Artem V. Melnykov

Revival of high order fluorescence correlation analysis: generalized theory and biochemical applications

Analysis of high-order correlations in fluorescence fluctuation spectroscopy was developed in the late 1980s but since then has been replaced by alternative brightness analysis methods. However, high-order correlation has important advantages in many experiments. We present a new cumulant-based formalism of high-order correlation that greatly simplifies data analysis. The new formalism is used to derive general expressions for variance of high-order correlations that show good agreement with experiment in a model system of fluorescently labeled DNA oligomers. A simulation of binary systems in which both diffusion time and brightness are varied illustrates clearly that high-order analysis has better sensitivity to brightness than fluorescence correlation spectroscopy (FCS). These results have implications for analysis of isomerization reactions and dual-beam FCS with flow. We also demonstrate that high-order correlations can detect photobleaching in the observation volume. The application of this formalism to many FCS-based experiments allows more accurate analysis in addition to describing more molecular parameters.

New mechanisms that regulate Saccharomyces cerevisiae short peptide transporter achieve balanced intracellular amino acid concentrations

The budding yeast Saccharomyces cerevisiae is able to take up large quantities of amino acids in the form of di‐ and tripeptides via a short peptide transporter, Ptr2p. It is known that PTR2 can be induced by certain peptides and amino acids, and the mechanisms governing this upregulation are understood at the molecular level. We describe two new opposing mechanisms of regulation that emphasize potential toxicity of amino acids: the first is upregulation of PTR2 in a population of cells, caused by amino acid secretion that accompanies peptide uptake; the second is loss of Ptr2p activity, due to transporter internalization following peptide uptake. Our findings emphasize the importance of proper amino acid balance in the cell and extend understanding of peptide import regulation in yeast. Copyright

Investigation of Nanoscopic Phase Separations in Lipid Membranes Using Inverse FCS

Measurement of the sizes of nanoscopic particles is a difficult challenge, especially in two-dimensional systems such as cell membranes. We have extended inverse fluorescence correlation spectroscopy (iFCS) to endow it with unique advantages for measuring particle size from the nano- to the microscale. We have augmented iFCS with an analysis of moments of fluorescence fluctuations and used it to measure stages of phase separation in model lipid bilayer membranes. We observed two different pathways for the growth of phase domains. In one, nanoscopic gel domains appeared first and then gradually grew to micrometer size. In the other, the domains reached micrometer size quickly, and their number gradually increased. These measurements demonstrate the value of iFCS measurements through their ability, to our knowledge, to provide new information about the mechanism of lipid phase separation and potentially about the physical basis of naturally occurring nanodomains such as lipid rafts.

Effect of loop composition on the stability and folding kinetics of RNA hairpins with large loops.

RNA hairpins are ubiquitous structural elements in biological RNAs, where they have the potential to regulate RNA folding and interactions with other molecules. There are established methods for predicting the thermodynamic stability of an RNA hairpin, but there are still relatively few detailed examinations of the kinetics of folding. Nonetheless, several recent studies indicate that hairpin folding does not proceed via a simple two-state model. Here, we monitor fluorescence from hairpins constructed as molecular beacons in ensemble, fluorescence correlation spectroscopy, and stopped-flow experiments to describe the folding of RNA hairpins with long (15 nucleotide) loops. Our results show that folding of these hairpins occurs through more than two states and that the mechanism of folding includes a fast intermediate phase observed on the tens of microseconds time scale and a slow phase, attributed to formation of the native folded hairpin loop and stem, observed on the milliseconds time scale. The composition of the RNA loop determines the time scale of intermediate and native folded states. Hairpins with a polyuracil loop sequence exhibit slower relaxation of the intermediate state and faster relaxation of the native folded state when compared to that of hairpins with cytosine or adenine in the loop. We hypothesize this composition dependence could be attributed to nucleobase stacking in cytosine and adenine containing regions of the loop, which would be absent in hairpins containing polyuracil loops. Such base stacking could destabilize the intermediate folds, thereby speeding the relaxation of the intermediate relative to similar sized hairpins with no base stacking in the loop. Likewise, the lower intermediate stability could prolong the relaxation of the native folded state.

FCS In Closed Systems And Application For Membrane Nanotubes

In the present study, we developed the fluorescence correlation spectroscopy theory for closed systems with either periodic or reflective boundaries. The illumination could be any arbitrary function. We tested our theory with simulated data of both boundary conditions. We also tested the theory with experimental data of membrane nanotubes, whose circular direction is a closed system. The result shows that the correlation function for nanotubes falls between 1D and 2D diffusion model. The fitting with our model gives an accurate recovery of the diffusion time and nanotube radius. We also give some examples of single molecule experiments for which our theory can be potentially useful.

Confidence Intervals for Estimation of the Concentration and Brightness of Multiple Diffusing Species

Understanding the effects of myofibroblasts on the electrical and mechanical functions of the heart is important to understanding the long-term consequences of cardiac fibrosis and myocardial infarction, but the effects are difficult to quantify. Cardiac fibroblasts convert from their quiescent state to the larger and contractile myofibroblasts phenotype under conditions of hypertension and following myocardial infarction. The excess fibrous connective tissue produced by myofibroblasts stiffens heart muscle and the presence of the MFs disrupts the normal pattern of electrical excitation of the cardiomyoctyes. In a model tissue system we have developed in our laboratories (engineered heart tissues, EHTs), overgrowth of myofibroblasts leads to an increase in average (tonic) contractile stress and, eventually, a shut-down of periodic or twitch contractile stress. To understand how myofibroblast/cardiomyoctye interactions relate to conduction and contraction changes in EHTs, a series of coupled mechanical and electrophysiological simulations and experiments were performed.Copyright