STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells.

Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes.

Reversible photoswitching enables single-molecule fluorescence fluctuation spectroscopy at high molecular concentration.

We demonstrate that photoswitchable markers enable fluorescence fluctuation spectroscopy at high molecular concentration. Reversible photoswitching allows precise control of the density of fluorescing entities, because the equilibrium between the fluorescent ON- and the dark OFF-state can be shifted through optical irradiation at a specific wavelength. Depending on the irradiation intensity, the concentration of the ON-state markers can be up to 1,000 times lower than the actual concentration of the labeled molecular entity. Photoswitching expands the range of single-molecule detection based experiments such as fluorescence fluctuation spectroscopy to large entity concentrations in the micromolar range.

Can biochemical properties serve as selective pressure for gene selection during inter-species and endosymbiotic lateral gene transfer?

During the evolution of endosymbiosis, only one orthologous gene, either from the invader or the invaded genome, is preserved. Genetic and environmental factors are usually invoked to explain this gene preference. How biochemical parameters can play a role in the selection of genes that code for enzymes that constitute a metabolic pathway is explored. Simple Michaelis-Menten-like enzymes are considered whose kinetic parameters are randomly generated to construct two parallel homologous pathways to account for the contributions of the invaded and the invader. Steady-state fluxes as targets of natural selection are focused. Enzymes are eliminated one by one so that the total flux through the pathway is least disturbed. Analysis of the results, done by different criteria, indicate that the maximal velocities, both forward and backward, are more influential in selection than the respective Michaelis constants. This inclination disappears as metabolite concentrations are increased. It is shown that kinetic selection criteria can result in a mosaicism of enzymes in the same pathway in terms of their genetic origin. Analysis of the results using the control coefficient paradigm disclosed an expected robust correlation between flux control coefficients of enzymes and their selective elimination. Similar analyses, performed for the case of single gene transfer or for gene replication with subsequent mutation, yielded essentially similar results. The results conform with the phenomenon of genetic mosaicism found in phylogenetic analyses of single or double endosymbioses and lateral gene transfer.