1. Circadian Rhythms in Rho1 Activity Regulate Neuronal Plasticity and Network Hierarchy 
Afroditi Petsakou, Themistoklis P. Sapsis, Justin Blau 
Cell 
Volume 162, Issue 4, Pages (August 2015)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 Rho1 Prevents s-LNv Projections from Expanding
(A) Confocal images of s-LNv projections from Pdf > CD8::GFP flies stained with antibodies to GFP (green) and PDF (blue) at ZT12 and ZT24. 3D reconstructions (rainbow images) were generated using the MATLAB script (see Experimental Procedures) with colors indicating depth in the z axis (blue to red represents posterior to anterior). White dots show the area quantified. 1 pixel = 0.12 μm and z-step is 1 μm. Graphs on right quantify 3D spread and axonal volume using the MATLAB script.
(B) Top: induction of Rho GTPase transgenes. Flies were raised at 19°C and entrained in LD cycles at 19°C for at least 3 days before shifting to 30°C at ZT12. Flies were dissected 12 hr later (ZT24∗) and stained with anti-PDF. Confocal images of s-LNv projections and their 3D reconstructions as above at ZT24∗ for Control (Pdf, tub-Gal80ts > CD8::GFP), Rho1 (Pdf, tub-Gal80ts > Rho1), Cdc42 (Pdf, tub-Gal80ts > Cdc42CA) and Rac1 (Pdf, tub-Gal80ts > Rac1). Graphs quantify parameters of s-LNv projections from control and Rho1-induced flies.
(C) Top: diagram of induction. Flies were handled and stained as in Figure 1B except dissection was at ZT15∗. Confocal images and 3D reconstructions as above for TrpA1 + LacZ (Pdf, tub-Gal80ts > TrpA1, LacZ) and TrpA1 + Rho1 (Pdf, tub-Gal80ts > TrpA1, Rho1). Control flies were Pdf, tub-Gal80ts > myrRFP. Graphs quantify s-LNv projections.
Error bars show SEM. Statistical comparisons are with Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Significance was also verified with ANOVA. (See also Figure S1.)
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4. Figure 2 Inducing Rho1 Locks s-LNv Projections in a Dusk-like State
Flies were raised and entrained as in Figure 1B. Rho1 was induced starting either at dawn with brains fixed at ZT12∗ or starting at dusk with brains fixed at ZT24∗. Confocal images of s-LNv projections and their 3D reconstructions as in Figure 1 from control (Pdf, tub-Gal80ts > myrGFP + myrRFP), Rho1 (Pdf, tub-Gal80ts > Rho1 + myrRFP), and Rho1DN flies (Pdf, tub-Gal80ts > Rho1DN). s-LNv projections were stained with anti-PDF and quantified as in Figure 1B. 1 pixel = 0.12 μm and z-step is 1 μm. Error bars show SEM. Statistical comparisons are with Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n.s., non-significant. Significance was also verified with ANOVA. (See also Figure S2.)
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5. Figure 3 Circadian Oscillations in Rho1 Activity in s-LNv Axons
(A) Confocal images of s-LNv axons from flies expressing Rho1-activity sensor (Pdf>PKNG58AeGFP, green), eGFP (Pdf>eGFP, gray), or myrRFP (Pdf>myrRFP, red) stained with anti-GFP or anti-RFP. Graph shows average fluorescence levels in LD measured with Fiji with ZT0 data replotted at ZT24.
(B) Confocal images of s-LNv cell bodies from flies expressing Rho1-activity sensor with average GFP levels plotted on the right.
(C) Confocal images of s-LNv axons and termini from control (Pdf>PKNG58AeGFP) and per0 flies (per0; Pdf > PKNG58AeGFP) with average GFP levels plotted on the right.
Error bars show SEM. Statistical comparisons are with Student’s t test. ∗∗p < 0.01, ∗∗∗p < 0.001; n.s., non-significant.
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6. Figure 4
Rho1 Regulates an Output Pathway Important for Circadian Behavior and Seasonal Adaptation
(A) Left: actograms show locomotor activity in DD of control (Pdf, tub-Gal80ts > myrRFP) and Rho1-inducible flies (Pdf, tub-Gal80ts > Rho1) for 7 days at 25°C (gray) or 30°C (pink). Right: average rhythm power at 25°C and at 30°C in DD (also see Table S1).
(B) Flies were entrained to LD cycles at 19°C and then transferred to DD at 30°C. Confocal images of s-LNv cell bodies (left panels) and DN1 clock neurons (right panels) from control (Pdf, tub-Gal80ts > +) and Rho1-induced flies (Pdf, tub-Gal80ts > Rho1) stained with antibodies to VRI (green) and PDF (blue) at CT5 and CT17 on day 3 in DD. Graphs show average VRI fluorescence. The phase of the oscillation in DN1s was significantly different between genotypes (p < 0.01, ANOVA).
(C) Left: actograms of Pdf, tub-Gal80ts > sggwt,myrRFP and Pdf, tub-Gal80ts > sggwt,Rho1 flies at 30°C in DD. Right: graph shows average period of rhythmic flies (also see Table S1).
(D) Locomotor activity of control (Pdf, tub-Gal80ts > myrGFP, gray), Rho1- (Pdf, tub-Gal80ts > Rho1, red) and Rho1DN-induced flies (Pdf, tub-Gal80ts > Rho1DN, blue) in winter (10L:14D) or summer (14L:10D) light conditions.
Error bars show SEM. Statistical comparisons are with Student’s t test (A–C) and ANOVA (D). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n.s., non-significant. (See also Figure S3.)
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7. Figure 5
Rho1 Controls Rhythmic Myosin Light-Chain Phosphorylation in s-LNv Axons
(A) Confocal images of s-LNv axons at ZT12 and ZT24 stained with antibodies against P-MLC (green) and PDF (blue). Fluorescent intensity was measured with Fiji, and the normalized average is plotted on the right.
(B) Confocal images of s-LNv axons at ZT24∗ from Pdf, tub-Gal80ts > + and Pdf, tub-Gal80ts > Rho1 flies stained and analyzed as in (A).
(C) Confocal images of s-LNv projections stained with anti-PDF (blue) and their 3D reconstructions as in Figure 1 from flies with Rho1-induced (Rho1) or both Rho1 and Mbs-induced (Rho1 + Mbs). Graphs show average 3D spread and axonal volume for control (Pdf, tub-Gal80ts > myrRFP + myrGFP), Rho1 (Pdf, tub-Gal80ts > Rho1 + myrRFP), Rho1 + Mbs (Pdf, tub-Gal80ts > Rho1 + Mbs), and Mbs (Pdf, tub-Gal80ts > Mbs + myrRFP) induced flies. 1 pixel = 0.12 μm and z-step is 1 μm.
(D) Locomotor activity of control (Pdf, tub-Gal80ts > myrGFP, gray) and Mbs-induced (Pdf, tub-Gal80ts > Mbs, purple) flies as in Figure 4D.
Error bars show SEM. Statistical comparisons are with Student’s t test (A–C) and ANOVA (D). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n.s., non-significant. (See also Figure S4 and Table S1.)
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8. Figure 6
Pura Is a Clock-Regulated Rho1 GEF that Activates Rho1 in s-LNv Axons at  Dusk and Is Required for Seasonal Adaptation
(A) Confocal images of s-LNv axons at CT12 and CT24 on day 1 in DD from control (Pdf > PKNG58AeGFP + corazoninRNAi) and Pdf > PuraRNAi flies (Pdf > PKNG58AeGFP + PuraRNAi) expressing the Rho1-activity sensor (green) as in Figure 3. Graph shows the average fluorescence intensity of the Rho1-sensor in s-LNv axons from the above genotypes and from Pdf > PurashRNA flies (Pdf > PKNG58AeGFP + PurashRNA).
(B) s-LNv projections were stained with anti-PDF and quantified as in Figure 1 from control (Pdf, tub-Gal80ts > myrRFP) and PuraRNAi flies (Pdf, tub-Gal80ts > PuraRNAi) and entrained and shifted to 30°C for 12 hr as in Figure 2.
(C) Confocal images of s-LNv projections stained with PDF (blue) and their 3D reconstructions from flies with either Rho1 (Pdf, tub-Gal80ts > Rho1 + myrRFP) or Rho1 and PuraRNAi (Pdf, tub-Gal80ts > Rho1 + PuraRNAi) induced for 12 hr and fixed at ZT24∗. Graphs show the average 3D spread and axonal volume. 1 pixel = 0.12 μm and z-step is 1 μm. Error bars show SEM.
(D) Locomotor activity of control (Pdf, tub-Gal80ts > myrGFP, gray) and PurashRNA-induced flies (Pdf, tub-Gal80ts > PurashRNA, orange) as in Figure 4D.
Error bars show SEM. Statistical comparisons are with Student’s t test (A–C) and ANOVA (D). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n.s., non-significant. (See also Figure S5 and Table S1.)
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9. Figure 7 Pura and Rho1 Regulate Synaptic Plasticity
(A) Confocal images of s-LNv projections at ZT2∗ and ZT14∗ from control (Pdf, tub-Gal80ts > brpshort-strawberry + LacZ), Rho1 (Pdf, tub-Gal80ts > brpshort-strawberry + Rho1), and PuraRNAi (Pdf, tub-Gal80ts > brpshort-strawberry + PuraRNAi) flies. Flies were raised at 19°C and entrained in LD cycles at 19°C for at least 3 days before shifting to 30°C, starting at ZT12 or ZT24. Flies were dissected 14 hr later at ZT2∗ and ZT14∗, respectively, and brains stained with αDsRed (gray) to visualize Brp. Graph shows average numbers of active zones.
(B) Confocal images of LNd clock neurons from control (Pdf, tub-Gal80ts > myrRFP), TrpA1 + LacZ (Pdf, tub-Gal80ts > TrpA1, LacZ), and TrpA1 + Rho1 (Pdf, tub-Gal80ts > TrpA1, Rho1) flies. Flies were raised and entrained in LD at 19°C before shifting to 30°C for 3 hr at ZT12 and dissecting 3 hr later at ZT15∗. Brains were stained for VRI (green), TIM, and PDP1. Quantification was as in Figure 4B.
(C) Confocal images of s-LNv projections at ZT2∗ (top) and ZT14∗ (bottom) from control (Pdf, tub-Gal80ts > DenMark + LacZ), Rho1 (Pdf, tub-Gal80ts > DenMark + Rho1CA) and PurashRNA (Pdf, tub-Gal80ts > DenMark + PurashRNA) flies. Flies were handled as in (A), and brains were stained with αDsRed (gray). Quantification of average DenMark fluorescence levels was performed with Fiji.
Error bars show SEM. Statistical comparisons are with Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n.s., non-significant. (See also Figure S6.)
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10. Figure S1
s-LNv Structural Plasticity Quantification and the Effect of Inducing Rac1, Cdc42, Fas2, and TrpA1 in s-LNvs, Related to Figure 1
(A) 3D reconstruction of s-LNv axonal termini showing how the local z- and y axis deviations σZ(x), σY(x) are calculated for every x value along the 3D mean curve (dashed line). The MATLAB script only quantifies the spread dorsal to the ∼90° turn of the LNv projections. The z- and y axis spread is the average of all local σZ(x), σY(x) respectively.
(B) Green and blue bars show the average spread of s-LNv projections in x, y and z axes from projections stained with GFP and PDF respectively. Quantification was with the MATLAB script. Error bars show SEM and data are an average of 12 brains per time point from 2 independent experiments.
(C) Bars show PDF fluorescence quantified in s-LNv axons from Rho1 and Control flies with genotypes and assay as in Figure 1B. Quantification as in Figure S1B. There was no significant difference in fluorescence (p > 0.05).
(D) s-LNv projections from Control (Pdf, tub-Gal80ts > CD8::GFP), Rac1 (Pdf, tub-Gal80ts > Rac1) and Cdc42 flies (Pdf, tub-Gal80ts > Cdc42CA) were quantified as in Figures 1A and S1B at ZT24∗ after 12hr induction and stained with PDF. Data are an average of 18 brains from two independent experiments. Rac1 and Cdc42 induction increases s-LNv projections in the x- and y-axes (p < 0.001).
(E) Representative confocal images of s-LNv projections and their 3D reconstructions at ZT24∗ for Control (Pdf, tub-Gal80ts > CD8::GFP) and Fas2 flies (Pdf, tub-Gal80ts > Fas2) 12hr after inducing UAS transgene expression. Quantification was as above. White dots show the area quantified. Each dataset is an average of 21 brains from two independent experiments. Fas2 induction for 12hr does not change the spread in the x- or y-axes or the 3D spread and axonal volume (p > 0.05), but reduces spread in the z-axis (p < 0.001).
(F) Bars show 3D spread and axonal volume of s-LNv projections from Control (Pdf, tub-Gal80ts > myrRFP, myrGFP) and TrpA1 flies (Pdf, tub-Gal80ts > TrpA1, myrRFP). Flies were raised and entrained in LD at 19°C. Brains were dissected at either ZT15 or ZT15∗ after 3hr induction at 30°C. Axonal volume and 3D spread of TrpA1 and Control flies were similar at ZT15 (p > 0.05) but were significantly different from each other at ZT15∗ (p < 0.001). Each dataset is an average of brains per genotype from two independent experiments.
Error bars show SEM. All statistical comparisons are with Student’s t test. 1 pixel = 0.12 μm, z-step = 1 μm, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s.: non-significant.
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11. Figure S2
Structural Plasticity of s-LNv Projections at 25°C in Rho1 and Control Flies, Related to Figure 2
Flies were raised and entrained at 19°C as in Figure 2. Rho1 and Control flies were shifted to 25°C (when Gal80 is still active) for 12hr starting either at dawn with brains fixed at dusk (ZT12) or starting at dusk and fixed at dawn (ZT24). Representative confocal images of s-LNv projections and their 3D reconstructions at ZT12 and ZT24 from Control (Pdf, tub-Gal80ts > myrRFP) and Rho1 flies (Pdf, tub-Gal80ts > Rho1). s-LNv projections were stained with anti-PDF and quantified as in Figure 1B. Each dataset is an average of brains from 2 independent experiments. Rhythms in the structure of s-LNvs were present in Control and Rho1 flies (p < 0.01, Student’s t test).
1 pixel = 0.12 μm and z-step is 1 μm. Statistical comparisons shown are with Student’s t test. Significance was verified with ANOVA. White dots show the area quantified. Error bars show SEM. ∗∗p < 0.01
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12. Figure S3
Molecular Clock Oscillations in Rho1-Induced Flies and Seasonal Adaptation, Related to Figure 4
(A) Graph shows average TIM levels in s-LNvs from 11 brains per time point from 2 independent experiments. Flies underwent the same temperature regime as in Figure 4B. No significant difference in TIM oscillations between control and Rho1-induced s-LNvs (p > 0.05, Student’s t test and ANOVA).
(B) Graph shows average PDP1 levels in DN1s from 6-9 brains per time point from 2 independent experiments. Flies went through the same temperature regime as Figure 4B. Statistical comparisons per time point are with Student’s t test and reveal differences between genotypes.
(C) The locomotor activity of Control (gray line, genotype as in 4D) corazoninRNAi (Pdf, tub-Gal80ts > corazoninRNAi, black line) and Rho1DN flies (Pdf, tub-Gal80ts > Rho1DN, blue line) was recorded in summer (14L:10D) for 5 days at 30°C. Normalized activity plots show averages of flies per genotype on days 4 and 5. Morning anticipation (ZT19-ZT24) for corazoninRNAi flies was not significantly different (p > 0.05) from Control flies, but was significantly different from Rho1DN flies (p < 0.01, ANOVA) during summer.
(D) The locomotor activity of Control (gray line, genotype as in 4D) and Pdf01 flies (green line) was recorded in winter (10L:14D) or summer (14L:10D) for 5 days at 30°C. Normalized activity plots show averages of flies per genotype as in Figure S3C. Morning anticipation (ZT19-ZT24) for Pdf01 flies was significantly different from Control flies during winter and summer (p < 0.001, ANOVA).
Error bars show SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
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13. Figure S4
Myosin Light Chain Phosphorylation Is Clock-Regulated and Downstream of Rho1 in s-LNv Axons, Related to Figure 5
(A) Flies were stained with antibodies against P-MLC and PDF as in Figure 5 and fluorescent intensities measured with Fiji. Data are normalized and show P-MLC levels at ZT12 and ZT24 from Control (Canton S) and per0 flies. P-MLC levels are rhythmic in Control flies (p < 0.01, Student’s t test) but not in per0 mutant flies (p > 0.05, Student’s t test). Data show 11 brains from two independent experiments.
(B) Left: Representative, double-plotted, scaled actograms from flies in DD for 10 days at 25°C for: Control (Pdf > myrRFP + myrGFP), Pdf > Rho1 flies overexpressing Rho1 and myrRFP and flies overexpressing both Rho1 and Mbs (Pdf > Rho1 + Mbs). Right: Average power of the rhythm is shown for the 3 genotypes on the left and for Pdf > Mbs flies that overexpress Mbs and myrRFP. Co-expressing Mbs with Rho1 rescues the low power rhythms of flies overexpressing Rho1 alone (p < 0.001, Student’s t test). Data are an average of flies (see Table S1).
(C) Representative confocal images of s-LNv projections and their 3D reconstructions at ZT12∗ for Control (Pdf, tub-Gal80ts > myrRFP) and Mbs (Pdf, tub-Gal80ts > Mbs) flies after 12hr induction. White dots show the area quantified as in Figure 1. Data are an average of brains from two independent experiments. Mbs induction for 12hr significantly increases 3D spread and axonal volume at ZT12∗ compared to Control (p < 0.01, Student’s t test).
Error bars show SEM. Significance was verified with ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s.: non-significant.
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14. Figure S5 PuraRNAi and PurashRNA Validation, Related to Figure 6
(A) Confocal images of the four s-LNv cell bodies from flies in which a UAS-destabilized GFP transgene was expressed with either a Pura-Gal4 enhancer trap (Pura > dsGFP, top) or Pdf-Gal4 (Pdf > dsGFP, bottom). Flies were stained with antibodies to GFP (green) and PDF (data not shown). White dots show s-LNvs. Graphs on the right shows the average normalized GFP fluorescent intensity. GFP levels are higher at ZT14 than ZT2 (p < 0.01) for Pura-Gal4 but there is no difference between time points for Pdf-Gal4. The Pura > dsGFP dataset has 17 brains per time point from 3 independent experiments and Pdf >-dsGFP has 12 brains per time point from 2 independent experiments.
(B) qPCR of RNA from whole heads of Control (w1118), elav > PuraRNAi and elav > PurashRNA. Pura probes detect all Pura transcripts and values were normalized to Calmodulin (CaM) levels. Data are an average of at least 2 biological replicates and 3-4 technical replicates. PuraRNAi and PurashRNA reduce Pura RNA levels compared to Controls (p < 0.05, p < 0.01 respectively).
(C) Locomotor activity was recorded from flies for 10 days in DD at 25°C. Representative double-plotted scaled actograms are shown on top with average power of rhythms on the bottom. Control flies (Pdf > myrRFP + myrGFP) had significantly stronger rhythms than Pdf > Rho1 flies constitutively expressing Rho1 (Pdf > Rho1 + myrRFP, p < 0.001). The weak power of Pdf > Rho1 flies was rescued by co-expressing PuraRNAi (Pdf > Rho1 + PuraRNAi, p < 0.001). Pdf > Rho1CA flies expressing a constitutively active GEF-independent Rho1 (Pdf > Rho1CA + myrRFP) had weaker rhythms than control flies (p < 0.001) and this was unchanged by co-expressing PuraRNAi (Pdf > Rho1CA + PuraRNAi, p > 0.05) flies per genotype (see Table S1).
(D) P-MLC levels were measured as in Figure 5B at ZT24∗ for Control flies (Pdf-Gal4 / +; tub-Gal80ts / +), flies with Rho1-induced (Pdf, tub-Gal80ts > Rho1 + myrRFP) and flies with Rho1 and PuraRNAi co-induced (Pdf, tub-Gal80ts > Rho1 + PuraRNAi). P-MLC fluorescent intensities divided by area were measured with Fiji, normalized and plotted in the graph. Data are an average of 7 brains. Reducing Pura expression overrides the increase in P-MLC seen when inducing Rho1 (p < 0.05) and made P-MLC levels no different from Control (p > 0.05).
(E) Graphs show average VRI levels in s-LNvs and LNds from 12 brains per time point in 2 independent experiments. Flies went through the same temperature regime as Figure 4B. There is no significant difference in VRI oscillations in s-LNvs between Pdf, tub-Gal80ts > myrGFP and Pdf, tub-Gal80ts > PurashRNA flies (p > 0.05). However, VRI levels at ZT24 in LNds are significantly higher in Pdf, tub-Gal80ts > PurashRNA flies than in controls (p < 0.01), indicating altered molecular clock oscillations.
Error bars show SEM. All statistical comparisons are with Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s.: non-significant.
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15. Figure S6
Co-localization of BRP and PDF in s-LNv Termini, Related to Figure 7
s-LNv projections at ZT2∗ showing Brpshort-strawberry (gray or red) as in Figure 7A, co-stained with PDF antibody (blue) from Pdf, tub-Gal80ts > brpshort-strawberry + LacZ flies. The data show that BRP puncta at ZT2∗ co-localize with PDF in s-LNv termini. Grayscale image on the left is the same image as Figure 7A.
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