Cellular characterisation of small Open Reading Frame function in Drosophila melanogaster

As our knowledge of the genome expands, so does our understanding of the characteristics of what we define as genes. Small Open Reading Frame (smORF) genes have eluded gene annotation until very recently, and evidence is mounting that these very short nucleotide sequences encode functional peptides that are ≤100 amino acids in size. From work conducted in the fruit fly, our lab has successfully characterised three Drosophila smORFs, of which two have been shown to have a function in higher vertebrates, including humans. The functional characterisation of one of these conserved smORF encoded peptides (SEPs), Hemotin, is presented in this thesis. Though the overall number is still low compared to the abundance of potential smORF-encoding genes in Drosophila, the information gathered here allows us to speculate on the wider role of smORF peptides through cell-based imaging studies conducted on Drosophila cells. Here, I will discuss the various techniques that can and should be employed in order to study the functions of SEPs. Chapter III describes the various phenotypic studies conducted on the Hemotin smORF which is expressed in Drosophila haemocytes, and are integral to the fruit fly immune system. This study showed that connecting subcellular localisation of an SEP to a direct functional assay in cells can reveal functional characteristics of the peptide for further study. Chapter IV details the results from a tagging-transfection assay, which began initially as a way to independently corroborate the translation of smORF mRNAs that were assessed as such by Ribosome Profiling. This experiment resulted in the discovery of several mitochondrial-localised SEPs in Drosophila S2 cells, opening the door for the direct functional assay described in Chapter V. The results from a small-scale RNAi screen conducted on the mitochondria of S2 cells provided a reliable read-out for functionality of a large proportion of the smORFs that were screened. This assay can potentially be used as a phenotypic read-out of mitochondrial-SEP function in any cell or tissue type. Elucidation of smORFs and the functions of the peptides that they encode will help us to expand the Drosophila proteome, along with providing evidence of their functionality across every organism in which they are found. Considering that characterised SEPs play very important roles in physiology and health, it is time for smORFs to be acknowledged as the important genomic elements that they are

Hemotin, a regulator of phagocytosis encoded by a small ORF and xonserved across metazoans

Translation of hundreds of small ORFs (smORFs) of less than 100 amino acids has recently been revealed in vertebrates and Drosophila. Some of these peptides have essential and conserved cellular functions. In Drosophila, we have predicted a particular smORF class encoding ~80 aa hydrophobic peptides, which may function in membranes and cell organelles. Here, we characterise hemotin, a gene encoding an 88aa transmembrane smORF peptide localised to early endosomes in Drosophila macrophages. hemotin regulates endosomal maturation during phagocytosis by repressing the cooperation of 14-3-3ζ with specific phosphatidylinositol (PI) enzymes. hemotin mutants accumulate undigested phagocytic material inside enlarged endo-lysosomes and as a result, hemotin mutants have reduced ability to fight bacteria, and hence, have severely reduced life span and resistance to infections. We identify Stannin, a peptide involved in organometallic toxicity, as the Hemotin functional homologue in vertebrates, showing that this novel regulator of phagocytic processing is widely conserved, emphasizing the significance of smORF peptides in cell biology and disease

Activation of Drosophila hemocyte motility by the ecdysone hormone

Summary Drosophila hemocytes compose the cellular arm of the fly's innate immune system. Plasmatocytes, putative homologues to mammalian macrophages, represent ∼95% of the migratory hemocyte population in circulation and are responsible for the phagocytosis of bacteria and apoptotic tissues that arise during metamorphosis. It is not known as to how hemocytes become activated from a sessile state in response to such infectious and developmental cues, although the hormone ecdysone has been suggested as the signal that shifts hemocyte behaviour from quiescent to migratory at metamorphosis. Here, we corroborate this hypothesis by showing the activation of hemocyte motility by ecdysone. We induce motile behaviour in larval hemocytes by culturing them with 20-hydroxyecdysone ex vivo. Moreover, we also determine that motile cell behaviour requires the ecdysone receptor complex and leads to asymmetrical redistribution of both actin and tubulin cytoskeleton

Hemotin modulates PI(3)P formation by repressing 14-3-3ζ-mediated activation of PI(3)K68D kinase.

<p>(A–I) Distribution of FYVE-positive compartments in <i>ex vivo</i> hemocytes (see also J). Yellow dashed lines indicate cell body area, and arrowheads indicate enlarged FYVE compartments. Scale bar (5 μm). (A) Wild-type hemocytes. (B) <i>hemo</i>^<i>A4</i> mutants show enlarged FYVE compartments (arrowheads), (C) this phenotype is rescued by <i>mtm</i> gain of function (<i>UAS-mtm</i>). (D) Hemocytes overexpressing <i>14-3-3ζ</i> (<i>UAS-14-3-3ζ</i>) or (E) the <i>PI3K68D</i> Kinase (<i>UAS-PI3K68D</i>) also show enlarged FYVE compartments (arrowheads), similar to those observed in <i>hemo</i>^<i>A4</i> mutants. (F) Enlarged FYVE compartments are also observed by the reduction of <i>mtm</i> (<i>mtm-RNAi</i>) in hemocytes. (G) Reduction of <i>PI3K68D</i> kinase function by RNAi rescues the enlarged-FYVE phenotype produced by overexpression of <i>14-3-3ζ</i>. (H) Reducing the function of <i>14-3-3ζ</i> (<i>14-3-3ζ-RNAi</i>) rescues the enlarged FYVE compartment produced by overexpression of <i>PI3k68D</i> or (I) by reduction of <i>mtm</i>. (J) Quantification of the FYVE OAI in primary hemocytes (see Fig 6, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s001" target="_blank">S1 Data</a>). Knocking down the <i>mtm</i> function (mtm-RNAi) produces enlarged FYVE compartments as shown with <i>hemo</i>^<i>A4</i> mutants. Both overexpression of <i>mtm</i> (<i>UAS-mtm</i>) and haploinsufficiency of <i>PI3K68D</i> (<i>Df(3)PI3K68D</i>/+), rescue the <i>hemo</i>^<i>A4</i> mutant FYVE phenotype. Overexpression of <i>PI3K68D</i> (<i>UAS-PI3K68D</i>) mimics the <i>hemo</i>^<i>A4</i> mutant FYVE phenotype, and this is rescued by overexpression of <i>hemo</i> full-length transcript (<i>UAS-hemoFL</i>). Reduction of <i>14-3-3ζ</i> function (<i>14-3-3ζ-RNAi</i>) corrects the <i>mtm</i> loss of function and <i>PI3K68D</i> gain of function FYVE phenotypes. The enlarged FYVE compartments produced by over-expression of <i>14-3-3ζ</i> (<i>UAS-14-3-3ζ</i>) are corrected by knocking down <i>PI3K68D</i> function (<i>PI3K68D-RNAi</i>). One-way ANOVA test indicated that means of samples are significantly different [F(12,508) = 14.01, <i>p</i> < 0.0001]. Post hoc Bonferroni’s multiple comparison test showed that <i>hemo</i>^<i>A4</i>, <i>UAS-14-3-3ζ</i>, <i>UAS-mtm-RNAi</i>, <i>UAS-PI3K68D</i> were significantly different to wild-type, whereas the rest of genotypes were not (<i>n</i> ≥ 19, <i>p</i> < 0.005). Error bars represent SEM. (K) In hemocytes, Hemo-GFP peptides colocalize with PI3K68D Kinase in intracellular compartments (arrowheads), presumably early endosomes. (K) PI3K68D-Cherry expression. (K’) Hemo-GFP, (K”) merge image. Scale bar (10 μm). (L) Western Blot of a Pull down experiment from hemocytes expressing PI3K68D-GFP and HA-14-3-3ζ revealing a protein interaction between PI3K68D and 14-3-3ζ. Supplemental data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s007" target="_blank">S6 Fig</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s001" target="_blank">S1 Data</a>.</p

Stannin and Hemotin replicate each other’s functions and counteract 14-3-3ζ function during endosomal maturation.

<p>(A) Mouse macrophage-like RAW264.7 cells treated with control-scrambled siRNAs and labelled with the acidic pH-sensitive Lysotracker (Red). Scale bar (5 μm). (B) RAW264.7 cells treated with two Fluorescein isothiocyanate (FITC)-labelled <i>snn</i> siRNAs (green) (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s006" target="_blank">S5 Fig</a>) and stained with Lysotracker. Note the highly enlarged lysosomal compartment in <i>snn</i> siRNA-treated cells (see C). Scale bar (5 μm). (C) Lysosomal OAI, revealed by Lysotracker, in nontransfected or control siRNA and <i>snn</i> siRNA-treated RAW264.7 cells, showing that <i>snn</i> siRNA-treated RAW 264.7 cells have significantly larger lysosomal compartments than control samples (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s001" target="_blank">S1 Data</a>). The graph shows averages of three independent experiments. One-way ANOVA test showed that samples were significantly different [F(2,780) = 185.6, <i>p</i> < 0.0001]. Post hoc Bonferroni multicomparison test showed that the siRNA-<i>snn</i> sample was significantly different to nontransfected and siRNA-control samples (<i>n</i> ≥ 240, <i>p</i> < 0.05). On average, <i>snn</i> siRNA-treated cells show a reduction in <i>snn</i> expression of 60% relative to nontreated cells, whereas cells treated with control siRNA only show a reduction of 0.8% (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s006" target="_blank">S5A Fig</a>). Error bars represent SEM. (D–D”) Hemo-GFP and Snn-FLAG peptides expressed in hemocyte-like <i>Drosophila</i> Kc165 cells (20) using the <i>Act5-Gal4</i> driver. Hemo-GFP (green) (D’) and Snn-FLAG (red) (D”) peptides colocalize in intracellular vesicles and punctate organelles (arrows). Scale bar (5 μm). (E) Colocalisation of Hemo-GFP peptides and Nt-tagged HA-14-3-3ζ protein in intracellular compartments in <i>ex-vivo</i> hemocytes. (E’) Hemo-GFP. (E”) HA-14-3-3ζ. (E”‘) Merged image. UAS constructs were driven with <i>He-Gal4</i>. Scale bar (10 μm). (F) Pull down of myc-14-3-3ζ with Hemo-GFP in transfected <i>Drosophila</i> Kc167 cells. Myc-14-3-3ζ interacts with Hemo-GFP but not with a GFP-only control. Molecular weight is indicated in kilodaltons Retention of Hemo-GFP and GFP is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s006" target="_blank">S5C Fig</a>. (G) Vacuole OVI measurements (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#sec009" target="_blank">Materials and Methods</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s001" target="_blank">S1</a> Data) in primary hemocytes. Expression of human <i>snn</i> (<i>UAS-snn</i>) rescues the <i>hemo</i>^<i>A4</i> vacuolation phenotype to a similar extent as the rescue observed by the <i>UAS-hemoFL</i> and <i>hemo-ORF</i> constructs. Reducing the dosage of <i>14-3-3ζ</i> (in a heterozygous null <i>14-3-3ζ</i> ^<i>12BL</i><i>/+</i> background, labelled <i>14-3-3ζ −/+</i>) reduces the <i>hemo</i>^<i>A4</i> vacuolation phenotype. Conversely, overexpression of <i>14-3-3ζ</i> (<i>UAS-14-3-3ζ</i>) in hemocytes induces the formation of larger vacuoles. The induction of vacuoles by excessive 14-3-3ζ is reversed by simultaneous overexpression of <i>hemo</i> full-length transcript (<i>UAS-hemoFL</i>) or overexpression of the human Stannin peptide (<i>UAS-snn</i>) but not by the expression of the control <i>UAS-GFP</i> construct. One-way ANOVA test showed that the means were significantly different [F(9,365) = 14.26, <i>p</i> < 0.0001]. Post hoc multiple comparison Bonferroni’s test showed that <i>hemo</i>^<i>A4</i>, <i>UAS-14-3-3ζ</i> and <i>UAS-14-3-3ζ;UAS-GFP</i> samples were significantly different than wild-type, whereas the rest were not (<i>n</i> ≥ 20, <i>p</i> < 0.05). Error bars represent SEM. (H) Measurement of the occupied FYVE area index (OAI) in <i>ex vivo</i> prepupal hemocytes (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#sec009" target="_blank">Materials and Methods</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s001" target="_blank">S1</a> Data). Overexpression of human Snn peptide (<i>UAS-snn</i>) rescues the <i>hemo</i>^<i>A4</i>-enlarged FYVE compartments. Similarly, reducing <i>14-3-3ζ</i> function by expression <i>14-3-3ζ-RNAi</i> restores the size of <i>hemo</i>^<i>A4</i> mutant FYVE-organelles to wild-type. Conversely, overexpression of <i>14-3-3ζ</i> (<i>UAS-14-3-3ζ</i>) mimics the <i>hemo</i>^<i>A4</i> mutant FYVE phenotype. The overexpression <i>14-3-3ζ</i>-phenotype is reversed by coexpression with <i>hemo</i> full-length transcript (<i>UAS-hemoFL</i>). UAS constructs were driven with <i>He-Gal4</i>. One-way ANOVA test showed that the means of samples were significantly different [F(8,346) = 23.15, <i>p</i> < 0.0001]. Multiple comparison post hoc Bonferroni’s test indicated that <i>UAS-14-3-3ζ</i> and <i>hemo</i>^<i>A4</i> were significantly different than wild-type whereas the rest of the samples were not (<i>n</i> ≥ 20, <i>p</i> < 0.05). Error bars represent SEM. (I–N) Intracellular distribution of FYVE-positive (green) compartments in <i>ex vivo</i> prepupal hemocytes (see also H). Scale bar (5 μm). (I) In wild-type FYVE-positive organelles appear as small rings and punctae. (J) In <i>hemo</i>^<i>A4</i> mutant hemocytes, FYVE compartments contain larger rings than wild-type (arrowheads). (K) Expression of Hemo-ORF peptide (<i>UAS-hemo-ORF</i>) rescues the enlarged <i>hemo</i>^<i>A4</i> mutant FYVE compartments. (L) Expression of <i>snn</i> (<i>UAS-snn</i>) reduces the <i>hemo</i>^<i>A4</i> FYVE phenotype. (M) Overexpression of <i>14-3-3ζ</i> (<i>UAS-14-3-3ζ</i>) produces enlarged FYVE compartments (arrowheads). (N) Reducing <i>14-3-3ζ</i> function with RNAi rescues the <i>hemo</i>^<i>A4</i> mutant FYVE phenotype. UAS constructs were driven with <i>He-Gal4</i>. Yellow dashed lines indicate the cell body. Supplemental data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s006" target="_blank">S5 Fig</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s007" target="_blank">S6 Fig</a>, and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s001" target="_blank">S1 Data</a>.</p

Identification and phenotypical characterisation of the <i>hemotin</i> gene.

<p>(A) <i>hemo</i> genomic locus including the <i>hemo</i>, <i>CG7691</i>, <i>fray</i>, and <i>fruitless</i> genes (blue arrows). The <i>hemo</i>^<i>A4</i> deletion (red bar) was generated by FRT-mediated recombination using the <i>P{RS3}fray</i>^{<i>CB-0706-3</i>} and the <i>P-Bac{WH}fru</i>^{<i>f02684</i>} transposable elements (blue triangles). Transcript models are represented under their respective genes, orange boxes represent coding exons, whereas gray boxes indicate noncoding exons (untranslated regions, UTRs). <i>hemo</i>^<i>A4</i> completely removes <i>hemo</i> and <i>CG7691</i> plus the first noncoding exons of <i>fray</i> and <i>fruitless</i>. The <i>P{PZ}fray</i>^<i>07551</i> insertion is a lethal <i>fray</i> allele [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.ref025" target="_blank">25</a>] and was used for genetic complementation experiments between <i>hemo</i>^<i>A4</i> and <i>fray</i>. (B) Top: Ribosomal profiling reads obtained from polyribosomes from S2 cells (Poly-Riboseq;(3)) mapped to the <i>hemo</i> full-length transcript (<i>hemoFL</i>). <i>hemo</i>-ORF is translated more efficiently than ORF2 (<i>hemo</i>-ORF RPKM: 29.4, coverage: 0.9 ORF; ORF2 RPKM: 6.6, coverage: 0.7. Note that the reads per kilobase of transcript per million mapped reads [RPKM] value of ORF2 is below the 11.8 cut-off to be considered translated [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.ref003" target="_blank">3</a>]). Bottom: schematic representation of other constructs used in this manuscript. <i>hemo</i>-ORF is a minigene consisting of an mRNA fragment truncated immediately after the <i>hemo</i>-ORF stop codon, ORF2 consists of a mini-gene construct carrying the ORF2 sequence only, including 6 nt upstream of its start codon (to conserve its endogenous Kozak sequence). <i>hemo</i>-GFP (green fluorescent protein) is a <i>hemo</i>-ORF-GFP fusion construct in which the GFP sequence (devoid of a start codon) was cloned into the <i>hemoFL</i> construct, immediately downstream and in frame with <i>hemo</i>-ORF (devoid of a stop codon) (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#sec009" target="_blank">Materials and Methods</a>). (C) Pattern of expression of <i>hemo</i> in germ band-retracted embryos revealed by <i>in situ</i> hybridisation. <i>hemo</i> is specifically expressed in embryonic hemocytes (arrows; compare with D) in the head, amnioserosa, and dispersed along the body. (D) Spatial distribution of embryonic hemocytes at germ band retraction stage revealed by <i>in situ</i> hybridisation of hemocyte-specific <i>croquemort</i> (<i>crq</i>) gene, showing similar distribution in the head, amnioserosa, and along the body (arrows). (E) Cluster of early embryonic hemocytes of the cephalic region expressing the <i>hemo</i> transcript revealed by FISH (fluorescent in situ hybridisation). Some hemocytes show drop-shape morphologies (asterisk) and membrane projections such as filopodia (arrows). (F) Embryonic hemocytes labelled with <i>crq-Gal4;UAS-GFP</i> expression from the head region displaying similar cellular morphologies (arrows and asterisk) as those in E. (G–H) White prepupal thoracic hemocytes revealed by <i>crq-Gal4;UAS-GFP</i> expression in wild-type (G) and <i>hemo</i>^<i>A4</i> mutants (H). In <i>hemo</i>^<i>A4</i> mutants, hemocytes display enlarged vacuoles within the cytoplasm (arrowheads), with larger occupied area index (OAI). Scale bar (50 μm). (I–N) hemocytes observed <i>ex vivo</i> [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.ref015" target="_blank">15</a>] showing Tubulin (green) and Actin (red) cytoskeletons and nuclei (2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, DAPI) with its corresponding orthogonal projection of confocal microscopy z-stacks (above inset) showing only tubulin cytoskeleton (green) and DAPI (blue) staining in the nucleus (n). Scale bar (5 μm). (I) Wild-type hemocyte. (J) <i>hemo</i>^<i>A4</i> mutant hemocyte shows large disruptions of the tubulin cytoskeleton that appear as rounded vacuoles (arrows; arrowhead in inset). (K) Knocking down the expression of <i>hemo</i> with a <i>UAS-hemo-RNAi</i> construct phenocopies the vacuolation phenotype (arrows and arrowhead in inset). (L) Expression of <i>hemo</i> full length transcript (<i>UAS-hemoFL</i>) rescues the vacuolated <i>hemo</i>^<i>A4</i> phenotype. Expression of <i>hemo</i>-ORF only (M) also rescues the <i>hemo</i>^<i>A4</i> mutant vacuolation. (N) Expression of ORF2 does not rescue the <i>hemo</i>^<i>A4</i> mutant vacuolated phenotype (arrows and arrowhead in inset). (O) Vacuolation measurements in <i>ex vivo</i> primary pre pupal hemocytes. <i>hemo</i>^<i>A4</i> mutant hemocytes show significantly higher occupied volume index (OVI) (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#sec009" target="_blank">Materials and Methods</a>) than wild-type. Rescue experiments show that the vacuolation phenotype is specific to the peptide encoded by <i>hemo</i>-ORF. All upstream activating sequence (UAS) constructs were driven by <i>crq-Gal4</i>. Error bars represent standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA test indicating that samples were significantly different [F(9,486) = 9.5, <i>p</i> < 0.0001]. A post hoc Bonferroni multicomparison test showed that <i>hemo</i>^<i>A4</i>, <i>UAS-hemo-RNAi</i>, <i>UAS-ORF2-hemo</i>^<i>A4</i>, <i>UAS-hemoFS</i> (expressing a <i>hemo</i> full-length transcript containing frameshifts in <i>hemo</i>-ORF and ORF2)-<i>hemo</i>^<i>A4</i> and <i>CG7691</i> genomic fragment (GF)-<i>hemo</i>^<i>A4</i> were significantly different than wild-type. The <i>UAS-hemoFL-hemo</i>^<i>A4</i>,<i>UAS-hemo-ORF-hemo</i>^<i>A4</i>,<i>UAS-hemoGFP-hemo</i>^<i>A4</i> and <i>fray</i>^<i>PZ</i><i>/hemo</i>^<i>A4</i> were not significant to wild-type (<i>n</i> ≥ 24, <i>p</i> < 0.05). Supplemental data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s001" target="_blank">S1 Fig</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s001" target="_blank">S1 Data</a>.</p

Model for the role of Hemotin in phagocytic processing.

<p>Simplified models of endosomal maturation, modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.ref018" target="_blank">18</a>] depicting the role of the proteins and markers analysed in this work. A) Wild-type endosomal trafficking is regulated by different phosphorylation states of PI. Phosphorylation of PI into PI(3)P is achieved in early endosomes by the class II or class III PI3 kinases PI3k68D and Vps34, respectively. In late endosomes PI(3)P is again phosphorylated to produce PI(3,5)P2. This phosphorylation step allows late endosomes to progress into degradation allowing lysosomes to fuse to late endosomes to produce multivesicular bodies. This trafficking progression can be reversed by dephosphorylation of PI(3)P or PI(3,5)P2 by myotubulurin phosphatases. Hemotin and Stannin are functional homologues that localise to early endosomes, where they bind and repress 14-3-3ζ. Our genetic and biochemical data indicates that 14-3-3ζ binds the PI3K68D kinase and promotes its function, perhaps by directly increasing its enzymatic activity, or indirectly by promoting its correct localisation in early endosomes. Since Hemotin antagonises 14-3-3ζ, it indirectly reduces the development of early endosomes through PI3K68D. B) The absence of Hemotin produces an excess of 14-3-3ζ function, which results in an excess of PI3K68D function and leads to an increase in endocytic vesicles containing PI(3)P, as detected by expansion of the area occupied by the FYVE marker. These abnormal vesicles display an abnormal maturation during phagocytosis, with excessive co-expression of early lysosomal markers (such as FYVE) and late ones (Lysotracker and Rab7), and a slower and less intense acidification of their contents, as revealed by the pHrodo pH marker.</p

The Hemotin peptide is required for proper endosomal maturation in hemocytes.

<p>(A–A”) Distribution of acidic organelles in <i>hemo</i>^<i>A4</i> mutant <i>ex vivo</i> hemocytes revealed by the expression of LAMP1-GFP lysosomal marker. The intracellular vacuoles that disrupt the beta-tubulin cytoskeleton (A, A”; red) accumulate LAMP1-GFP positive compartments (A, A’; green). Compare with wild-type in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s003" target="_blank">S2A–S2A” Fig</a>. Scale bar (5 μm). (B–B”) Distribution of the endosomal marker FYVE (PI(3)P binding zinc finger domain, early endosomal marker, named after being found in Fab1, YOTP, Vac1, EEA1) (green) (B, B’) and Lysotracker (red) (B, B”) organelles in a <i>hemo</i>^<i>A4</i> mutant <i>ex vivo</i> hemocyte showing enlarged intracellular compartments coexpressing FYVE and Lysotracker. Scale bar (5 μm). (C–C”) Wild-type <i>ex vivo</i> hemocyte labelled as in (B), showing little overlap between early endosome-FYVE positive (green) (C,C’) and lysosomal (red) (C,C”) compartments. (D) Quantification of the FYVE OAI in <i>ex vivo</i> hemocytes (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#sec009" target="_blank">Materials and Methods</a>). <i>hemo</i>^<i>A4</i> mutants display a significantly larger FYVE area than wild-type. This phenotype is rescued by the expression of the <i>hemo</i> full length transcript (<i>UAS-hemoFL</i>) and is specific to <i>hemo</i>-ORF function, as it is also rescued by the expression of the <i>hemo</i>-ORF mini gene (<i>UAS-hemo-ORF</i>) or C-terminal-tagged <i>hemo</i>-GFP <i>(UAS-hemo-GFP</i>). No rescue was observed by a <i>CG7691</i> genomic fragment (<i>CG7691-GF</i>), or with a <i>hemo</i> full-length transcript containing a frameshift in the <i>hemo</i>-ORF (<i>UAS-hemoFS</i>), or with the ORF2 mini gene (<i>UAS-ORF2</i>). All UAS constructs were driven with He-Gal4. Error bars represent SEM. One-way ANOVA analysis shows that there is a statistically significant difference between these groups [F(7,286) = 27.12, <i>p</i> < 0.0001]. Post hoc comparisons using Bonferroni test indicated that the mean score of <i>hemo</i>^<i>A4</i>, <i>UAS-hemo-ORF2-hemo</i>^<i>A4</i>, <i>UAS-hemoFS-hemo</i>^<i>A4</i>, and <i>CG7691-GF-hemo</i>^<i>A4</i> did significantly differ from wild-type (<i>p</i> < 0.05), whereas <i>UAS-hemoFL-hemo</i>^<i>A4</i>, <i>UAS-hemoORF-hemo</i>^<i>A4</i>, and <i>UAS-hemoGFP-hemo</i>^<i>A4</i> did not. (E) Analysis of the overlap between FYVE-positive early endosomal and Lysotracker-positive compartments using Pearson’s correlation coefficient in wild-type and <i>hemo</i>^<i>A4</i> mutant hemocytes. In <i>hemo</i>^<i>A4</i> hemocytes, there exist significantly more intracellular compartments displaying FYVE and Lysotracker colocalisation than in the wild-type as shown by a Two-tailed Mann-Whitney test (<i>n</i> ≥ 17; <i>p</i> < 0.05). Error bars represent SEM. (F) Statistical analysis of the Pearson’s coefficient measurements of Hemotin-GFP (Hemo-GFP) with early endosomal (FYVE-cherry) and lysosomal (Lysotracker) markers. Tagged-Hemotin peptides are significantly enriched in early endosomal compartments in comparison with lysosomes as shown by a two tailed Mann-Whitney test (<i>n</i> ≥ 20, <i>p</i> < 0.05). Error bars represent SEM. (G–G”) Localisation of Hemo-GFP peptides (green)(G’) and the endosome FYVE marker (red)(G”) in hemocytes <i>(He-Gal4;UAS-hemo-GFP</i>). A substantial part of Hemo-GFP pattern colocalizes with FYVE-positive compartments (G) (arrows). Scale bar (5 μm). (H–H”) Distribution of Hemo-GFP peptides (green) (H’) and the lysosomal marker (lysotracker; red) (H”) in hemocytes. Only a small overlap exists between Hemo-GFP compartments and lysosomes (H) (arrow). Supplemental data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s003" target="_blank">S2 Fig</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s007" target="_blank">S6 Fig</a>, and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s001" target="_blank">S1 Data</a>.</p

<i>hemotin</i> is involved in phagocytic processing and is necessary for optimal bacterial clearance and life span.

<p>(A–B) Time-lapse imaging showing the phagocytic trafficking of pHrodo-labelled bacterial particles (arrowhead, red) in <i>ex vivo</i> hemocytes expressing the early endosomal marker FYVE-GFP (green) with the <i>He-Gal4</i> driver in wild-type (A) or <i>hemo</i>^<i>A4</i> mutants (B) (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s012" target="_blank">S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s013" target="_blank">S2</a> Videos). (t0) represents the time when the particle docks into the cell membrane, displaying a relatively faint intensity. By t = 6 minutes (min), the pHrodo particles are in FYVE-positive early endocytic vesicles in both wild-type (A) and <i>hemo</i>^<i>A4</i> mutant hemocytes (B). By t = 22 min in the wild-type hemocyte (A), the FYVE signal around the particle is dramatically reduced, while the intensity of the pHrodo signal increases, suggesting that the vesicle has progressed into a PI3P-depeleted and acidified endolysosome. In the <i>hemo</i>^<i>A4</i> mutant hemocyte (B), the FYVE signal around the particle is still visible at t = 44 min, showing an increased prevalence of PI(3)P in this vesicle, and therefore an extended early endocytic phase. However, the intensity of the pHrodo signal is lower than in wild-type, indicating a delay in the acidification of the endocytic vesicles (See also C and D). The insets show a magnification of the specific particles. Scale bars = 5 μm. (C) Magnified raw images from the particles shown in (A) and (B), showing the region of interest (ROI) used to quantify their fluorescent intensity. The integrated intensity read-out for each time point is indicated in red. (D) Quantification of fluorescent intensity of pH-sensitive pHrodo particles undergoing phagocytosis in wild-type (blue) or <i>hemo</i>^<i>A4</i> mutant (red) hemocytes. Average integrated intensity per pixel is represented for each time point. Error bars represent SEM. A two-way ANOVA analysis with Bonferroni post test indicates that the difference between these curves is significantly different from t = 16.5 min (<i>n</i> = 8, <i>p</i> < 0.001). (E) Quantification of FYVE prevalence on pHrodo particles undergoing phagocytosis in wild-type (blue) or <i>hemo</i>^<i>A4</i> mutant (red) hemocytes. The FYVE signal remains significantly longer in <i>hemo</i>^<i>A4</i> mutants (mean = 46.06 +/− 5.3 min) than wild-type (mean = 19.87 +/− 2.1 min) as indicated by a one-tailed unpaired <i>t</i> test (<i>n</i> = 11, <i>p</i> < 0.0003). Error bars represent SEM. (F–I) Visualisation of bacterial up-take and processing <i>in vivo</i>. Dorsal vessel-associated adult hemocytes (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#sec009" target="_blank">Materials and Methods</a>) expressing FYVE-GFP (green) driven by <i>He-Gal4</i> from wild-type (F, G) or <i>hemo</i>^<i>A4</i> mutant flies (H, I) infected with mCherry-expressing <i>E</i>. <i>coli</i> bacteria (red) (OD600 = 0.05) and dissected 20 min (F, H) or 120 min (G, I) postinjection. Wild-type and mutant flies exhibit a similar number of bacterial cells per hemocyte after 20 min; however, this number increases in mutant flies after 120 min, whereas it remains constant in wild-type flies, suggesting that mutant hemocytes accumulate undigested bacterial cells. Yellow dashed lines represent the cell body outline. (J) Quantification of bacterial uptake. <i>hemo</i>^<i>A4</i> mutant hemocytes contain a similar number of bacterial cells as wild-type after 20 min but significantly more bacterial cells after 120 min. Average number of bacterial cells per hemocyte is represented in the <i>y</i>-axis. (<i>n</i> = 30, <i>p</i> < 0.0001). (K) Visualisation of bacterial load from either wild-type or <i>hemo</i>^<i>A4</i> mutant 30days (d)old adult fly homogenates. Each spot represents the bacterial colonies grown from an individual male fly. Each homogenate was plated in decreasing dilution (1:1 or 1:10). Note the higher density of bacterial colonies in the spots from <i>hemo</i>^<i>A4</i> mutants compared to wild-type. (L) Quantification of bacterial colonies grown from single adult fly homogenates. <i>hemo</i>^<i>A4</i> mutants contain significantly more bacteria than wild-type, both at 10 (<i>n</i> = 12, <i>p</i> < 0.0054) or 30 d old (<i>n</i> = 8, <i>p</i> < 0.0078). Error bars represent SEM. (M) <i>hemo</i>^<i>A4</i> mutants (red) have a reduced viability over time compared to wild-type (blue), with a median life-span of 23 +/− 6 d, compared to 49 +/− 3 d for wild-type. The addition of antibiotics (penicillin-streptomycin) to the food media significantly increases the median life span of <i>hemo</i>^<i>A4</i> mutants to 38 +/− 6 d, as determined by a paired <i>t</i> test (<i>p</i> < 0.05). For each condition, five different replicates were analysed for a total of 50 flies. Error bars represent SEM. Supplemental data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s004" target="_blank">S3 Fig</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s012" target="_blank">S1 Video</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s013" target="_blank">S2 Video</a>, and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002395#pbio.1002395.s001" target="_blank">S1 Data</a>.</p