Download Visualizing_the_mechanical_activation_of_Src

Yingxiao Wang et al.
Presented by Matthew Loper
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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
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Create Src reporter complex
◦ Src specific
◦ Allows real-time visualization of Src activity in live
cells
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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.
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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
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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
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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
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Added fibronectincoated beads to human
umbilical vein endothelial
cells (HUVECs)
◦ Binds to integrins causing
coupling to cytoskeleton
◦ Applied 300 pN force with
optical tweezers
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Immediate distal
and slower wave
propagation of
Src activation
◦ Wave propagated
at a speed of 18.1
+/- 1.7 nm s-1
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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.”