Effects on circadian locomotor activity of altering AC3 in M pacemakers.

<p>(A) Representative locomotor behavior of flies that are heterozygous for the <i>AC3</i> locus (<i>Df</i>(2L)<i>DS6</i>). (B) Representative locomotor behavior of flies that combine a knockdown of <i>AC3</i> by RNAi together with a deficiency for the <i>AC3</i> locus. (C) Representative locomotor behavior of flies over-expressing <i>AC3</i>. (D) Representative locomotor behavior of flies over-expressing <i>PDF-R</i> and over-expressing <i>AC3</i>. Morning anticipation index was calculated as (sum of activity 3 h before lights-on)/(sum of activity 6 h before lights-on). The average morning anticipation index was calculated from three replicates for each genotype. Error bars denote SEM. *** <i>p</i><0.001 (compared with control). Statistical analysis of morning anticipation is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001337#pbio-1001337-t001" target="_blank">Table 1</a>, and behavioral outcomes for DD are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001337#pbio-1001337-t002" target="_blank">Table 2</a>.</p

Effects of over-expressing <i>AC</i> isoforms on different receptor signaling systems in M pacemakers.

<p>(A) Effects of over-expressing diverse ACs on M cell responses to neuropeptide PDF. (B) Effects of over-expressing <i>AC3</i> on M cell responses to neuropeptide DH31. (C) Effects of over-expressing <i>AC3</i> on M cell responses to dopamine. All genotypes include <i>Pdf</i>-gal4;<i>Epac1camps</i>. Error bars denote SEM. *** <i>p</i><0.001 (compared with control).</p

Data collection of FRET responses and transgenic RNAi screen of ACs potentially coupled to PDF receptor in M cell pacemakers.

<p>(A) Raw FRET imaging data (CY/CC) collected for 10 min (each trace represents an individual cell recorded as an ROI) to show FRET loss in response to a bolus of PDF (marked by arrow) and recoveries to baseline. (B) The scatter plot represents the data shown in 1A and retains its color-coding. For each trace, the maximal deflection from its value at the initial time point is computed as “percent FRET loss” and represented as a single point. Error bars represent SEM. (C) Double-stranded RNAi directed against 11/12 genes known to encode known adenylate cyclases in the <i>Drosophila</i> genome. All genotypes include <i>Pdf</i>-gal4;<i>Epac1camps</i> and one copy of UASRNAi (except for control). Error bars denote SEM. *** <i>p</i><0.001, ** <i>p</i><0.01 (compared with control).</p

Effects of manipulating <i>G_sα60A</i> and AC3 levels in E cell subgroup.

<p>(A) PDF responses in PDF-R expressing LNd cells (E cells). Flies with the severe PDF-R mutation <i>han⁵³⁰⁴</i> show no response to PDF. (B) PDF responses in PDF-R expressing LNd cells (E cells). Both knockdown and over-expression of <i>G_sα60A</i> significantly reduce PDF responses in E cells. (C) PDF responses in PDF-R expressing LNd cells (E cells) in genotypes that most severely disrupt M cell PDF responses. Knockdown (<i>Df</i>(2L)/<i>AC3</i>RNAi) and over-expression of AC3 do not affect E cell PDF responses. All genotypes include <i>Mai179</i>-gal4;<i>Epac1camps</i>. Error bars denote SEM. *** <i>p</i><0.001 (compared with control).</p

DD behavioral outcomes grouped by genotype.

<p>Periods are calculated using chi-squared periodigram. Flies with a power <10 were scored as arrhythmic.</p

Quantification of Morning Anticipation Index for LD behavior.

<p>Statistical analysis of morning anticipation behavior calculated as (total activity 3 h before lights-on)/(total activity 6 h before lights-on) for genotypes shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001337#pbio-1001337-g008" target="_blank">Figure 8</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001337#pbio.1001337.s005" target="_blank">Figure S5</a>. Average morning anticipation was calculated from three replicates. <i>Pdf⁰¹</i> genotype represents <i>Pdf</i>-null mutants, which have been widely studied and serve as an example of a total lack of morning anticipation.</p>*<p><i>p</i><0.05,</p>**<p><i>p</i><0.01,</p>***<p><i>p</i><0.001. <i>ns</i>, not significant.</p

A conditional transgenic RNAi test of AC involvement in PDF signaling in M cell pacemakers.

<p>(A) Temperature sensitive gal80 was used to induce knockdown in adult cells only. Flies were raised at 18°C and moved to 29°C for 6 h (inactive), to allow for readable levels of <i>Epac1camps</i> sensor, or >36 h (active). Adult induction of <i>AC3</i>RNAi (gal80ts;<i>AC3</i>RNAi (active)) significantly reduces the PDF response. Adult induction of <i>AC76E</i>RNAi shows no significant difference from control. (B) Genetic confirmation of <i>AC3</i> involvement was performed using two independently generated RNAi lines against <i>AC3</i> (GD:<i>AC3</i> and TRiP:<i>AC3</i>) as well as flies that are deficient for the <i>AC3</i> gene region (<i>Df</i>(2L)<i>DS6</i>). (C) DH31 responses in M cells from flies with a knockdown of <i>AC3</i> in combination with <i>Df</i>(2L)<i>DS6</i>. All genotypes include <i>Pdf</i>-gal4;<i>Epac1camps</i>. Error bars denote SEM. *** <i>p</i><0.001 (compared with control).</p

Effects on PDF responses following RNAi knockdown of scaffolding protein RNAs in M cells.

<p>(A) PDF responses of M pacemakers in flies expressing <i>nervy</i> RNAi. (B) DH31 responses of M pacemakers expressing <i>nervy</i> RNAi. (C) PDF responses of M pacemakers expressing <i>AC3</i> and <i>nervy</i> RNAi. All transgenic lines are significantly different (<.001) from control and internal comparisons are highlighted by bracketed lines. All genotypes include <i>Pdf</i>-gal4;<i>Epac1camps</i>. Error bars denote SEM. *** <i>p</i><0.001 (compared with control).</p

Genetic rescues of <i>AC3</i> knockdown and over-expression effects in M cells.

<p>(A) Rescuing the loss of function state. Flies were raised at 25°C and moved to 18°C as adults for 12–15 h before imaging to reduce levels of <i>AC3</i> over-expression. The effect of this schedule on the effects of <i>AC3</i> knockdown (TRiP:<i>AC3</i>RNAi) and <i>AC3</i> over-expression (UAS-<i>AC3</i>) is shown. The ability of over-expressing <i>AC78C</i> (TRiP:<i>AC3</i>/UAS<i>AC78C</i>) and <i>AC3</i> (TRiP:<i>AC3</i>RNAi/UAS<i>AC3</i>) to reverse the knockdown effect of <i>AC3</i> RNAi are also shown. (B) Rescuing the gain of function state. Two <i>PDF-R</i> over-expression genotypes were tested for their ability to affect <i>AC3</i> over-expression: a UAS construct (UAS<i>PDF-R</i>;UAS<i>AC3</i>) and a construct in which <i>PDF-R</i> is driven by its endogenous promotor (UAS<i>AC3</i>;<i>PDF-R</i>myc). For comparison the effects of co-misexpressing a heterologous neuropeptide receptor is also shown (UAS<i>DH31R</i>/UAS<i>AC3</i>). All genotypes include <i>Pdf</i>-gal4;<i>Epac1camps</i>. Error bars denote SEM. *** <i>p</i><0.001, * <i>p</i><.05 (compared with control).</p

A model for a circadian signalosome comprised of preferential PDF-R:AC3:nervy coupling.

<p>M pacemakers respond to dopamine, DH31, and PDF-R through G_sα-coupled receptors: Activation of each receptor lead to increases in cAMP levels. Both DH31 and PDF receptors signal through <i>G_sα60A</i>, however AC3 alterations affect PDF signaling without affecting DH31 responses. We propose that PDF signals through AC3 to affect circadian function but that dopamine and DH31 couple to other AC isoforms. Likewise, the AKAP nervy preferentially associates with the PDF-R:AC3 signaling complex; other AKAPs support the G_sα-coupled receptors that mediate responsiveness to DA and DH31.</p