Program Project Goals

Cell-matrix adhesions between the cell and the extracellular matrix are mechanosensitive multiprotein assemblies that transmit force across the cell membrane and regulate biochemical signals in response to changes in the mechanical environment. These combined functions in force transduction, signaling and mechanosensing are crucial for cell behaviors in development, homeostasis and disease.

Mechanotransduction at cell-matrix adhesions has been tackled primarily at the level of individual proteins, provided details of force-induced activation (possibly involving unfolding and exposure of binding sites under force), binding partners, dissociation constants, rough spatial localization, and atomic structures of domains. Much less is known about the interactions between these proteins in situ, how they vary between different types of adhesions, how they are regulated and how they determine signaling outputs. The major challenge is to establish approaches that can capture adhesions at high structural detail in defined functional states while components working as a three-dimensional ensemble within a living cellular.  Clearly, insights across multiple structural levels are acutely needed to establish the molecular bases of mechanotransduction.

Towards this end and under the auspices of this Program Project, we are combining cutting edge high-resolution light and electron microscopy imaging modalities with state-of-the-art computational techniques and a seamless integration with biological, biochemical and biophysical approaches.  The central hypothesis of this Program Project is that cells respond to forces through distinct structural adaptations within their mechanosensitive hubs. These hubs  form, mature and disassemble continuously, processes driven by physical forces that originate from endogenous myosin activity or external forces from the extracellular matrix.  However derived form our hypothesis, we expect that the spectrum of adhesion types results from a spatially and temporally tightly regulated force field, which is dynamically adjusted during migration and also to the extracellular mechanical environment.

Here, we propose to establish an approach for systematic and quantitative derivation of structure-function correlates in physiologically relevant environments.  We will be generating and linking data together that span five orders of magnitude (Ångstrom to tens of microns) in length scale and five orders of magnitude (milliseconds to minutes) in time scale. Therefore, by studying adhesions during cell migration, we will detail how force variation are dynamically altering adhesions’ structures, composition, and signaling.

Our  Workflow: The approach includes generating and linking data that span five orders of magnitude using a three pronged approach, working our way from in-vitro reconstituted system using biochemically-defined assemblies, generating molecular models by tying information from high-resolution techniques  the to unbiased identification of these assemblies in a whole cell environment at defined functional states.

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For example: (A) qFSM fluorescence used to localize the regions of interest (B-D, bar in D = 2μm). This correlation approach also allows correlating dynamic information obtained by live-cell imaging, and (B) the underlying structure. (E) Surface representation of a cryo-tomogram from a region in the lamellipodium (blue in D). (F). Surface representation of a cryo-tomogram from a region in the lamella (red in D). The actin-network morphology appears markedly different. Ribosomes, clathrin, actin filaments, microtubules and Arp2/3-mediated actin branches can be readily identified in the reconstructions. We use the structural information of these assemblies previously obtained from reconstituted systems to aid analysis of the extracted motifs (adapted from  Hanein and Horwitz, Curr. Opin. Cell Biol. 2012, PubMed ).

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GM 

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