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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. 

The essential cytoskeletal protein actin and its func- tions are paramount for motility, communication, and locomotive processes in eukaryotic cells. Detection and quantification of actin protein is of great interest for in vitro studies potentially eluci- dating unknown cellular mechanisms affecting drug responses with an extension to the study of disease states (e.g., study of neurodegenerative disorders). To this end, development of biomedical platforms and biosensors plays an important role in providing reliable and sensitive devices to study such intracellular constructs. Here, we present for the first time the microfabrica- tion, characterization, testing, and electrical/interfacial modeling of a microfluidic biosensor for actin protein characterization. The device allows for the interaction and characterization of actin bundles using electrochemical impedance spectroscopy (EIS). The device was tested with 1 μM and 8 μM actin bundles concentrations producing shifts in impedance response in the significant biological frequency of 1 kHz from 17 to 30 kOhm (k). Interfacial capacitance and electrical modeling showed that at increasing actin bundles concentrations, the distance from the electrode to the diffusion region (Debye length) was reduced from 386 to 136, and from 1526 to 539 Å. Inter- facial capacitance was evaluated for 1 μM concentration at two dielectric constants (εr = 5 and 78) resulting in 3.8 and 15.6 mF/m2 respectively. Similarly, for 8 μM concentration, interfacial capacitance resulted in 10.1 and 43.3 mF/m2 for the same values of εr. Based on these theoretical calculations, the interface model could accurately predict the quantification of the actin bundles previously elucidated by the experimental EIS method. 

The structural and mechanical properties of actin bundles are essential to eukaryotic cells, aiding in cell motility and mechanical support of the plasma membrane. Bundle formation occurs in crowded intracellular environments composed of various ions and macromolecules. Although the roles of cations and macromolecular crowding in the mechanics and organization of actin bundles have been independently established, how changing both intracellular environmental conditions influence bundle mechanics at the nanoscale has yet to be established. Here we investigate how electrostatics and depletion interactions modulate the relative Young’s modulus and height of actin bundles using atomic force microscopy. Our results demonstrate that cation- and depletion-induced bundles display an overall reduction of relative Young’s modulus depending on either cation or crowding concentrations. Furthermore, we directly measure changes to cation- and depletion-induced bundle height, indicating that bundles experience alterations to filament packing supporting the reduction to relative Young’s modulus. Taken together, our work suggests that electrostatic and depletion interactions may act counteractively, impacting actin bundle nanomechanics and organization. 

The mechanical and structural properties of actin cytoskeleton drive various cellular processes, including structural support of the plasma membrane and cellular motility. Actin monomers assemble into double-stranded helical filaments as well as higher-ordered structures such as bundles and networks. Cells incorporate macromolecular crowding, cation interactions, and actin-crosslinking proteins to regulate the organization of actin bundles. Although the roles of each of these factors in actin bundling have been well-known individually, how combined factors contribute to actin bundle assembly, organization, and mechanics is not fully understood. Here, we describe recent studies that have investigated the mechanisms of how intracellular environmental factors influence actin bundling. This review highlights the effects of macromolecular crowding, cation interactions, and actin-crosslinking proteins on actin bundle organization, structure, and mechanics. Understanding these mechanisms is important in determining in vivo actin biophysics and providing insights into cell physiology.