Distribution of SSS and Shaker immunoreactivity in the adult fly brain Whole–mount brain samples of iso31 (wild–type– a–d) flies were stained with an antibody to SSS (a, c) or to Shaker (b, d). Appropriate mutant lines (sss^Δ40 for anti–SSS, e, and Shaker^Df for anti–Shaker, f) are also shown. Maximal intensity projections of 1–μm confocal sections of the anterior third (ant–a, b) or posterior third (post–c, d) of the brain are shown for wild–type brains. For mutant brains, maximal projections of the entire brain are shown. The bracket and arrows point to the mushroom bodies (MB), anterior optic tubercle (AOT), superior protocerebrum (SP), and the antennal nerve (AN). SSS and Shaker expression in the posterior third of the brain included fibers from a group of visual projection neurons (VPN fiber) sending processes to the lobula plate (Lo P) of the optic lobe. Representative brains are shown, taken from at least three independent experiments. For iso31 brains, n=21 for anti–SSS and n=12 for anti–Shaker. For sss^Δ40 brains, n=16 and for Shaker^Df brains, n=10. Scale bar, 50 μm.

Altered Shaker expression and localization in sss mutants Shaker immunostaining of control iso31 (a, c) and sss^Δ40 (b, d) whole–mount adult brains is shown. Maximal intensity projections from 1–μm sections from the entire brain are shown for (a) and (b), while a single 1–μm section from the posterior aspect of the brain including the protocerebral bridge is shown for (c) and (d). A 2x magnified inset is shown for (d). Shaker immunostaining in whole–mount adult thoracic ganglia of iso31 (e,g) and sss^Δ40 (f, h). (g) and (h) are 2x magnified images from the boxed areas in (e) and (f), respectively. Structures are labeled as follows: alpha and alpha’ (α/α’) and gamma (γ) lobes of the mushroom bodies, antennal nerve (an), processes of a group of visual projection neurons (vpn), and the central connective (cc). Representative images are shown, from at least three independent experiments. n=10 and n=11 for iso31 and sss^Δ40 brains, respectively. n=9 for iso31 and sss^Δ40 thoracic ganglia. Scale bar in (a) for (a–d), (e) for (e,f), and (g) for (g,h), 50 μm

Cell–autonomous rescue of the sss phenotypes at the Drosophila larval NMJ (a) mEJP frequencies for sss^P1 (black bars) and Shaker^Df (gray bar) larvae were significantly increased relative to background controls (white bar). The UAS–sss transgene (gray bars) significantly decreased mEJP frequencies in the sss^P1 mutant background only when combined with the elav–GAL4 driver.
(b) Time–to–peak I_A current (t_peak) was significantly greater in sss^P1 (black bars) than in background controls (white bar). The UAS–sss transgene (gray bars) significantly decreased I_A t_peak in the sss^P1 mutant background only when paired with the 24B–Gal4 driver.
(c) Representative traces of I_A current illustrating the decrease in current magnitude and delayed time–to–peak in sss^P1 mutant larvae. Dashed line is from wild–type; solid line is from mutant muscle.
(d) I_A density in larval muscle could be partially rescued over a range of voltages in sss^P1 mutants bearing a UAS–sss transgene under control of the muscle–specific driver 24B–Gal4, compared to background and driver controls. Rescue was not observed using the pan–neuronal driver elav–Gal4 coupled to UAS–sss. Values for n are the same as in (b). *P < 0.05; **P < 0.01.

Rescue of the sleep phenotype of sss mutants with a UAS–sss transgene (a) Daily sleep amounts for female sss^P1 mutant flies with a UAS–sss transgene and either an elav–Gal4 or sss–Gal4 driver (white bar) were markedly increased relative to sss^P1 mutants with either the transgene or the driver alone (black bar). Heterozygous (het) sss^P1 flies served as a wild–type control (gray bar). Successful rescue of ether–induced leg shaking is indicated by “+” at the bottom. In this and subsequent figures, error bars indicate SEM. n ≥ 30 for each genotype. **P < 0.0001.
(b) Rescue of the sleep phenotype by expression of sss in cholinergic neurons. For each Gal4 driver, daily sleep amounts are presented for female sss^P1 mutant flies carrying either the driver alone (black bar) or for those carrying both the driver and a UAS–sss transgene (white bar). Rescue of ether–induced leg shaking is shown for each driver as successful (“+”) or unsuccessful (“−”). n ≥ 30 for each genotype, except for TH–Gal4 driver control, for which n = 12. *P < 0.01; **P < 0.0001.

Shaker and SSS specifically affect each other’s expression (a) Shaker was reduced in sss^P1 mutants, and SSS was reduced in Shaker^Df (Sh^Df) mutants. Shaker and SSS levels were not reduced in other short–sleeping mutants, DAT^fumin and Cl^jrk. Head extracts of background control and mutant flies of indicated genotypes were analyzed by Western blotting using antibodies to Shaker and SSS. Shaker^Df and sss^P1 mutant flies were used as negative controls, and antibody to Actin was used to control for loading. Representative blots from 4 independent experiments are shown in a and b.
(b) Eag expression was not affected in sss mutants. Head extracts of eag^sc29 and sss^P1 mutants, as well as background control, were analyzed by Western blotting using antibodies to Eag and Actin (for loading control).

Rescue of Shaker expression in sss mutants by transgenic expression of sss (a–d) OK107–Gal4 was used to direct expression of GFP (a, c) or sss (b, d), and GFP or Shaker expression was examined, respectively. Since Shaker was enriched in synapse–rich neuropil, GFP fused to synaptogamin (syt–GFP), which is targeted to the synapse, was used. GFP expression was seen in the mushroom bodies (a), but not in the group of visual projection neurons (VPNs) sending processes to the optic lobe (c). Transgenic expression of sss using OK107–Gal4 increased Shaker expression in the mushroom bodies (b), but not in the optic lobe (d).
(e–h) vGlut–Gal4 directed GFP expression in the visual projection neurons and the optic lobe (g), but not the mushroom bodies (e). Transgenic expression of sss using vGlut–Gal4 increased Shaker expression in the optic lobe (h), but not in the mushroom bodies (f). Arrows point to the fiber bundles formed by the VPNs. Maximal intensity projections of seven 1–μm sections from the anterior of the brain are shown for (a), (b), (e), and (f), and a single 1–μm section from the posterior of the brain at the level of the protocerebral bridge is shown for (c), (d), (g), and (h). Representative brains are shown, taken from two independent experiments. n=5 or 6 for all genotypes. Scale bar in (a) for (a,b,e,f) and (c) for (c,d,g,h), 50 μm

SSS modulates Shaker function.
(a) Representative normalized currents measured at 15 mV from a holding potential of −100 mV in excised patches of HEK–tsA cells transfected with cDNA encoding the ShakerB (ShB) isoform in the absence or presence of sss.
(b) Representative normalized currents measured by two–electrode voltage clamp at 20 mV from a holding potential of −70 mV in oocytes expressing the ShakerD (ShD) isoform in the absence or presence of sss.
(c) Time to reach peak current (t_peak) following a step change to 15 mV from a holding potential of −100 mV in HEK-tsA cells or following a step change to 20 mV from a holding potential of −70 mV in oocytes. n=5 for ShakerB alone, n=6 for ShakerB + sss, n=14 for ShakerD alone, n=14 for ShakerD + sss; **P < 0.01.
(d) Extracts from oocytes injected with cRNA derived from ShakerD, sss, or both were immunoprecipitated (IP) with an antibody to Shaker and analyzed by Western blotting (WB) using an antibody to SSS. Prior to immunoprecipitation, 5% of cell extracts (Input) were analyzed separately using antibodies to SSS and Shaker. A non–specific band (*) served as a loading control.