ABSTRACT
Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent ("caveolar-like") mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a "vital marker" for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.
Subject(s)
Antigens, CD , Cell Membrane/metabolism , Clathrin/metabolism , Globosides/pharmacokinetics , Golgi Apparatus/metabolism , Lactosylceramides/pharmacokinetics , Adaptor Proteins, Signal Transducing , Boron Compounds/pharmacokinetics , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Caveolin 1 , Caveolins/metabolism , Cells, Cultured , Dynamins , Endocytosis/physiology , Endosomes/metabolism , Fibroblasts/cytology , Fluorescent Dyes/pharmacokinetics , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Gangliosidoses/metabolism , Green Fluorescent Proteins , Humans , Indicators and Reagents/pharmacokinetics , Intracellular Signaling Peptides and Proteins , Luminescent Proteins/pharmacokinetics , Mutagenesis/physiology , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Transport/physiology , Skin/cytologyABSTRACT
The effect of the membrane fluidity of lactosylceramide (LacCer)-bearing liposomes on their liver uptake was investigated in rats. Liposomes consisting of phosphatidylcholine (PC): cholesterol:dicetylphosphate:LacCer (7:2:1:1, molar ratio) were prepared with various fluidities using dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC) and egg PC. These liposomes were all equally stable in serum and were small enough to pass freely through the fenestrae and be taken up easily by liver cells. The LacCer modification of DPPC-liposomes markedly facilitated blood clearance, whereas no enhancing effect of LacCer was observed with egg PC- and DMPC-liposomes. Tissue distribution studies showed the preferential liver uptake of LacCer-bearing DPPC-liposomes, which was largely compatible with the rapid clearance induced by the LacCer modification. In addition, electron spin resonance (ESR) spectroscopic analysis revealed that the LacCer modification of DPPC-liposomes significantly enhanced the order parameter S, indicating that LacCer-bearing DPPC-liposomes were the most rigid of those used in this study. These observations suggest that the membrane fluidity of liposomes in vivo is a crucial factor for their preferential liver uptake.