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./nWe 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./nIn 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./nCentral 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./nLastly, 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.

Advisor: Fletcher, Daniel A.