Final Exam: May 4th, 9-11am DCL 3211

Last Class: Genetic Engineering 1. Restriction nucleases 2 Last Class: Genetic Engineering 1. Restriction nucleases 2. DNA labeling 3. Accurate Nucleic acid hybridization, Northern/Southern Blot, Microarray 4. Molecular cloning, DNA replication by vector 5. Gene sequencing 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

Resolution Calculation

Two ways to get contrast

Four Types of light microscopy Bright field, phase contrast, differential interference contrast, Dark-field microscopy

Fluorescence Microscope

Fluorescent Dyes

Blue: DNA; Green: microtubules; Red: centrimere Fluorescent image Blue: DNA; Green: microtubules; Red: centrimere

Immunofluorescence

Immunostaining

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 TEM DIC SEM

Summary of Visualizing Cells Transmitted lights Fluorescence Electron microscopy

Fluorescence Proteins and Live Cell Imaging

A Cell and A City

Track Molecular Motions

Jellyfish and GFP Osamu Shimomura discovered GFP in 1962 Shimomura O, et al, 1962. J. Cell. Comp. Physiol.

Dr. Douglas Prasher Prasher DC, et al. 1992. Gene

GFP and its labeling strategy Recombinant Gene Target Molecule GFP Transcription Translation Recombinant Protein GFP Target Molecule 510 nm 488 nm Wang et al. Annual Review in Biomedical Engineering, 2008

Martin Chalfie Chalfie M, et al. 1994. Science Inouye S, Tsuji FI. 1994. FEBS Lett.

Passive Applications of GFP GFP-microtubules

The Discovery of DsRed (discosoma, coral reef from Indo-pacific) 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 Letters Giepmans, BN. et al. 2006. Science

Multiple color visualization 2

Photoactivatable Fluorescence Proteins Lukyanov, KA. et al. 2005. Nature Rev Mol Cell Biology

Photoactivatable Fluorescence Proteins UV UV PA-FP PA-FP PS-FP PS-FP C UV Blue Dronpa Dronpa Wang et al. Annual Review in Biomedical Engineering, 2008

Photoactivatable Proteins Dronpa

Circularly Permutated Proteins cpFP B N C Inserted Domain Stimulator Domains for interaction A FP 144 145 cpFP C 145-238 1-144 Breakage Site N Wang et al. Annual Review in Biomedical Engineering, 2008

Calcium Oscillation in Heart

Technologies utilizing FPs Fluorescence Lifetime Microscopy (FLIM) Chromophore Assisted Laser Inactivation (CALI) Fluorescence Resonance Energy Transfer (FRET) Applications of FRET Biosensors

Fluorescence Lifetime Microscopy (FLIM) A Excitation Emission Frequency Domain Fluorescence Intensity Time  B Fluorescence Intensity Time Excitation Emission Time Domain Wang et al. Annual Review in Biomedical Engineering, 2008

Chromophore Assisted Laser Inactivation (CALI) FP FP ROS Target Molecule 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 A 527 nm 433 nm 476 nm EYFP ECFP EYFP ECFP 433 nm B 527 nm 433 nm 476 nm EYFP ECFP EYFP ECFP 433 nm C 433 nm 476 nm 433 nm FRET 527 nm EYFP ECFP ECFP EYFP Wang et al. Annual Review in Biomedical Engineering, 2008

FRET-Based Biosensors Ras and Rap1 Calcium Miyawaki, et al 1997, Nature Mochizuki, et al 2001, Nature Tyrosine Kinase Abl Ting, et al 2001, PNAS

Why Src? The first protein tyrosine kinase discovered. Src plays a significant role in: Cell polarity Adhesion Focal adhesion dynamics Lamellipodia formation Migration Mechanotransduction Cancer development

Design Strategy ECFP(1-227) SH2(from c-Src) Substrate EYFP Linker 433 nm Weak FRET 490 nm 433 nm Strong FRET 527 nm Src Activation Phosphatase

The Src kinase induces a FRET response of the Src reporter Emission spectra of the Src reporter -Src CFP YFP +Src Emission Intensity Arbitrary Units Wavelength (nm)

EGF induced FRET responses in HeLa Cells Ratio (CFP/YFP) 0.4 0.3

The Src reporter with CFP and YFP monomers ECFP(1-227) SH2(from c-Src) Substrate EYFP Linker A206K 0.5 0.35 A206K Zacharias, D. A. et al, Science, 2002

Construction of membrane-tethered Src reporter MGCIKSKRKDNLNDDE mCFP SH2 substrate mYFP Plasma Membrane mCFP mYFP GC Zacharias, D. A. et al, Science, 2002 0.5 0.3

Application of Mechanical Stimulation by Using Laser Tweezers Physical Principle of Laser Tweezers F1 F F2

Polystyrene beads were coated with fibronectin and positioned on cells Optic Lens Light Fibronectin Bead Cell Body (with Src reporters) F Integrins Actin

Polystyrene beads were coated with fibronectin and positioned on cells

Pulling Polylysine-coated beads did not have significant effects on FRET 0.55 0.35 FRET

Pulling Fibronectin-coated Beads induced a directed and long-range Src activation 0.44 0.22 Overlay Force

Pulling Fibronectin-coated Beads induced a directed propagation of Src activation 0.52 0.25

Cytochalasin D Treated The directed and long-range activation of Src is dependent on cytoskeleton-integrity 0.44 0.25 Nocodazole Treated 0.45 0.25 Cytochalasin D 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.