Selective Caveolin-1–dependent Endocytosis of Glycosphingolipids

Abstract

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.

Isoform and Splice-Variant Specific Functions of Dynamin-2 Revealed by Analysis of Conditional Knock-Out Cells

“…We were unable to detect significant inhibition of CTB internalization in our Dyn2 KO cells ( Figure 5A), although we confirmed findings of others that the overexpression of a dominant negative mutant Dyn2K44A blocks CTB uptake in our fibroblasts ( Figure 5B). Similar results were obtained using BODIPY-LacCer, which is reported to be a more specific probe for caveolae-mediated endocytosis (Singh et al, 2003; Figure 5C). Because overexpressed Dyn2K44A will dominantly interfere with the function of both endogenous Dyn2 and -1 isoforms, the more potent effect of dominantnegative overexpression suggests that the remaining Dyn1 may be sufficient to support CTB uptake in the Dyn2 KO cells.…”

Caveolin-2 Is Required for Apical Lipid Trafficking and Suppresses Basolateral Recycling Defects in the Intestine ofCaenorhabditis elegans

“…To confirm that cav-2 mutants have defects in apical trafficking, we exposed animals to BODIPY-tagged lactosylceramide (BODIPY FL C5 LacCer, Invitrogen). It has previously been shown that glycosphingolipid analogues are internalized through a predominantly caveolin-dependent route in human cells (Singh et al, 2003;Mayor and Pagano, 2007;Singh et al, 2007). In wild-type animals exposed to 5 M BODIPY-LacCer for 2 h, fluorescent LacCer accumulates in a punctate pattern in the intestine.…”

Membrane lipids and vesicular traffic

“…By contrast, the preferential uptake of glycolipids by caveolae [31] requires lipid-based aggregation [32]. Glycolipids are used as endocytotic receptors by toxins, viruses and bacteria.…”