ENDOCYTOSIS AND SORTING OF GSLS

Glycosphingolipids (GSLs) play important roles in a wide variety of cell functions, including cell-cell interactions, cell growth and differentiation, and signal transduction. GSLs can interact with cholesterol to form membrane microdomains, and data from many studies suggest that the plasma membrane (PM) GSL and cholesterol composition are tightly regulated. To better understand this regulation we have been studying the endocytosis and intracellular sorting of fluorescent GSL analogs in detail.

Major Findings

• GSL analogs (e.g., BODIPY-LacCer) are selectively internalized via caveolae in multiple cell types. See Figure 1.
• GSL analogs rapidly merge with markers for the clathrin pathway at early endosomes where they are fractionated into two pools-one pool is transported to the Golgi apparatus, while the second is recycled to the PM. See Figure 2.
• The endocytic itinerary of BODIPY-LacCer has been dissected using DN constructs of various rab GTPases. See Figure 3.
• Caveolar endocytosis is stimulated by acute treatment of cells with BODIPY-LacCer, C8-LacCer, naturally-occurring GSLs, or cholesterol. See Figure 4 (QT Movie).

Open Questions for Future Study

• What are the molecular mechanism(s) responsible for selective caveolar internalization of GSL analogs via caveolae?
• Can we visualize GSL microdomains at the PM of living cells? What controls their size and dynamics? How do such domains relate to specific signaling events on the cytosolic surface of the PM?
• Are endocytosis and secretion of GSLs or other lipids "coupled processes"?

Selected References

• Puri V, Watanabe R, Singh RD, Dominguez M, Brown JC, Wheatley CL, Marks DL, Pagano RE. Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways. J Cell Biol. 2001;154:535-547.
• Sharma DK, Choudhury AK, Singh R, Wheatley CL, Marks DL, Pagano RE. Glycosphingolipids internalized via caveolar-related endocytosis rapidly merge with the clathrin pathway in early endosomes and form microdomains for recycling. J Biol Chem. 2003;278:7564-7572.
• Singh RD, Puri V, Valiyaveettil JT, Marks DL, Bittman R, Pagano RE. Selective caveolin-1-dependent endocytosis of glycosphingolipids. Mol Biol Cell. 2003;14:3254-3265.
• Sharma DK, Brown JC, Choudhury A, Peterson TE, Holicky E, Marks DL, Simari R, Parton RG, Pagano RE. Selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol. Mol Biol Cell. 2004;15(7):3114-3122.