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First published online 16 September 2008
doi: 10.1242/jcs.030163

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TM9SF4 is required for Drosophila cellular immunity via cell adhesion and phagocytosis

¹ CEA, iRTSV, LTS, 38054 Grenoble, France
² INSERM U873, 38054 Grenoble, France
³ Université Joseph Fourier, 38000 Grenoble, France
⁴ Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen AB24 2TZ, UK
⁵ Minatec, Grenoble Institute of Technology, LMPG, 38054 Grenoble, France
⁶ Centre Médical Universitaire, Département de Physiologie Cellulaire et Métabolisme, Université de Genève, CH-1211 Geneva 4, Switzerland

View larger version (15K): [in this window] [in a new window]   Fig. 1. The nonaspanin family in Drosophila melanogaster. Similarity tree of TM9 proteins in D. melanogaster (Dm; CG7364, CG9318, CG10590) compared with human (Hs; TM9SF1-TM9SF4) and D. discoideum (Dd; PHG1A, PHG1B, PHG1C).

View larger version (20K): [in this window] [in a new window]   Fig. 2. TM9SF4 gene map. (A) The TM9SF4 gene produces one transcript of 2.6 kb which contains one coding sequence (coloured in grey). The insertion point for {lacW}CG7364k07245 is 94 bp upstream of the ATG translation start. One 1.4 kb deletion (TM9SF41) was recovered encompassing the transcription start site and the N-terminal part of the corresponding protein. (B) The deletion creates a null allele as visualised by northern analysis of TM9SF4 transcripts in control w1118 (lane 1) compared with mutant TM9SF41 (lane 2) flies. (C) Developmental northern blot. Lane 1, embryos; lane 2, third instar larvae; lane 3, pupae; lane 4, adult. (D) TM9SF4 transcripts were quantified by real-time PCR from total RNAs extracted from either the whole third instar larvae (L3), the gut (Gut), the fat body (FB) or the larval circulating plasmatocytes (He). Results are mean ± s.d.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Survival rate of infected Drosophila flies. 5- to 7-day-old males, previously raised at 25°C, were infected with indicated bacteria, either by septic injury onto the thorax with a thin needle previously dipped into the indicated bacterial solution (A,C-G) or by oral ingestion (B). Survival rate was followed at 25°C except in the case of S. aureus (20°C) as indicated. (A-F) Survival of TM9SF4 mutant flies and TM9SF4/Df(2L)b82a2 compared with control w1118 (w), Rev45 flies or dTAK12 (TAK1) mutant flies affected in the Imd pathway. (G) The number of colony forming units (CFUs), plotted in logarithmic scale, was calculated from bacteria isolated from infected flies.

View larger version (43K): [in this window] [in a new window]   Fig. 4. Expression of genes encoding antimicrobial peptide is not affected in TM9SF4 flies. (A,C) Expression of Attacin (Att), Diptericin (Dipt), Drosocin (Dro) and Drosomycin (Drs) as indicated, and of Actin (Act) which served as a loading control, was detected by northern blot. Expression of Diptericin, Drosomycin and Defensin (Def) by quantitative real-time PCR (B,D,E) was performed with total RNA isolated from 5- to 7-day-old flies. Control (w1118, Rev45) or mutant [TM9SF4, TM9SF4/Df(2L)b82a2, TAK12] flies were sacrificed before infection (NI) or at several time points (in hours) following infection. (A) Northern analysis of antimicrobial gene expression in E. cloacae-infected flies. Expression level of all antimicrobial encoding is similar in TM9SF4 mutants compared with Rev45 flies. Note that, because of different genetic background, w1118 flies expressed slightly higher levels of antimicrobial peptides transcripts than Rev45 flies, although both strains displayed similar resistance to infection. (B) Quantitative analysis of Diptericin expression level in either E. cloacae- or K. pneumoniae-infected TM9SF4 mutant and Rev45 control flies. (C) Northern analysis of Drosomycin expression in Enterococcus faecalis TM9SF4 mutant and w1118 (w) control infected flies. (D) Quantitative analysis of the expression level of Drosomycin in Micrococcus luteus-infected flies. (E) Quantitative analysis of Defensin expression in Rev45 and TM9SF4/Df(2L)b82a2 transheterozygous flies (TM9SF4/Df). These flies are deficient for TM9SF4 and hemizygous for the Defensin locus. In B,D and E, results are expressed as the fold induction compared with non-infected flies. Post-infection times in hours are indicated below each histogram.

View larger version (64K): [in this window] [in a new window]   Fig. 5. In vivo engulfment of GFP-labelled K. pneumoniae by Drosophila haemocytes. Dorsal view of Rev45 (A,C) and TM9SF41 mutant (B,D) fly abdomen injected with GFP-expressing K. pneumoniae at 3 hours (A,B) and 5 hours (C,D) post injection time. Arrowheads in A-D indicate the position of clustered haemocytes. Arrows in D indicate extracellular fluorescence associated with haemolymph.

View larger version (33K): [in this window] [in a new window]   Fig. 6. TM9SF4 mutant larval haemocytes have defective phagocytosis and encapsulation. (A) Circulating plasmatocytes were isolated from third instar larvae and incubated for 15 minutes with fluorescent latex beads. The internalisation of FITC-latex beads was observed following addition of quenching Trypan Blue solution. (B) Using the same procedure as in A, the internalisation rate of FITC-labelled beads or E. coli or S. aureus, was calculated as the number of internalised particles per haemocyte from 300-500 haemocytes. A phagocytic rate of 100% was attributed to control Rev45 cells in each experiment. The results are the mean ± s.d. of three independent experiments. A significant difference (Student's t-test, P<0.03) was found in phagocytic rate for latex beads and E. coli, but not S.aureus between Rev45 and TM9SF4 mutant cells (left panel). Directed expression of TM9SF4 mainly in the haemocyte lineage through the srpGal4 driver line (srpGal4/Y; TM9SF41; UASTM9SF4/+ larvae) partially rescued the phagocytic properties of circulating plasmatocytes (right panel) (P<0.01, Student's t-test). (In this experiment, larvae were raised at 18°C.) (C) Encapsulation rate of control (w1118, Rev45) and mutant (TM9SF4) larvae following wasp parasitisation. The total number of parasitised larvae examined is indicated on the top of each histogram, the number in parenthesis indicates the number of larvae presenting a dark capsule. Experiments were performed at 24°C and 29°C as indicated.

View larger version (50K): [in this window] [in a new window]   Fig. 7. Impaired lamellipodia formation and defective actin reorganisation in TM9SF4 mutant macrophages. Circulating plasmatocytes were isolated from wild-type (A,C) or mutant TM9SF4 (B,D) third instar larvae and allowed to spread for 15 minutes in a glass coverslip chamber. (A,B) Phase contrast. (C,D) Reflection interference contrast microscopy. Arrowhead indicates the loss of adhesive belt; arrow indicates the white area that represents more distant contacts. (E-I) Confocal analysis of actin network in isolated larval macrophages. Texas-Red-phalloidin fluorescent labelling revealed polymerised actin (E,H) and nuclei were stained with Hoechst 33258 (F,I); overlays are shown in G,J. Control cells are regularly sized and round (E-G), whereas most TM9SF4 mutant cells have a larger area and differentiate long actin-stained filopodia (H-J). (K) The surface of the cytoskeleton network was calculated from 500-1000 cells. Mutant TM9SF4 cells were 2.3-fold larger than Rev45 control cells (P<0.0001, Student's t-test). Cell size was partially rescued by expression of TM9SF4 cDNA in the haemocyte lineage. A significant difference between TM9SF4 mutant and srpGal4; TM9SF4;UAS-TM9SF4/+ rescued plasmatocytes was found (P<0.004, Student's t-test) (larvae raised at 18°C). Scale bars: 10 µm.

View larger version (11K): [in this window] [in a new window]   Fig. 8. TM9SF2 and TM9SF4 are required for phagocytosis in S2 cells. S2 cells were untreated (Control) or treated for 3 days with siRNA GFP (Green fluorescent protein), TM9SF4, TM9SF2, PGRP-LC or Dscar, either alone or in combination (TM9SF2+4), as indicated. Cells were then incubated for 10 minutes with FITC-labelled E. coli (A) or FITC-labelled S. aureus (B) and the internalised fluorescence was measured in the presence of external Trypan Blue quenching solution. The phagocytosis index was quantified as the percentage of fluorescence-positive cells multiplied by the mean fluorescence of these cells. We counted 10,000-20,000 cells from each sample. A phagocytosis index of 100% was attributed to control cells. The results presented are the mean ± s.d. of three experiments. Significant differences were observed using Student's t-test between: (A) TM9SF4 and control (P<0.003), TM9SF2 and control (P<0.02), PGRP-LC and control (P<0.001), Dscar and control (P<0.001); and (B) TM9SF2 and control (P<0.001), TM9SF2+4 and TM9SF2 (P<0.05), Dscar and control (P<0.001).

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