2mit gene structure and protein sequence analysis

D. melanogaster 2mit (FBgn0260793) is organized in 2 exons and 1 intron and maps in an opposite transcriptional orientation within the 11^th intron of the tim2 locus (Figure 1A [7]). 2mit conceptual translation originates a 1141 aa protein (NP_650258) with a predicted molecular weight of ~125 kDa, a theoretical pI of 6.53 and a slightly hydrophilic Grand Average of Hydropathicity (GRAVY) value of -0.396. A transmembrane domain spanning 931-953 residues was recognized by different software tools with the N- and C-terminal regions localized in the extracellular space and cytoplasmic compartment, respectively. The N-terminal region was found to contain a signal peptide with a cleavage site at position 29. These analyses strongly suggest that the mature form of the 2MIT protein is ~122 kDa in size, 1113 aa long, and a type I single-pass transmembrane protein. The 902 aa N-terminus is suggested to be exposed to the extracellular (or luminal) space, while the 187 aa C-terminus is cytoplasmic (Figure 1B). The extracellular region is suggested to be composed of a structured domain and a long disordered region. The latter is characterized by low complexity sequences (residues 522-755 and 795-911) with a Thr-rich domain spanning 530-705 residues and a partially overlapping Ala-rich domain between 552-617 residues. The cytosolic domain is predicted to be prevalently disordered and contains a Ser-rich domain. In this region, the ELM server recognized several functional motifs, such as binding sites for 14-3-3 protein/s, Forkhead-associated (FHA) 1 and 2 factors, and a phosphorylation site for protein kinase A (PKA; Figure 1B).

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Figure 1. tim2-2mit host-nested gene organization in D. melanogaster.

(A) Schematic representation of the tim2 locus showing positions and intron-exon structures for the four nested sequences (CG34308, BK002510, 2mit and AY118619) with only 2mit representing a protein coding gene. Arrowheads show positions of PB (indicated with c or f) or MB transposons in different insertional strains. (B) Diagram showing the organization of protein domains contained in 2MIT. The signal peptide (yellow), the LRR domain with 16 repeat units (blue) and their N- and C- flanking regions (orange), the transmembrane region (TM, purple), and the presence of Ala-, Thr-, Ser-rich domains (green) are shown both for the extracellular and cytoplasmic portions of the protein (drawing not to scale). The 3D-structure at the top represents the 2MIT LRR repeats of the Toll-like receptor 3 crystal structure (PDB code: 2A0Z, chain A). The predictions of sequence features (low complexity and disorder) are listed at the bottom as grey and black rectangles. Linear motifs found with ELM with their approximate position are indicated by black vertical bars.

https://doi.org/10.1371/journal.pone.0076351.g001

FlyBase reported a second 2MIT protein isoform predicted to be 13 residues longer, possibly originating from translational stop codon read-through [9]. The C-terminal region of this longer 2MIT isoform (1155 aa) contains a TRG_endocytic motif, which is implicated in vesicular trafficking of different molecules (Figure 1B).

A scan of the N-terminal sequence against domain and protein signature databases, such as Pfam and PROSITE, revealed the presence of some LRRs which correspond to structural units (with a LxxLxLxxN/CxL conserved pattern) consisting of a β-strand and an α-helix. Since LRR domains are organized in series, they can form non-globular, crescent-shaped structures, which create a solvent-exposed, elongated, and concave surface of parallel β-strands, acting as a scaffold for interactions with other proteins [10]. The LRR domain was modeled using a MANIFOLD approach [11], which combines the prediction of secondary structures and results obtained by different repeat prediction methods. The first step was to identify the correct number of repeated units. RADAR, TRUST, and Repetita tools revealed different numbers of repeats. A structural alignment of all predicted repeated units was built in order to calculate a consensus pattern and to identify other missing repeats in the region spanning between the 80 and 530 amino acid positions. It was thus possible to recognize 16 repeats matching the consensus xxxLxxLxLxxNxLxxLpxxoFxx sequence that is typical for the LRR domain (Figure 2A). Each repeat contained hydrophobic, conserved positions (mostly Leu residues), predicted to be buried internally and to have a structural role. Other polar/charged residues are likely exposed to the solvent and are probably involved in protein-protein interactions.

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Figure 2. Structural analysis of the D. melanogaster 2MIT LRR domain.

(A) Identification and structural alignment of the LRR units. The sequence of the LRR N-flanking region forming three hypothetical α-helices is outlined at the top. The main LRR repeats are aligned based on the structural correspondence between residues. A schematic cartoon used to depict the β-strand and the α-helix is shown above, and the consensus sequence is highlighted below the repeats. The bottom shows the LRR C-flanking region sequence with connecting lines between cysteines forming disulfide bonds. (B) Two dimensional representation of LRR repeats with β-strand and α-helix from N- to C-terminus. The consensus sequence is shown inside circles representing single residues. Conserved hydrophobic positions (pink circles) face the internal side of the repeat, while conserved polar residues (blue circles) map on the surface.

https://doi.org/10.1371/journal.pone.0076351.g002

Most LRR proteins contain flanking regions that are an integral part of the LRR domain. In the D. melanogaster 2MIT protein two terminal variable regions flank the LRR-NT and LRR-CT repeats. These regions usually have a capping role which protects the first and last repeats, but may also have a functional role. The LRR-NT is predicted to be disordered, contains low complexity regions, and includes a cluster of charged residues between the 64 and 91 positions. Secondary structure analysis of the LRR-NT revealed that there are three hydrophilic α-helices which may form a N-terminal cap. The LRR-CT is estimated to be about 70 residues long and contains four Cys that may form disulfide bonds (Figure 2A). The template search identified the structure of the Toll-like receptor 3 (PDB code: 2A0Z, chain A) as the most structurally similar to the 2MIT LRR domain (Figure 2B).

2mit expression during development

2mit is transcribed throughout D. melanogaster development in two mRNA isoforms of ~5.4 and 3.8 kb, carrying a unique 5’ UTR and two length-differing 3’ UTRs (353 and 1892 nt excluding the polyA segment [7]). In situ hybridization experiments on whole-mount embryos detected a diffuse 2mit expression pattern at the blastoderm stage (stage 5; Figure 3A). During segmentation (stage 15), 2mit mRNA localized in the developing Central and Peripheral Nervous Systems (CNS, PNS; Figure 3C, D).

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Figure 3. 2mit mRNA expression in w¹¹¹⁸ flies.

(A-D) Embryos at different developmental stages hybridized with the antisense 2mit probe. (A) Stage 5. (B) Stage 9. (C-D) lateral and frontal views of stage 15. (E) Negative control showing a stage 15 embryo hybridized with the 2mit sense probe. Bar in (E) represents 25 µm for (A)-(E). (F) Northern blot from wild-type adult heads sampled every 3 h in 12:12 LD conditions. 2mit indicates the single 2mit transcript revealed in adult heads. rp49 represents the rp49 housekeeping mRNA. M: RNA Ladder molecular marker. (G) 2mit mRNA levels [mean ± standard error of the mean (SEM)] sampled every 4 h in 12:12 LD (white bars) and DD (black bars) conditions. For each condition, 3 replicates were performed. In 12:12 LD, significant variations in 2mit mRNA levels were detected (F_5,10=7.89, p <0.01). In DD, no significant modifications in 2mit mRNA levels were detected (F_5,12= 0.56 p= 0.76). (H-N) 2mit mRNA localization in whole-mount adult brains sampled at ZT 0. (H-L) Wild-type adult brains hybridized with the 2mit antisense probe. (H) 2mit mRNA signals are visible at the level of the Kenyon cells (arrow) and in the different lobes of the MBs (image shows a ~6 µm Z-projection along the antero-posterior axis). (I) and (L) ~5 µm Z-projections obtained from the same brain showing 2mit mRNA signals in the Kenyon cells (arrows; I) and in the EB (L). (M) 2mit^c03963 adult brain hybridized with the 2mit antisense probe. Weak signals are detected in the MB lobes and EB. (N) Wild-type adult brain hybridized with the 2mit sense probe (negative control). (M) and (N) are ~10 µm Z-projections. The following abbreviations are used: ol: optic lobe; al: antennal lobe; mb: mushroom bodies; mbl: mushroom bodies lobes; eb: ellipsoid body; sg: subesophageal ganglion; cg: central ganglion. α/ α’: vertical mushroom bodies lobes; β, β’, γ: medial mushroom bodies lobes. Bar in (N) represents 50 µm for (H), (M) and (N), and 25 µm for (I), (L), and the 2X magnification inset in (M).

https://doi.org/10.1371/journal.pone.0076351.g003

In adult heads of wild-type flies that underwent 12 h: 12 h light:dark cycles (12:12 LD conditions, with ZTs 0 and 12 corresponding to lights-on and -off, respectively), Northern blot analyses revealed the presence of a single transcript corresponding to the ~5.4 kb 2mit mRNA isoform, suggesting that at least in that anatomical structure the longer mRNA variant is the most represented 2mit transcript (Figure 3F). To evaluate potential circadian variations in 2mit expression levels, quantitative PCR (QPCR) experiments were performed on adult heads, sampled every 4 h during the day, in both 12:12 LD and constant darkness (DD) regimes. Under 12:12 LD conditions, 2mit transcript levels showed an oscillating profile with a slight but significant variation over the 24 h cycle (F_5,10= 7.89 p <0.01; Figure 3G). In particular, we revealed an increase in 2mit mRNA levels at approximately the end of the night/beginning of the day, between ZT 20 and ZT 0, and a reduction at ZT 8 (Newman-Keuls post-hoc test: ZT 8 vs ZT 0 or ZT 20p<0.05). After two days of DD, 2mit expression became constitutive (F_5,12= 0.56 p= 0.76, not significant; Figure 3G).

2mit mRNA localization pattern was evaluated by in situ hybridization experiments on whole-mount adult fly brains sampled at ZT 0, when 2mit expression was known to be high. Specific hybridization signals were observed mainly in the MBs and EB of the central complex (Figure 3H-L), structures primarily involved in learning, memory, and locomotor activity control [12,13]. In the MBs, 2mit mRNA staining was observed in the neuronal somata (Kenyon cells) and at the level of both the vertical (α/α’) and medial (β, β’, γ) lobes, representing compact axonal structures (Figure 3H, I). Additional 2mit expression was visualized in the sub-esophageal ganglion (SOG; Figure 3H). Diffuse and weak signals were noted in both optic and antennal lobes (OLs, ALs; Figure 3H), probably as a result of non-specific hybridization staining. In fact, similar signals were observed when the 2mit sense probe was used in the same regions of the negative controls (Figure 3N).

Our 2mit mRNA analyses are consistent with high-throughput expression data from FlyBase indicating that the highest 2mit expression levels occur during embryogenesis, between 14 and 20 h after fertilization, and that transcription is restricted to nervous system structures at both larval and adult stages. Moreover, a recent study listed 2mit (CG 17319) among those genes preferentially expressed in the MBs [14].

2mit is involved in courtship conditioning memory but not in learning

We previously demonstrated that the c03963 transposon insertion line (Exelixis Gene Disruption Project), carrying a PiggyBac (PB) element ~20 kb upstream of the 2mit ATG start codon, was characterized by a ~50% 2mit mRNA decrement and normal tim2 mRNA levels estimated at the third larval stage (L3 [7];; Figure 1A). After out-crossing with w¹¹¹⁸ flies for eight generations, a ~50% 2mit mRNA depletion and unaffected tim2 mRNA levels compared to w¹¹¹⁸ controls were confirmed in c03963 homozygous L3 (2mit^c03963) by QPCR (Table 1). We also analyzed mRNA levels of the other internally transcribed sequences in the tim2 locus, showing that the PB insertion in 2mit^c03963 homozygous L3 did not cause any significant modifications in their expression compared to w¹¹¹⁸controls (mRNA levels in 2mit^c03963 and w¹¹¹⁸ individuals: CG34308: 1.13 ± 0.33 vs 1 ± 0.0; F_1,6= 0.15 p= 0.71; BK002510: 1.27 ± 0.57 vs 1 ± 0.0; F_1,6= 0.22 p= 0.64; AY118619: 0.94 ± 0.17 vs 1 ± 0.0; F_1,6= 0.93 p= 0.37; all p values are not significant). We next determined 2mit mRNA levels in 2mit^c03963 homozygous adult heads, showing ~20% 2mit mRNA transcription levels compared to those of w¹¹¹⁸ controls (Table 1). In addition, a weak 2mit mRNA signal was detected in 2mit^c03963 homozygous adult brains by in situ hybridization experiments. In particular, we observed faint 2mit staining in the neuronal fibers of the EB and MB lobes (Figure 3M). No evident 2mit mRNA signals were detected in the brain region where MB cell bodies are located (Figure 3M), as probably they are under the detection limit of the in situ hybridization technique performed in this study.

2 mit^c03963 homozygous flies were viable and fertile. No lethal phenotypes were observed during post-embryonic development (Table 2). Adult 2mit^c03963 homozygous flies did not display any gross morphological abnormalities and their overall brain organization was similar to that of wild-type flies. In addition, they did not show any impairment in light perception, evaluated as phototactic behavior (Table S1), as well as in locomotor activity (Figure S1).

2mit mRNA levels were also analyzed in five other strains (f00075, f06803, MB03271, MB08962, and MB08132, Exelixis Gene Disruption Project; Figure 1A) carrying PB or Minos (MB) transposons in proximity or within the 2mit gene. None of these strains showed significant modifications in 2mit mRNA levels (Table 1); thus they were excluded from subsequent analyses.

Given the specific brain 2mit mRNA hybridization pattern, we decided to assess whether 2mit plays a role in the behavioral plasticity associated with learning and memory. Therefore, 2mit^c03963 homozygous adult males were analyzed using the courtship conditioning assay [15,16]. This test is based on natural sexual behavior and measures the reduction in courtship levels of male flies which have previously courted non-receptive, mated females [17]. For each genotype, we measured the Courtship Index (CI), defined as the time spent by a male courting an anesthetized virgin female during a 10 min observation period, in conditioned and sham males [15,16]. In OR-R control flies, the CIs of conditioned males were significantly reduced compared to those of sham individuals, indicating short-term memory (STM) formation (Mann-Whitney U test: OR-R: p<0.01; Figure 4A). As expected, w¹¹¹⁸ sham males showed lower CI levels compared to OR-R sham controls under light conditions since white-eyed, vision-defective males have difficulty tracking females during courtship [18]. However, w¹¹¹⁸ males showed STM formation, as the CIs of conditioned males were significantly lower with respect to those of sham controls (Mann-Whitney U test: w¹¹¹⁸: p< 0.05; Figure 4A). Similar results were also described by another study on flies carrying the w¹¹¹⁸ allele in a cantonized strain (w CantonS [19]). Therefore, we considered the w¹¹¹⁸ flies a suitable control to study memory phenotypes using the courtship conditioning paradigm in 2mit^c03963 homozygous flies, which essentially have a w¹¹¹⁸ genetic background, except for the PB transposon insertion detectable by a mini-w⁺ marker gene.

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Figure 4. Memory and learning in 2mit^c03963 homozygous mutant flies.

(A) Comparison between Courtship Indices in sham (white bars) and conditioned (black bars) males for different genotypes. w¹¹¹⁸ and OR-R represent control strains; 2mit^- : 2mit^c03963 homozygous mutant flies; dnc¹: dunce¹ memory mutant flies; tim2 ^c06976/+ and tim2 ^f00297/+ heterozygous flies for two different tim2^- insertional mutant alleles. Data are expressed as mean ± SEM, with the number of tested flies indicated above each bar. Mann-Whitney U test revealed significant differences between the CIs of the conditioned and sham males in w¹¹¹⁸, OR-R, tim2 ^c06976/+, tim2 ^f00297/+: p< 0.05 for all genotypes. No significant differences were identified comparing the CIs of the conditioned and sham males in 2mit^c03963 and dunce¹ flies (2mit^c03963: p=0.38; dunce¹: p=0.32). (B) Learning evaluated as a training index (ratio between CIs for the last (CI_f) and the first (CI_i) 10 min of the training period) in conditioned 2mit^c03963 and w¹¹¹⁸ males (Mann-Whitney U test: p=0.60). 2mit^- : 2mit^c03963 homozygous mutant flies. *: significant difference; ns: not significant difference.

https://doi.org/10.1371/journal.pone.0076351.g004

The CIs of 2mit^c03963 sham males resulted similar to those of OR-R sham controls (Mann-Whitney U test: p= 0.36, not significant; Figure 4A), indicating that 2mit^c03963 homozygous males possess normal virgin female perception. However, in 2mit^c03963 flies, the CIs of conditioned males were not significantly different from those of sham controls (Mann-Whitney U test: p= 0.38, not significant; Figure 4A). Analogous results were obtained after comparing the CIs of sham and conditioned males in the classic memory mutant dunce (dnc¹), used as negative control (Mann-Whitney U test: p= 0.32, not significant; Figure 4A). Since during the test we did not observe evident impairments in courtship behavior and any abnormality in locomotor activity of 2mit^c03963 flies (Figure S1), these data suggest the presence of memory defects in 2mit^c03963 mutant males.

In addition, when tim2^-/+ heterozygous males for two different alleles (tim2^c06976 and tim2^f00297) have been analyzed, the CIs of conditioned males resulted significantly lower compared to those of sham controls (Mann-Whitney U test: p< 0.05 for both tim2 ^c06976/+ and tim2 ^f00297/+ individuals; Figure 4A), suggesting that the tim2 depletion in tim2^-/+ heterozygous flies does not cause memory impairment.

To understand whether 2mit^c03963 memory defects were associated with training (learning) deficiencies, we determined the training index (TI), defined as the ratio between CIs during the final (CI_f) and initial 10 min (CI_i) of the training period [15,16], in conditioned 2mit^c03963 mutant and w¹¹¹⁸ control males. TI values ≤ 0.5 are generally typical of wild-type flies, while TIs > 0.5 are characteristic of conditioning defective individuals [16]. Both 2mit^c03963 and w¹¹¹⁸ flies exhibited TIs < 0.5 and the comparison between 2mit^c03963 and w¹¹¹⁸ did not show any significant difference (Mann-Whitney U test: p= 0.6, not significant; Figure 4B), indicating that 2mit^c03963 males reduced their courtship behavior in the presence of a mated female, thus displaying learning activity.

In order to determine whether memory defects in 2mit^c03963 flies were related to 2mit depletion, we generated three independent transgenic lines (2mitO^F8, 2mitO^M4, 2mitO^M14) for a UAS-2mitHA cDNA chimeric construct designed for 2mit over-expression studies. The presence of a 2MIT-HA chimeric protein was visualized by Western blot from flies in which 2MIT-HA over-expression was ubiquitously activated using an Actin5C-Gal4 (ActGal4) driver in a wild-type 2mit⁺ genetic background (Figure 5A). We identified a ~150 kDa band in the three ActGal4> 2mitO lines that was absent in the negative control (ActGal4>CyO) and likely represents 2MIT-HA. The discrepancy in molecular weight between the visualized band and the one expected from the estimation of 2MIT-HA theoretic weight (~122 kDa) may have resulted from anomalous detergent binding and denaturation in SDS-PAGE migration, which for transmembrane proteins may explain till ± ~45% molecular weights variations [20]. The three ActGal4>2mitO lines overexpressed different degrees of the 2MIT-HA protein, with higher levels in ActGal4>2mitO^F8flies and lower ones in ActGal4>2mitO^M14 individuals.

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Figure 5. 2mit over-expression in adult flies.

(A) 2MIT-HA Western blot on adult bodies of ActGal4>2mitO^F8, ActGal4>2mitO^M4, ActGal4>2mitO^M14 and negative control ActGal4>+, in a wild-type 2mit⁺ background. 2MIT-HA indicates the ~150 KDa 2MIT-HA form revealed in all ActGal4>2mitO over-expressing lines. HSP70 represents ~70 KDa Heat Shock Protein 70, used as loading control. Four replicates were performed. (B) 2mit mRNA relative levels (mean ± SEM) in dissected brains of three independent elavGal4>2mitO; 2mit ^c03963 lines (F8, M4 and M14) and in w¹¹¹⁸ controls. Plot of 9 replicates. F_3,31= 552 p< 0.0001; Newman-Keuls post-hoc test: each elavGal4>2mitO; 2mit ^c03963 line vs w¹¹¹⁸ controls p< 0.001. (C) Courtship Indices in sham (white bars) and conditioned (black bars) males for the elavGal4>2mitO, 2mit ^c03963 lines (F8, M4 and M14) and relative negative controls elavGal4>+; 2mit^c03963; and + > 2mitO ; 2mit^c03963 (F8, M4 and M14). 2mit^- indicates the 2mit^c03963 allele. Data are expressed as mean ± SEM, with the number of tested flies indicated above each bar. The CIs of conditioned flies were not significantly different from those of sham males in the negative control lines (Mann-Whitney U test: elavGal4>+; 2mit^c03963: p= 0.24 ; + >2mitO^F8, 2mit^c03963: p= 0.81; + >2mitO^M4 ; 2mit^c03963: p= 0.33; + >2mitO^M14 ; 2mit^c03963: p= 0.43), but were significantly reduced in elavGal4>2mitO, 2mit ^c03963 lines. The number of asterisks indicates the significance level: **: p < 0.005; ***: p < 0.0001; ns: not significant.

https://doi.org/10.1371/journal.pone.0076351.g005

Both 2mit mRNA and 2MIT-HA chimeric protein productions were then evaluated in adult brains of flies over-expressing the UAS-2mitHA construct at the level of the MBs (using the OK107Gal4 driver) in a wild-type 2mit⁺ genetic background. In OK107Gal4>2mitO brains, 2mit mRNA and 2MIT-HA protein signals co-localized in the Kenyon cells and axonal lobes of the MBs (Figure S2).

Using genetic crossing and the pan-neuronal elavGal4 driver, we generated three elavGal4>2mitO lines, over-expressing 2MIT at the CNS level in a mutant 2mit^c03963 background (elavGal4>2mitO^F8, 2mit^c03963; elavGal4>2mitO^M4 ; 2mit^c03963 and elavGal4>2mitO^M14; 2mit^c03963). 2mit mRNA levels were then checked in dissected adult brains by QPCR, which revealed 2mit mRNA over-expression in all three elavGal4>2mitO; 2mit^c03963 transgenic lines compared to w¹¹¹⁸ controls, with higher values in elavGal4>2mitO^F8, 2mit^c03963 flies (~ 9-fold higher) and lower in elavGal4>2mitO^M14 ; 2mit^c03963 individuals (~ 2-fold higher; Figure 5B).

In order to evaluate 2MIT’s ability to rescue the 2mit ^c03963mutant memory phenotype, we determined the CIs of conditioned and sham male flies for the three elavGal4>2mitO, 2mit^c03963 lines (elavGal4>2mitO^F8, 2mit^c03963; elavGal4>2mitO^M4; 2mit^c03963 and elavGal4>2mitO^M14; 2mit^c03963) and relative controls (elavGal4>+; 2mit^c03963; +>2mitO^F8, 2mit^c03963; +>2mitO^M4; 2mit^c03963; +>2mitO^M14; 2mit^c03963; Figure 5C). In all three elavGal4>2mitO, 2mit^c03963 lines, the CIs of conditioned males were significantly reduced with respect to those of sham individuals, indicating a substantial STM rescue (Mann-Whitney U test: elavGal4>2mitO^F8, 2mit^c03963: p< 0.005; elavGal4>2mitO^M4; 2mit^c03963: p< 0.0001; elavGal4>2mitO^M14; 2mit^c03963: p< 0.005). Memory defects were maintained in negative control flies carrying the elavGal4 driver or the UAS-2mitHA construct alone in a mutant 2mit^c03963 background, with the CIs of conditioned males similar to those of sham flies in all control lines (Mann-Whitney U test: elavGal4>+; 2mit^c03963: p= 0.24; + >2mitO^F8, 2mit^c03963: p= 0.81; + >2mitO^M4 ; 2mit^c03963: p= 0.33; + >2mitO^M14 ; 2mit^c03963: p= 0.43; all p values are not significant). Taken together, these data suggest that 2mit is required for Drosophila memory formation.

2mit downregulation alters memory phenotype

To confirm the involvement of 2mit in STM formation, we produced three independent transgenic lines (2mit KD^6.1; 2mit KD^16.2; 2mit KD^61.1) carrying a UAS-construct for 2mit knock-down (KD) analyses. No lethal phenotypes were observed when 2mit KD was generally induced in both neuroblasts and neurons (with the l(3)-31Gal4 driver) or in mature neurons (with the elavGal4 driver) during embryonic development (Table 2). Pan-neuronal elavGal4>2mit KD produced different levels of 2mit mRNA depletion in dissected adult brains of the three transgenic lines. In fact, 2mit expression levels ranged from 0.17 ± 0.01 in 2mit KD^61.1 to 0.34 ± 0.01 in 2mit KD^6.1 compared to 1.00 ± 0.19 of w¹¹¹⁸ controls (Figure 6A).

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Figure 6. 2mit knockdown in adult flies.

(A) 2mit mRNA relative levels (mean ± SEM) in the dissected brains of three independent elavGal4>2mitKD (6.1; 16.2; 61.1) and w¹¹¹⁸ controls. Plot of 6 replicates. F_3,20= 1571, p<0.0001; Newman-Keuls post-hoc test: each elavGal4>KD line vs w¹¹¹⁸ controls: p< 0.001. (B) Courtship Indices in sham (white bars) and conditioned (black bars) males for elavGal4>2mitKD (6.1; 16.2; 61.1) and appropriate controls [elavGal4>+; +> 2mit KD (6.1; 16.2; 61.1)]. Mann-Whitney U test showed no significant differences between the CIs of sham and conditioned males in all the elavGal4>2mit KD (elavGal4>2mit KD^6.1.: p= 0.17; elavGal4>2mit KD^16.2: p= 0.13; elavGal4>2mit KD^61.1: p= 0.88). Significant differences between the CIs of conditioned and sham males were found in the control lines elavGal4> +; +> 2mit KD⁶¹. ; +> 2mit KD^16.2 ; +> 2mit KD^61.1 ; +> 2mit KD^61.1. (C) Courtship Indices in sham (white bars) and conditioned (black bars) males for OK107Gal4>2mitKD (6.1; 16.2; 61.1), c772Gal4>2mitKD (6.1; 16.2; 61.1), MB247Gal4>2mitKD (6.1; 16.2; 61.1) lines, and the control lines OK107Gal4>+, c772Gal4>+, MB247Gal4>+. Mann-Whitney U test showed no significant differences between the CIs of sham and conditioned males in two out of three OK107Gal4>2mit KD lines (OK107Gal4>2mit KD^16.2: p= 0.18; OK107Gal4>2mit KD^61.1: p= 0.36), in all the c772Gal4>2mit KD flies (c772Gal4>2mit KD⁶¹. : p= 0.67; c772Gal4>2mit KD^16.2: p= 0.72; c772Gal4>2mit KD^61.1: p= 0.42) and in all the three MB247Gal4>2mit KD lines (M247Gal4>2mit KD⁶¹. : p= 0.88; MB247Gal4>2mit KD^16.2: p= 0.12; MB247Gal4>2mit KD^61.1: p= 0.27). Significant differences between the CIs of conditioned and sham males were found in the control lines OK107Gal4>+, c772Gal4>+, MB247Gal4>+ and in the OK107Gal4>2mit KD⁶¹. transgenic line. In (B) and (C) data are expressed as mean ± SEM, with the number of tested flies indicated above each bar. The number of asterisks indicates the significance level: *: p < 0.05; **: p < 0.005; ***: p < 0.0001; ns: not significant.

https://doi.org/10.1371/journal.pone.0076351.g006

The STM phenotype in the elavGal4>2mit KD lines was then analyzed (Figure 6B). The pan-neuronal 2mit mRNA KD affected STM formation, since the CI values of conditioned flies resulted comparable to those of sham males in all of the elavGal4>2mit KD lines (Mann-Whitney U test: elavGal4>2mit KD^6.1 : p= 0.17; elavGal4>2mit KD^16.2: p= 0.13; elavGal4>2mit KD^61.1: p= 0.88; all p values are not significant). Moreover, significant differences were observed in the CIs of conditioned and sham flies in all negative controls (Mann-Whitney U test: elavGal4> +: p< 0.05; +> 2mit KD^6.1 : p< 0.0001; +> 2mit KD^16.2 and +> 2mit KD^61.1 : p< 0.005), suggesting once again that 2mit plays a specific role in the Drosophila memory phenotype.

We then evaluated the CIs of conditioned and sham males in transgenic flies, in which 2mit was silenced mainly in the whole MB structure, using the OK107- and c772Gal4 drivers, or in α, β, and γ MB lobes, with the MB247Gal4 driver [21]. Additional 2mit KD could be produced in other brain regions, since these drivers result weakly active also in the OLs and ALs (OK107Gal4), the OLs, ALs, EB and SOG (c772Gal4), and the OLs and glia cells (MB247Gal4) [21].

In two out of three OK107Gal4>2mit KD transgenic lines (2mit KD^16.2 and 2mit KD^61.1) and in all of the c772Gal4>2mit KD and MB247Gal4> 2mit KD lines (2mit KD^6.1 , 2mit KD^16.2, and 2mit KD^61.1), the CIs of conditioned and sham males were not statistically different, indicating altered memory formation (Mann-Whitney U test: with the OK107Gal4 driver: 2mit KD^16.2: p= 0.18; 2mit KD^61.1: p= 0.36; with the c772Gal4 driver: 2mit KD^6.1 : p= 0.67; 2mit KD^16.2: p= 0.72; 2mit KD^61.1: p= 0.42; with the MB247Gal4 driver: 2mit KD^6.1 : p= 0.88; 2mit KD^16.2: p= 0.12; 2mit KD^61.1: p= 0.27; all p values are not significant; Figure 6C). On the contrary, normal STM was observed in all the appropriate negative controls (+> 2mit KD lines; Figure 6B; Mann-Whitney U test: OK107Gal4> +: p<0.005; c772Gal4> + and MB247Gal4> +: p< 0.05; Figure 6C). The third OK107Gal4>2mit KD^6.1 transgenic line did not show any impairment in the STM phenotype, since in that case the CIs of conditioned males were significantly reduced from those of sham controls (Mann-Whitney U test: p< 0.05; Figure 6C). The absence of STM defects in that line could have been due to an inefficient 2mit downregulation generated by the combination of the OK107Gal4 driver and the 2mit KD transgene, specifically in OK107Gal4> 2mit KD^6.1 flies. In fact, when 2mit KD was pan-neuronally activated in 2mit KD^6.1 flies by elavGal4, a 2mit mRNA down-regulation in dissected brains and parallel defects in STM were detected. In addition, the use of both c772- and MB247Gal4 drivers produced STM deficiencies in 2mit KD^6.1 flies.

Finally, no STM deficiencies were detected when 2mit silencing was induced in different neurons of the central complex, using the c232- and 52YGal4 drivers. In fact, the CIs of conditioned males were significantly reduced compared to those of sham controls for all these 2mit KD-driver combinations (Mann-Whitney U test: c232Gal4> + and c232Gal4> 2mit KD^6.1: p< 0.005; c232Gal4> 2mit KD^16.2: p< 0.0001; c232Gal4> 2mit KD^61.1: p< 0.05; 52YGal4>+ and 52YGal4> 2mit KD^6.1 : p<0.005; 52YGal4> 2mit KD^16.2: p< 0.05; 52YGal4> 2mit KD^61.1: p< 0.0001; Figure S3). These data rule out the R3 and R4d neurons (active in the c232Gal4 driver [22]), as well as the R1, R3 and R4 neuronal cells and the subtype of F neurons connecting the fan-shaped body to the EB (active in the 52YGal4 line [22]) as possibly involved in the 2mit–mediated STM phenotype.

The nested 2mit gene does not show a functional relationship with its timeless2 host gene

A functional relationship could be shared by nested and host genes [5]. In Drosophila, homozygous tim2^-/tim2^- flies die very early during pupal development. As heterozygous tim2^-/+ adult flies are characterized by a modified light synchronization response of the circadian clock [7], we investigated whether 2mit shares similar functions in the adult brain. Since the natural ls/s polymorphism in the circadian clock gene tim1 significantly influences circadian light responses [23,24], we initially determined the ls-tim1/s-tim1 genotype, showing that both 2mit^c03963 and w¹¹¹⁸ flies were homozygous for the ls-tim1 variant. Subsequently, we analyzed 2mit^c03963 and w¹¹¹⁸ adult male responses to 20 min light pulses given at different times during the night (ZTs: 13, 15, 17, 19, 21, and 23), generating a phase response curve (PRC). As expected, w¹¹¹⁸ control flies showed delayed or advanced phase shifts in locomotor activity when pulsed at the beginning (ZTs: 13, 15, and 17) or at the end (ZTs: 21 and 23) of the night, respectively (Figure 7). The PRC profile of 2mit^c03963 flies was similar to that of w¹¹¹⁸ controls (genotype X time (ZT) interactions: F_5,434=1.76, p=0.12, not significant; Figure 7), suggesting that 2mit is not involved in light synchronization of the adult fly circadian clock.

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Figure 7. Phase response curve of 2mit^c03963 and w¹¹¹⁸ flies.

Analysis of variance genotype X time interactions (ZT): F_5,434=1.76 p=0.12, not significant. Advance and delay phase shift responses are represented respectively as positive and negative values. 2mit^-: 2mit^c03963 homozygous mutant flies. Data are expressed as mean ± SEM; (Ns) and [Ns] indicate the number of 2mit^c03963 and w¹¹¹⁸ flies analyzed, respectively.

https://doi.org/10.1371/journal.pone.0076351.g007

Drosophila melanogaster 2MIT orthologs and phylogenesis

Searching for 2MIT orthologs with tBlastN (using D. mel 2MIT as a query) against non-redundant sequences identified 2MIT conservation in 12 Drosophilidae genomic sequences released in 2007 (D. melanogaster, D. simulans, D. sechellia, D. yakuba, D. erecta, D. ananassae, D. pseudoobscura, D. persimilis, D. willistoni, D. mojavensis, D. virilis, and D. grimshawi; Drosophila 12 Genomes Consortium 2007, http://rana.lbl.gov/drosophila/), in 4 Lepidoptera (Manduca sexta and Bombyx mori moths and Danaus plexippus and Heliconius melpomene butterflies) and in the following insect species: Culex quinquefasciatus, Apis mellifera, Aedes aegypti, Pediculus humanus, and Acyrthosiphon pisum. Due to the presence of gaps in the assembly of contigs, the multi-alignment process of 2MIT orthologs showed an incomplete coverage in 8 Drosophilidae species (D. simulans, D. sechellia, D. yakuba, D. erecta, D. ananassae, D. pseudoobscura, D. persimilis, and D. willistoni) and in all the non-Drosophilidae species, except for A. mellifera. It was possible to extend the 2mit coding region for Drosophilidae and Lepidoptera, using the Augustus gene prediction tool [25] on available whole-genome-shotgun sequences, and a full-length 2mit coding sequence was obtained (Table 3). To corroborate gene prediction results, each identified full-length 2mit coding region was then subjected to a Blast vs dbEST search in order to verify at least partial coverage by specific expressed sequence tags (ESTs). We subsequently extended analyses to 9 new available Drosophilidae sequenced genomes released in 2013 [including only whole-genome-sequences (wgs) data; https://www.hgsc.bcm.edu/content/drosophila-modencode-project] and ran the Augustus gene prediction tool for each contig identified by tBlastN. 2MIT orthologs were detected in D. eugracilis, D. rhopaloa, D. biarmipes, D. bipectinata, D. elegans, D. ficusphila, D. kikkawai, D. takahashii, and D. miranda (Table 3). However, it was not possible to obtain a full-length 2mit coding region for C. quinquefasciatus, A. aegypti, P. humanus and A. pisum, since no additional sequence annotations were available on the National Center for Biotechnology Information (NCBI) database; thus they were excluded from subsequent analyses.

We generated a 2MIT ortholog multi-alignment for the 21 Drosophilidae, 4 Lepidoptera and Apis mellifera species, detecting a significant degree of conservation (Table 3 and File S1). In all 2MIT proteins, the N-terminal signal peptide, LRR domains, and transmembrane helix were identified (File S1). In Drosophilidae, both the disordered central region and C-terminal domain were longer compared to those of other 2MIT orthologs. Moreover, the 2MIT putative LRR domains of Lepidoptera and A. mellifera were characterized by the presence of two additional repeated units.

Maximum Likelihood analysis produced a 2MIT phylogenetic tree which mirrored the species tree. Among Drosophilidae, 2MIT phylogenesis followed both subgenus and subgroup classification of the Drosophila genus (Figure 8A).

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Figure 8. 2MIT Phylogenetic analysis.

(A) Unrooted phylogenetic tree of 2MIT protein in 21 Drosophilidae, 4 Lepidoptera and Apis mellifera. Statistical support for nodes on the trees was evaluated by the bootstrapping values (×500) shown in each branch point obtained by the Maximum-Likelihood method included in the MEGA 5.0 Software. (B) Colored bars at the right side of the phylogenetic tree represent binding motifs recognized by different factors in 2MIT ortholog cytoplasmic regions. CLV_NDR_NDR1: N-arginine dibasic convertase site; CLV_PCSK_KEX2_1: subtilisin-like proprotein convertases cleavage site; LIG_14-3-3_3: 14-3-3 ligand site; MOD_PKA_2: PKA Phosphorylation site; LIG_FHA_1 and LIG_FHA_2: FHA phosphopeptide ligands; MOD_N-GLC_1: N-glycosylation site; TRG_ER_diArg_1: di Arginine retention/retrieving signal; TRG_ENDOCYTIC_2: sorting signal motif; LIG_EVH1_1: EVH1 ligands.

https://doi.org/10.1371/journal.pone.0076351.g008

Within the 2MIT cytoplasmic region, the ELM server [26] identified different binding motifs common to almost all species (Figure 8B). Descriptions for these linear motifs can be found on the ELM web-site. In particular, in all 2MIT orthologs we detected recognition sites for N-arginine dibasic convertase (CLV_NDR_NDR1) and subtilisin-like pro-protein convertases (CLV_PCSK_KEX2_1), known to be involved in post-transduction maturation of several target proteins, as well as ligand sites for 14-3-3 protein/s (LIG_14-3-3_3) and a PKA phosphorylation site (MOD_PKA_2), implicated in fundamental cellular processes such as signal transduction and cell-cycle control. Moreover, at least one ligand site for FHA factors (LIG_FHA_1 and/or LIG_FHA_2), a protein domain involved in many signaling processes, and one intracellular sorting signal motif (TRG_ENDOCYTIC_2 and/or TRG_ER_diArg_1) were found in all the 2MIT ortholog C-terminal regions analyzed.

Analyses of tim2-2mit chromosomal organization in Drosophilidae, Lepidoptera and Apis mellifera

Comparative analyses of the D. melanogaster 2mit-harboring chromosomal region with those of the 12 Drosophila species sequenced in 2007 made it possible to identify conserved synteny using a 200kb-gene sliding window. Specifically, in all 12 species, 2mit was found to localize in an opposite transcriptional orientation within the tim2 11^th intron. Moreover, 2mit was always identified in an opposite transcriptional direction in the same contig of the tim2 gene in the 9 newly sequenced Drosophila species released in 2013, which presented only wgs data. Analyses of the A. mellifera genome database (release 4.5) showed that the 2mit honey bee (Am 2mit) ortholog maps internally to the Am tim2 intron 14 on opposite strands. However, the tim2-2mit host-nested gene structure was not maintained in the 4 lepidopterous species (EnsemblMetazoa, release 19).

Finally, we analyzed the D. melanogaster genomic region around the c03963 transposon, using a sliding window of approximately 400 nucleotides both upstream and downstream of the insertion site, searching for potential transcription factors (TFs). In particular, we identified a 6 bp TF binding site specific for the zinc-finger neuronal SNAIL repressor within the 11^th intron of the D. melanogaster tim2 gene (5'CAGGTG3', mapping ~20 kb upstream of the 2mit coding region). Comparative analyses performed on the 21 Drosophilidae identified SNAIL binding site conservation in the tim2 11^th intron in all species with the exception of D. virilis, D. ananassae, D. grimshawi, and D. rhopaloa, which are characterized by incomplete assembly coverage in the region upstream of 2mit.

Drosophila stocks and maintenance

Flies were raised on a standard agar-yeast-sucrose medium at 23°C in 12:12 LD. w¹¹¹⁸, sine oculis¹, dunce¹, l(3)-31Gal4/TM6B, Actin5C-Gal4/CyO, elavGal4/CyO, OK107Gal4, and c232Gal4 strains were obtained from the Bloomington Drosophila Stock Center (http://flystocks.bio.indiana.edu). c772- and MB247Gal4 lines were received from C. Helfrich-Foerster, University of Wuerzburg (Wuerzburg, Germany) and the 52YGal4 driver from J.D. Armstrong, University of Edinburgh (Edinburgh, Scotland, UK). Insertional strains c03963, f00075, f06803 (carrying a PB transposon) and MB03271, MB08132, MB08962, MB08132 (carrying a MB element) were obtained from the Exelixis Drosophila Stock Collection (drosophila.med.harvard.edu/). In these strains, PB or MB transposon insertions map in the tim2 intron 11. In particular, in MB08132 and f06803, insertions localize in the 2mit 3’ UTR; in MB08962, the insertion maps in the unique 2mit intron; in c03963, MB03271, and f00075 strains upstream of the 2mit gene. PBtim2^c06976 (tim2^c06976) and PBtim2^f00297 (tim2^f00297) flies, carrying the PB transposon in the tim2 5’ UTR and intron 8, respectively, were originally obtained from the Exelixis Drosophila Stock Collection and were characterized in [7].

2mitO and 2mit KD construct production and transgenic line generation

A 3453 bp 2mitHA chimeric construct (2mitO), characterized by 3423 bp 2mit full-length cDNA followed by an in-frame 27 bp HA (haemagglutinin) tag sequence and a stop codon, was generated for 2mit over-expression studies. Using specific primers (Table S2), 2mitO cDNA was initially amplified in four 5’–3’ serial fragments of 792, 1056, 1046 and 739 bp in length, which were cloned in a pCR ^®II-TOPO^® vector (Invitrogen) and checked for errors by sequencing. The four fragments were then digested with appropriate restriction enzymes (Table S2), obtaining 5’–3’ 768 bp NotI-BamHI, 980 bp BamHI-SalI, and 964 bp SalI-NdeI consecutive 2mit cDNA fragments and a 736 bp NdeI-XhoI segment, coding the 3’ 2mit region followed by 27 bp HA sequence. These segments were sequentially subcloned in a pBluescript® II S/K (+/-) vector (Invitrogen), obtaining the 3453 bp NotI-XhoI 2mitO cDNA, which was then transferred into a pUAST vector.

For KD studies, the UAS-2mit RNAi construct (2mit KD) was generated as in [55], using a 1234 bp fragment of 2mit cDNA (positions 2756-3989 in NM_142001.2) without off-target effects as predicted by a bioinformatic program of the Vienna Drosophila RNAi Center (VDRC, http://stockcenter.vdrc.at/). The 1234 bp cDNA fragment was amplified with the primers listed in Table S2.

Both 2mitO and 2mit KD transgenic lines were obtained by P-element-mediated transformation following standard procedures [56]. Three independent 2mitO transgenic lines were generated by the Drosophila Embryo Injection Service (Transflier, University of Ferrara, Ferrara, Italy): 2mitO^F8 [insert on 3^rd chromosome (Chr)], 2mitO^M4 (insert on 2^nd Chr), 2mitO^M14 (insert on 2^nd Chr). Three independent 2mit KD transgenic lines were obtained in our laboratory: 2mit KD⁶¹. (insert on 3^rd Chr), 2mit KD^16.2 (insert on 2^nd Chr) and 2mit KD^61.1 (insert on 2^nd Chr). Insert localization along polytene chromosomes was determined by in situ hybridization [55].

Generation of Drosophila lines for molecular and behavioral analyses

Molecular and behavioral analyses were performed on the 2mit^c03963 strain obtained by out-crossing c03963 flies into a w¹¹¹⁸ background for at least eight generations and on w¹¹¹⁸ controls.

To evaluate the effects of 2mit over-expression in the 2mit^c03963 homozygous mutant background, using CyO/Sco; MKRS/TM6B balancing stock, we initially generated the elavGal4/CyO; 2mit^c03963 strain, and 2mitO M4/CyO; 2mit^c03963 and 2mitO M14/CyO; 2mit^c03963 lines, carrying elavGal4 or 2mitO constructs over a CyO balancer and the homozygous PB c03963 transposon insertion on the 3^rd Chr. The 2mitO^F8, 2mit^c03963 strain, characterized by the presence of both 2mitO and PB c03963 inserts on the 3^rd Chr was generated via genetic recombination in F1 females, obtained by mating homozygous 2mitO^F8 and 2mit^c03963 parental lines and crossed to MKRS/TM6B balancing stock. Red-eyed 2mitO^F8, 2mit ⁺ /TM6B or 2mitO^F8, 2mit ^c03963/TM6B F2 flies were singly mated with w; MKRS/TM6B flies and checked for recombination via PCR, using specific primers to identify the PB c03963 insertion (Table S2). One F3 recombinant 2mitO^F8, 2mit^c03963 /TM6B line was then selected for subsequent studies. The effects of 2mit over-expression in a 2mit^c03963 homozygous mutant background were evaluated on elavGal4>2mitO; 2mit^c03963 flies and compared to elavGal4>+; 2mit^c03963 and +>2mitO; 2mit^c03963 relative controls, obtained by mating 2mit^c03963 homozygous flies with those bearing the elavGal4 driver or 2mitO construct alone in the 2mit^c03963 background.

2 mit KD effects were analyzed in Gal4-driven 2mit KD flies compared to Gal4>+ and +>2mit KD flies obtained by mating w¹¹¹⁸ flies with individuals carrying either a Gal4 driver or the 2mit KD construct alone.

Evaluation of tim2 depletion effects was performed on heterozygous tim2^-/+ for two different tim2^- alleles (tim2 ^c06976and tim2^f00297) obtained by mating tim2^- /TM6B flies to w¹¹¹⁸ flies.

RNA isolation, QPCR, and Northern blotting

Total RNA was obtained from L3, 3-5 day-old adult heads and dissected brains. 3-5 day-old flies raised in 12:12 LD conditions or after 3 days in DD were sampled at 3 or 4 h intervals. Adult heads were separated from bodies according to [57]. Brains were dissected in PBS, fixed in 4% paraformaldehyde in PBS for 30 min at 4°C and washed three times for 10 min in PBS. Total RNA was extracted from samples using Trizol™ Reagent (Invitrogen) following the manufacturer’s protocol. The cDNA was synthesized from 1 µg of total RNA using SSII Reverse Transcriptase (Invitrogen) and an Oligo(dT)₂₀ primer. QPCR reactions were performed in a 10 µl reaction volume, containing 200 nM of specific primers (Table S2), 5 µl GoTaq® qPCR Master Mix (Promega) and ~30 ng of cDNA per sample. QPCR was performed in triplicate and repeated three times on an ABI7500 system (Applied Biosystem), with the following amplification profile: 95°C for 2 min, 40 cycles of two-step amplification (95°C for 25 sec and 60°C for 60 sec), and melting curve (60–90°C with a heating rate of 0.5 °C/10 sec). To evaluate differences in gene expression we chose a relative quantification analysis based on the standard curve method [58]. Levels of expression were compared with those of an endogenous control transcript (rp49) that did not appear to be differentially expressed under our experimental conditions.

Northern blotting was carried out as in [59], using a ~1.7 kb 3’ 2mit probe (3040-4722 positions in NM_142001.2) and rp49 full-length cDNA (U92431) as standard.

In situ mRNA hybridization and immunohistochemistry

mRNA in situ hybridization on embryos was carried out as in [60] with Fluorescein-labelled 980 nt antisense and sense 2mit RNA probes (2060-3039 positions in NM_142001.2). The 2mit sense probe was used as negative control. Hybridization signals were detected using an alkaline phosphatase-conjugated anti-Fluoroscein antibody (1:2000, Roche) and NBT/BCIP substrates (Roche).

Biotin labeled RNA antisense and sense ~1.7 kb 2mit RNA probes (3040-4722 in NM_142001.2) were made for mRNA hybridization on whole-mount adult brains using the Biotin RNA Labelling Mix (Roche). The 2mit sense probe was used as negative control. In situ hybridization experiments were performed on 3-5 day-old adult brains collected at ZT 0, in 12:12 LD conditions. Sample collection, tissue fixation, and mRNA in situ hybridization procedures were performed as described in [7]. Samples were hybridized at 65°C overnight with 100 ng probe. Probe detection was performed using TSA^TM Signal Amplification kit (PerkinElmer) following the manufacturer’s instructions, incubating samples at 4°C overnight in Streptavidin (1:100) and 3 h in tyramide solution (TSA^TM Cyanine 3 System). To visualize the presence of both 2mit mRNA and 2MIT-HA chimeric protein in OK107Gal4>2mitO adult brains, in situ hybridization protocols did not include treatments with Proteinase K [7]. After in situ procedures, brains have been incubated at 4°C for 3 days with a rabbit anti-HA antibody (1:600; Sigma) and at 4°C overnight with a goat anti-rabbit IgG-Alexa 488 (1:250; Invitrogen). Samples were mounted in Vectashield H-1000 (Vector Laboratories) and microscopic analyses were performed using a Leica TCS SP5 II confocal microscope (Leica Microsystems). At least 10 brains for each genotype were analyzed. For each brain, optical sections (Z-series) were taken at 0.5 µm intervals. Post-acquisition analysis and Z-stack construction were performed with Fiji, an open source image processing package based on ImageJ (http://fiji.sc/wiki/index.php).

Western Blotting

Adult fly whole-bodies were homogenized in extraction buffer as in [61]. After 2 min of sonication, β-dodecyl maltopyranoside detergent was added (1% final concentration). Samples were placed on a rotating wheel for 1 h at 4°C to allow membrane solubilization. According to [62], 0.2 volumes of 5% sodium deoxycholate were added and samples were incubated 10 min on ice. Lysates were centrifuged twice (2800g, 4°C); supernatants were diluted in LDS loading buffer (Invitrogen) and DTT 1 M (0.73 X final concentration; Sigma) and placed 10 min at 70°C. SDS-PAGE was performed using 3-8% NuPAGE® Tris-Acetate pre-cast gel (Invitrogen). After blotting, nitro-cellulose membranes (Trans-Blot Transfer Medium; Bio-Rad) were incubated with a rabbit anti-HA antibody (1:1000; Sigma) and a goat anti-rabbit IgG-HRP (1:1000; Santa Cruz Biotecnology Inc.). Positive immunoreactivity was visualized using the ECL detection system.

Evaluation of ls/s-tim1 haplotypes

The single 294 Guanosine insertion/deletion polymorphism existing in the D. melanogaster tim1 gene was determined as in [24] using the Amplification of Refractory Mutations System (ARMS) PCR method on single flies. Primers are listed in Table S2.

Egg-to-adult viability

For each genotype, vitality test experiments were performed collecting ~100-300 embryos and counting developing L3, pupae, and adult flies.

Phototactic behavior

Phototactic behavior was tested in a maze consisting of a series of Y and T tubes (4 mm external diameter) interconnected by transparent 1.5 cm long plastic tubes similar to those proposed to test geotaxis behavior by [63]. Each maze had a single entrance at one side and eight terminal ends at the other. The eight terminal ends were closed with funnel traps, as described in [63]. Mazes were placed horizontally in a box, with black internal walls and a white LED light (130 lux) placed in a corner, corresponding to one of the maze terminal edges. Before testing, flies were maintained in dark conditions. Analyses were performed at the ZT 0-2 time interval on 3-5 day-old males. During each trial, 10 flies were placed at the entrance of the maze and after 1 h each fly received a score from 0 to 7, reflecting the number of positive choices towards the light source.

Locomotor activity analyses

3–5 day-old flies were individually transferred into transparent tubes (1.5 cm diameter and 4.5 cm length). Tubes were placed inside a black box under white light (141 lux) and locomotor activity was recorded during a 10 min period for each fly. Data were processed by AnyMaze software (Stoelting, Wood Dale, IL, USA). The following parameters were evaluated: total distance moved (m; sum of the distance between each point in the track); average speed (mm/sec); total number of immobility episodes (number of transitions from mobility to an immobile state exceeding 2 sec); total immobility time (sec; sum of duration of each immobility episode). Recordings were performed from ZT 1 to ZT 7.

Circadian locomotor analyses

Locomotor activity was recorded using the Drosophila Activity Monitoring System© (DAMSystem, Trikinetics Inc., Waltham, MA, USA). PRCs were obtained subjecting flies to 20 min light pulses (400 lux) delivered at ZTs 13, 15, 17, 19, 21, and 23. Phase changes were calculated as described in [64]. Positive and negative values represent advanced and delayed phase shift responses, respectively.

Memory analyses

Courtship conditioning assays were performed as described in [15,16]. Conditioned male flies and sham controls for each genotype were analyzed. Briefly, 4 day-old virgin males, previously kept alone as soon as they eclosed, were individually placed with a 4-5 day-old wild-type OR-R mated female for a 1 h training period (conditioned males) or were kept alone (sham controls) in a mating chamber (8 mm diameter and 3 mm high). Males were individually transferred to a new mating chamber and, within 5-10 min, were tested for 10 min with a CO₂-anesthetized wild-type OR-R virgin female, collected that day. Male courtship activities towards virgin or mated females were videotaped under uniform white light (141 lux). The first and last 10 min of the 1 h training period and the 10 min test were inspected to record male courtship behavior. Males which copulated during the training period or courted less than 1 min during the first 10 min of training period were excluded from analyses. If males copulated during the test period or an anesthetized virgin awakened from anesthesia, the observation period was concluded. A courtship index (CI), defined as the amount of time a male spent courting during the 10 min test period, was calculated for each conditioned and sham male. The training index (TI), defined as the ratio between CIs in the final 10 min (CI_f) and initial 10 min (CI_i) of the training period, was calculated [16].

Statistical analyses

All molecular and behavioral data, except for those regarding memory analyses, were analyzed by parametric one- or two-way analysis of variance (ANOVA) and Neuman-Keuls post hoc test. CI data did not approximate normal distributions, evaluated with Lilliefors (Kolmogorov-Smirnov) and Shapiro-Wilk tests, even after arcsine, arcsine squared, or arcsine square root transformation. Therefore, they were non-parametrically analyzed using the Mann-Whitney U test for pair-wise comparisons. Analyses were performed using the Statistica 5.0 package (Statsoft Inc.).

Bioinformatic tools

Sequence accession and annotation were performed through the FlyBase web platform (release FB2013_03; http://flybase.org; 12 Genomes Consortium 2007, http://rana.lbl.gov/drosophila/; modENCODE project, https://www.hgsc.bcm.edu/content/drosophila-modencode-project) or by the EnsemblMetazoa database (http://metazoa.ensembl.org/index.html). D. melanogaster 2mit nucleotide and amino acid sequences were compared with the non-redundant sequences available at the NCBI using BLAST and tBLASTN algorithms. The identified contigs were then analyzed with the Augustus gene prediction tool [25].

A multiple protein sequence alignment was obtained by using the MAFFT program (http://mafft.cbrc.jp/alignment/server/) and subjected to phylogenetic analysis by Maximum Likelihood method, via MEGA 5 software [65]. Genetic distances were calculated using the Jones–Taylor–Thornton algorithm [66] and statistical support for nodes on the tree was evaluated using bootstrapping (500 iteration cycles) [67]. Searching for transcription factor binding sites around c03963 transposon mapping region was performed using the ConSite tool [68].

Sequence feature analysis

2MIT protein sequence (SwissProt accession ID: Q9VFY9) annotation was obtained from the Annie server (http://annie.bii.a-star.edu.sg), which integrates the prediction from several computational tools, such as CAST and SEG for low complexity region detection and SAPS for the analysis of amino acid composition. The transmembrane topology prediction was derived from the consensus of tools provided by Annie: HMMTOP, PHOBIUS, and TMHMM. We employed an integrative bioinformatic approach combining sequence and domain database searches with the consensus from predictions of protein structural features. The 2MIT sequence was used as a query to scan the domain databases InterPro and Pfam; the secondary structure was predicted using a consensus approach [69]. Prediction of intrinsic disorder and the presence of signal peptides was assessed using ESpritz [70] and SignalP [71], respectively. The N-terminal sequence of 2MIT was scanned for the presence of repeated units using a combination of different classes of repeat prediction methods: RADAR [72], TRUST [73], and Repetita [74]. The predicted repeats were manually aligned in order to calculate the consensus pattern which defines the repeats in the 2MIT protein and to identify further mis-predictions [75]. The C-terminus was scanned for functional linear motifs using the ELM server [26].

Alignment construction

The structural template for the 2MIT LRR domain was found using MANIFOLD based on sequence and secondary structure. Initial alignments were generated through systematic parameter variation from an ensemble of similar alternatives. Given the problematic nature of repeated sequences, the best initial alignment was used as a starting point only. Manual refinement was performed using knowledge obtained from the structural alignment of the different predicted repeats. Knowledge of key residues and secondary structure was used to anchor the aligned repeats.

Molecular modeling

The model for the 2MIT LRR domain was constructed using the HOMER server (URL: http://protein.cribi.unipd.it/), which uses the conserved parts of the structure to generate a raw model, which is then completed by modeling the divergent regions with a fast divide and conquer method [76]. Side chains were placed with SCWRL3 [77] and the energy was evaluated with FRST [78]. The final models were subjected to a short steepest descent energy minimization with GROMACS [79] to remove energy hotspots before calculating the electrostatic surface with APBS [80]. The structure is visualized using PyMOL (DeLano Scientific, URL: http://pymol.sourceforge.net/).