(phospholipid flip-flop/aminophospholipid translocase/phosphatidylserine/photobleaching/lipid traffic)

The lateral diffusion of fluorescent phospho- lipids in cultured Chinese hamster lung fibroblasts was exam- ined by modulated fringe pattern photobleaching. When cells were labeled and maintained at 7?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-dia- zol-4-yl-amino)caproyl) phosphatidylcholine (C6-NBD- PtdCho) both diffused with the same apparent lateral diffusion coeffi'cient (D1 - 0.3 x 10-9 cm2/s). By contrast, the phos- phatidylserine derivative {1-acyl-2-(6-(7-nitrobenz-2-oxa-1,3- diazol-4-yl-amino)caproyl) phosphatidylserine (C6-NBD-Ptd- Ser)} gave rise to two diffusional components: a slow compo- nent, D1, analogous to that measured with the choline- containing lipids, and a fast component (D2 - 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 regis- tered at different intervals, we calculated that =50% of the plasma membrane sphingomyelin is recycled in less than 30 min in Chinese hamster fibroblasts by an ATP- and microtu- bule-dependent process.