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Rigidity sensing at the leading edge through α v β 3 integrins and RPTP α

Rigidity sensing at the leading edge through α v β 3 integrins and RPTP α. Guoying Jian, Angela H. Huang, Yunfei Cai, Monica Tanase, and Michael P. Sheetz Department of Biological Sciences at Columbia University. Embryogenesis and stem cell differentiation.

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Rigidity sensing at the leading edge through α v β 3 integrins and RPTP α

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  1. Rigidity sensing at the leading edge through αvβ3 integrins and RPTPα Guoying Jian, Angela H. Huang, Yunfei Cai, Monica Tanase, and Michael P. Sheetz Department of Biological Sciences at Columbia University

  2. Embryogenesis and stem cell differentiation mechanical properties of extracellular matrix (ECM) genes hormones cell behavior chemical signals intercellular interactions

  3. Elasticity of ECM determines fibroblasts’ shape rigid substrate soft substrate

  4. Shape of fibroblasts determines their fate DEATH!

  5. Idealized Model of a Fibroblast on a 2D ECM nucleus cell membrane stress fibers (actin microfilaments) ECM focal adhesion complexes (FACs) ECM protein

  6. ARG GLY ASP fibronectin Various protein actin Focal Adhesion Complex • Integrins interact with specific ligands in the ECM. • FACs can be viewed as organelles that serve as structural scaffolds. β3 αv outside inside cell membrane integrin

  7. Compartmentalization of biochemical activities • Force exposes activation sites in the focal adhesion complex. • Cells use solid-state biochemistry to increase efficiency of chemical reactions by no longer being limited by diffusion rates.

  8. What molecules in the FAC are essential for elasticity-induced change in cell morphology? ARG GLY ASP fibronectin β3 αv Various protein

  9. RPTPα is essential for sensing of fibronectin elasticity WT WT RPTPα-/- RPTPα-/- rigid substrate soft substrate

  10. RPTPα • A receptor-like protein tyrosine phosphatase. • Forms a complex with αvβ3 at the leading edge of the fibroblast lamellipodia.

  11. Can RPTPα sense matrix rigidity through αvβ3 integrins that are not physically attached to it? fibroblast FAC (αvβ3 clusters) RPTPα fibronectin

  12. Laser trap assay fibroblast laminin Fmigration Flaser

  13. Laser trap assay • The rigidity of beads was proportional to the laser power: • ‘soft’ = .02 pN/nm • ‘rigid’ = .18 pN/nm • Assay was performed at the leading edge and 2 μm from the leading edge. • Cells should form stronger bonds with fibronectin with the ‘rigid’ laser trap.

  14. Possible outcomes of laser trap assay Cells can sense rigidity at the leading edge but not 2 μm away RPTPα must be physically linked to αvβ3 Cells can sense rigidity both at the leading edge and 2 μm away αvβ3 can chemically signal RPTPα

  15. Results of laser assay

  16. Major conclusions • RPTPα is critical for the αvβ3 mediated fibronectin matrix rigidity sensing process in fibroblasts. • RPTPα must be physically linked to αvβ3. • Fibronectin rigidity sensing occurs at the leading edge. • A rapid application of 10 pN can elicit the rigidity response.

  17. Larger implications • Many important biochemical cascades are regulated by mechanical cues. • In order to create human tissues in vitro, we must dynamically mimic the in vivo environment. • Stem cell differentiation is unlikely to be guided only by chemical and biological cues.

  18. Questions?

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