1. Last Class: Genetic Engineering 1. DNA labeling 2. 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

2. Visualizing Cells

3. Resolving Power

4. Light Microscope

5. Interference between light waves

6. Two ways to get contrast

7. Resolution Calculation

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

9. Immunostaining

10. Fluorescence Microscope

11. Fluorescent Dyes

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

13. Immunofluorescence

14. Confocal Fluorescence Microscopy

15. The difference between conventional and confocal microscopes

16. 3D reconstruction from confocal images

17. Transmission Electron microscopy (TEM, resolution 0.002 nm)

18. A root-tip cell under electromicroscopy

19. The scanning electron microscope (SEM)

20. Stereocillia from a hair cell SEM TEM DIC

21. Summary of Visualizing Cells 1.Transmitted lights 2.Fluorescence 3.Electron microscopy

22. Fluorescent Proteins and Live Cell Imaging

23. A Cell and A City

24. Track Molecular Motions

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

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

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

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

29. Passive Applications of GFP GFP-microtubules

30. Sergey A. Lukyanov The Discovery of DsRed (discosoma, coral reef from Indo-pacific) Matz MV, et al. 1999. Nature Biotech.

31. Roger Y. Tsien Tsien RY. 1998, Ann Rev Biochem. Tsien RY. 2005, FEBS Letters Giepmans, BN. et al. 2006. Science

32. Multiple color visualization 2

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

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

35. Photo-activatable Proteins Dronpa

36. A FP 144145 cpFP C 145-238 1-144 Breakage Site FP N C N cpFP B N C N C C Inserted Domain Stimulator Domains for interaction Circularly Permutated Proteins Wang et al. Annual Review in Biomedical Engineering, 2008

37. Calcium Oscillation in Heart

39. 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

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

41. FP Target Molecule ROS FP Chromophore Assisted Laser Inactivation (CALI) Wang et al. Annual Review in Biomedical Engineering, 2008

42. Spy on their Actions! FRET

43. The Principle of Fluorescence Resonance Energy Transfer (FRET) When the fluorophores are far apart: No FRET ExcitationEmission When fluorophores are close: FRET occurs ExcitationEmission FRET

44. The General Design of FRET-based Fluorescent Probes ECFP EYFP ECFP A 433 nm 476 nm 433 nm 527 nm FRET 476 nm 433 nm 527 nm EYFP 433 nm C B 476 nm 433 nm ECFPEYFP ECFP 527 nm ECFP EYFP Wang et al. Annual Review in Biomedical Engineering, 2008

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

46. 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.

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

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

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

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

51. Construction of membrane-tethered Src reporter MGCIKSKRKDNLNDDE mCFPSH2substratemYFP mCFPmYFP GC Plasma Membrane 0.5 0.3 Zacharias, D. A. et al, Science, 2002

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

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

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

55. Polystyrene beads were coated with fibronectin and positioned on cells

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

57. 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

58. 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.

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