This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Last Class: Genetic Engineering
1. DNA labeling2. Accurate Nucleic acid hybridization,
3. Fluorescence Resonance Energy Transfer (FRET)4. Applications of FRET Biosensors
B
Flu
ore
scen
ceIn
ten
sity
Time
Excitation Emission
Time DomainA
Excitation Emission
Frequency Domain
Flu
ore
scen
ceIn
ten
sity
Time
Wang et al. Annual Review in Biomedical Engineering, 2008
Fluorescence Lifetime Microscopy (FLIM)
FP
Target Molecule
ROS FP
Chromophore Assisted Laser Inactivation (CALI)
Wang et al. Annual Review in Biomedical Engineering, 2008
Spy on their Actions!
FRET
The Principle of Fluorescence Resonance Energy Transfer (FRET)
When the fluorophores are far apart: No FRET
Excitation Emission
When fluorophores are close: FRET occurs
Excitation Emission
FRET
The General Design of FRET-based Fluorescent Probes
EC
FP
EYFP
EYFP
ECFPECFP
A
433 nm
476 nm433 nm
527 nm
FRET
476 nm 433 nm527 nm
EYFP
433 nm
C
B476 nm433 nm
433 nm
ECFP EYFP
EYFP
ECFP
527 nmE
CF
P
EY
FP
Wang et al. Annual Review in Biomedical Engineering, 2008
FRET-Based BiosensorsCalcium Ras and Rap1
Miyawaki, et al 1997, Nature
Mochizuki, et al 2001, Nature
Ting, et al 2001, PNAS
Tyrosine Kinase Abl
Why Src?
Src plays a significant role in:
• Cell polarity
• Adhesion
• Focal adhesion dynamics
• Lamellipodia formation
• Migration
• Mechanotransduction
• Cancer development
The first protein tyrosine kinase discovered.
Design Strategy
Weak FRET
Phosphatase
Strong FRET
433 nm
527 nm
433 nm
490 nm
ECFP(1-227) SH2(from c-Src) Substrate EYFPLinker
Src Activation
Em
iss i
on
Inte
ns i
ty
Arb
itra
ry U
nit
s
Wavelength (nm)
-Src
+Src
Emission spectra of the Src reporter
The Src kinase induces a FRET response of the Src reporter
CFP YFP
EGF induced FRET responses in HeLa Cells
Rat
io (
CF
P/Y
FP
)
0.4
0.3
The Src reporter with CFP and YFP monomers
ECFP(1-227) SH2(from c-Src) Substrate EYFPLinker
A206K A206K
A206K
A206K
Zacharias, D. A. et al, Science, 2002
0.5
0.35
Construction of membrane-tethered Src reporter
MGCIKSKRKDNLNDDE mCFP SH2 substrate mYFP
mCFP mYFPGC
Plasma Membrane
0.5
0.3
Zacharias, D. A. et al, Science, 2002
Application of Mechanical Stimulation by Using Laser Tweezers
F1
F2
F
Physical Principle of Laser Tweezers
Pulling Polylysine-coated beads did not have significant effects on FRET
0.55
0.35
FRET
Bead
Polystyrene beads were coated with fibronectin and positioned on cells
Cell Body
(with Src reporters)
F
Optic Lens
Light
Integrins
Fibronectin
Actin
Polystyrene beads were coated with fibronectin and positioned on cells
Pulling Fibronectin-coated Beads induced a directed propagation of Src activation
0.52
0.25
The directed and long-range activation of Src is dependent on cytoskeleton-integrity
0.45
0.25
Cytochalasin D Treated
0.44
0.25
Nocodazole Treated
Summary
FRET-based biosensors can allow the detection of various biochemical signals with high tempo-spatial resolution in live cells, including the signal transduction in response to mechanical stimulation.
Pulling Fibronectin-coated Beads induced a directed and long-range Src activation