Last Class: Genetic Engineering
1. DNA labeling2. Accurate Nucleic acid hybridization,
Northern/Southern Blot, Microarray 3. Gene sequencing
4. Restriction nucleases 5. Molecular cloning, DNA replication by vector
6. polymerase chain reaction 7. Monitoring Gene expression
8. the application of genetic engineering: Detect proteins and protein-protein interactions, library
screening, gene mutation
• Visualizing Cells
Resolving Power
Light Microscope
Interference between light waves
Two ways to get contrast
Resolution Calculation
Four Types of light microscopyBright field, phase contrast, differential
interference contrast, Dark-field microscopy
Immunostaining
Fluorescence Microscope
Fluorescent Dyes
Fluorescent imageBlue: DNA; Green: microtubules; Red: centrimere
Immunofluorescence
Confocal Fluorescence Microscopy
The difference between conventional and confocal microscopes
3D reconstruction from confocal images
Transmission Electron microscopy (TEM, resolution 0.002 nm)
A root-tip cell under electromicroscopy
The scanning electron microscope (SEM)
Stereocillia from a hair cell
SEM
TEMDIC
Summary of Visualizing Cells
1. Transmitted lights2. Fluorescence
3. Electron microscopy
Fluorescent Proteins and Live Cell Imaging
A Cell and A City
Track Molecular Motions
Jellyfish and GFP
Osamu Shimomuradiscovered GFP in 1962
Shimomura O, et al, 1962. J. Cell. Comp. Physiol.
Dr. Douglas Prasher
Prasher DC, et al. 1992. Gene
Target Molecule GFP
A B
Transcription Translation
488 nm510 nm
Recombinant Gene
Recombinant Protein
GFPTarget Molecule
GFP and its labeling strategy
Wang et al. Annual Review in Biomedical Engineering, 2008
Martin Chalfie
Chalfie M, et al. 1994. ScienceInouye S, Tsuji FI. 1994. FEBS Lett.
Passive Applications of GFP
GFP-microtubules
Sergey A. Lukyanov The Discovery of DsRed (discosoma, coral reef from
Indo-pacific)
Matz MV, et al. 1999. Nature Biotech.
Roger Y. Tsien
Tsien RY. 1998, Ann Rev Biochem.Tsien RY. 2005, FEBS LettersGiepmans, BN. et al. 2006. Science
Multiple color visualization
2
Photoactivatable Fluorescence Proteins
Lukyanov, KA. et al. 2005. Nature Rev Mol Cell Biology
A BUV
PA
-FP
PA
-FP
UV
PS
-FP
PS
-FP
C UV
Dro
np
a
Blue D
ron
pa
Photoactivatable Fluorescence Proteins
Wang et al. Annual Review in Biomedical Engineering, 2008
Photo-activatable ProteinsDronpa
A
FP
144 145
cpF
P
CcpFP
145-238 1-144
Breakage Site
FP
N C
N cpF
P
cpF
P
cpF
P
cpF
P
B
NC
NC
C
Inserted Domain
Stimulator
Stimulator
Domains for interaction
Circularly Permutated Proteins
Wang et al. Annual Review in Biomedical Engineering, 2008
Calcium Oscillation in Heart
Technologies utilizing FPs
1. Fluorescence Lifetime Microscopy (FLIM)2. Chromophore Assisted Laser Inactivation (CALI)
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
Overlay
Force
0.44
0.22