Download ppt - Yingxiao Wang et al. Presented by Matthew Loper. Investigate cellular response to mechanical stimuli ◦ How mechanical stimuli are transmitted into biochemical

Yingxiao Wang et al.Presented by Matthew Loper

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

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

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

Yang et al.

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

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

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

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

Immediate distal and slower wave propagation of Src activation◦ Wave propagated

at a speed of 18.1 +/- 1.7 nm s-1

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

“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.”