Analyses for Molecular Interactions in Living Cells

1. Analyses for Molecular Interactions in Living Cells Chi-Wu Chiang, Ph.D. Institute of Molecular Medicine College of Medicine National Cheng Kung University

2. Can these signaling networks be observed in living cells? Adopted from 2003-2004 catalog, Cell Signaling Technology, Inc.

3. Traditional methods to detect protein-protein interactions Yeast two-hybrid assay Mammalian two-hybrid assay Co-immunoprecipitation Affinity purification Co-localization by immunostaining

4. Seeing is believing

5. Fluorescent proteins Aequorea fluorescent protein (AFP) variants Green fluorescent protein (GFP) Enhanced green fluorescent protein (EGFP) Yellow fluorescent protein (YFP) Enhanced yellow fluorescent protein (EYFP) Cyan fluorescent protein (CFP) Enhanced cyan fluorescent protein (ECFP) From jellyfish Aequorea victoria

6. Fluorescent probes Red fluorescent protein (RFP) from Discosoma genus, Reef coral DsRED, DsRED2, DsRED-express, mRFP1 Tended to be tetrameric DsRED-monomer is a new RFP (45 amino acid substitutions of DsRED) without property of forming tetramer

7. Excitation Max nm Emission Max nm Protein 476 ECFP 439 484 510 EGFP 529 512 EYFP DsRED2 563 592 Excitation and Emission Spectra of Fluorescent Proteins

8. The structure of GFP GFP is an 11-stranded b-barrel threaded by an a-helix running up the axis of the cylinder . The chromophore is attached to the a-helix and is buried almost perfectly in the center of the cylinder, which has been called a b-can Ser65, Tyr66, and Gly67 are key residues to form chromophore

9. Creation of monomeric fluorescent probes by mutagenesis Interface disrupting mutation

10. Compare the wtGFP to EGFP Mutations in several residues, such as Ser65, Ala206, Leu221, Phe223 Increase in stability and brightness Dimerization at high concentrations was overcome

11. Compare EYFP to EGFP YFP was rationally designed on the basis of the GFP crystal structure to red-shift the absorbance and emission spectra with respect to EGFP and other green fluorescent variants YFP is much brighter than EGFP but is more sensitive to low pH and high halide concentrations

12. Factors affect the efficiency using the Fluorescence probes Photostability -----caused by photobleaching PH sensitivity -----most of the first-generation probes are acid sensitive Oligomerizing property ----using AFPs with Ala206Lys mutation ----using newest DsRED varients, such as mRFP1 Perturbation of intracellular conditions ----introduction of the fluorescent probes may perturb the cellular component of interest

13. Finding more fluorescent probes More mutated variants From Renilla mulleri Renilla mulleri GFP, with narrow excitation and emission spectrua From Anemonia sulcata dsFP593

14. Small molecule probes The biarsenical-tetracysteine system Based on: Membrane-permeant fluorescein derative with two As substitutents, named FIAsH Interaction of a single arsenic with a pair of thio groups is well known 1,2-ethanedithiol (EDT) as a 1,2-dithiol antidotes to prevent non-specific labeling in cells CCPGCC Nat. Rev. Mol. Cell Bio.3, 906-918 (2002).

15. The biarsenical-tetracysteine system Analogues of FIAsH have different excitation and emission spectra FIAsH ReAsH CHoXAsH

16. Current Methods in detecting protein-protein interactions in living cells Fluorescence resonance energy transfer (FRET) Bioluminescence resonance energy transfer (BRET) Biomolecular luminescence/fluorescence complementation Fluorescent protein complementation b-galactosidase/Luciferase complementation

17. FRET Fluorescence (or Forster) Resonance Energy Transfer Energy transfer between two fluorophores within distance on nanometer scales FRET is the radiationless transfer of excited-state energy from an initially excited donor to an acceptor Absorption Emission

18. Factors impact the rate of FRET Proper spectral overlap of the donor and acceptor The orientation factor, κ2, is given by κ2= (cosθT − 3cosθdcosθa) r6 r, the distance between the two fluorophores FRET is inversely proportional the distance between the fluorophores o < 10 nm or < 80 A apart

19. Compare a small fluorescent molecule-tagged FRET to a large fluorescent molecule-tagged FRET Larger fluorescent proteins Small fluorescent chemicals Free orientation and FRET is only limited by the distance factor Limited orientation and spatially restricted, however, FRET is sensitive to orientation, distance, and conformation of two interacting molecules

20. The best pair of fluorophores are CFP and YFP

21. Basic designs for analysis of molecular interactions by FRET CFP YFP

22. Applications for monitoring molecular interactions in Living cells • Intermolecular FRET-based indicators • G protein subunits dissociation • Transcription factor homo- and heterodimerization • Ras and Rap1 activation • b. Intramolecular FRET-based indicators • Caspase activation • Calcium flux sensor • Kinase activation

23. Applications for monitoring molecular interactions in Living cells Monitor protein-protein interaction Monitor intramolecular conformational change

24. FRET applications Calcium sensor (Cameleon) Kinase activation sensor M13, a peptide binds to calmodulin in calcium-dependent manner

25. Methods for FRET analysis in Living cells Analyses by Fluorescence Spectrophotometry Cells, treated or not treated, suspension In PBS Spectrofluorimeter measurement

26. Spectrofluorimeter measurement Featured with Excitation light source, arc lamp, in UV or visible Emission detector, such as photon counter or charged coupled device (CCD) Scan full spectrum periodically, using filter sets and crystal counter< 1 second

27. Detecting 14-3-3 dimerization in Living cells 430nm EYFP FRET ECFP 530nm 14-3-3 14-3-3 14-3-3

28. Imaging molecular interactions in single cells using FRET ECFP EYFP 14-3-3 Living cells in medium (no phenol red) With or without stimuli 14-3-3 Fluorescence microscopy or Confocal laser scanning microscopy

29. FRET measured by Inverted Fluorescence Microscopy Featured with • Fluorescence illumination (HBO100 or HBO50) • Fluorescence optics (Plan-Neofluar 10x, 20x, 40x oil, 63x oil, 100x oil) • 4 FRET filter cubes (CFP, YFP, FRET, Bleach), highly motorized • Digital camera • Computer • FRET image analysis software • Microscope Setup software

30. Laser scanning confocal microscopy

31. Spatio-temporal images of growth-factor-induced activation of Ras and Rap1 Nature 411, 1065 - 1068 (2001); NAOKI MOCHIZUKI et al. Ras, small G protein Activation of RAS by GTP binding Regulated by guanine nucleotide exchange factor (GEF), the activator And by GTPase activating protein (GAP), the inactivator Spatial: of the space Temporal: of the time

32. Ras activation near plasma membrane, whereas Rap1 activation near the perinuclear region

33. Bioluminescence Resonance Energy Transfer BRET Naturally, In jellyfish, blue-light emitting aequorin can promote GFP to excite green light In the current BRET system, In the presence of a substrate, bioluminescence from the luciferase excites the acceptor fluorophore 530 nm EYFP A 480 nm Rluc B Substate for Rluc Similar to FRET but avoid the photon excitation damage

34. A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins Proc. Natl. Acad. Sci. USA. 1999 January 5; 96 (1): 151–156 By Yao Xu et al. Whether bacterial circadian proteins form dimers to function?

35. Biomolecular fluorescence complementation (BiFC) Origin from classical studies of intragenic complementation of the lacZ locus of E. coli, demonstrating that fragments of b-galactosidase that have no enzyme activity can associate spontaneously to generate an active complex GFP fragments fused to peptide sequences capable of producing an antiparallel coiled coil produced flurescent complexes in vitro and in E. coli

36. Biomolecular luminescence/fluorescence complementation Fluorescence complementation Cut GFPN GFPC GFP (155-238) (1-154) Protein A Protein B If Yes A interacts with B?

37. bZIP:basic region-leucine zipper bZIP family members, such as FOS and JUN

38. Biomolecular luminescence complementation Luciferase complementation A interacts with B A B A B C Luc N Luc C Luc N Luc light luciferin

39. PNAS 101:12288-12293, 2004 The kinase mTOR is inhibited by FKBP in a rapamycin-dependent manner FRB: rapamycin-binding domain of the mTOR fused to NLuc FKBP:FK506-binding protein 12 fused to CLuc

40. From Cell Biology to application in Biomedicine by FRET and BRET Uncover the molecular interactions in living cells and living animals in a spatial and temporal manner Molecular diagnosis Screening drugs in a high throughput way

41. References Xu, Y. et al. A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins. Proc. Natl. Acad. Sci. USA 96, 151-156 (1999) Mochizuki, N. et al. Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature 411, 1065-1068 (2001) Jin Zhang et al. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Bio.3, 906-918 (2002). Hu, C. –D., Chinenov, Y., and Kerppola, T. K. Visualization of interactions among bZIP and Rel family proteins in living cells using biomolecular fluorescence complementation. Mole. Cell 9, 789-798, 2002 Jares-Erijman,E. A., & Jovin, T. M. FRET imaging. Nat. Biotechnol. 21, 1387-1395 (2003) Miyawaki, A. Visualization of the spatial and temporal dynamics of intracellular signaling. Develop. Cell 4, 295-305 (2003) Kathryn E. Luker et al. Kinetics of regulated protein–protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. PNAS 101, 12288-12293 (2004)