Plasma Membrane Fluidity Affects Transient Immobilization of Oxidized Phospholipids in Endocytotic Sites for Subsequent Uptake

Abstract

Oxidized phospholipids in serum initiate severe pathophysiological responses during the process of atherogenesis. On the cellular level it is known that these lipids induce apoptosis; however, the uptake mechanism remains enigmatic. We investigated here the behavior of the fluorescent oxidized phospholipid 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-NAlexa647-ethanolamine (PGPE-Alexa647) in the plasma membrane of various cell lines. The probe was taken up by the cells unspecifically via caveolae or clathrin-coated pits. Interestingly, we found the uptake to be facilitated by the overexpression of the scavenger receptor class B type I. Ultra-sensitive microscopy allowed us to follow the uptake process at the single molecule level; we observed rapid diffusion of PGPE-Alexa647 in the plasma membrane, interrupted by transient halts with duration of ϳ0.9 s at endocytotic sites. Scavenger receptor class B type I overexpression yielded a pronounced increase in the single molecule mobility, and in consequence an increased frequency of immobilization. Alternatively, the plasma membrane fluidity could also be increased by treating cells with high levels of the unlabeled oxidized phospholipid 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine; also in this case, the immobilization frequency of PGPE-Alexa647 was concomitantly increased. The data demonstrate the relevance of plasma membrane properties for uptake of oxidized phospholipids, and indicate a novel indirect mechanism for the control of endocytosis. Oxidation of low density lipoprotein (LDL)2 is known to be a key step in atherogenesis, leading to inflammation, proliferation, and apoptosis of cells of the arterial wall. These effects are largely exerted by oxidatively fragmented phospholipids, which are highly exchangeable between cells, tissues, and lipoproteins (1-5). In particular, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) has been identified in minimally modified LDL and has been reported to elicit a wide range of pathophysiological responses in vascular cells, e.g. the activation of apoptotic signaling pathways (6). Fast uptake of synthetic PGPC analogues to the perinuclear region of vascular smooth muscle cells has been reported (7) and discussed in view of its ability to activate acid sphingomyelinase (8). The uptake mechanism and sites of action, however, still remain enigmatic.PGPC carries an acyl fragment in the sn-2 position, which represents the oxidation product of an unsaturated acyl chain. This hydrophilic side chain is expected to be folded to the polar region of the membrane, yielding a structure best described by a lysolipid-like inverted cone (7). Addition of such nonbilayer lipids to model membranes is known to bend lipid monolayers, and it may well assist in the formation of highly curved structures also in cell membranes (9). In turn, such lipids are expected to become energetically trapped and thereby enriched in bent membrane regions (9 -12). It can therefore be expected that local enrichment of PGPC may exert dramatic chan...

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