Fig. 1: Production of C. elegans muscle exophers is upregulated during pathogen infection.

a Confocal images of C. elegans expressing RPN-5::wrmScarlet (proteasome subunit, red) and MOM::GFP (mitochondrial outer membrane marker, green). Images were acquired using a Leica STELLARIS confocal microscope in LIGHTNING mode to enhance resolution. All five panels originate from the same animal and were generated from the same confocal z-stack. (i) Differential interference contrast (DIC) image aligned with a single confocal optical section, showing overall body morphology and the position of exophers. (ii) Overview fluorescence image of the mid-body region, illustrating the distribution of exophers along the body-wall muscles. (iii) Magnified view of two mitochondria-containing exophers from the region boxed in panel 2, with dimensions indicated to show their size variation and mitochondrial content. (iv) Three-dimensional surface rendering of the corresponding z-stack generated in LAS X 3D Viewer (Leica Microsystems), highlighting the morphology and spatial arrangement of exophers relative to surrounding tissues. (v) Depth-coded 3D heatmap representation of the same reconstruction. The color scale denotes the z-position of structures within the volume, with cooler colors (blue/cyan) indicating elements closer to the imaging plane and warmer colors (yellow/red) marking deeper regions. Blue arrows indicate mitochondria-containing exophers, and white arrows mark exophers emerging from body-wall muscles. This depth information emphasizes the extracellular location and three-dimensional topology of exophers. Scale bar: 20 μm. Representative of at least 23 animals from two independent replicates. b Experimental Setup: Synchronized AD1 worms were exposed to Pseudomonas aeruginosa (PA14) and Serratia marcescens (Db10), or to Escherichia coli (OP50) for 3 hours at 25 °C, followed by transfer to OP50-seeded plates. Exopher production was assessed on the second day of adulthood (AD2). Created in BioRender. Kolodziejska, K. (https://BioRender.com/xsyp3oe. c Short time infection with P. aeruginosa PA14 or S. marcescens Db10 leads to increased exopher production (n = 60, 58 and 59 worms (for respective columns), N = 2 independent experiments). d Investigated innate immunity pathways: Various innate immunity pathways were assessed for their roles in regulating exopher production. e Worms deficient in key immune response pathways exhibited changes in exopher levels (n = 360, 90, 90, 90 and 90 worms (for respective columns), N = 3 independent experiments; n = 180, 120 and 91 worms (for respective columns), N = 3-4 independent experiments). f–h Infection-induced exopher production is not dependent on the (f) insulin signalling (n = 60, 58, 59, 56, 54, 58, 55, 50 and 60 worms (for respective columns), N = 2 independent experiments), (g) p38 MAPK (n = 60, 58, 59, 37, 55 and 50 worms (for respective columns), N = 2 independent experiments; n = 58, 54, 54, 54, 55 and 55 worms (for respective columns), N = 2 independent experiments), or (h) TGF-β pathways (n = 60, 58, 59, 54, 56 and 45 worms (for respective columns), N = 2 independent experiments). i Exopher production in response to S. marcescens Db10 infection is fully dependent on the IPR pathway, while production in response to P. aeruginosa PA14 infection is partially dependent on this pathway (n = 58, 54, 54, 51, 51 and 56 worms (for respective columns), N = 2 independent experiments). j Exposure to S. marcescens Db10, but not P. aeruginosa PA14, results in embryo accumulation in the uterus (n = 75, 55 and 55 worms (for respective columns), N = 3-4 independent experiments). Data information: The data shown in graphs (f–i) were collected together and share the same controls but are presented in separate panels to improve visualization and enhance clarity. Baseline differences between (e, f–i) arise from methodological variations which are detailed in the Materials and Methods section “Scoring of Muscle Exophers and Fluorescence Microscopy”. Differences in basal exopher levels observed among wild-type controls are associated with the use of different transgenes for exopher visualization, as detailed in Supplementary Data 3. Data are presented as medians with interquartile ranges (c, e–j). Non-significant p values (p > 0.05) are highlighted in pink, while significant p values (p < 0.05) are shown in blue. Statistical significance was assessed using the Kruskal-Wallis test with Dunn’s multiple comparisons test.