BUSCADOR RECOLECTA

All images are super-resolution single confocal sections except B, which is a projection of super-resolution confocal sections. (A, B) In the trachea of wild-type embryos, Reb and Kkv do not colocalise, and they show a complementary pattern (A’-A”’) at the local subcellular level. (C) In salivary gland of embryos expressing Reb, the patterns of Kkv and Reb are complementary. (D) Models for the role of kkv and exp/reb in chitin deposition. Kkv oligomerises in complexes that localise to the apical membrane (as proposed in [2]). In the absence of exp/reb activity, Kkv can polymerise chitin from sugar monomers (discontinuous red lines), but it cannot translocate it because the channel is closed, and polymerised chitin remains in the cytoplasm. In addition, Kkv is not homogeneously distributed. Exp/Reb form a complex with other proteins, which localises to the apical membrane. The presence of Exp/Reb complex regulates Kkv apical distribution and activity. In model 1, we propose that a factor/s recruited by Exp/Reb (Factor X) can induce a posttranslation or conformational modification to Kkv protein that opens the channel promoting translocation of chitin fibers to the extracellular domain. In model 2, we propose that a factor/s recruited by Exp/Reb (Factor X’) can induce changes in membrane composition/curvature that will then promote a conformational change in Kkv that opens the channel to translocate chitin. These membrane changes lead to Kkv shedding extracellularly. In model 3, we propose that Exp/Reb complex can bind and relocalise Factor X”, which normally inhibits Kkv-translocating activity. This neutralises the activity of Factor X” allowing chitin translocation. Scale bars: 5 μm., Peer reviewed

All images are projections of confocal sections, of super-resolution microscopy. (A, B) Kkv localises apically in the trachea of wild-type embryos (A) and in absence of exp reb (B). (C, D) The localisation of Kkv is apical also in presence of exp ΔMH2 in trachea (C) and in presence of MH2-exp in salivary glands (D). (E, F) At stage 14, in wild-type embryos (E) and in embryos deficient for exp and reb (F), Kkv is present in the apical membrane and in many intracellular vesicles (yellow arrowheads). (G) At stage 16, in wild-type embryo, Kkv apical distribution follows the pattern of taenidial folds and intracellular vesicles are mostly absent. (H) At stage 16, in exp reb mutant embryos, Kkv is apical but shows altered distribution pattern. (I, J) At stage 15, in control embryos, Kkv pattern is apical and covers the whole membrane leaving minimal spatial gaps (I); instead, in exp reb mutant embryos, Kkv distribution changes to a less organised pattern at the apical membrane (J). (K) Three different types of spatial distribution within a selected area. The positions of the defined objects can be random and exhibit characteristics of attraction (clustered pattern) or repulsion (regular pattern). The F-Function tends to be larger (≈1) for clustered patterns and smaller (≈0) for regular. The G-Function tends to be smaller (≈0) for clustered and larger (≈1) for regular patterns. (L) Kkv punctae (magenta) on the apical cell area marked by Armadillo (green) in the trachea of a control embryo. (L’) Positions of Kkv punctae on the selected area marked by black dots. (L”) Random pattern of distribution for the same area created by the spatial statistics 2D/3D image analysis plugin. (M) The corresponding observed F and G functions (blue) are displayed above and below the reference simulated random distributions (black) and the 95% confidence interval (light gray), respectively, indicating a nonrandom spatial pattern. (N) SDI histogram for the F-Function of the control (blue) and the Df(exp reb) samples. A significant difference between the frequency distributions for each group of individuals has been observed. (Kolmogorov–Smirnov D = 0.5833, p < 0.05) (N’) SDI histogram for the G-Function of the control (blue) and the Df(exp reb) samples. Statistical analysis of the distributions did not reveal significant differences between the two groups of individuals for this parameter (Kolmogorov–Smirnov D = 0.25, p > 0.05). (O) Kkv punctae (magenta) on the apical cell area marked by Armadillo (green) in the trachea of a exp reb mutant embryo. (O’) Positions of Kkv punctae on the selected area marked by black dots. (O”) Random pattern of distribution for the same area created by the spatial statistics 2D/3D image analysis plugin. (P) The corresponding observed F and G functions (blue) are displayed above and below the reference simulated random distributions (black), respectively. Both curves largely overlap with the 95% confidence interval (light gray), indicating a tendency towards a random spatial pattern. (Q) Frequency distribution histograms for the nearest neighbour distances between Kkv punctae in control (blue) and exp reb mutant samples. The distribution of values between the two groups is found significantly different (Kolmogorov–Smirnov D = 0.2036, p < 0.005). The underlying data for quantifications can be found in the S1 Data. Scale bars A-J: 10 μm; L, O: 2 μm., Peer reviewed

All images are single confocal sections except A-C, which are projections of confocal sections. (A, A’) In trachea of wild-type embryos, Kkv is present in the apical region (blue arrows) and in intracellular vesicles (yellow arrowheads). (B) In kkv mutants unable to polymerise chitin, Kkv is not properly localised. (C) GFP-Kkv localises to the apical region (blue arrows) and in intracellular vesicles (yellow arrowheads). (D) When reb and GFP-kkv are coexpressed in salivary glands, Kkv is present in the apical membrane (blue arrow), in intracellular vesicles (yellow arrowhead), and also in punctae in the lumen (pink arrowheads). This is clearly observed in orthogonal sections (D’). (E) These luminal punctae corresponded to membranous structures. (F, F’) In contrast, when GFP-kkv is expressed alone, luminal punctae are absent, and Kkv is only found apically (blue arrow) and in intracellular vesicles (yellow arrowhead). (G) Luminal punctae (pink arrowheads) are also observed in the trachea of embryos overexpressing reb and GFP-kkv. (H) When endocytosis is prevented, the coexpression of reb and GFP-kkv in salivary glands still leads to formation of Kkv luminal punctae (pink arrowheads). (I) Quantifications of the number of intracellular Kkv vesicles in salivary glands when expressing GFP-kkv (I’), reb and GFP-kkv (I”), and GFP-kkvΔCC. n is the number of salivary glands analysed. The underlying data for quantifications can be found in the S1 Data. Scale bars: 10 μm., Peer reviewed

All images show salivary glands. (A, C, E-L, N, O) Show single confocal sections and (B, D, M) show projections of several sections. (A-A’) The concomitant expression of reb and GFP-kkv leads to luminal chitin deposition (blue arrow in orthogonal section in A’). (B, C) Coexpression of expΔMH2 and GFP-kkv produces intracellular chitin punctae, some of which partially colocalise with GFP-Kkv vesicles (yellow arrowhead) while others do not (red arrowhead). GFP-Kkv vesicles without chitin are also observed (green arrowhead). Note the accumulation of chitin in the apical domain (white arrow in C) that is not deposited extracellularly in the lumen (blue arrow in orthogonal section in C’). (D) In Rab5DN background, intracellular chitin punctae are still present (white arrowheads). (E-L) Analysis of the nature of GFP-Kkv vesicles and chitin punctae using markers Golgin245 (E-F), Hrs 27–4 (G-H), Arl8 (I-J), and Rab11 (K-L); arrowheads indicate colocalisation between Kkv and each specific marker. (M, N) All GFP-Kkv vesicles colocalise with the membrane marker CD4-mIFP (white arrowheads), and few of them also with chitin (orange arrowheads); single chitin punctae do not colocalise with CD4-mIFP (red arrowheads). (O-O”) Frames from live imaging movie show that partially colocalising GFP-Kkv and chitin punctae (yellow arrow) can separate from each other; however, many GFP-Kkv (green arrow) and chitin puncta (red arrow) do not colocalise. Scale bars A-D, M: 10 μm; E-L, N-N”‘: 1 μm; O-O”: 5 μm., Peer reviewed

(A) Schematic representation of Kkv protein (CD, catalytic domain; WGTRE; CC, coiled-coil domain). (B, C, G, I, J) Show projections of confocal sections and (D-F, H, K-N) show single confocal sections. (B) The overexpression of GFP-kkvΔWGTRE in a kkv mutant background does not rescue the absence of extracellular chitin deposition (white arrow, note the absence of CBP) and the protein accumulates in a generalised pattern. (C-D) The overexpression of GFP-KkvΔWGTRE does not produce intracellular chitin punctae, neither in trachea at early stages (C-C’) nor in salivary glands (D). (E-E”‘) GFP-kkvΔWGTRE colocalise with the ER marker KDEL. (F) GFP-KkvΔWGTRE does not colocalise with the marker FK2. (G) The overexpression of GFP-kkvΔCC in a kkv mutant background rescues the lack of extracellular chitin deposition in the trachea (note the presence of CBP staining). (H, I) The simultaneous expression of reb and GFP-kkvΔCC in salivary glands produces ectopic extracellular chitin (H), and no defects in trachea (I). (J) The overexpression of reb in trachea leads to morphogenetic defects. (K, L) Overexpressed GFP-Kkv localises mainly apically (orange arrowheads) although a bit of the protein can be detected in the basal region (yellow arrowheads). (M, N) Apical accumulation of overexpressed GFP-KkvΔCC is less conspicuous. (O) Quantifications of accumulation of GFP-Kkv and GFP-KkvΔCC in apical versus basal region. n corresponds to the number of ratios analysed (apical/basal ratio per cell), and brackets indicate the number of embryos used. Ratios were obtained from the apical (orange line in K) and basal (yellow line in K) domains of single cells in trachea and salivary glands. The underlying data for quantifications can be found in the S1 Data. Scale bars B, C, G, I, J: 25 μm; D-F, H, K-N: 10 μm., Peer reviewed

(A, B) Parental (Par), and Ndufs4−/− RAW 264.7 cells were left untreated or treated with LPS (100 ng/ml). Supernatants were collected at 8 hours for measurement of cytokine concentrations by ELISA (A). Cells were collected at 4 hours for quantification of cytokine transcripts using real-time PCR (expressed as fold increases versus untreated parental cells) (B). (C) Representative flow cytometry plots (left) showing phagocytosis of FITC labeled heat killed E. coli (HKEC) and bar graph representing % of fluorescent cells and the MFI values (right). Ctrl, control. Ns, not significant; *, P <0.05; **, P <0.01; ***, P<0.005; ****, P<0.001. Each point represents a biological replicate. In all experiments, 4 to 5 different Ndufs4−/− clones were used. Data are shown as the mean ± SEM., Peer reviewed

Native gels were incubated with NADH (as a substrate), and nitro blue tetrazolium (NBT, as the electron acceptor). CI activity is shown in purple., Peer reviewed

1D-BNE showing OXPHOS complexes I to IV (CI-CIV) and SCs. Western blot analysis was performed using antibodies against CI (NDUFA9), CIII (Core 2), CIV (COX5A), and CV (ATP5A). Asterisks (*) indicate lower molecular weight SC. Loading control, CII subunit SDHA., Peer reviewed

(A) Activities of MRC complex (I–IV) were assayed spectrophotometrically, and the results were normalized to citrate synthase (CS) activity in mitochondria isolated from parental (Par) and Ndufs4−/− RAW 264.7 cells. (B) Left panel, a representative experiment showing OCR in RAW 264.7 sublines before and after the sequential addition of oligomycin (2.6 μM), FCCP (1 μM), and a combination of rotenone (Rot) and antimycin A (AA) (1 μM). Right panel, basal respiration, maximal respiration, and ATP production. Ns, not significant; *, P <0.05; **, P <0.01; ***, P<0.005; ****, P<0.001. Each point represents a biological replicate. Data are shown as the mean ± SEM., Peer reviewed

(A) Structural model for ovine (Ovis aries) mitochondrial complex I (CI, Ref: 5LNK) [17] displaying the location of the NDUFS4 subunit (colored in black). UCSF ChimeraX (v1.5) [18] was used for visualization. (B) General Cas9 HITI strategy to ablate Ndufs4. Cas9 produces a double stranded DNA break at specific target sequences within the first exon of Ndufs4. Cas9 also excises the HITI donor by cleaving the same target sequence flanking the blasticidin cassette to be inserted. The excised HITI donor is ligated into the genomic site through the NHEJ pathway. The forward integration depicted in the scheme is “locked” and cannot be processed further. A reverse integration could be corrected by continuous excision/repair cycles in virtue of flanking target sites reconstitution. Grey pentagons represent Cas9/gRNA target sequences. Black lines within pentagons indicate Cas9 cleavage sites. (C) Whole cell lysates from each resultant knockout cell line were analyzed by Western blotting using antibodies against Ndufs4 or β-actin (loading control). Correctly targeted clones were named Ndufs4−/− followed by a serial number; Par, parental cell line., Peer reviewed