Phospholipid transmembrane domains and lateral diffusion in fibroblasts.

Abstract

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 7C, the fluorescence remained localized at the plasma membrane. N-[6-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyllsphingosylphosphocholine (C6-NBD-SphPCho) and 1-acyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproylJ phosphatidylcholine (C6-NBDPtdCho) both diffused with the same apparent lateral diffusion coefficient (D, 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)caproylJ phosphatidylserine (C6-NBD-PtdSer)} gave rise to two diffusional components: a slow component, D1, analogous to that measured with the cholinecontaining 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 cholinecontaining lipids and the aminolipid. However, with C6-NBDSphPCho, a fraction of very slowly diffusing or quasiimmobilized 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 =50% of the plasma membrane sphingomyelin is recycled in less than 30 min in Chinese hamster fibroblasts by an ATP-and microtubule-dependent process.The existence of lateral and/or transverse lipid domains in membranes is often inferred from the properties of fluorescent lipids incorporated into model systems (1-3) or in cell membranes (4-8). Domains are generally associated with regions of distinct fluidity, sometimes corresponding to a pure gel or a pure liquid-crystalline state. However, domains do not automatically imply lipid phase immiscibility per se but may also be delimited by barriers. For example, tight junctions restrict the lateral diffusion of lipids in polarized cells (9), whereas protein clusters would restrict that of plastoquinones in chloroplasts (10). Similarly, the hydrophobic core of all membranes constitutes a barrier to the passage of phospholipids from one leaflet to the other. In red blood cells, ESR and fluorescence techniques were used to demonstrate a fluidity difference between inner and outer leaflets of the plasma membrane without implying gel formation (11-13). The difference was attributed to the specific lipid composition of each leaflet, in particular to the greater degree ofunsaturation ofthe lipid chains in the inner monolayer (14).All eukaryotic cells seem to have a plasma membrane with an asymmetrical phospholipid distribution (for a review, see ref. 15). In the present article, we show that the asymmetrical transverse lipid composition of V79 cultured Chinese hamster fibroblast membranes...

Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement

“…Thus lower local microviscosity (Morrot et al, 1989;Tanaka and Ohnishi, 1976) and a faster lateral diffusion (Morrot et al, 1986;Rimon et al, 1984) have been ascribed to the lipids in the cytoplasmic erythrocyte leaflet. Similar properties exist also in other cells, such as fibroblasts (El-Hage Chahine et al, 1993). Studies with artificial membranes reconstituted from the characteristic phospholipids of each monolayer demonstrated that differences in microviscosity and diffusibility were primarily due to the phospholipid composition of the membrane (Cribier et al, 1990).…”

“…Apparently, this absence of diffusion might be explained by sphingomyelin-cholesterol interactions, as depleting the erythrocyte membrane in cholesterol stimulated sphingomyelin transmembrane movement (our unpublished data). This absence of sphingomyelin diffusion was also detected in other cells, namely hamster fibroblasts (El-Hage Chahine et al, 1993).…”

Detection of Lipid Domains in Model and Cell Membranes by Fluorescence Lifetime Imaging Microscopy of Fluorescent Lipid Analogues

“…We noted that the plasma membrane-associated lifetime of C6-NBD-PS is slightly shorter than that of C6-NBD-PC. This might be explained again by the fact that a significant fraction of the PS analogue has been transported to the cytoplasmic leaflet sensing the reduced lipid packing of this leaflet as previously shown by biochemical and biophysical studies (36,37).…”

“…Taking into account that a lateral diffusion coefficient of 10 Ϫ7 cm 2 /s is an upper estimate, domains resolvable by FLIM could be even smaller. Typical values for lateral diffusion of lipid analogues in plasma membranes are between 1 ϫ 10 Ϫ9 cm 2 /s and 5 ϫ 10 Ϫ9 cm 2 /s as measured for fibroblasts (36) and red blood cells (37), respectively. Hence, FLIM should be appropriate to capture even very small lipid domains as long as they are stable for at least 30 ns.…”

Basic Fibroblast Growth Factor Modulates the Aminophospholipid Translocase Activity Present in the Plasma Membrane of Bovine Aortic Endothelial Cells

“…This marked and unexpected stability in the labeling of the plasma membrane we are describing for the vascular endothelium, with no significant probe internalization, appears to be very useful for the present study. It is another example that the balance between plasma membrane labeling and probe internalization can strongly vary from one cell type to the other (Martin and Pagano, 1987;El Hage Chahine et al, 1993;Kok et al, 1990;Kobayashi and Arakawa, 1991 ;Sleight and Abanto, 1989).…”