1. Volume 27, Issue 16, Pages 2431-2441.e3 (August 2017)
Drosophila CRY Entrains Clocks in Body Tissues to Light and Maintains Passive Membrane Properties in a Non-clock Body Tissue Independent of Light 
Parul Agrawal, Jerry H. Houl, Kushan L. Gunawardhana, Tianxin Liu, Jian Zhou, Mark J. Zoran, Paul E. Hardin 
Current Biology 
Volume 27, Issue 16, Pages e3 (August 2017)
DOI: /j.cub
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2. Current Biology 2017 27, 2431-2441.e3DOI: (10.1016/j.cub.2017.06.064)
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3. Figure 1
The GFP-cry Transgene Rescues Light-Induced Phase Shifts in cry03 Flies
Representative double-plotted actograms for w1118 (+), w1118;;cry03 mutant (cry03), and w1118;GFP-cry;cry03 rescue (GFP-cry;cry03) flies are shown. Flies were subjected to LD cycles for 3 days, transferred to DD, and exposed to a 15-min light pulse at either CT15 (A) or CT21 (B) on DD day 1. White boxes, lights-on period; black boxes, lights-off period; horizontal lines, consecutive days of activity recording; vertical black bars, fly activity; blue bars, activity peaks before the light pulse; yellow bars, activity peaks after the light pulse; pink asterisk, light pulse. The height of black bars indicates relative activity level. See also Table S1.
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4. Figure 2 GFP-CRY Is Expressed in a Subset of Brain Pacemaker Neurons
w1118;GFP-cry;cry03 (GFP-cry;cry03) and w1118;;cry03 (cry03) flies were collected at the indicated times on DD day 1, immunostained, and imaged by confocal microscopy as described (see STAR Methods).
(A) 72- and 78-μm z stacks of left hemispheres from GFP-cry;cry03 and cry03 brains, respectively, immunostained with CLK (red) and GFP (green) antibodies. Merged CLK and GFP images (CLK+GFP) are shown as yellow. Large ventrolateral neurons, lLNvs; small ventrolateral neurons, sLNv; dorsolateral neurons, LNd, dorsal neuron 1 s, DN1; dorsal neuron 2 s, DN2; dorsal neuron 3 s, DN3; non-clock central brain neurons (nc); non-clock ventral brain neurons (nv).
(B) 68-, 72-, and 76-μm z stacks of the left hemisphere from GFP-cry;cry03 brains immunostained with GFP (green) antibody. Asterisk indicates non-specific antibody staining.
(C) Quantification of GFP positive pacemaker neurons based on analysis of at least six brain hemispheres at CT24 using ImageJ.
Error bars indicate SEM. See also Figure S1 and STAR Methods.
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5. Figure 3
GFP-CRY Levels in Peripheral Tissues from Adults Are Reduced by Light
(A) Malpighian tubules (MTs), intestine (posterior midgut), and fat body (FB) dissected from w1118;GFP-cry;cry03 (GFP-cry;cry03) and w1118;;cry03 (cry03) flies entrained in LD and collected at CT1 on DD day 3 in the presence or absence of a 1-hr light pulse were immunostained with CLK (red) and GFP (green) antibodies and imaged as a projected Z series by confocal microscopy. Merged CLK and GFP images (CLK+GFP) are shown as yellow.
(B) GFP-CRY was detected with GFP antibody in MTs from GFP-cry;cry03 flies collected at the indicated time points during LD and imaged as in (A).
(C) GFP-CRY was detected with GFP antibody in MTs from GFP-cry;cry03 flies collected at the indicated time points during day 1 of DD and imaged as in (A). Relative nuclear and cytoplasmic GFP-CRY levels were calculated as a nuclear:cytoplasmic ratio (N:C) (see STAR Methods) ± SEM. GFP-CRY showed a significant (p < 0.05) rhythm in N:C ratio that peaks at CT22 and troughs at CT10. See also STAR Methods.
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6. Figure 4 GFP-CRY Triggers Light-Dependent TIM Degradation in MTs
w1118;GFP-cry;cry03 (GFP-cry;cry03) and w1118;;cry03 (cry03) flies were entrained to LD cycles, subjected to either a 1-hr (D–I and M–R) or no-light pulse (A–C and J–L) at ZT15 and ZT21, and collected at ZT16 (A–I) or ZT22 (J–R), respectively. MTs were immunostained with GFP (green) and TIM (red) antibodies and imaged as in Figure 3. Merged GFP and TIM (GFP+TIM) images are shown as yellow. See also Figure S2.
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7. Figure 5 GFP-CRY Interacts with TIM but Not CLK and PER at ZT0.5
(A) GFP-CRY complexes from the bodies of w1118;GFP-cry;cry03 (GFP-cry;cry03) flies collected at ZT0.5 were immunoprecipitated using GFP nanobeads (see STAR Methods), and western blots containing input, flow through (FT), and immunoprecipitate (IP) samples were probed with GFP, TIM, CLK, and PER antisera.
(B) CLKV5 complexes from the bodies of w1118;GFP-cry;CLKV5-cry03 (GFP-cry;CLKV5-cry03) flies collected at ZT0.5 were immunoprecipitated using V5 antibody, and western blots containing input, FT, and IP samples were probed with V5, PER, CRY, and GFP.
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8. Figure 6
GFP-CRY Is Expressed in LSGs and Alters Their Passive Membrane Properties
(A) LSGs dissected from w1118;GFP-cry;cry03 (GFP-cry;cry03) and w1118;;cry03 (cry03) L3 larvae collected at the indicated time points in LD were immunostained with GFP antibody (green) and imaged as a Z series by confocal microscopy.
(B) Representative traces of resting membrane potential (RMP) recordings of LSG cells from GFP-cry;cry03 and cry03 larvae collected at ZT0.5.
(C) Boxplots showing RMP of LSG cells from w1118, GFP-cry;cry03, and cry03 larvae collected at ZT0.5. Error bars represent positive and negative SEM values.
(D) Traces showing responses to – and +3-nA-current injection steps in LSG cells from GFP-cry;cry03 and cry03 larvae collected at ZT0.5.
(E) I/V curves generated from the injection of – and +3-nA current into LSG cells from w1118, GFP-cry;cry03, and cry03 larvae collected at ZT0.5.
(F) Representative traces showing input resistance (Ri) after injection of −3-nA current in LSG cells from w1118, GFP-cry;cry03, and cry03 larvae collected at ZT0.5.
(G) Boxplots showing Ri in LSG cells from w1118, GFP-cry;cry03, and cry03 larvae collected at ZT0.5. a, the mean Ri in w1118 and GFP-cry;cry03 is significantly higher (p < 0.001) than in cry03 (see STAR Methods for statistical analysis). Error bars represent positive and negative SEM values.
(H) Boxplots of RMP and Ri from w1118, GFP-cry;cry03, and cry03 LSGs at the indicated time points. ZT, zeitgeber time; CT, circadian time. White boxes, lights on; gray boxes, lights off. b, the mean RMP in cry03 at ZT11.5 is significantly (p = 0.013) higher than GFP-cry;cry03 at ZT11.5; c, the mean Ri in GFP-cry;cry03 at ZT0.5 is significantly (p < 0.001) higher than in cry03 at ZT0.5; d, the mean Ri in GFP-cry;cry03 at ZT11.5 is significantly (p = 0.025) higher than in cry03 at ZT11.5; e, the mean Ri in GFP-cry;cry03 at CT0.5 is significantly (p < 0.001) higher than in cry03 at CT0.5; f, the mean Ri in GFP-cry;cry03 at CT0.5 is significantly higher (p = 0.034) than in GFP-cry;cry03 at ZT0.5. Error bars represent positive and negative SEM values.
See also Figures S3 and S4.
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9. Figure 7
CRY Collaborates with K+ Channel Subunits in LSGs to Mediate Changes in Ri
RMP and Ri responses were measured, plotted, and analyzed as described in STAR Methods.
(A) Boxplots of RMP and Ri in LSG cells from Hyperkinetic (Hk1, Hk2), Shaker (Sh5), ether-a-go-go (eag), cacophony (cacTS), paralytic (parats1), Drosophila Sodium Channel 1 (DSC1a) mutants and w1118;GFP-cry;cry03 (GFP-cry;cry03) larvae collected at ZT0.5.
(B) Boxplots of RMP and Ri in LSG cells from heterozygous Hyperkinetic (Hk1/+, Hk2/+), Shaker (Sh5/+), ether-a-go-go (eag/+), and cry (cry03/+) mutants and transheterozygous cry03/Hk1, cry03/Hk2, cry03/Sh5, and cry03/eag mutants collected at ZT0.5. a, the mean Ri in GFP-cry;cry03 is significantly (p < ) higher than in Hk1, Hk2, Sh5, and eag; b, the mean RMP in cry03/Hk2 is significantly (p = 0.020) higher than in cry03/+; c, the mean Ri in GFP-cry;cry03 is significantly (p < ) higher than in cry03/Hk1, cry03/Hk2, cry03/Sh5, and cry03/eag; d, the mean Ri in GFP-cry;cry03 is significantly (p = 0.024) higher than in Hk1/+; e, the mean Ri in Hk1/+ is significantly (p = 0.001) higher than in cry03/Hk1; f, the mean Ri in Hk2/+ is significantly (p = 0.004) higher than in cry03/Hk2; g, the mean Ri in eag/+ is significantly (p = 0.040) higher than in cry03/eag; h, the mean Ri in cry03/+ is significantly (p = 0.030) higher than in cry03/Hk1; i, the mean Ri in cry03/+ is significantly (p = 0.040) higher than in cry03/Hk2; j, the mean Ri in cry03/+ is significantly (p = 0.040) higher than in cry03/Sh5; k, the mean Ri in cry03/+ is significantly (p = 0.040) higher than in cry03/eag.
Error bars represent positive and negative SEM values. See also Figure S5.
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