Epithelial cells constitute a paradigm for cell polarity. Most striking is the segregation of their plasma membrane into two functionally and biochemically distinct domains, an apical and a basolateral membrane, each with characteristic protein and lipid compositions. The apical membrane is highly enriched in (glyco)sphingolipids, which are involved in ordering the bilayer.  Sphingolipids preferentially associate with cholesterol to form so-called lipid rafts and a raft-clustering mechanism for carrier formation has been proposed to be responsible for the favorable localization of sphingolipids to the apical membrane domain.
The aim of my work is two-fold. The first one is to ascertain a functional connection between the cellular lipid composition and sphingolipid-associated processes in polarized MDCK cells. Sphingolipid levels are manipulated through interference with their synthesis using metabolic inhibitors. The analysis of sphingomyelin levels reveals lipid species-dependent regulation of synthesis.  Different culturing conditions have been established that maintain cell polarity and integrity while efficiently reducing raft lipid content, as determined by quantitative mass spectrometry. Under these conditions, apical transport of the raft-associated HA protein is significantly reduced, and apical plasma membrane condensation, assessed with the order-sensing dye C-laurdan, becomes compromised. These observations support a functional role of sphingolipid-cholesterol-enriched microdomains in membrane trafficking and consequently in apical membrane order. Further, they lay the groundwork for correlative studies of cellular lipidomics and membrane organization.
The second aim is to analyze the mechanism of action of the four-phosphate adaptor protein 2 (FAPP2), a proposed component of the apical transport machinery using in vivo and in vitro approaches.