Correlation analyses reveal differential diffusion behavior of eisosomal proteins between mother and daughter cells.

Eisosomes are nanoscale plasma membrane domains shaped as furrow-like invaginations. InSaccharomyces cerevisiaethese relatively immobile and uniform structures are mainly composed of two cytoplasmic proteins Pil1 and Lsp1. The present work uses fluctuation of fluorescence signals and analytical methods to determine Pil1 and Lsp1 dynamics at different subcellular locations. Using scanning techniques and autocorrelation analysis we determine that the cytoplasmic pools of Pil1 and Lsp1 behave mainly by passive diffusion. Single-point FCS experiments performed at several subcellular locations reveal that Pil1 mobility is faster in daughter cells. Furthermore, pair correlation function analysis indicates a rapid dynamic of Pil1 near the plasma membrane of growing yeast buds, where the membrane is expected to be actively assembling eisosomes.

Dengue Virus Capsid-Protein Dynamics in Live Infected Cells Studied by Pair Correlation Analysis.

It has become increasingly evident that unveiling the mechanisms of virus entry, assembly, and virion release is fundamental for identifying means for preventing viral spread and controlling viral disease. Due to virus mobility and structural and/or functional heterogeneity among viral particles, high spatiotemporal resolution single-virus/single-particle techniques are required to capture the behavior of viral particles inside infected cells.In this chapter, we present fluorescence imaging analysis methods for studying the mobility of fluorescently labeled dengue virus (DENV) proteins in live infected cells. Some of the most recent Fluorescence Fluctuation Spectroscopy (FFS) methods will be presented and, in particular, the pair Correlation Functions (pCF) approach will be discussed. The pCF method does not require individual molecule isolation, as in a particle-tracking experiment, to capture single viral protein behavior. In this regard, image acquisition is followed by the spatiotemporal cross-correlation function at increasing time delays, yielding a quantitative view of single-particle mobility in intact live infected cells.We provide a general overview and a practical guidance for the implementation of advanced FFS techniques, and the pair Correlation Functions analysis, as quantitative tools to reveal insights into previously unreported DENV mechanisms. We expect this protocol report will serve as an incentive for further applying correlation imaging studies in virology research.

Modeling the intracellular dynamics of the dengue viral infection and the innate immune response.

The interplay between the dengue virus and the innate immune response is not fully understood. Here, we use deterministic and stochastic approaches to investigate the dynamics of the interaction between the interferon-mediated innate immune response and the dengue virus. We aim to develop a quantitative representation of these complex interactions and predict their system-level dynamics. Our simulation results predict bimodal and bistable dynamics that represent viral clearance and virus-producing states. Under normal conditions, we determined that the viral infection outcome is modulated by the innate immune response and the positive-strand viral RNA concentration. Additionally, we tested system perturbations by external stimulation, such as the direct induction of the innate immune response by interferon, and a therapeutic intervention consisting of the direct application of mRNA encoding for several interferon-stimulated genes. Our simulation results suggest optimal regimes for the studied intervention approaches.