Emerging concepts of ganglioside metabolism

Ten years ago, we wrote a Review on lipid rafts and signalling in the launch issue of Nature Reviews Molecular Cell Biology. At the time, this field was suffering from ambiguous methodology and imprecise nomenclature. Now, new techniques are deepening our insight into the dynamics of membrane organization. Here, we discuss how the field has matured and present an evolving model in which membranes are occupied by fluctuating nanoscale assemblies of sphingolipids, cholesterol and proteins that can be stabilized into platforms that are important in signalling, viral infection and membrane trafficking.

When internalized receptors and other cargo are destined for lysosomal degradation, they are ubiquitinated and sorted by the ESCRT complexes 0, I, II, and III into multivesicular bodies. Multivesicular bodies are formed when cargo-rich patches of the limiting membrane of endosomes bud inward by an unknown mechanism and are then cleaved to yield cargo-bearing intralumenal vesicles. The biogenesis of multivesicular bodies was reconstituted and visualized using giant unilamellar vesicles, fluorescent ESCRT-0, I, II, and III complexes, and a membrane-tethered fluorescent ubiquitin fusion as a model cargo. ESCRT-0 forms domains of clustered cargo but does not deform membranes. ESCRT-I and II in combination deform the membrane into buds, in which cargo is confined. ESCRT-I and II localize to the bud necks, and recruit ESCRT-0-ubiquitin domains to the buds. ESCRT-III subunits localize to the bud neck and efficiently cleave the buds to form intralumenal vesicles. Intralumenal vesicles produced in this reaction contain the model cargo but are devoid of ESCRTs. The observations explain how the ESCRTs direct membrane budding and scission from the cytoplasmic side of the bud without being consumed in the reaction.