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Gelsolin is a calcium (Ca²⁺) dependent, pH sensitive actin-binding protein that regulates actin filament dynamics to remodel the actin cytoskeleton. It is known that gelsolin binding induces conformational changes of actin filaments, leading to filament severing. However, the influence of physiological conditions, such as pH variations, on gelsolin-mediated filament severing activities, mechanics and conformations remains unclear despite their role in actin-actin interactions. Using Total Internal Reflection Fluorescence (TIRF) microscopy imaging and pyrene fluorescence assays, we demonstrate that filament severing efficiencies by gelsolin are enhanced in acidic conditions. In addition, analysis of filament thermal fluctuations using TIRF reveals that gelsolin binding stiffens actin filaments. Furthermore, we show that gelsolin binding induces conformational changes in filaments by measuring the filament half-pitch using high resolution Atomic Force Microscopy imaging. Together, our results suggest that pH modulation plays a key role in gelsolin-mediated filament severing activities, bending mechanics, and conformational changes, which have implications in many cellular processes including cell motility and morphogenesis. 

Despite considerable advances in recent years, challenges in delivery and storage of biological drugs persist and may delay or prohibit their clinical application. Though nanoparticle-based approaches for small molecule drug encapsulation are mature, encapsulation of proteins remains problematic due to destabilization of the protein. Reverse micelles composed of decylmonoacyl glycerol (10MAG) and lauryldimethylamino-N-oxide (LDAO) in low-viscosity alkanes have been shown to preserve the structure and stability of a wide range of biological macromolecules. Here, we present a first step on developing this system as a future platform for storage and delivery of biological drugs by replacing the non-biocompatible alkane solvent with solvents currently used in small molecule delivery systems. Using a novel screening approach, we performed a comprehensive evaluation of the 10MAG/LDAO system using two preparation methods across seven biocompatible solvents with analysis of toxicity and encapsulation efficiency for each solvent. By using an inexpensive hydrophilic small molecule to test a wide range of conditions, we identify optimal solvent properties for further development. We validate the predictions from this screen with preliminary protein encapsulation tests. The insight provided lays the foundation for further development of this system toward long-term room-temperature storage of biologics or toward water-in-oil-in-water biologic delivery systems.