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First published online 22 September 2009
doi: 10.1242/jcs.051557

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Alternate raft pathways cooperate to mediate slow diffusion and efficient uptake of a sphingolipid tracer to degradative and recycling compartments

¹ Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Singapore 138648
² Department of Chemistry, National University of Singapore, Singapore 117543
³ School of Biological Sciences, Nanyang Technological University, Singapore 637551

^* Author for correspondence (rskraut{at}ntu.edu.sg)

Accepted 3 August 2009

Several cholesterol-dependent cellular uptake pathways involving microdomain-resident sphingolipids have been characterized, but little is known about what controls the further intracellular trafficking routes of those domains. Here, we present evidence that the uptake and intracellular trafficking of a recently described sphingolipid-binding probe, the sphingolipid binding domain (SBD) peptide, is mediated by two parallel cooperating mechanisms requiring flotillin, dynamin and cdc42, which act in concert to direct a distinct surface behavior and trafficking itinerary. Diffusion measurements of SBD at the cell surface by fluorescence correlation spectroscopy suggest that cdc42- and flotillin-associated uptake sites both correspond to domains of intermediate mobility, but that they can cooperate to form low-mobility, efficiently internalized domains. Interestingly, we find that the choice of uptake mechanism affects subsequent trafficking of SBD, as does cholesterol content. Interference with one or other uptake pathway acts as a toggle switch for the trafficking of SBD to recycling endosomes or endolysosomes, whereas both of these pathways are bypassed if cholesterol is reduced. The data are in accordance with a scenario in which SBD mirrors the trafficking response of raft-borne lipids towards a degradative or recycling target. In summary, we suggest that both the surface behavior of a cargo and its subsequent trafficking are determined by a combination of endocytic accessory proteins and the cholesterol content of different membrane compartments.

Key words: FCS, SBD, Microdomain, Raft, Sphingolipid, Trafficking

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