Eye pigmentation defects of blos1 mutant flies. (A) Representative light microscopic images of unstained eye sections obtained from adult wild-type (Canton-S) and homozygous blos1^ex2 and blos1^ex65 mutants. Images pairs were selected to show longitudinal and cross-sections of the ommatidia, and were acquired under identical illumination and digital camera settings. Scale bars: 20 μm. (B) Absorption spectra of red and brown pigments extracted from the heads of 2–3-day-old males of the Canton-S and blos1^ex2 fly lines. (C) Quantification of the contents of brown pigments (open bars) and red pigments (filled bars) in the eyes of males of the indicated genotypes. Values are expressed as percentages of the pigment content of Canton-S flies and represent means ± SD. One-way ANOVA followed by Dunnett's tests comparing each sample versus the corresponding Canton-S control: **P < 0.01.

Electrophysiological analyses of glutamatergic neuromuscular junctions from larvae of the wild-type line Canton-S (CS) or homozygous for mutations in the genes encoding BLOC-1 subunit 1 (blos1^ex2) or the δ subunit of AP-3 (g²). (A) Representative traces of spontaneous miniature excitatory junction potentials (mEJPs). (B) Representative evoked junction potentials (EJPs). (C and D) Recordings like those shown in (A) were used to measure the amplitude (C) and frequency (D) of 70 consecutive mEJP events per experiment. (E) EJP amplitude was measured for the last 75 events of a series of 100 stimulations at 2 Hz. (F) The quantal content was estimated as the average amplitude of EJPs divided by the mean amplitude of mEJPs. Bars represent means ± SEM of at least seven independent experiments. One-way ANOVA followed by Tukey's test: ***P < 0.001 versus CS.

Genetic interactions between blos1^es2 and mutant alleles of pigment granule group genes. Flies carrying mutant alleles of the genes encoding the Rab-family member Lightoid (ltd¹), its putative guanine-nucleotide-exchange factor Claret (ca¹), subunits of the AP-3 complex (rb¹, cm¹, g² and g^53d) or the Hps5 subunit of BLOC-2 (p^p) were crossed with flies carrying the blos1^es2 allele to obtain homozygous double mutants and a homozygous g²; blos1^ex2; p^p triple mutant. (A) Quantification of red pigments extracted from the heads of 2–3-day-old male flies homozygous for the mutant alleles indicated on the left and for normal (open bars) or mutant (filled bars) alleles of blos1. Values are expressed as percentages of the pigment content of Canton-S flies and represent means ± SD. One-way ANOVA followed by Bonferroni comparison of selected group pairs: NS, not significant; ***P < 0.001. (B–D) Eye color phenotypes of selected double mutants in comparison with the corresponding single mutants.

Structural requirements for the phenotypic enhancement effects caused by misexpression of Auxilin in the eyes of flies deficient in subunits of BLOC-1 or AP-3. (A) Schematic representation of the domain organization of Auxilin from Drosophila melanogaster fused to monomeric red fluorescence protein (mRFP), and of transgenic constructs used in this study. FL, full length; CBM or C, clathrin-binding-motif-containing region. Dashed segments denote deleted regions. The approximate locations of pairs of DLL and DPF motifs, which in some constructs were mutated to DAA and APA respectively, are indicated. (B) The heads of transgenic flies carrying the Auxilin-mRFP fusion constructs depicted in (A), with (+) or without (−) the GMR-GAL4 driver, were homogenized and processed for immunoblotting using antibodies to the mRFP domain or to the endogenous Hsp70 protein (the latter as a loading control). (C–F) Quantitative analyses of the effects of Auxilin-mRFP contruct misexpression on the red pigment content of 2–3-day-old male flies of the wild-type (CS) genetic background (E) as well as homozygous for the BLOC-1 subunit mutant allele blos1^ex2 (C) or the AP-3 subunit mutant alleles or^49h (D) and g² (F). Each data point represents the mean ± SD of red pigment values obtained per Auxilin-mRFP contruct transgene in the presence of the GMR-GAL4 driver, normalized to those obtained for the same transgene in the absence of the driver. Data points labeled as ‘−’ represent red pigment values obtained for flies carrying the GMR-GAL4 driver without any Auxilin-mRFP construct. In cases where two or more independent insertions of the same Auxilin-mRFP construct were available for analysis, bars represent the overall weighted averages. One-way ANOVA analysis: P < 0.0001 for the data sets shown in (C), (D) and (F); not significant differences between the data groups shown in (E). See Supplementary Materials, Table S2 for the results of post hoc statistical tests.

Sequence analysis and mutant alleles of the fly orthologue of human BLOS1. (A) Alignment of the primary sequences of BLOS1 from Homo sapiens (Hs) and the product of the blos1 gene from Drosophila melanogaster (Dm). Identical amino acid residues are highlighted. (B) Schematic representation of the organization of the blos1 gene, indicating the two exonic regions that compose the ORF, the insertion site of a P-element in the fly line EY06269 and the segments deleted (dotted lines) in the blos1^ex2 and blos1^ex65 mutant alleles. (C) Analysis by agarose gel electrophoresis of PCR products obtained by amplification of genomic DNA from the indicated fly lines, using primers derived from sequences about 206 bp upstream and 1156 bp downstream of the P-element insertion site. Molecular size markers were run on both sides of the gel. Notice the reduced sizes of the PCR products obtained from homozygous blos1^ex2 and blos1^ex65 flies. Due to the large size of the inserted P-element (∼10 kb), no PCR product was obtained under these conditions for the EY06269 line.

Representative transmission electron micrographs of eye sections from adult wild-type and blos1 mutant flies. (A–H) Cross-sections of ommatidia, each of them containing seven photoreceptors and separated from the others by a sheath of pigment cells. In wild-type flies (A–D), the cells of the sheath contain numerous electron-dense pigment granules. In homozygous blos1^ex2 flies (E–H), the ommatidia appear normal in size and number of photoreceptors, but the number and electron density of pigment granules in the sheath cells are significantly reduced. Rectangles in (A) and (E) denote areas shown at higher magnification in (B) and (F), respectively. Likewise, the regions highlighted in (B) and (F) are shown at higher magnification in (C and D) and (G and H), respectively. Scale bars: 5 μm (A and E), 2.5 μm (B and F) and 1 μm (C, D, G and H).

Abnormal behavior of blos1 mutant flies. (A and B) Adult male flies (5 ± 2 days after eclosion) of the Canton-S (CS) control line, or carrying mutant alleles that render them deficient in BLOC-1 subunit 1 (blos1^ex2), the AP-3 complex subunit δ (g²) or the ABC transporter White (w*), were placed in groups of 25 in plastic Petri dishes, under conditions of high humidity and controlled temperature, and visually monitored for a 10 min period. (A) The male-to-male courtship index was calculated as the number of males displaying courting behaviors in ‘courtship chains,’ not counting the first male in each chain. (B) Physical contacts were assessed by counting the number of times that a male fly touched another during the 10 min period, regardless of the type of behavior displayed. See Materials and Methods for further experimental details. Bars represent means ± SEM of 10 independent experiments. One-way ANOVA followed by Bonferroni comparison of selected group pairs: NS, not significant; *P < 0.05; **P < 0.01.

Screening for genetic modifiers of the eye pigmentation phenotypes of fly mutants deficient in BLOC-1 subunit 1 (blos1^ex2) or in AP-3 subunit σ3 (or^49h). (A and B) Quantification of red pigments extracted from the heads of male flies homozygous for the mutant alleles blos1^ex2 (A) and or^49h (B) and carrying P-element insertions for GAL4-dependent misexpression of the products of the indicated candidate genes. Two independent P-element insertions nearby Rab11, labeled as ‘1’ and ‘2’, were used. Controls included Canton-S (CS) flies and homozygous mutant flies devoid of P-elements for GAL4-dependent misexpression (–) and either lacking (open bars) or carrying (filled bars) the GMR-GAL4 driver. Values are expressed as percentages of the pigment content of Canton-S flies and represent means ± SD. (C) Additional analyses were performed as in (A) and (B) to test whether the effects observed for the constructs targeting auxilin and Rab11 (P-element insertion 1) were dependent on the presence of the GMR-GAL4 driver. One-way ANOVA followed by Bonferroni comparison of selected group pairs: NS, not significant; **P < 0.01; ***P < 0.001. (D) Eye color phenotypes of g² mutant male flies carrying the UAS-aux transgene for misexpression of Auxilin, the GRM-GAL4 driver, or both. See Supplementary Materials, Table S1 for the corresponding quantitative analysis.