Gelsolin-mediated actin filament severing in crowded environments.

Gelsolin is a calcium-regulated actin binding protein that severs and caps actin filaments. Gelsolin's severing activity is important for regulating actin filament assembly dynamics that are required for cell motility as well as survival. The majority of in vitro studies of gelsolin have been performed in dilute buffer conditions which do not simulate the molecular interactions occurring in the crowded intracellular environment. We hypothesize that crowding results in greater gelsolin severing activity due to induced conformational changes in actin filaments and/or gelsolin. In this study, we evaluated the effects of crowding on gelsolin-mediated actin filament severing and gelsolin binding to actin filaments in crowded solutions, utilizing total internal reflection fluorescence (TIRF) microscopy and co-sedimentation assays. Our data indicates that the presence of crowders causes a decrease in the rate of gelsolin severing as well as a decrease in the amount of gelsolin bound to actin filaments, with greater effects caused by the polymeric crowder. Despite the severing rate decrease, gelsolin-mediated filament severing is increased in the presence of crowders. Understanding the crowding effect on gelsolin-mediated actin filament severing offers insight into the interactions between gelsolin and actin that occur inside the crowded cytoplasm.

SDS-PAGE for Monitoring the Dissolution of Zinc Oxide Bactericidal Nanoparticles (Zinkicide) in Aqueous Solutions.

Zinkicide is a systemic bactericidal formulation containing protein-size fluorescent zinc oxide-based nanoparticles (nano-ZnO). Previous studies have shown that Zinkicide is effective in controlling citrus diseases. Its field performance as an antimicrobial agent has been linked to the bioavailability of zinc ions (Zn2+) at the target site. It is therefore important to monitor Zn2+ release from Zinkicide so that application rates and frequency can be estimated. In this study, we present a simplistic approach designed to monitor Zinkicide nanoparticle dissolution rates in water and acidic buffer solutions using traditional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The evolution of nano-ZnO in the polyacrylamide gel scaffolds was studied by exciting the sample with UV light and detecting the fluorescence of nano-ZnO. Fluorescence intensities measured with this assay allowed for quantitative analysis of molecular weight changes of nano-ZnO in citrate buffer, a surrogate of citrus juice. Our results demonstrated that citrate buffer induced the greatest degradation of Zinkicide. Fluorescence intensity fluctuations were observed over time, indicating interactions of citrate with the surface of nano-ZnO. These findings provide a new approach to quantify the dissolution of nanoparticles in simulated environments, even when other analytical methods lack sensitivity because of the small size of the system (≈4 nm).

Molecular dynamics study of interactions between polymorphic actin filaments and gelsolin segment-1.

The assembly of protein actin into double-helical filaments promotes many eukaryotic cellular processes that are regulated by actin-binding proteins (ABPs). Actin filaments can adopt multiple conformations, known as structural polymorphism, which possibly influences the interaction between filaments and ABPs. Gelsolin is a Ca2+ -regulated ABP that severs and caps actin filaments. Gelsolin binding modulates filament structure; however, it is not known how polymorphic actin filament structures influence an interaction of gelsolin S1 with the barbed-end of filament. Herein, we investigated how polymorphic structures of actin filaments affect the interactions near interfaces between the gelsolin segment 1 (S1) domain and the filament barbed-end. Using all-atom molecular dynamics simulations, we demonstrate that different tilted states of subunits modulate gelsolin S1 interactions with the barbed-end of polymorphic filaments. Hydrogen bonding and interaction energy at the filament-gelsolin S1 interface indicate distinct conformations of filament barbed ends, resulting in different interactions of gelsolin S1. This study demonstrates that filament's structural multiplicity plays important roles in the interactions of actin with ABPs.