Glycosphingolipids Internalized via Caveolar-related Endocytosis Rapidly Merge with the Clathrin Pathway in Early Endosomes and Form Microdomains for Recycling

Abstract

We have previously demonstrated that glycosphingolipids are internalized from the plasma membrane of human skin fibroblasts by a clathrin-independent, caveolarrelated mechanism and are subsequently transported to the Golgi apparatus by a process that is dependent on microtubules, phosphatidylinositol 3-kinase, Rab7, and Rab9. Here we characterized the early steps of intracellular transport of a fluorescent glycosphingolipid analog, BODIPY-lactosylceramide (LacCer), and compared this to fluorescent transferrin (Tfn), a well established marker for the clathrin pathway. Although these two markers were initially internalized into separate vesicles by distinct mechanisms, they became co-localized in early endosomes within 5 min. These results demonstrate that glycosphingolipid-containing vesicles derived from caveolar-related endocytosis fuse with the classical endosomal system. However, in contrast to Tfn, internalization and trafficking of LacCer was independent of Rab5a, a key regulator of transport to early endosomes. By taking advantage of the monomer/excimer properties of the fluorescent lipid analog, we were also able to visualize LacCer segregation into distinct microdomains of high (red emission) and low (green emission) concentrations in the early endosomes of living cells. Interestingly, the high concentration "red" microdomains co-localized with fluorescent Tfn upon exit from early endosomes and passed through Rab11-positive "recycling endosomes" prior to being transported back to the plasma membrane. These results together with our previous studies suggest that glycosphingolipids internalized by caveolar endocytosis are rapidly delivered to early endosomes where they are fractionated into two major pools, one that is transported via late endosomes to the Golgi apparatus and the other that is returned to the plasma membrane via the recycling compartment.

Membrane lipids and vesicular traffic

“…In addition, the spontaneous segregation of lipid mixtures into different phases appears to be one of the basic mechanisms by which proteins and lipids are sorted towards their Endocytic lipid sorting. Glycolipids (with probably sphingomyelin and cholesterol) aggregate in the plasma membrane and the endosomes, and recycle to the plasma membrane via recycling endosomes and partially via late endosomes and Golgi [34]. Sphingolipids and cholesterol may be present as sizeable domains only when stabilized, e.g.…”

“…Lipid rafts seem to play a major role in receptor signaling and endocytosis (not necessarily via caveolae), a process possibly regulated by integrins [33]. The glycolipid rafts appear to concentrate in the pathways from the early endosomes to the recycling endosomes and to the late endosomes, whence the rafts reach the Golgi [34], a transport pathway followed by glycolipid-binding toxins en route to the ER [35]. Transport of both sphingolipids and cholesterol to the Golgi is interrupted under conditions of lipid storage in endocytotic organelles (as seen in many lysosomal storage diseases) [36].…”

Selective Caveolin-1–dependent Endocytosis of Glycosphingolipids

“…One well-characterized marker for caveolar internalization is SV40 virus that colocalizes with GFP-tagged caveo-lin-1 (cav-1) during endocytosis (Pelkmans et al, 2001); however, the intracellular itinerary and slow rate of transport of SV40 virus differs from that of some other cargo internalized via caveolae (Gleizes et al, 1996;Orlandi and Fishman, 1998;Lobie et al, 1999;Sharma et al, 2003). These and other observations suggest the possibility that several forms of caveolar endocytosis may exist (Mineo and Anderson, 2001; Le and Nabi, 2003).…”

Insights in cellular uptake mechanisms of pDNA–polycationic amphiphilic cyclodextrin nanoparticles (CDplexes)

“…CIE includes several pathways which share a cholesterol dependency (32), among which caveolin mediated endocytosis Cav-ME is probably the most thoroughly characterized. In this case the membrane of the formed vesicles, which are called cavicles, typically contains caveolin-1 (33,34). Selective inhibition of the different internalization pathways has been shown to be a powerful tool to investigate uptake mechanisms of nanoparticles and infer the relevance of the blocked pathway in the internalization mechanism (32,(35)(36)(37)(38)(39)(40)(41)(42)(43).…”