Transcript Analyses for Molecular Interactions in Living Cells

Analyses for Molecular
Interactions in Living Cells
Chi-Wu Chiang, Ph.D.
Institute of Molecular Medicine
College of Medicine
National Cheng Kung University
Can these signaling networks be observed in
living cells?
Adopted from 2003-2004 catalog, Cell Signaling Technology, Inc.
Traditional methods to detect protein-protein
interactions
Yeast two-hybrid assay
Mammalian two-hybrid assay
Co-immunoprecipitation
Affinity purification
Co-localization by immunostaining
Seeing is believing
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
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
Excitation and Emission Spectra of Fluorescent Proteins
Protein
ECFP
EGFP
EYFP
DsRED2
Excitation
Max nm
Emission
Max nm
439
484
476
512
529
563
592
510
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
Creation of monomeric fluorescent
probes by mutagenesis
Interface disrupting mutation
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
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
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
Finding more fluorescent probes
More mutated variants
From Renilla mulleri
Renilla mulleri GFP, with narrow excitation and emission
spectrua
From Anemonia sulcata
dsFP593
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).
The biarsenical-tetracysteine system
Analogues of FIAsH have different excitation and
emission spectra
CHoXAsH
FIAsH
ReAsH
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
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
Emission
Absorption
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
Compare a small fluorescent moleculetagged FRET to a large fluorescent
molecule-tagged FRET
Small fluorescent chemicals
Free orientation and FRET
is only limited by the
distance factor
Larger fluorescent proteins
Limited orientation and spatially
restricted, however, FRET is
sensitive to orientation, distance,
and conformation of two
interacting molecules
The best pair of fluorophores are
CFP and YFP
Basic designs for analysis of molecular
interactions by FRET
CFP
YFP
Applications for monitoring molecular interactions
in Living cells
a. 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
Applications for monitoring molecular interactions
in Living cells
Monitor protein-protein interaction
Monitor intramolecular conformational change
FRET applications
Calcium sensor (Cameleon)
M13, a peptide binds to calmodulin in
calcium-dependent manner
Kinase activation sensor
Methods for FRET analysis in Living cells
Analyses by
Fluorescence Spectrophotometry
Cells, treated or not treated, suspension
In PBS
Spectrofluorimeter measurement
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
Detecting 14-3-3 dimerization in Living cells
430nm
EYFP
FRET
ECFP
530nm
14-3-3
14-3-3
14-3-3
FRET1433ECFP/1433EYFP
160000
1433ECFP1
1433ECFP0.5/1433EYFP
2.5
1433ECFP0.75/1433EYF
P2.2
1433ECFP1/1433EYFP2
120000
100000
80000
60000
1433EYFP2
40000
20000
pcDNA3
Wavelength
548
540
532
524
516
508
500
492
484
476
468
0
460
Emission intensity
140000
Imaging molecular interactions in single cells
using FRET
ECFP
14-3-3 EYFP
14-3-3
Living cells in medium (no phenol red)
With or without stimuli
Fluorescence microscopy or
Confocal laser scanning microscopy
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
Laser scanning confocal microscopy
(1) optical
fibers
(7,8,9)
secondary
dichroic
(2)
collimators
beam
splitters
(3) beam
combination
(10) pinhole
diaphragm
(4) main
dichroic
beam
(11)
emission
filters
splitter
(12) photomultiplier
tubes
(5) scanner
mirrors
(13) neutral
filters
(6) scanning
lens
(14) monitor
diode
Spatio-temporal images of growth-factorinduced activation of Ras and Rap1
NAOKI MOCHIZUKI et al.
Nature 411, 1065 - 1068 (2001);
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
Ras activation near plasma membrane, whereas Rap1
activation near the perinuclear region
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
B
Rluc
Substate for Rluc
Similar to FRET but avoid the photon excitation damage
A bioluminescence resonance
energy transfer (BRET) system:
Application to interacting circadian
clock proteins
By Yao Xu et al.
Whether bacterial
circadian proteins
form dimers to
function?
Proc. Natl. Acad. Sci. USA. 1999 January 5; 96 (1): 151–156
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
Biomolecular luminescence/fluorescence
complementation
Fluorescence complementation
Cut
GFP
GFPN GFPC
(1-154) (155-238)
A interacts with B? If Yes
Protein A
Protein B
bZIP:basic region-leucine zipper
bZIP family members, such as FOS and JUN
Biomolecular luminescence complementation
Luciferase complementation
A interacts with B
A
B
N Luc C Luc
A B
N LucC Luc
luciferin
light
PNAS 101:12288-12293, 2004
The kinase mTOR is inhibited by FKBP in a rapamycindependent manner
FRB: rapamycin-binding domain of the mTOR fused to NLuc
FKBP:FK506-binding protein 12 fused to CLuc
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
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)