One of the central problems of cell biology is the mechanism of membrane assembly. This project has addressed the problem by focusing on the physico-chemical properties of the lipid components, and examining self-assembly mechanisms for the lipid bilayer structure of membranes. An early discovery of this approach was the finding that the total lipid mixture of membrane lipids, extracted from cells and suspended in water, spontaneously form unilamellar structures, but only at the physiological temperature of the donor cell. This phenomenon has been observed for a variety of cell types including bacteria, squid axons, erythrocytes, and brain tissue, and for a wide range of physiological temperatures (15-60 C). The present research period has shown (1) that the unilamellar structure that forms at physiological temperatures is a critical state, and (2) that the mechanism of forming the critical unilamellar state is operating during assembly of membranes. (1) Bilayers, formed with lipids extracted from E. coli cellls that were cultured at three temperatures 25, 29 and 32 deg C, were doped with a fluorescent phospholipid. Using fluorescence recovery after photobleaching (FRAP), diffusion coefficients of the labeled phospholipid were measured as a function of temperature in each of the three bilayer preparations. A diffusion anomaly, manifested as a diffusion maximum, was found in each at the growth temperature of the cells from which the lipids were extracted. Anomalous diffusion behavior as observed in E. coli bilayers is characteristic of a critical state. (2) Membrane assembly in embryos of Lytechinus pictus was examined at three incubation temperatures: 10, 16 and 23 deg C by measuring the lipid composition of the yolk bodies and nascent membranes of the embryos until the blastula stage of development. During embryogenesis no de novo lipid synthesis occurs. The total lipid composition of the embryo was unchanged, and remained the same as in unfertilized eggs at each of the incubation temperatures. However, the lipid composition of the newly formed membranes changed with the incubation temperature. Moreover, the lipids from the nascent membranes were shown to exhibit properties of the critical unilamellar state at the incubation temperature of the embryo. Thus, membrane assembly during embryogenesis of this sea urchin occurs from a metabolically static pool of lipids to form the critical membrane state.
