Understanding how cells sense and respond to the mechanical properties of their tissue microenvironment('mechanosense') would enhance our ability to control stem cell differentiation, enabling advances inregenerative medicine, and to develop drugs to disrupt tumor metastasis, the cause of the vast majority ofcancer deaths. Despite the importance of extracellular matrix stiffness in controlling cell fate and behavior, themolecular mechanisms underlying mechanosensing remain unknown. Cells interact with the extracellularmatrix (ECM) through focal adhesions and associated stress fibers, actin-rich structures which enable a cell topull on its surroundings via myosin II motor protein mediated contractility. Focal adhesions contain more than150 different proteins, forming a highly complex interaction network whose composition changes dramaticallyin response to contractile forces which are modulated by the stiffness of a cell's environment. The proposedstudies will dissect a primary mechanism underlying this compositional remodeling: how dozens of proteinscontaining motifs known as LIM domains are recruited to focal adhesions and stress fibers when they areunder tension. Although the role of most LIM proteins in focal adhesions and stress fibers has not been wellcharacterized, substantial cytological evidence suggests that one family member, the protein zyxin,accumulates at sites where the actin cytoskeleton is deformed or damaged in a LIM domain-dependentmanner. I therefore hypothesize that myosin motor activity, which is known to distort actin cytoskeletons invitro, generates binding sites for LIM domains in cells when contractility is high on stiff ECMs. This proposalbuilds upon an innovative application of electron tomography, which I previously adapted to quantify thedistributions of single molecules in reconstructed three-dimensional maps of heterogeneous networks ofinteracting proteins. I will employ this technology to characterize the distortions which myosin motor activitygenerates in reconstituted cytoskeletal networks. This will reveal for the first time the structural consequencesof a physiological force generator on the actin cytoskeleton in molecular detail. I will then uncover which ofthese distortions act as binding sites for LIM domains using zyxin as a model, and how the interaction isformed. Finally, I will stringently establish which LIM proteins are mechanosensitively recruited to focaladhesions and stress fibers in cells, and how they also recognize deformed cytoskeletal structures. Developingpharmacological inhibitors of force-dependent LIM protein accumulation, a key aspect of a cell'smechanosensitive response to its microenvironment, will be accelerated by identifying and establishing thestructural details of the interaction between force-sensitive LIM domains and their receptors on the actincytoskeleton. My studies will also provide insight into how the prevalence of modular LIM domains in the focaladhesion and stress fiber protein-protein interaction network enables a large-scale mechanosensitive responseby the alteration of a single class of interactions.
The goal of this project is to uncover the molecular workings of a key system which allows cells in the body to react to the mechanical properties of their localmicroscopic environments. A specific group of proteins that contain a common element accumulate at force-generating cellular structures when they come under tension; which occurs in response to a stiff environment. Understanding this process will enhance our ability to develop ways to inhibit it; which may lead to improved therapies for metastatic cancer and novel ways to control stem cell differentiation for regenerative medicine.
