fig

fig. activates the immune deficiency (Imd) pathway in the fat body [2, 3, 4]. The signal from PGRP-LC is transmitted via the receptor-bound scaffolding protein Imd [5, 6]. Imd activation induces a signalling cascade resulting in the Relish (NF-B like transcription factor)-dependent activation of stress and immune response genes including those encoding antimicrobial peptides (AMPs) such as and (Phg1A, B, C), in the yeast (TMN1-3) and in flies Pexidartinib (PLX3397) (TM9SF2, TM9SF3, TM9SF4) and 4 members in humans (TM9 superfamily proteins TM9SF1 to TM9SF4) [10, 14, 15, 16, 17]. They are found in the endosomal compartments of yeast, and human cells where they possibly contribute to cell migration, vesicular transport, endocytic trafficking and autophagy [11, 12, 17, 18, 19, 20, 21]. In TM9SF4/Phg1A is required for the phagocytosis and killing of bacteria [16, 22, 23]. Moreover, the two TM9 proteins Phg1A and Phg1B synergistically contribute to the expression and/or localization of transmembrane proteins [14, 24]. The function of TM9SF4 in phagocytosis is conserved in human immune cells, where TM9SF4 overexpression contributes to enhanced phagocytic activity of metastatic tumour cells [13, 25], and in [15, 26]. In mutant macrophages and These two TM9 proteins interact with PGRP-LC and co-localize with the receptor in both intracellular punctate structures and at the plasma membrane. TM9SF4, but not TM9SF2, is required for PGRP-LC localization at the cell surface, which might account for the specific function of TM9SF4 in internalization of Gram-negative bacteria. Moreover, and to a lesser extent mutant flies showed constitutive activation of AMP gene expression, suggesting a negative Pexidartinib (PLX3397) regulatory function of these two TM9 proteins on the unstimulated receptor. Since expression of both TM9SF2 and TM9SF4 inhibits PGRP-LC but not Imd signalling Pexidartinib (PLX3397) activity, mediated by their overexpression in S2 cells, these two TM9 proteins likely directly prevent inappropriate PGRP-LC signalling activity by interacting with the receptor. Materials and Methods Fly Strains Flies were raised at 25C. The null mutant is described in the report of Bergeret et al. [15]. P[UAS-PGRP-LCx-Flag] (lines 16B and 77A) are described in the report of Schmidt Mouse monoclonal to DDR2 et al. [27]. The P[EP]CG9318EP2088 designed in this study as was obtained from the Exelixis Collection at the Harvard Medical School (https://drosophila.med.harvard.edu/). The transgenic lines P[UAS-TM9SF2-GFP] and P[UAS-TM9SF4-GFP] were obtained by germ-line-mediated integration using standard methods. With regard to the FLPout GAL4/UAS method, spontaneous activation of the GAL4 transcription factor without heat shock has been reported by Hennig et al. [28]. Cell Culture S2 cells were maintained in Schneider’s medium supplemented with 10% heat-inactivated fetal calf serum (Invitrogen). Gene inactivation was achieved as described by Clemens et al. [29]. The methodology and primers used are described in the legend to online supplementary figure 2 (for all online suppl. material, see www.karger.com/doi/10.1159/000365112). Activation of the promoter in S2 cells and induction of AMP genes in vivo were monitored as described by Thevenon et al. [30]. DNA Constructs cDNA clones for (LD44273) and (GH02822) were purchased from Drosophila Genomics Resource Center (DGRC). The following primer sets were used for PCR amplification: TM9SF2 forward, 5-ggggtaccATGATCCTGCTATCCGGA?CTT-3, TM9SF2 reverse, 5-ctagtctagaATCCACCTTGACAAC?ACTGTA-3; TM9SF4 forward, 5-ggggaattcCACTCCCACACA?CCACCAACA-3, and TM9SF4 reverse, 5-gcggatccGTCGATC?TTCACAGCTCCGTA-3. Full-length PCR products were cloned into pAc5.1/V5/HisB vector (Invitrogen) or pAc-GFP vectors, allowing for the expression of corresponding tagged proteins. Full-length and truncated pAc-PGRP-LC-V5 constructs and pAc-Imd-V5 constructs were made from the corresponding pMT vectors described in the report of Choe et al. [5]. Immunoprecipitation Co-immunoprecipitation of GFP-tagged TM9 protein with V5- or Flag-tagged PGRP-LC was performed following standard procedures. Immunofluorescence Microscopy and Clonal Analysis Immunofluorescence microscopy of S2 cells and Pexidartinib (PLX3397) dissected fat body were performed as described by Bergeret et al. [15] and Pexidartinib (PLX3397) Taillebourg et al. [31], respectively. Fluorescence-Activated Cell Sorting Analysis S2 cells were transfected with pAc-PGRP-LC-V5 and double-stranded RNA (dsor dsS2 cells. TM9SF2-GFP and TM9SF4-GFP proteins localized both in intracellular round structures and at the cell membrane (fig. 2a, b, c, d). Similar staining was observed for PGRP-LC-V5, with about half of the cells showing mainly membrane-bound PGRP-LC (fig. 2e-g) and the second half presenting both membrane-bound and cytosolic staining (fig. 2f, h). These experiments revealed a close co-localization of each TM9SF protein with PGRP-LC (fig. 2i-l). We conclude from.