Fragility and Mechanosensing in a Thermalized Cytoskeleton Model with Forced Protein Unfolding

We describe a model of cytoskeletal mechanics based on the force-induced conformational change of protein cross-links in a stressed polymer network. Slow deformation of simulated networks containing cross-links that undergo repeated, serial domain unfolding leads to an unusual state — with many cross-links accumulating near the critical force for further unfolding. This state is robust to thermalization and does not occur in similar protein unbinding based simulations. Moreover, we note that the unusual configuration of near-critical protein cross-links in the fragile state provides a physical mechanism for the chemical transduction of cell-level mechanical strain and extra-cellular matrix stiffness

Light-Triggered Myosin Activation for Probing Dynamic Cellular Processes

Shining light on myosin: The incorporation of a caging group onto the essential phosphoserine residue of myosin by protein semisynthesis enables light-triggered activation of the protein (see picture). Caging eliminates the myosin activity, but exposure to 365 nm light restores its function to native levels. The caged protein can also be introduced into cells to facilitate studies of myosin with precise spatial and temporal resolution.American Heart Association (Fellowship)National Institutes of Health (U.S.) (NIH Cell Migration Consortium (GM064346))National Institute of General Medical Sciences (U.S.) (Biotechnology Training Grant

Discovery and characterization of small molecules that target the Ral GTPase

The Ras-like GTPases RalA and B are important drivers of tumor growth and metastasis. Chemicals that block Ral function would be valuable as research tools and for cancer therapeutics. Here, we used protein structure analysis and virtual screening to identify drug-like molecules that bind a site on the GDP-form of Ral. Compounds RBC6, RBC8 and RBC10 inhibited Ral binding to its effector RalBP1, Ral-mediated cell spreading in murine fibroblasts and anchorage-independent growth of human cancer cell lines. Binding of RBC8 derivative BQU57 to RalB was confirmed by isothermal titration calorimetry, surface plasma resonance and 15N-HSQC NMR. RBC8 and BQU57 show selectivity for Ral relative to Ras or Rho and inhibit xenograft tumor growth similar to depletion of Ral by siRNA. Our results show the utility of structure-based discovery for development of therapeutics for Ral-dependent cancers

Measuring, modelling, and dissecting the cellular mechanical response

While understanding cells\u27 responses to mechanical stimuli is seen as increasingly important for understanding cell biology, the underlying mechanisms of how cells sense and interpret mechanical variables are still unknown. The ubiquity of mechanosensitive processes suggests a physically based mechanism. The combination of rheological measurements and modeling has been used for centuries to study the response of non-living materials to applied stresses. However cell theological measurements are limited by a myriad of confounding effects including: cytoskeletal heterogeneity, ATP-dependent processes and cell regional variations. Here a novel formalism for interpreting microrheology data in the presence of ATP dependent processes is created and a suite of single cell microrheological techniques to control for all of these variables is developed. This approach allows the determination of the consensus mechanical picture of the cell that is suitable for modeling, isolates mechanically distinct sub-cellular structures, evaluates the predictions of cell mechanical models, and identifies key molecular determinants of cellular mechanics. Specifically, two mechanically distinct structures corresponding to the cell cortex and perinuclear region are found. Both regions exhibit shear moduli with similar weak power-law frequency dependence at low frequency, that transition to ω 3/4 at high frequencies. However the cortical region is actin dominated, while the microtubles are a key component of the perinuclear region. Furthermore by analyzing the observed cytoskeletal fluctuations and heterogeneity we find that no current cell mechanics model is sufficient for describing this data. Based on similarities with in vitro systems of actin and cross-linking proteins, we develop a mechanical model based on protein domain unfolding and evaluate its behavior with simulations. A unique state, where proteins accumulate on the cusp of unfolding, suggesting a mechanosensing role, is found. An analogous state is observed in other models that produce rheology similar to that observed cellularly. As the folding state of a protein is readily detected biochemically, we hypothesize that by modulating binding of signaling species, unfolding cross-link domains function as the fundamental biochemical transducers of cytoskeletal deformation

Towards a dynamic understanding of cadherin-based mechanobiology

Cadherin-based cell-cell adhesions are a primary determinant of tissue structure. For several decades, it had been thought that the primary function of these ubiquitous structures was to resist external mechanical loads. Here we review recent evidence that cadherins also couple together the force-generating actomyosin cytoskeletons of neighbouring cells, serve as potent regulators of the actomyosin cytoskeleton, and activate diverse signalling pathways in response to applied load. In considering the force sensitivity of the molecular-scale processes that mediate these events, we propose a dynamic picture of the force-sensitive processes in cell-cell contacts. This quantitative and physical understanding of the mechanobiology of cadherin cell-cell junctions will aid endeavours to study the fundamental processes mediating the development and maintenance of tissue structure

The "Stressful" Life of Cell Adhesion Molecules: On the Mechanosensitivity of Integrin Adhesome.

Cells have evolved into complex sensory machines that communicate with their microenvironment via mechanochemical signaling. Extracellular mechanical cues trigger complex biochemical pathways in the cell, which regulate various cellular processes. Integrin-mediated focal adhesions (FAs) are large multiprotein complexes, also known as the integrin adhesome, that link the extracellular matrix (ECM) to the actin cytoskeleton, and are part of powerful intracellular machinery orchestrating mechanotransduction pathways. As forces are transmitted across FAs, individual proteins undergo structural and functional changes that involve a conversion of chemical to mechanical energy. The local composition of early adhesions likely defines the regional stress levels and determines the type of newly recruited proteins, which in turn modify the local stress distribution. Various approaches have been used for detecting and exploring molecular mechanisms through which FAs are spatiotemporally regulated, however, many aspects are yet to be understood. Current knowledge on the molecular mechanisms of mechanosensitivity in adhesion proteins is discussed herein along with important questions yet to be addressed, are discussed