Steric regulation of tandem calponin homology domain actin-binding affinity.

Tandem calponin homology (CH1-CH2) domains are common actin-binding domains in proteins that interact with and organize the actin cytoskeleton. Despite regions of high sequence similarity, CH1-CH2 domains can have remarkably different actin-binding properties, with disease-associated point mutants known to increase as well as decrease affinity for F-actin. To investigate features that affect CH1-CH2 affinity for F-actin in cells and in vitro, we perturbed the utrophin actin-binding domain by making point mutations at the CH1-CH2 interface, replacing the linker domain, and adding a polyethylene glycol (PEG) polymer to CH2. Consistent with a previous model describing CH2 as a steric negative regulator of actin binding, we find that utrophin CH1-CH2 affinity is both increased and decreased by modifications that change the effective "openness" of CH1 and CH2 in solution. We also identified interface mutations that caused a large increase in affinity without changing solution "openness," suggesting additional influences on affinity. Interestingly, we also observe nonuniform subcellular localization of utrophin CH1-CH2 that depends on the N-terminal flanking region but not on bulk affinity. These observations provide new insights into how small sequence changes, such as those found in diseases, can affect CH1-CH2 binding properties

The Mechanoregulation of the Actin Cytoskeleton

The actin cytoskeleton is essential for maintaining mechanical integrity of cells and tissues and for providing structural support during dynamic processes including migration, endocytosis and cytokinesis. From a molecular perspective, it consists of (1) actin monomers polymerized in double helical filamentous structures and (2) an ensemble of regulatory proteins that regulate shape and function of actin structures. From a mechanics perspective, the cytoskeleton is a dynamic entity that can generate force while being subject to various load perturbations. Though molecular understanding of actin networks is extensive, our understanding of how molecular signaling is converted to force output and how force input feeds back into molecular activity remains limited. The goal of this dissertation is to investigate how the interplay between molecular and mechanical attributes of the actin cytoskeleton results in desired cellular activity and physiological phenotypes. We first focus on the leading edge of migrating cells where nucleation of branched actin structures is involved in membrane protrusion. In chapter 2, we investigate the effect of the biochemical composition of these structures on the observed dynamic properties of network growth. To do so, we reconstitute branched actin network assembly using a minimal set of essential proteins (i.e. nucleation promoting factors, ARP2/3 and capping proteins) and evaluate their role over a broad range of concentrations. We find that in the absence of opposing force, changes in the nanomolar range of soluble protein concentration significantly modulates architectural and kinetic properties of nucleating actin structures. In cells, branched actin networks do not just transmit forces in the form of protrusion but also resists opposing load imposed by the membrane and other physical barriers. In chapter 3, we use atomic force microscopy to study the impact of external force on the biochemical composition and mechanical properties of reconstituted branched actin structures. Interestingly, we find that mechanical loading alters network density and composition, which in turn modulates its bulk mechanical properties and renders it stiffer, more powerful and efficient. Central to assembly and function of actin networks is the activity of actin binding proteins. We next extend our investigation to ask whether forces on actin filaments can influence actin binding protein (ABP) localization and activity in the cytoskeleton. Despite sharing the same cytoplasm, ABPs in cells spatially segregate and differentially regulate actin structures. In the context of the leading edge of migrating cells, cofilin binds and severs filaments in the lamellipodia, whereas tropomyosin is secluded as it binds and stabilizes filaments in the lamellum. In chapter 4, we hypothesize that these proteins are mechanosensitive and show that cofilin preferentially binds to network structures subject to compression whereas tropomyosin favors relaxed structures. Lastly, in chapter 5, we explore the sensitivity of calponin homology domain-containing proteins to the mechanical state of actin filaments. We focus our study on wild type and mutated versions of the utrophin actin binding domain, which is used as a universal actin marker. Using a multiscale biophysical approach, we show that mutant utrophin can selectively bind highly stressed actin filaments in vitro and in cells. We use this mutant to develop a ratiometric actin mechanosensor for mapping physiological forces in-vivo which provides a new tool for exploring mechanoregulation of cellular processes. Overall, the findings in this dissertation provide direct evidence for the importance of mechanical perturbations in regulating structure and function of the actin cytoskeleton

Steric regulation of tandem calponin homology domain actin-binding affinity.

Tandem calponin homology (CH1-CH2) domains are common actin-binding domains in proteins that interact with and organize the actin cytoskeleton. Despite regions of high sequence similarity, CH1-CH2 domains can have remarkably different actin-binding properties, with disease-associated point mutants known to increase as well as decrease affinity for F-actin. To investigate features that affect CH1-CH2 affinity for F-actin in cells and in vitro, we perturbed the utrophin actin-binding domain by making point mutations at the CH1-CH2 interface, replacing the linker domain, and adding a polyethylene glycol (PEG) polymer to CH2. Consistent with a previous model describing CH2 as a steric negative regulator of actin binding, we find that utrophin CH1-CH2 affinity is both increased and decreased by modifications that change the effective openness of CH1 and CH2 in solution. We also identified interface mutations that caused a large increase in affinity without changing solution openness, suggesting additional influences on affinity. Interestingly, we also observe nonuniform subcellular localization of utrophin CH1-CH2 that depends on the N-terminal flanking region but not on bulk affinity. These observations provide new insights into how small sequence changes, such as those found in diseases, can affect CH1-CH2 binding properties

Biased localization of actin binding proteins by actin filament conformation.

The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1-CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells. We demonstrate that the binding kinetics of CH1-CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs and other binding proteins. These findings suggest that conformational changes of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop

Force Feedback Controls Motor Activity and Mechanical Properties of Self-Assembling Branched Actin Networks

Branched actin networks–created by the Arp2/3 complex, capping protein, and a nucleation promoting factor– generate and transmit forces required for many cellular processes, but their response to force is poorly understood. To address this, we assembled branched actin networks in vitro from purified components and used simultaneous fluorescence and atomic force microscopy to quantify their molecular composition and material properties under various forces. Remarkably, mechanical loading of these self-assembling materials increases their density, power, and efficiency. Microscopically, increased density reflects increased filament number and altered geometry, but no change in average length. Macroscopically, increased density enhances network stiffness and resistance to mechanical failure beyond those of isotropic actin networks. These effects endow branched actin networks with memory of their mechanical history that shapes their material properties and motor activity. This work reveals intrinsic force feedback mechanisms by which mechanical resistance makes self-assembling actin networks stiffer, stronger, and more powerful