This document summarizes fluorescent biosensors for intracellular signaling molecules. It describes various types of biosensors including GFP-based sensors that change brightness or use FRET to report on analytes like pH, calcium, cyclic nucleotides. Translocation sensors are described that detect protein movement from cytosol to membranes or nuclei in response to signals. Photoconvertible proteins like Dendra are presented as tools to visualize protein degradation. The mechanisms and applications of FRET-based, single fluorescent protein, and circularly permuted fluorescent protein sensors are outlined. Examples demonstrate calcium, kinase, and redox signaling biosensors. Imaging with HyPer revealed hydrogen peroxide production occurs locally near receptor tyrosine kinases and does not diffuse far within cells. A dual
5. Translocation Biosensors – Tools for Signaling
Pathway Analysis
Translocation of a proteins from the cytosol to receptor
complexes at the plasma membrane:
- kinase receptors activation
Akt3-GFP in untreated cells
(upper) and in activated cells
(lower).
Akt3 is a serine/threonine kinase
that plays a key in regulating cell
survival, insulin signaling,
angiogenesis and tumor formation.
6. Translocation Biosensors – Tools for Signaling
Pathway Analysis
Translocation of a proteins from cytosol to nucleus:
- transcription factor activation
- caspase activation (apoptose)
Translocation of HIF-1a – GFP fusion protein from the
cytosol (a) to the nucleus (b) in response to PDT.
Photodynamic therapy-mediated hypoxia-independent activation of the
hypoxia inducible factor 1a (HIF-1a) pathway.
7. Dendra - a monomeric mutant of greento-red photoconvertible FP from
Dendronephthya
Dendronephthya sp.
300
400
500
600
Wavelength, nm
Gurskaya et al. Nat. Biotech. 2006
700
8. Visualization of target protein degradation using
Dendra2 photoactivatable protein
Green fluorescence
intensity depends on
both protein synthesis
and degradation
Expression of
Dendra2-tagged protein
Dendra2 photoconversion
in whole cell
Time-lapse
Red fluorescence
intensity depends only
on protein degradation
t0
t1 ...
tn
Quantification
Red 1
fluorescence
0.5
1/2
Time
9. FRET-based biosensors
Molecular sensors (pH, Ca2+, cAMP, kinases, redox, H2O2, etc.) on the base of FRET or
brightness changes.
(a) Biosensors based on a ligand-dependent protein–protein interaction. Cameleons (based
on a fusion of calmodulin and M13) and GTPase biosensors (based on a fusion of the GTPase
and its effector) fall into this category. (b) Post-translational modification biosensor (i.e., for
a kinase). (c) Protease substrate-type biosensor. (d) Biosensor based on conformational
change of a single protein.
11. Genetically encoded fluorescent sensor of ERK activity
The MAPK family is a class of serine/threonine kinases that includes the ERK, p38, and JNK
subfamilies. Members of the ERK subfamily are essential for numerous, diverse physiological
functions, including cellular differentiation, proliferation and neuronal plasticity, and their
activities are up-regulated in many cancers.
Fluorescence lifetime images of HEK293 cells
transfected with EKARcyto before and after (12 min)
addition of EGF (100 ng/ml).
12. Tyrosine Phosphorylation of Cytoplasmic Domain of
EGFR Monitored by FRET
Lippincott-Schwartz, Snapp and Kenworthy Nat. Rev. Mol. Cell Biol. 2:444, 2001
13. Tyrosine Phosphorylation of Cytoplasmic Domain of
EGFR Monitored by FRET
Verveer et al., Science 290:1567, 2000
14. Single FP-based biosensors
Molecular sensors (pH, Ca2+, cAMP, kinases, redox, H2O2, etc.) on the base of FRET or
brightness changes.
(a) Single FP biosensor based on intrinsic (i.e., pH) sensitivity. (b) Single FP biosensor
based on the extrinsic sensitivity (i.e., Ca2+) of a genetically fused domain (i.e.,
calmodulin). (c) GCaMP X-ray crystal structure.45 Linker regions that were not visible
in the crystal structure are represented with dashed lines.
16. FP biosensor structure and imaging
Molecular sensors (pH, Ca2+, cAMP, kinases, redox, H2O2, etc.) on the base of FRET or
brightness changes.
0s
10 s
10.8 s
11.3 s
(A) GCaMP2, a calcium indicator
constructed with a circularly permutated
EGFP fused to calmodulin and the
calmodulin-binding domain of myosin
light chain kinase (M13 domain) in the
absence of calcium.
(B) GCamP2 structure when bound to
calcium.
(C–F) Widefield fluorescence calcium
imaging in the cytosol of HeLa cells
expressing a calcium biosensor. (C) Real
color image of two cells, t = 0,
histamine (10 M) added; (D)
pseudocolored ratio image of two HeLa
cells as a calcium wave initiates in the
upper cell, t = 10 s. (E–F) The calcium
wave propagates through the cytoplasm
of both cells.
23. Research questions:
-Which cellular compartment is responsible for H2O2
production in RTKs signaling?
-When H2O2 appears?
-What is the diffusion distance of H2O2 within the cell?
33. H2O2 production by HeLa-Kyoto cells
stimulated with EGF
Mishina et al, ARS 2011
34. H2O2 production by HeLa-Kyoto cells
stimulated with EGF
Scale 5 m
Mishina et al, ARS 2011
35. H2O2 production by HeLa-Kyoto cells
stimulated with EGF
-H2O2 production/Nox activity
co-localizes with activated RTK
-H2O2 does not diffuse for a
long distance
-Nox activity translocates from
the PM to the endosomes
Mishina et al, ARS 2011
42. H2O2 production at the ER surface imaged using
HyPer-TA
Mishina et al, ARS 2011
43. Single system controls H2O2 production in fibroblasts
Co-localizes with PTP-1B
phosphatase
Co-localizes with active
RTK
Mishina et al, ARS 2011
44. -Which cellular compartment is responsible for NADPH
oxidases activation and H2O2 production in RTKs signalling?
Epithelial cells – Endosomes, ER membrane;
Fibroblasts – Plasma membrane, ER membrane.
45. -What is diffusion distance of H2O2 within the cell?
Estimated to be ~1m or even less. However, may vary in
different cell types.