g(m1)-ganglioside and pyrene

Biomimetic particles supporting lipid bilayers are becoming increasingly important to isolate and reconstitute protein function. Cholera toxin (CT) from Vibrio cholerae, an 87-kDa AB5 hexameric protein, and its receptor, the monosialoganglioside GM1, a cell membrane glycolipid, self-assembled on phosphatidylcholine (PC) bilayer-covered silica particles at 1 CT/5 GM1 molar ratio in perfect agreement with literature. This receptor-ligand recognition represented a proof-of-concept that receptors in general can be isolated and their function reconstituted using biomimetic particles, i.e., bilayer-covered silica. After incubation of colloidal silica with small unilamellar PC vesicles in saline solution, pH 7.4, PC adsorption isotherms on silica from inorganic phosphorus analysis showed a high PC affinity for silica with maximal PC adsorption at bilayer deposition. At 0.3 mM PC, fluorescence of pyrene-labeled GM(1) showed that GM(1) incorporation in biomimetic particles increased as a function of particles concentration. At 1 mg/mL silica, receptor incorporation increased to a maximum of 40% at 0.2-0.3 mM PC and then decreased as a function of PC concentration. At 5 microM GM(1), 0.3 mM PC, and 1 mg/mL silica, CT binding increased as a function of CT concentration with a plateau at 2 mg bound CT/m2 silica, which corresponded to the 5 GM(1)/1 CT molar proportion and showed successful reconstitution of receptor-ligand interaction.

Topics: Adsorption; Animals; Biomimetics; Cattle; Chemical Phenomena; Chemistry, Physical; Cholera Toxin; G(M1) Ganglioside; Hot Temperature; Infection Control; Lipid Bilayers; Liposomes; Membrane Lipids; Particle Size; Phosphatidylcholines; Phospholipids; Pyrenes; Receptors, Cell Surface; Silicon Dioxide; Vibrio cholerae

A new fluorescence ratio imaging technique aiming to monitor the lateral distribution of pyrene-labeled lipids in the membranes through visualization of the excimer/monomer (E/M) intensity ratio, has been set up, studying the association of a fluorescent derivative of GM1 ganglioside (pyreneGM1) to rat cerebellar granule cells in culture. The imaging results show that the mean E/M ratio value, under experimental conditions leading to the association of pyreneGM1 with the plasma membrane, is significantly higher in neuritic processes than in cell bodies and, moreover, locally distributed in patches. Fluorescence antisotropy imaging of the fluorescent probe TMA-DPH (trimethylaminodiphenylhexatriene) shows the presence of domains having different fluidity and that the average fluidity is higher in cell bodies than in neuritic processes. Additional experiments using TMA-DPH as a marker of fluid-phase pinocytosis, suggest that the rate of endocytosis is comparable in the two regions of the cell. Taken together, on the one hand these data indicate that the fluorescent ganglioside is present in a more clustered state at the level of neuritic processes than in cell bodies and locally distributed in patches of different size and enrichment. On the other hand, they point out the potentiality of the excimer formation imaging technique to study membrane behavior and dynamics of pyrene-labeled lipids, with a particular insight into their aggregative properties.

Topics: Animals; Cells, Cultured; G(M1) Ganglioside; Glycolipids; Image Processing, Computer-Assisted; Membrane Lipids; Microscopy, Fluorescence; Microscopy, Video; Pyrenes; Rats; Rats, Sprague-Dawley