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. Author manuscript; available in PMC: 2016 Jun 29.
Published in final edited form as: Curr Biol. 2015 Jun 18;25(13):1717–1726. doi: 10.1016/j.cub.2015.05.037

Figure 3. TARA Regulates Sleep in Neurons.

Figure 3

(A) Western blot showing a marked reduction of TARA in tara1/s132 mutants compared with control flies. Head extracts of control flies and tara mutants (lanes 1 & 2) as well as S2 cell extracts transfected with an empty UAS vector or a UAS-tara construct under the control of actin-Gal4 (lanes 3 & 4) were probed with a polyclonal antibody to TARA. The band that corresponds to TARA can be readily recognized by the upregulation in S2 cells transfected with tara cDNA. # denotes non-specific labeling or a degradation product of TARA. MAPK was used to control for loading. (B) Maximal-intensity projection of confocal slices of the adult brain showing widespread expression of TARA::GFP. Scale bar: 50 μm. (C) Representative confocal sections of tara::GFP adult brains costained with antibodies to GFP and ELAV or REPO (neuronal or glial marker, respectively). Each panel shows a single confocal slice of a region ventral to the mushroom bodies. Scale bar: 10 μm. (D) Pan-neuronal knockdown of tara markedly reduces sleep. Pan-neuronal elav-Gal4 or nsyb-Gal4 was used to drive a combination of two UAS-tara RNAi constructs and UAS-dcr2 (elav > tara RNAi and nsyb > tara RNAi, respectively). Flies harboring the two UAS-tara RNAi constructs and UAS-dcr2 without a driver (+ > tara RNAi) and those harboring a driver and UAS-dcr2 (elav > + or nsyb > +) served as controls (n=31–58). (E) Adult-stage expression of tara partially rescues the tara short-sleeping phenotype. Daily sleep is presented for females of the indicated genotypes in the absence (white bar) or presence (black or red bar) of RU486 (n=24–32). Data from parental control flies show that RU486 by itself did not affect sleep. (F) Knockdown of tara in cholinergic neurons reduces sleep. For each Gal4, total daily sleep of females expressing tara RNAi under the control of the driver (black bar) was compared to parental controls (white bar) (n=30–173). The sleep phenotype of flies in which tara was knocked down in dopaminergic neurons was not determined due to lethality. Mean ± SEM is shown. ***p < 0.001, Dunnett post hoc test relative to both parental controls (D), t test with Bonferroni correction (E), Tukey post hoc test relative to both parental controls (F). See also Figure S3.