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Letter
Optogenetic inhibition of G#q protein signaling reduces calcium oscillation stochasticity Pimkhuan Hannanta-anan, and Brian Y. Chow ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/ acssynbio.8b00065 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 28, 2018
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GPCR
Ca2+ R2-box
helix Effector signaling
CIBN
Targeting Component
Enzymatic Component
G*q
Gq
x
-bo
R2
linker CRY2
R2-box
Enzymatic Component
Ca2+/Effector signaling
2
helix
opto-RGS2
R2-box
CRY
Pi
GDP
Targeting Component
Light
CRY2
Inactive
Gq* GTP
Ligand
CIBN
Gq
Active
Human RGS2 (hRGS2) helix
CIBN
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
ACS Synthetic Biologyc.
b.
Ligand
box
R2-
Ca2+/Effector signaling
Figure 1. Design of optogenetic RGS2 (opto-RGS2). (a) RGS2 terminates activated Gαq signaling as a GAP (GTPase accelerating protein). (b) Opto-RGS2 splits the catalytic box and N-terminal amphipathic helix of human RGS2, and fuses them respectively to cryptochrome (CRY2-PHR) and its interaction partner, CIBN. (c) CRY2-R2box is sequestered in the cytosol in the dark. Optogenetic hetero-dimerization reconstitutes a functional membrane-localized complex.
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Fluid delivery & perfusion stand
∆F/F
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Light, ρ = 0.42 Dark mutant, ρ = 0.15
Weakly inhibited
λ = 470nm (constant)
Ca2+ (∆F/F0) Amplitude
b.
a.
5
∆ F/F
Strongly
Ca2+ (∆F/F0) Amplitude
Change in cytosolic mCherry, F/F0
Strongly
CRY2-R2box translocation
Ca2+ suppression
Weakly inhibited
Ca2+ (∆F/F0) Amplitude
0
Ca2+ suppression
Dark mutant 1 4 0 2 In Out 0 100 200 300 400 500 600 3 time (s) 3 Carbachol 2 4 λ = 470nm 3 1 (constant) 2 5 1 Microscope 0 6 opto-RGS2 stimulation 0 0.8 0.9 1.0 1.1 & imaging 7 Change in cytosolic mCherry, F/F 0 100 200 300 400 500 600 time (s) 8 CRY2-R2box translocation 9 10 e. Machine Learning Prediction d. f. n.s. Non*** 11 *** responsive Responsive 12 6 1.10. 6 Light, ρ = 0.37 True Label 13 5 or Experimental 5 1.05 Classification 14 Weakly inhibited 4 1.00 Strongly inhibited 15 4 3 0.95 16 3 2 17 2 0.90 1 18 1 0.85 19 0 0 0.80 20 0 2000 4000 0 2000 4000 6000 WT hRGS2 Light Dark CRY2-R2box expression CRY2-R2box expression 21 opto-RGS2 (mCherry Intensity, AU) (mCherry intensity, AU) 22 23 2. Opto-RGS2 functional determinants in transfected HEK cells. (a) Experimental setup for single-cell functional assays Figure of 24optogenetic suppression of carbachol-induced oscillations. (b) GCamp6f imaging traces of exemplar 30 uM carbachol (CCh)-induced 25 calcium oscillations and optogenetic suppression. (Top) Red = Illuminated opto-RGS2 dark mutant or optically insensitive apoprotein control. (Bottom) Blue = Illuminated opto-RGS2 or flavin holoprotein. (c) Correlation analysis between oscillation suppres26 sion and CRY2-R2box translocation (inverse of change in cytosolic mCherry intensity (F/F0)). ρ = Spearman’s rank correlation 27 coefficient. Cells with strong calcium suppression (calcium amplitude lower than 0.5, beneath the dashed line) are considered “strongly28inhibited”. (d) Correlation analysis between oscillation suppression and CRY2-R2box expression. (e) Machine learning-predicted decision boundary for discriminating cells likely to suppress calcium levels robustly (predicted responsive, gray-shaded area) based 29 on translocation efficiency and CRY2-R2box expression level. True Label = Classification from experimental data shown in panels c-d 30 as training set. The boundary was optimized to maximize the true positive prediction with 80% precision, where 80% of “predicted 31 responsive” cells are “strongly inhibited” experimentally. (f) Box-and whisker plot of calcium oscillation peak amplitude in wild-type HEK 32 cells, constitutively active hRGS2 cells, opto-RGS2 dark mutant, and opto-RGS2 cells selected by the machine learning criteria from 33 panel e (dataset distinct from the training set). N = 174-367. 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 ACS Paragon Plus Environment 50 51
1.5
d = 0.33 ***
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Light
b.
Pre
e. 1.5
d = 0.39 ***
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Light
c. Pre
Post
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Fluorescence (AU)
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Amplitude ratio [Post:Pre]
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** Normalized Transcription
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Frequency ratio [Post:Pre]
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R2helix-CIBN
Duty Ratio ratio [Post:Pre]
a.
Fluorescence (AU)
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300
WT opto-RGS2 hRGS2
1.6 1.2 0.8 0.4 0.0
SERCA2b
IP3R3
IP3R1
Figure 3. Inhibitory regulation of calcium oscillations in opto-RGS2 cell lines. (a) Fluorescence micrographs of light-induced translocation of mCherry-tagged CRY2-R2box (red) in opto-RGS2 cell lines. (top left) GFP-tagged R2helix-CIBN is membrane- and nuclear-localized, consistent with known R2helix distribution patterns. (top right) CRY2-R2box in the cytoplasm of the same cell, (bottom left) membrane localization 30 seconds after brief (1 s) blue light stimulation, and (bottom right) thermal reversion back to the cytoplasm in the dark (900 s post-illumination). Scale = 5 um. (b) Example X-rhodamine calcium imaging traces of dynamic suppression of 100 uM CCh-induced oscillations in response to a single epoch of blue light. Optogenetic dampening primarily decreased the frequency. (c) Same as panel b, except where optogenetic dampening primarily decreased the amplitude. (d) Single epoch of blue light dynamically reduced oscillation frequency. Post-illumination parameter values were normalized to pre-illumination values of the same oscillation in the same cell, as in panel a. (*** p < 0.001, d = Cohen’s effect size) (e) Same as panel d, except for amplitude. (f) Same as panel d, except for duty ratio. (g) Quantitative PCR measurements of SERCA2b and IP3R transcripts. SERCA2b level was altered in hRGS2 cells to compensate for long-term reduction in calcium load (p < 0.01), but not in opto-RGS2 cells.
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Interspike interval (ISI, T)
Stochasticity α = s.d.(T) / (Tav)
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Ligand
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PIP3
Gq*
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s.d.(T) (s)
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α → 1, Stochastic CaM 1 60 Gq PLC ??? Increased 2 GPCR 40 CaM IP R feedback & IP 3 synchronicity 20 SERCA 4 0 Ca α
