Organization Of Membrane Domains Research Articles

Application of three-dimensional molecular hydrophobicity potential to the analysis of spatial organization of membrane domains in proteins: I. Hydrophobic properties of transmembrane segments of Na+, K(+)-ATPase.

A new computer-aided molecular modeling approach based on the concept of three-dimensional (3D) molecular hydrophobicity potential has been developed to calculate the spatial organization of intramembrane domains in proteins. The method has been tested by calculating the arrangement of membrane-spanning segments in the photoreaction center of Rhodopseudomonas viridis and comparing the results obtained with those derived from the X-ray data. We have applied this computational procedure to the analysis of interhelical packing in membrane moiety of Na+, K(+)-ATPase. The work consists of three parts. In Part I, 3D distributions of electrostatic and molecular hydrophobicity potentials on the surfaces of transmembrane helical peptides were computed and visualized. The hydrophobic and electrostatic properties of helices are discussed from the point of view of their possible arrangement within the protein molecule. Interlocation of helical segments connected with short extramembrane loops found by means of optimization of their hydrophobic/hydrophilic contacts is considered in Part II. The most probable 3D model of packing of helical peptides in the membrane domain of Na+, K(+)-ATPase is discussed in the final part of the work.

Hormonal Responsiveness by Immature Rabbit Uterine Epithelial Cells Polarizedin Vitro*

This paper reports the development of an in vitro cell culture system of polarized uterine epithelial (UE) cells from the immature rabbit. UE cells from immature rabbit were cultured on EHS (Engelbreth-Holm-Swarm) matrix-coated semipermeable filter inserts in a serum-free, phenol red-free defined medium. The cells in primary culture attached, proliferated, formed monolayers, and exhibited structural and functional polarity. Structural differentiation was validated by ultrastructural features, such as apical microvilli, intercellular junctions, desmosomes, and polar organization of apical vs. basal membrane domains. Trans-monolayer epithelial resistance, a reflection of functional tight junctions, preferential basal uptake of [35S]methionine, polarized distribution of labeled secretory proteins, and increased secretory activity marked by preferential apical secretion (greater than 90%) of proteins were indices of functional polarity. With the development and maintenance of polarized state by the UE cells, some of the newly synthesized proteins were sorted and vectorially secreted into their distinct compartments. De novo synthesis and exclusive apical secretion of uteroglobin (a progesterone-induced protein in vivo) by the polarized UE cells occurred in response to progesterone treatment in vitro. The hormone responsiveness was maintained for a prolonged period under these conditions. A culture system in which the UE cells maintain functional and morphological polarity and sustain their hormonal responsiveness facilitates the study of regulation of specialized functions by the UE cells that are regulated by steroid hormones and their interactions with the other uterine cell types.

The organization of cell surface membrane domains

Cell surface membranes are based on a fluid lipid bilayer and models of the membranes' organization have emphasised the possibilities for lateral motion of membrane lipids and proteins within the bilayer. Two recent trends in cell and membrane biology make us consider ways in which membrane organization works against its inherent fluidity, localizing both lipids and proteins into discrete domains. There is evidence for such domains, even in cells without obvious morphological polarity and organization [Table 1]. Cells that are morphologically polarised, for example epithelial cells, raise the issue of membrane domains in an accute form.The technique of fluorescence photobleaching and recovery, FPR, was developed to measure lateral diffusion of membrane components. It has also proven to be a powerful tool for the analysis of constraints to lateral mobility. FPR resolves several sorts of membrane domains, all on the micrometer scale, in several different cell types.

Increased membrane heterogeneity in stimulated human granulocytes

TMA-DPH fluorescence decay in human PMN before and after stimulation with FMLP was studied using frequency domain fluorometry. Membrane heterogeneity was assessed by the width of the continuous distributions of lifetime values of Lorentzian shape used to describe the fluorescence decay. In non-stimulated granulocytes TMA-DPH fluorescence decay is characterized by two distributions of lifetime values centered at 6.5 and 1.0 ns and full width at half maximum of 0.3 and 1.2 ns, respectively. Within 15 min after stimulation, the center values of the two distribution components were 5.1 and 0.8 ns and the distribution width was 0.8 and 0.6 ns, respectively. These results indicate changes of membrane domain organization which can be ascribed to compositional changes redistribution of membrane components.

Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells.

The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.

The effects of dietary ( [formula omitted]) fatty acid supplementation on lipid dynamics and composition in rat lymphocytes and liver microsomes

Rats were fed diets devoid of ( n − 3) fatty acids (olive oil supplementation) or high in ( n − 3) fatty acids (fish oil supplementation) for a period of 10 days. In spleen lymphocytes and liver microsomes derived from animals fed fish oil diets, relatively high levels of ( n − 3) eicosapentaenoic (20:5), docosapentaenoic (22:5) and docosahexaenoic acids (22:6) were obtained compared to minimal levels when fed the olive oil diet. When the average lipid motional properties were examined by measuring the fluorescence anisotropy of diphenylhexatriene, no significant different was found between intact liver microsomes from animals fed the two diets. However, when lipid motion was examined in vesicles of phosphatidylcholine, isolated from the microsomes from fish oil fed animals (21.4% ( n − 3) fatty acids), the fluorescence anisotropy was significantly less than the corresponding phosphatidylcholine from olive oil fed animals (5.6% ( n − 3) fatty acids), indicating a more disordered or fluid bilayer in the presence of higher levels of ( n − 3) fatty acids. Phosphatidylethanolamine ( n − 3) fatty acids were also elevated after fish oil supplementation (41.3% of total fatty acids), compared to the level after olive oil supplementation (21.4%). The major effect of the fish oil supplementation was a replacement of ( n − 6) arachidonic acid by the ( n − 3) fatty acids and when this was ‘modeled’, using liposomes of synthetic lipids, 1- palmitoyl-2- arachidonyl(n − 6) or docosahexaenoyl(n − 3)- phosphatidylcholine , significant differences in lipid motional properties were found, with the docosahexaenoate conferring a more disordered or fluid lipid environment. Thus it appears that although lipid order/fluidity can be significantly decreased by increases in the highly unsaturated ( n − 3) fatty acid levels, alterations in membrane domain organization and/or phospholipid molecular species composition effectively compensated for the changes, at least as far as average lipid motional properties in the intact membranes was concerned.