Visualizing the mechanical activation of Src

1. Visualizing the mechanical activation of Src Yingxiao Wang et al. Presented by Matthew Loper

2. Study Goals • Investigate cellular response to mechanical stimuli • How mechanical stimuli are transmitted into biochemical signals through the cytoskeleton • Src known to regulate integrin-cytoskeleton interaction • Need a way to study Src response to mechanical stimuli

3. Study Outline • Create Src reporter complex • Src specific • Allows real-time visualization of Src activity in live cells • Attach beads to cell cytoskeleton • Apply a force to beads with laser-tweezer • Confirm that cytoskeleton is necessary for signal transduction

4. Flourescent Resonance Energy Transfer http://en.wikipedia.org/wiki/Image:FRET.PNG

5. Reporter Complex Yang et al.

6. Reporter Complex • Reporter highly specific to Src • Yes, FAK, EGFR, Abl, Jak2, ERK1 show ~2% CFP/YFP ratio change • Fyn, close cousin to Src, shows ~10% change

7. Reporter Complex • Test SH2-phosporylated substrate interaction by transfecting HeLa cells with Src, stimulating with EGF • Mutation of either Tyr 662 or 664 led to no FRET response • Mutation of Arg 175 to Val eliminated FRET • Phosphorylation of Tyr still occurs if just one Tyr is mutated but binding doesn’t • Neighboring amino acids are important for SH2 binding

8. Reporter Complex • CFP and YFP can form anti-parallel dimer • Introduced A206K mutations • No effect on spectral properties • Better response to Src • FRET response reversible by EGF washout

9. Force application • Added fibronectin-coated beads to human umbilical vein endothelial cells (HUVECs) • Binds to integrins causing coupling to cytoskeleton • Applied 300 pN force with optical tweezers

10. Force application • Immediate distal and slower wave propagation of Src activation • Wave propagated at a speed of 18.1 +/- 1.7 nm s-1

11. Cytoskeletal dependence • Beads coated with polylysine do not integrate into cytoskeleton and do not induce FRET response • Destruction of actin with cytochalasin D or microtubules with nocodazole blocked long range but not local FRET response

12. Force response direction “Applied force transmitted through cytoskeleton network to distal locations to activate Src…This directionality may release tension at desired locations and rearrange intracellular stress distributions, thus serving as a feedback mechanism for the cell to adapt to new mechanical environments.”

13. Proposed feedback mechanism

14. Questions and answers