UCSF

The dynamic actin cytoskeleton plays a key role in a number of cellular processes including motility and shape change. Composed of individual filaments polymerized from actin monomers, the actin cytoskeleton is organized into a branched and cross-linked dendritic network by a diverse set of actin binding proteins. Directed growth of dendritic actin networks by monomer addition, such as at the leading edge of a crawling cell, generates the mechanical forces necessary for deforming the membrane during cell motility, endocytosis, and phagocytosis. Dysfunctional actin network regulation is associated with metastatic cancers, immune system disorders, and bacterial infection and pathogenesis.

Significant biochemical work over the past four decades has culminated into the dendritic nucleation model for actin network growth. This model summarizes the role of the major actin binding proteins, and interactions among them, that form and maintain a growing, dendritic actin network in crawling cells. Though actin biochemistry has been well studied, the force-generating ability and mechanical properties of growing dendritic actin networks that produce dynamic cellular shape changes remain unclear. This dissertation presents development of a unique measurement system for the purpose of understanding the biophysics of dendritic actin network growth. An experimental platform was built around a custom differential atomic force microscope by adapting a method for reconstituting actin network growth from cell-free extract in vitro to measure network force production and mechanics. The results described here demonstrate that dendritic actin networks possess a built-in force feedback system that enables active remodeling to support increasing forces. In addition, these networks exhibit the ability to reversibly stress soften under large loads, thereby avoiding catastrophic failure and retaining their underlying network structure as a molecular scaffold. These results have implications for understanding how crawling cells navigate through the physical barriers of the extracellular matrix and connective tissue in vivo while feeling a wide range of compressive forces.