Pattern of distribution and cycling of SLOB, Slowpoke channel binding protein, in Drosophila
- Angela M Jaramillo†1,
- Xiangzhong Zheng†1, 2,
- Yi Zhou1,
- Defne A Amado1,
- Amanda Sheldon1,
- Amita Sehgal1, 2 and
- Irwin B Levitan1Email author
© Jaramillo et al; licensee BioMed Central Ltd. 2004
Received: 18 September 2003
Accepted: 27 January 2004
Published: 27 January 2004
SLOB binds to and modulates the activity of the Drosophila Slowpoke (dSlo) calcium activated potassium channel. Recent microarray analyses demonstrated circadian cycling of slob mRNA.
We report the mRNA and protein expression pattern of slob in Drosophila heads. slob transcript is present in the photoreceptors, optic lobe, pars intercerebralis (PI) neurons and surrounding brain cortex. SLOB protein exhibits a similar distribution pattern, and we show that it cycles in Drosophila heads, in photoreceptor cells and in neurosecretory cells of the PI. The cycling of SLOB is altered in various clock gene mutants, and SLOB is expressed in ectopic locations in tim 01 flies. We also demonstrate that SLOB no longer cycles in the PI neurons of Clk jrk flies, and that SLOB expression is reduced in the PI neurons of flies that lack pigment dispersing factor (PDF), a neuropeptide secreted by clock cells.
These data are consistent with the idea that SLOB may participate in one or more circadian pathways in Drosophila.
Five independent groups [1–5] recently conducted genome-wide microarray analyses to identify Drosophila transcripts that display circadian oscillations. Each group uncovered slob as a robustly cycling RNA transcript. SLOB, Slowpoke binding protein, is a key component of the Drosophila Slowpoke/SLOB/Leonardo dynamic protein complex . This complex is thought to affect membrane excitability, as electrophysiological recordings reveal that SLOB binding to the channel results in an increase in channel activity, whereas the addition of Leonardo to the SLOB/dSlo complex dramatically shifts the channel voltage range of activation to more depolarized potentials .
The circadian system consists of an input pathway, a central clock, and an output pathway . The clock itself is comprised of the transcription factors CLOCK (CLK) and CYCLE (CYC), which bind to the promoters of period (per) and timeless (tim), inducing their expression. The PER and TIM proteins heterodimerize and feed back to repress activity of CLK/CYC. Although the expression patterns of CLK and CYC are not known, the localization of PER and TIM has been important with respect to identifying neurons relevant for circadian behavioral rhythms . This molecular loop must transduce signals to surrounding cells to generate rhythmic behavior . Altering membrane excitability is a key mechanism for transducing neuronal information, and thus it is reasonable to suspect that the molecular feedback loop might communicate with ion channels.
Ion channels may be under direct transcriptional control of the clock genes or modulated by clock-controlled genes [10, 11]. Mounting evidence supports the importance of electrical activity for the propagation of circadian oscillations. For example, electrical silencing of clock neurons through targeted expression of potassium channels stops the oscillation of PER and TIM proteins and causes arrhythmicity in flies . The cyclic release of neuropeptides from clock cells  may be a direct consequence of a rhythmic fluctuation in membrane potential . Diurnal modulation of pacemaker potentials and calcium current, intracellular calcium levels and NMDA-evoked calcium currents have all been observed within a mammalian central clock, the suprachiasmatic nucleus (SCN) [11, 15, 16]. In addition, microarray screens have detected the cycling transcripts of ion channels such as Shaker, trpl and slowpoke [1, 3], and flies mutant in slowpoke have weak locomotor rhythms . These observations suggest that ion channels and their modulators may participate in circadian regulation.
We explored a role for SLOB in the circadian system and found that SLOB protein cycles in Drosophila heads during both light/dark and constant darkness conditions. SLOB oscillates in at least two discrete areas of the fly head, the photoreceptor cells and the PI neurons. The photoreceptors have their own peripheral circadian oscillator , whereas the PI neurons, large neurosecretory neurons, are suspected to play a role in the output pathway that drives rest:activity rhythms (Kaneko and Hall, 2000). Our results reveal differential effects of clock mutations on SLOB expression and cycling in these two regions. There is a significant decrease in SLOB levels in the PI neurons of Pdf 01 flies, thus implicating PDF as an upstream regulator of SLOB. SLOB also no longer cycles in the PI neurons of Clk jrk flies, supporting the idea that SLOB is a clock controlled protein. Together with the observation that flies overexpressing SLOB exhibit a breakdown of rest:activity patterns, these data are consistent with the idea that SLOB participates in circadian rhythms.
SLOB protein cycles in Drosophilaheads
To ensure that rhythms of SLOB expression are circadian and not driven only by external LD cycling we analyzed SLOB protein cycling in conditions of constant darkness. Flies were entrained for three days in LD, and then placed in constant darkness (DD) for two days. SLOB protein still cycles under these conditions, with a slightly dampened amplitude, but with the same overall phase (Figure 1B). A similar decrease in amplitude has been described for oscillations of other clock genes in DD [18, 19].
Expression of SLOB protein is influenced by clock genes
The cycling of SLOB was also analyzed in per 01 and tim 01 mutants during LD (Figure 2A) and DD (Figure 2C,2D). In both mutant lines, SLOB continues to cycle in LD with a phase similar to that of wild type (Figure 2A). SLOB may be regulated by a direct light-dependent mechanism, obviating the need for these clock genes in LD. As in the case of Clk jrk , there appears to be either dampened or no protein cycling in per 01 flies under DD conditions (Figure 2C). However, in tim 01 flies SLOB still cycles, but there is a shift in the phase of the oscillation in DD (Figure 2D). Instead of peaking at Circadian Time (CT) 10–14, SLOB now peaks at CT 2. This suggests that the regulation of SLOB by TIM may be different from that by PER, which is not unprecedented .
Expression pattern of slob transcript in Drosophilaheads
SLOB is expressed in the photoreceptors and the brain
Siegmund and Korge  performed a large scale analysis of peptidergic neurons of Drosophila larvae. Their study identified three distinct subsets of PI neurons (PI 1–3) that innervate the corpora cardiaca (a glandular tissue) and the aorta. To determine which subset of PI neurons is SLOB positive we used four of their enhancer trap lines. Mai 301, Kurs 58 and Kurs 45 GAL4 lines express GAL4 exclusively in subsets PI-1, PI-2 and PI-3, respectively. Mai 281 GAL4 expresses GAL4 in two subsets, PI-2 and PI-3. We crossed these GAL4 flies to a UAS-GFP transgenic line, and found by immunostaining that SLOB is expressed exclusively in subset PI-3 (Figure 4F,4G). SLOB does not colocalize with GFP from the enhancer fly lines that express GFP only in subsets PI-1 or -2 (Figure 4D,4E).
Figure 4H highlights components of the circadian system in order to illustrate the location of the PI neurons in relation to other clock gene expressing cells. The lateral neurons (LNs) as mentioned before consist of three clusters, a cluster of cells located dorsally (LNds), and two other ventrally located clusters differing in the size of their somata, large LNvs and small LNvs. Another group of clock gene expressing cells are the dorsal neurons (DNs), which consists of three subsets as well, DN1–3. This wholemount was immunostained for SLOB, PER and PDF. The LNs and DNs are immunostained red indicating the presence of PER. Projections from the LNvs are green due to PDF immunostaining. The PI neurons are the SLOB positive green cells.
The phase of SLOB protein cycling is different in the photoreceptor and PI neurons
Ectopic expression of SLOB in tim 01 flies
SLOB is regulated by a circadian output molecule, PDF
Overexpression of SLOB alters locomotor activity
We report here that the dSlo binding protein, SLOB, cycles in Drosophila heads. Microarray analyses reported slob transcript cycling with a peak at either ZT 15  or at CT 11 . We show by western analysis that SLOB protein peaks at ZT 10–14/CT 14, consistent with an earlier peak for the RNA. Under LD conditions, SLOB continues to cycle in per 01 , tim 01 and Clk jrk flies in phase with the oscillation of wild type flies. It was noted in one of the recent microarray studies  that there is a cluster of genes, named the apterous cluster, that shows rhythmicity in these three mutants during LD. This cluster has a characteristic peak at ZT 17 and includes such proteins as transcription factors, synaptic regulators and transporters. The genes in this group may be regulated not only by the circadian clock, but also by a light-dependent mechanism. However in DD, where light is no longer a factor, we find that SLOB does not cycle in Clk jrk and per 01 and exhibits an altered phase in tim 01 flies. One might expect tim 01 and per 01 flies to give similar results, but tim 01 flies may not be true genetic nulls . Consistent with the observation that SLOB does not cycle in Clk jrk flies during DD (Figure 2B) are microarray data from Ueda et al.  indicating that slob levels do not change in Clk jrk flies, and other microarray data from McDonald and Rosbash  demonstrating that slob levels are at mid-point in the Clk jrk mutants. Taken together these data demonstrate clearly that clock genes regulate slob mRNA and protein expression.
Within Drosophila adult heads, slob mRNA is present in the photoreceptors, optic lobe, the neurosecretory cells of the PI and the surrounding brain cortex. We also find prominent immunostaining for SLOB protein in the photoreceptor cells and the PI neurons. In situ hybridization experiments with larval brain revealed slob RNA in an area of the brain close to PDF-filled projections of the lateral neurons . This is consistent with our findings of slob transcript in the PI neurons and surrounding cortex.
The Drosophila eye expresses many of the major circadian genes, and is thought to contain an autonomous oscillator that presumably regulates an eye-specific function . In addition, the eye contributes to photic entrainment of the pacemaker LN cells . Interestingly, the SLOB binding partner, Slo is also expressed in the visual system including the eye, lamina, medulla and lobula . Ceriani et al.  have demonstrated that the RNA levels of both slob and slo cycle in phase in both LD and DD. The dSlo protein was also shown by western blot to cycle and peak at ZT 20 . This correlates with the cycling of SLOB in the photoreceptors, where SLOB peaks at ZT 14–21 (Figure 5A).
The PI region lies directly beneath the root of the ocellar nerve. The PI neurons have large, 15 μm diameter, cell bodies, and their axons project along the median bundle and then bifurcate . One of the branches proceeds ventrally and arborises in the dorsal tritocerebrum region, below the oesophagus. The other branch moves in a posterior direction and enters the cardiac recurrent nerve in the oesophageal canal. The PI neurons have an extensive network of endoplasmic reticulum and contain secretory granules, suggesting that they are neurosecretory cells . In insects, peptidergic neurons of the central nervous system regulate the synthesis of developmental hormones. The PI neurons, in particular, have been implicated in hormone production and release in various insects [28, 29]. Three subsets of PI neurons have been identified. We have identified the subgroup PI-3 to be the SLOB positive subset of PI neurons. Among the hormones identified in the PI neurons is insulin, and it has been proposed that the release of insulin into the hemolymph is essential for growth control and carbohydrate homeostasis . We have confirmed that the SLOB positive PI neurons are also insulin positive (data not shown).
The photoreceptors and the PI neurons express oscillating SLOB protein and intriguingly, the rhythms in the two neuronal types are not in phase with each other. The mechanisms responsible for these phase differences are not known. PER and TIM are expressed in the lateral neurons in the central brain, in glial cells of the optic lobes, and in the photoreceptor cells . PER and TIM protein have not been shown to be expressed in the PI neurons, although Kaneko and Hall  found that there is expression of GAL4 driven by the per promoter in PI neurons. The photoreceptors in contrast have all the traditional clock genes that might contribute to SLOB cycling [32–34]. The presence of these genes in the eye, but not in the PI, may contribute to the phase differences. In addition, it is reasonable to expect that there will be molecular differences in circadian regulation in different cell types. Furthermore, the subcellular localization of SLOB is different between the two areas (Figure 4A,4C). SLOB appears to be primarily cytoplasmic in the PI neurons, and nuclear in the photoreceptors.
How is SLOB regulated in the PI neurons? Interestingly, the PI neurons have been associated with behavioral rhythmicity and it has been hypothesized that this involves hormone release . In fact, the PI neurons of Teleogryllus commodus (crickets) have long been hypothesized to serve as a region of coupling between the circadian pacemakers and behavioral rhythms . A pathway for Drosophila proposed by Kaneko and Hall  suggests that oscillatory signals of the "master pacemaker" in the small LNvs first modulate the oscillatory mechanism or neuronal activity operating within neurons in the dorsal region. The DNs send their oscillatory signals to the PI, which may lead to rhythmic neurosecretory peptide release. It has been observed in the larval CNS that projections of the DNs terminate near the midline in the PI region . These DNs express both PER and TIM and hence may send robust oscillatory signals to downstream targets such as PI neurons. Interestingly, two neurons of the DN group express PER and TIM cycling antiphase to the other DNs and LNvs . Regardless of the precise role of the DNs, it is clear that this dorsal region of the brain is important for rest:activity rhythms. For example, PDF release and MAPK activity cycle specifically in this region, and both participate in behavioral rhythmicity [13, 37].
Clock mutants alter either SLOB protein oscillation or levels in both the eye and PI neurons. One striking observation is the ectopic or elevated expression of SLOB in the lamina of tim 01 flies. This suggests that TIM negatively regulates SLOB. Western analyses show that upregulation of SLOB does not occur in per 01 flies. Ectopic expression of PER occurs in double-time (dbt) flies that are mutant for a casein kinase 1ε involved in PER turnover . The interpretation in that case is that PER is synthesized in many cell types where its expression is normally undetectable due to destabilization by the kinase. A similar mechanism may account for elevated expression of SLOB in tim 01 flies. The observation that tim 01 alters SLOB expression while per 01 does not could suggest a pathway for slob regulation that is independent of PER. We also found that SLOB fails to cycle in the PI neurons of Clk jrk flies.
Perhaps most intriguing is the decrease of SLOB in the PI neurons of Pdf 01 flies. The oscillation of PDF is restricted to the dorsal projections emanating from the lateral neurons . Dorsal terminals of the LNs express abundant PDF early in the morning, which is indicative of a block in its release. Thus, PDF release is low during the day while SLOB is at its trough, consistent with PDF being a positive regulator of SLOB. In Figure 4H we see that PDF expressing terminals do not appear to contact the PI neurons. As discussed above, we hypothesize that PDF termini affect the DN, or alternatively, other neurons of the dorsal region, which in turn communicate with the PI neurons. Clk jrk flies lack PDF in the small ventral LNs, but still express it in the large LNs . This may account for the difference in the phenotype of Clk jrk and Pdf 01 mutants and would suggest a role for the large LNs in SLOB regulation.
The molecular oscillations of the circadian clock proteins result ultimately in behavioral rhythmicity . Our data demonstrate that the panneuronal expression of SLOB causes a delayed breakdown of rhythms. Breakdown after several days is characteristic of some circadian output mutants such as Pdf 01 flies . Likewise, panneuronal overexpression of PDF results in a delayed disruption of rhythms, and overexpression of PDF in the PI neurons results in a shortened period and an advance of the morning peak . disconnected (disco) flies, which lack LNs, also become arrhythmic only after several days in DD .
Using GAL4 drivers that direct overexpression of SLOB specifically to the eye or PI neurons, we found no obvious circadian locomotion phenotype. It is possible that these drivers are not strong enough, compared to the panneuronal driver elavc155. We note that the overexpression of PER and TIM with the tim-GAL4 driver results in a more severe phenotype than with per-GAL4, even though the expression pattern of the two drivers is similar , possibly because the per promoter is weaker . Alternatively, the specific drivers we used may not target all the behaviorally-relevant SLOB positive neurons in the eye and PI region. Any SLOB positive neurons that are not overexpressing SLOB are therefore wild type, and this may prevent rhythmicity breakdown. Similar explanations have been proposed for the lack of a phenotype when PER and TIM are overexpressed by the pdf-GAL4 driver although both genes cause arrhythmia when their overexpression is driven by the more widely expressed tim and per-GAL4 drivers [24, 42].
The widespread distribution of SLOB in the eye and brain suggests that other cells, in addition to or instead of the photoreceptor and PI neurons, may account for SLOB's apparent role in behavioral rhythms. The panneuronal overexpression of SLOB in other SLOB-expressing cells in the brain cortex may explain the rhythmic breakdown. Alternatively, ectopic expression of SLOB in neurons involved in locomotor rhythms might also account for the altered rhythmicity.
In this study, we have demonstrated that SLOB protein cycles in a circadian fashion in Drosophila heads. SLOB oscillates in two discrete areas of the fly head, the photoreceptor cells and the PI neurons. Our results reveal differential effects of clock mutations on SLOB expression and cycling in these two regions. There is a significant decrease in SLOB levels in the PI neurons of Pdf 01 flies, thus implicating PDF as an upstream regulator of SLOB. Along with the observation that flies overexpressing SLOB exhibit a breakdown of rest:activity patterns, these data suggest that SLOB is a clock controlled protein.
We showed previously that SLOB, along with dSlo and Leonardo, participates in a dynamic regulatory complex in presynaptic nerve terminals. Leonardo binding is dynamically regulated by phosphorylation of SLOB by the calcium/calmodulin-dependent protein kinase II (CAMKII) . Intriguingly, CAMKII has recently been implicated in circadian rhythmicity in vertebrates , and it is tempting to speculate that this regulatory complex participates in the fly circadian output pathway. Not only do dslo mutants have weak rhythms [1, 44], but leonardo mutants have defects in behavior, synaptic transmission and plasticity [45, 46]. Our data are consistent with the hypothesis that SLOB participates in circadian rhythmicity by regulating synaptic function and membrane excitability.
Fly stocks and germ line transformation
D. melanogaster strains Canton S (wild type), y w, ry, tim 01 , per 01 , Clk jrk , and Pdf 01 and transgenic fly strains were raised at 25°C on standard Drosophila medium. slob cDNA was cloned into a pUAST vector and P-element-mediated transformation was performed as described previously . The transformed lines were crossed to either elavC155 (provided by Leslie Griffith), GMR (provided by Konrad Zinsmaier) or Mai 281, Mai 301, Kurs 45, and Kurs 58 GAL4 (provided by Gunter Korge).
In situ hybridizations
The slob RNA antisense and sense probes were synthesized using the DIG RNA Labeling Mix (Boehringer Manheim). The sequence used for the RNA probes was made from basepairs 1142–1441 of the slob transcript. In situ hybridization on adult 12 μm head sections were done according to the protocols found at http://www.rockefeller.edu/labheads/vosshall/protocols.php with slight modifications. All hybridizations and washes were done at 55°C. Sections are developed in the dark for 3 days.
SLOB antibody purification
A GST-SLOB fusion protein was used to immunize rabbits as described previously . Polyclonal antibodies specific to SLOB were generated by purifying the serum using a combination of CNBr conjugated GST and CNBr conjugated GST-SLOB columns.
Western blotting and quantitation
Flies were entrained and collected in LD and DD conditions at four hour intervals. Fly heads were lysed in 1% CHAPS, 20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 120 mM NaCl, 50 mM KCl, 2 mM DTT and protease inhibitors (1 mM PMSF, 1 μg/mL each aprotonin, leupeptin, and pepstatin A (SIGMA)). Protein concentration was determined using the BioRad DC Protein Assay. 100 μg of protein was loaded on 4–15% polyacrylamide gradient gels and transferred to nitrocellulose membranes. After blocking with 5% nonfat milk in TBST (0.1% Tween 20 in Tris-buffered saline), the blots were probed with the appropriate primary and secondary antibodies. Enhanced chemiluminescence detection system (Amersham) was used to visualize the proteins. Film exposures of western blots were scanned using Bio Rad Molecular Analyst. The level of SLOB at each time point was calculated as the SLOB signal minus the background in each lane. The blots were stripped and reprobed with anti-MAP Kinase (Sigma). The ratio of SLOB to MAP Kinase was normalized and averaged between several westerns.
Brain whole mount: Flies were entrained in LD for three days and then transferred to DD. Fly heads were collected at given time points, fixed in 4% paraformaldehyde (PFA), and the brains were dissected and kept in cold PBS, and subsequently blocked with 6% normal donkey serum in phosphate-buffered saline (PBS)/0.3% Triton X-100 for one hour. Samples were then incubated with primary antibody at a dilution of 1:400 overnight at 4°C. After washing in PBS/0.3% Triton X-100 three times for 30 min each at room temperature, samples were incubated with the appropriate secondary antibody (Fluorescein (FITC)-conjugated AffiniPure Donkey Anti-Rabbit IgG and Texas Red dye-conjugated AffiniPure Donkey Anti-Rabbit IgG from Jackson ImmunoResearch) at a dilution of 1:500 in 3% normal donkey serum in PBS/0.3% Triton X-100 for 1 hour at room temperature, and washed in PBS three times for 30 minutes each. Brains were mounted onto slides with mounting medium (Vector H-1200). Wholemounts were visualized using fluorescence microscopy on a Leica DMIRE2.
Section: Flies were collected at given time points, mounted with mounting medium, and sectioned at 12 μm using a cryostat (Leica 3450). Sections were fixed with 4% PFA, washed in PBS/0.3% Triton X-100, and blocked with 6% normal donkey serum in PBS/0.3% Triton X-100 for 1 hour, and subsequently incubated with primary antibody in 6% normal donkey serum in PBS/0.3% Triton X-100 overnight at 4°C. The sections were then washed and incubated with secondary antibodies as described above.
The staining intensity of brain whole mounts and sections was assessed by blind scoring. A subjective intensity scale from zero to four was used, with zero being undetectable and four being maximal. Statistical analysis of average staining intensity scores was done using ANOVA and the Tukey HSD test.
Flies aged from 1–5 days were entrained for three days in 12 hr light/dark cycles at 25°C and then kept in constant darkness for 14 days. Activity was monitored by using the Trikinetics system. Individual flies were analyzed for rhythmicity based on their by chi-square periodogram and Fast Fourier Transform (FFT) values . The analyses were performed using ClockLab software.
This work was supported by grants from the National Institutes of Health to I.B.L. and A.S., an NRSA to A.M.J. and the US Army Medical Research Command to A.S. We are grateful to Hua Wen, Konrad Zinsmaier, Leslie Griffith and Joan Hendricks for helpful discussions and to Gunter Korge for his GAL4 lines.
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