UNC79 and UNC80, Putative Auxiliary Subunits of the NARROW ABDOMEN Ion Channel, Are Indispensable for Robust Circadian Locomotor Rhythms in <i>Drosophila</i>

<div><p>In the fruit fly <i>Drosophila melanogaster,</i> a network of circadian pacemaker neurons drives daily rhythms in rest and activity. The ion channel NARROW ABDOMEN (NA), orthologous to the mammalian sodium leak channel NALCN, functions downstream of the molecular circadian clock in pacemaker neurons to promote behavioral rhythmicity. To better understand the function and regulation of the NA channel, we have characterized two putative auxiliary channel subunits in <i>Drosophila</i>, <i>unc79 (aka dunc79)</i> and <i>unc80 (aka CG18437).</i> We have generated novel <i>unc79</i> and <i>unc80</i> mutations that represent strong or complete loss-of-function alleles. These mutants display severe defects in circadian locomotor rhythmicity that are indistinguishable from <i>na</i> mutant phenotypes. Tissue-specific RNA interference and rescue analyses indicate that UNC79 and UNC80 likely function within pacemaker neurons, with similar anatomical requirements to NA. We observe an interdependent, post-transcriptional regulatory relationship among the three gene products, as loss of <i>na, unc79,</i> or <i>unc80</i> gene function leads to decreased expression of all three proteins, with minimal effect on transcript levels. Yet despite this relationship, we find that the requirement for <i>unc79</i> and <i>unc80</i> in circadian rhythmicity cannot be bypassed by increasing NA protein expression, nor can these putative auxiliary subunits substitute for each other. These data indicate functional requirements for UNC79 and UNC80 beyond promoting channel subunit expression. Immunoprecipitation experiments also confirm that UNC79 and UNC80 form a complex with NA in the <i>Drosophila</i> brain. Taken together, these data suggest that <i>Drosophila</i> NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.</p></div

Transgenic rescue of <i>unc79</i> and <i>unc80</i> rhythmicity phenotypes.

1<p>Few flies survived to the end of DD; refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078147#pone-0078147-g006" target="_blank">Figures 6B,E</a> for LD phenotype.</p

NA, UNC79, and UNC80 proteins form a complex in the <i>Drosophila</i> head.

<p>Western blot analyses of immunoprecipitated complexes. Immunoprecipitations were performed from membrane preparations of adult <i>Drosophila</i> head extracts using the antibodies indicated (anti-NA, anti-UNC79, anti-UNC80, and anti-α-SPECTRIN; see <i>Materials and Methods</i>).</p

RNAi knockdown of <i>na, unc79,</i> or <i>unc80</i> in circadian pacemaker neurons decreases DD rhythmicity.

<p>RNAi knockdown of <i>na, unc79,</i> or <i>unc80</i> in circadian pacemaker neurons decreases DD rhythmicity.</p

RNAi knockdown of <i>na, unc79</i>, or <i>unc80</i> in all pacemaker neurons results in anticipation defects.

<p>Normalized activity profiles from adult male populations averaged over four days of LD entrainment. White bars represent light phase; black bars indicate dark phase. Error bars represent standard error of the mean. Arrows indicate morning anticipation (black) and evening anticipation (gray). The genotypes represented in the left panels are <i>timGAL4</i>/ +; UAS-<i>dcr2</i>/ +, heterozygous for the following insertions from the Vienna <i>Drosophila</i> RNAi Center (VDRC): (top panel) no RNAi  =  control strain <i>attp VIE-260B</i> (n =  55), MAI  =  1.5+/−0.1, EAI  =  2.6+/−0.1; (second panel) <i>na = </i> 103754 (n  =  43), MAI  =  0.5+/−0.1, EAI  =  0.4+/−0.1; (third panel) <i>unc79  = </i> 108132 (n =  44), MAI  =  0.3+/−0.1, EAI  =  0.3+/−0.1; (bottom panel) <i>unc80  = </i> 108934 (n =  42), MAI  =  0.7+/−0.1, EAI  =  0.3+/−0.1. Genotypes represented in the right panels are <i>pdfGAL4</i> UAS-<i>dcr2</i>/+ heterozygous for the same VDRC strains: (top panel) <i>attp VIE-260B</i> (n =  32), MAI  =  1.3+/−0.1, EAI  =  2.4+/−0.2; (second panel) <i>na = </i> 103754 (n  =  33), MAI  =  1.0+/−0.1, EAI  =  2.3+/−0.2; (third panel) <i>unc79  = </i> 108132 (n =  37), MAI  =  1.5+/−0.2, EAI  =  2.1+/−0.2; (bottom panel) <i>unc80  = </i> 108934 (n =  35), MAI  =  1.3+/−0.2, EAI  =  2.3+/−0.2. Both MAI and EAI differ significantly among the <i>timGAL4/ +</i>; UAS-<i>dcr2/</i> + genotypes (Kruskal-Wallis one-way ANOVA, 3 degrees of freedom, P<0.001), and each RNAi genotype exhibits significantly lower MAI and EAI than the control strain (Dunn’s method, P<0.05). MAI and EAI values for each <i>timGAL4</i> UAS-<i>dcr2</i> RNAi strain are either lower or not significantly different from values calculated from strong mutant alleles for the corresponding gene (Kruskal-Wallis one-way ANOVA, 1-2 degrees of freedom). No significant differences in MAI or EAI are observed among <i>pdfGAL4</i> UAS-<i>dcr2</i>/ + genotypes (Kruskal-Wallis one-way ANOVA, 3 degrees of freedom, P > =  0.146).</p

Transgenic rescue of <i>unc79</i> and <i>unc80</i> anticipation phenotypes.

<p>(A-H) Normalized activity profiles from adult males averaged over four days of LD conditions. White bars indicate light phase; black bars indicate dark phase. Error bars represent standard error of the mean. (A) <i>UAS-unc79MYC 23-24/+; unc79^x25</i> (n = 42), MAI  =  1.1+/0.2, EAI  =  0.5+/−0.1; (B) <i>timGAL4</i>/ <i>UAS-unc79MYC 23-24; unc79^x25</i> (n = 68), MAI  =  1.7+/−0.1, EAI  =  2.6+/−0.2; (C) <i>unc79^f01615/ unc79^x25</i> (n = 117), MAI  =  0.4+/−0.1, EAI  =  0.26+/−0.03; (D) <i>timGAL4</i>/ <i>+; unc79^f01615/ unc79^x25</i> (n = 74), MAI  =  1.1+/−0.1, EAI  =  1.6+/−0.1; (E) <i>UAS-HAunc80 1M unc80^x42/ unc80^x42</i> (n = 52), MAI  =  0.4+/−0.1, EAI  =  0.3+/−0.1; (F) <i>timGAL4</i>/ +; <i>UAS-HAunc80 1M unc80^x42/ unc80^x42</i> (n = 48), MAI  =  1.1+/−0.2, EAI  =  1.5+/−0.1; (G) <i>unc80^{GS12792 (UAS)}/ unc80^x42</i> (n = 71), MAI  =  0.5+/−0.1, EAI  =  0.3+/−0.1; (H) <i>timGAL4</i>/ <i>+; unc80^{GS12792 (UAS)}/ unc80^x42</i> (n = 43), MAI  =  1.7+/−0.1, EAI  =  1.1+/−0.1. MAI and EAI values differ significantly between each mutant (A, C, E, G) and the corresponding rescue genotype (B, D, F, H), as determined by Kruskal-Wallis one-way ANOVA (all comparisons P<0.001, 1 degree of freedom).</p

<i>Drosophila</i> NA, UNC79, and UNC80 exhibit an interdependent regulatory relationship.

<p>(A) Representative Western blot analyses, performed from adult <i>Drosophila</i> head extracts. The strains assayed were generated by backcrossing <i>na^e04385</i> (Lanes 1–2), <i>unc79^x25</i> (Lanes 3–4), <i>unc80^x42</i> (Lanes 5–6), or <i>unc80^GS12792</i> (Lanes 7–8) to <i>iso31</i> for 6–8 generations. NS  =  non-specific UNC79 bands; see <i>Materials and Methods</i> for more details. (B) Quantitation of NA, UNC79, and UNC80 protein levels in each mutant strain, as a percentage of the level observed in the corresponding wild-type strain. Black bars indicate NA protein, gray bars UNC79 protein, and white bars UNC80 protein. Error bars indicate standard error of the mean, determined from 3 independent experiments. NA, UNC79, and UNC80 protein levels are significantly lower in each mutant strain compared to the corresponding control strain, as determined by Student’s t-test (all P<0.01, except UNC80 levels in <i>na^e04358</i>, P  =  0.012).</p

Transgenic <i>unc79, unc80</i> or <i>na</i> expression produces increased protein levels in wild-type and mutant backgrounds.

<p>(A-C) Western blot analyses were used to label UNC79, UNC80, and NA proteins from adult head extracts; representative blots are shown. For all blots shown, lane 1  =  <i>elav</i>GAL4/+ (no UAS); lanes 3,5,7  =  <i>UAS/+</i> (no GAL4); lanes 2,4,6,8  =  <i>elavGAL4; UAS/+.</i> (A) Pan-neuronal expression of <i>UAS-unc79MYC 23-24</i> in wild-type (lane 2), <i>na^har</i> (lane 4), <i>unc79^x25</i> (lane 6), or <i>unc80^x42</i> (lane 8) backgrounds. (B) Expression of <i>UAS-HAunc80 1M</i> in wild-type (lane 2), <i>na^har</i> (lane 4), <i>unc79^x25</i> (lane 6), or <i>unc80^x42</i> (lane 8) backgrounds. (C) Expression of <i>UAS-na U3</i> in wild-type (lane 2), <i>na^har</i> (lane 4), <i>unc79^x25</i> (lane 6), or <i>unc80^x42</i> (lane 8) backgrounds.</p

<i>Drosophila unc79</i> and <i>unc80</i> mutants display defects in anticipatory behavior.

<p>(A-F) Normalized locomotor activity profiles from adult male populations, averaged over four days of 12 hr light: 12 hr dark (LD) entrainment conditions. White bars indicate light phase; black bars indicate dark phase. Error bars represent standard error of the mean. Arrows indicate morning anticipation (black) and evening anticipation (gray). All strains were backcrossed to <i>w¹¹¹⁸ (iso31)</i> for 6–8 generations. Morning anticipation index (MAI) and evening anticipation index (EAI) are indicated for each genotype (see Materials and Methods). (A) Representative wild-type strain (n = 52), MAI  =  1.5+/−0.1, EAI  =  2.1+/−0.1; (B) <i>na^e04385</i> (n = 75), MAI  =  0.8+/−0.2, EAI  =  0.5+/−0.1; (C) <i>unc79^f03453</i> (n = 44), MAI  =  0.5+/−0.1, EAI  =  1.0+/−0.1; (D) <i>unc79^x25</i> (n = 93), MAI  =  0.7+/−0.1, EAI  =  0.4+/−0.1; (E) <i>unc80^GS12792</i> (n = 64), MAI  =  0.7+/−0.2, EAI  =  0.5+/−0.1; (F) <i>unc80^x42</i> (n = 67), MAI  =  0.9+/−0.2, EAI  =  0.5+/−0.1. Comparison of MAI values using Kruskal-Wallis one-way ANOVA indicates a significant difference among groups (P<0.001, 5 degrees of freedom), with each of the mutant genotypes (B-F) differing from the wild-type strain (A; Dunn’s method, P<0.05). Significant differences among genotypes are also observed for EAI values (Kruskal-Wallis one-way ANOVA, P<0.001, 5 degrees of freedom). Each mutant strain again differs from wild-type, and <i>unc79^f03453</i> (C) also differs significantly from the other mutant genotypes (B, D-F; Dunn’s method, P<0.05). No significant differences in MAI or EAI are observed among the strong mutant alleles <i>na^e04385</i> (B), <i>unc79^x25</i> (D), <i>unc80^GS12792</i> (E) or <i>unc80^x42</i> (F), as determined by Dunn’s method.</p