PHOSPHOLIPID TRANSMEMBRANE DOMAINS AND LATERAL DIFFUSION IN FIBROBLASTS

The lateral diffusion of fluorescent phospholipids in cultured Chinese hamster lung fibroblasts was examined by modulated fringe pattern photobleaching. When cells were labeled and maintained at 7-degrees-C, the fluorescence remained localized at the plasma membrane. N-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl]sphingosylphosphocholine (C6-NBD-SphPCho) and 1-acyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl] phosphatidylcholine (C6-NBD-PtdCho) both diffused with the same apparent lateral diffusion coefficient (D1 almost-equal-to 0.3 x 10(-9) cm2/s). By contrast, the phosphatidylserine derivative {1-acyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl] phosphatidylserine (C6-NBD-PtdSer)} gave rise to two diffusional components: a slow component, D1, analogous to that measured with the choline-containing lipids, and a fast component (D2 almost-equal-to 2 x 10(-9) cm2/s). The fast component only exists in ATP-containing cells. It was shown to be associated with C6-NBD-PtdSer translocated to the inner leaflet. This indicates that the two leaflets form very different membranous domains. At higher temperature, the same difference in mobility was, observed between the choline-containing lipids and the aminolipid. However, with C6-NBD-SphPCho, a fraction of very slowly diffusing or quasi-immobilized probes gradually appeared with time. This could be attributed to sphingomyelin located in small organelles after internalization. From the amplitude of this component registered at different intervals, we calculated that almost-equal-to 50% of the plasma membrane sphingomyelin is recycled in less than 30 min in Chinese hamster fibroblasts by an ATP- and microtubule-dependent process.