways, vesicle escape and nuclear delivery appear to be supported by a clathrin-mediated pathway. As our previous efforts have focused on utilizing recombinant penton base proteins as delivery agents for therapeutics, these findings allow us to evaluate the use of the penton base as a cell entry and intracellular trafficking agent, and may be of interest concerning the development of vectors for efficient delivery of therapeutics to cells. Gene Therapy (2006) 13, 821-836.

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“…CTXB is enriched in biochemical lipid raft fractions and in caveolae (Fra et al, 1994;Parton et al, 1994;Parton, 1996;Smart et al, 1995;Schnitzer et al, 1996;Harder and Simons, 1997;Stauffer and Meyer, 1997;Henley et al, 1998;Orlandi and Fishman, 1998;Wolf et al, 1998;Lencer et al, 1999). Three GPI-anchored proteins, which are major components of detergentinsoluble raft fractions (Skibbens et al, 1989;Stefanova et al, 1991;Brown and Rose, 1992;Fiedler et al, 1993;Sargiacomo et al, 1993), were used as protein markers for lipid rafts.…”

Section: Plasma Membrane Distribution Of Lipid Raft Markers Detected mentioning

confidence: 99%

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

Kenworthy

Petranova

Edidin

2000

MBoC

406

311

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“Lipid rafts” enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.

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Membrane traffic and the cellular uptake of cholera toxin

Cited by 229 publications

References 152 publications

Selective Caveolin-1–dependent Endocytosis of Glycosphingolipids

Selective Caveolin-1–dependent Endocytosis of Glycosphingolipids

Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

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