The extracellular matrix (ECM) is the mechanical scaffold that determines the elasticity and tensile strength of organs and tissues and finely regulates their development by controlling cell adhesion and migration. The ECM is formed by modular proteins and polysaccharides knitted together by self-assembly and through interactions with the cell adhesion receptors of a variety of cell types. Mechanical forces play important roles in ECM assembly and function. ECM fibrils are pre-stretched up to four times their resting length and are thought to translate mechanical forces into biological signals through cryptic binding sites that are exposed by mechanical unfolding. However, nothing is known about the molecular basis of the mechanical extensibility and mechanical signaling of the molecules composing the ECM. The long term aim of this proposal is to determine the force driven conformational changes that allow the ECM molecules to extend under an applied force and turn this force into a cellular signal. Towards this aim we will combine cellular and molecular biological techniques together with state of the art force spectroscopy (AFM) techniques and GFP based fluorescence imaging techniques, capable of observing force driven conformational changes in single molecules. During our first grant period we propose to focus on fibronectin and heparin, abundant molecules which are thought to play crucial mechanical roles in the ECM and have a central function in general animal physiology and pathology. We will use force spectroscopy to examine the mechanical unfolding of native fibronectin and of selected fibronectin modules that are known to play important mechanical roles in matrix assembly. We will engineer recombinant fibronectin proteins designed with specific mechanical properties that then will be transfected into CHO cells for fibronectin secretion and matrix assembly. We will use novel GFP based energy transfer probes in order to measure the resting force per molecule and to determine if unfolding occurs in vivo. We will also use force spectroscopy to detect force driven conformations in matrix glycosaminoglycans, in particular of heparin. We will use GFP probes to examine the binding of fibronectin modules to heparin under a stretching force. Mechanical forces play a critical role in ECM assembly and function. The proposed experiments will investigate, for the first time, the molecular basis of matrix mechanics. The findings may be of great importance for organ and tissue engineering and wound repair.
| Garcia-Manyes, Sergi; Giganti, David; Badilla, Carmen L et al. (2016) Single-molecule Force Spectroscopy Predicts a Misfolded, Domain-swapped Conformation in human ?D-Crystallin Protein. J Biol Chem 291:4226-35 |
| Echelman, Daniel J; Alegre-Cebollada, Jorge; Badilla, Carmen L et al. (2016) CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks. Proc Natl Acad Sci U S A 113:2490-5 |
| Valle-Orero, Jessica; Eckels, Edward C; Stirnemann, Guillaume et al. (2015) The elastic free energy of a tandem modular protein under force. Biochem Biophys Res Commun 460:434-8 |
| Chen, Hu; Yuan, Guohua; Winardhi, Ricksen S et al. (2015) Dynamics of equilibrium folding and unfolding transitions of titin immunoglobulin domain under constant forces. J Am Chem Soc 137:3540-6 |
| Saqlain, Farees; Popa, Ionel; Fernández, Julio M et al. (2015) A novel strategy for utilizing voice coil servoactuators in tensile tests of low volume protein hydrogels. Macromol Mater Eng 300:369-376 |
| Perez-Jimenez, Raul; Alonso-Caballero, Alvaro; Berkovich, Ronen et al. (2014) Probing the effect of force on HIV-1 receptor CD4. ACS Nano 8:10313-20 |
| Alegre-Cebollada, Jorge; Kosuri, Pallav; Giganti, David et al. (2014) S-glutathionylation of cryptic cysteines enhances titin elasticity by blocking protein folding. Cell 156:1235-1246 |
| Solsona, Carles; Kahn, Thomas B; Badilla, Carmen L et al. (2014) Altered thiol chemistry in human amyotrophic lateral sclerosis-linked mutants of superoxide dismutase 1. J Biol Chem 289:26722-32 |
| Giganti, David; Alegre-Cebollada, Jorge; Urresti, Saioa et al. (2013) Conformational plasticity of the essential membrane-associated mannosyltransferase PimA from mycobacteria. J Biol Chem 288:29797-808 |
| Popa, Ionel; Kosuri, Pallav; Alegre-Cebollada, Jorge et al. (2013) Force dependency of biochemical reactions measured by single-molecule force-clamp spectroscopy. Nat Protoc 8:1261-76 |
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