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Vol. 14, Issue 8, 3254-3265, August 2003
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* Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation,
Rochester, Minnesota 55905;
Department of Chemistry and Biochemistry, Queens College, The City University
of New York, Flushing, New York 11367
Submitted December 11, 2002;
Revised March 14, 2003;
Accepted April 11, 2003
Monitoring Editor: Vivek Malhotra
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 (>80%) 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 GM1, 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 GM1, 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.
Abbreviations used: AF, AlexaFluor; BODIPY, boron dipyrromethenedifluoride; Cav-1, caveolin-1; CPZ, chlorpromazine; CtxB, cholera toxin, B-subunit; DF-BSA, defatted BSA; Eps15, EGFR pathway substrate clone 15; GalCer, galactosylceramide; GM1, ganglioside GM1; GSL, glycosphingolipid; HMEM, 10 mM HEPES-buffered minimal essential medium (pH 7.4); HMEMG+I, HMEM without glucose containing 5 mM NaN3 and 50 mM 2-deoxyglucose; HSFs, human skin fibroblasts; LacCer, lactosylceramide; MalCer, maltosylceramide; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; PC, phosphatidylcholine; PM, plasma membrane; RFs, rat fibroblasts; SL, sphingolipid; SM, sphingomyelin; So, sphingosine; Tfn, transferrin.
Corresponding author. E-mail address:
pagano.richard{at}mayo.edu.
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