1. Yingxiao Wang et al. Presented by Matthew Loper

2.  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.  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. http://en.wikipedia.org/wiki/Image:FRET.PNG

5. Yang et al.

6.  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.  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.  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.  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.  Immediate distal and slower wave propagation of Src activation ◦ Wave propagated at a speed of 18.1 +/- 1.7 nm s -1

11.  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. “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.”