BUSCADOR RECOLECTA

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

(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) Alignment of amino acids (aa) sequences of the isoform B of Exp (aa 356–433) and homologs; the blue square indicates 8 aa highly conserved, the red square includes 9 aa less conserved. (B-D) Show projections of confocal sections and (E-F) show single confocal sections. (B) The overexpression of expΔCM2 in an exp reb mutant background rescues the lack of extracellular chitin deposition. (C, D) The simultaneous expression of expΔCM2 and GFP-kkv produces morphogenetic defects in the trachea (arrowheads in C) and ectopic chitin deposition in the lumen of salivary glands (D). (E, F) Overexpressed Exp localises mainly in the apical region (orange arrowheads) with respect the basal domain (yellow arrowheads), while the apical accumulation of overexpressed ExpΔCM2 is less conspicuous (F). (G) Quantifications of accumulation of Exp and ExpΔCM2 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 E) and basal (yellow line in E) 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: 25 μm; D-F: 10 μm., Peer reviewed

The excerpted portion of the immunoblot shown in the figures is highlighted by a black box., Peer reviewed

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Salivary gland of a stage 15 embryo carrying GFP-kkv, ChtVisTomato, and expΔMH2 visualised from a lateral view using Drangofly 505 (Andor) with 63× oil objective and a 2× zoom. Images were taken every 3 s in one single Z-stack during 2 min. The movie shows a chitin particle detaching from a GFP-kkv vesicle., Peer reviewed

Table of Contents for the Supporting Information Section S1. Discussion for the DPPC and CHOL isotherms and their mixtures Section S2. Discussion for the CCM stability at the air-water interface Section S3. Thermodynamic study of the binary mixtures Section S4. Thermodynamic study of the ternary mixtures at the air-water interface., Peer reviewed

Summary of phenotypes of the different UAS constructs in wild-type (wt), exp reb mutant, and kkv mutant embryos in different overexpression conditions. The phenotypes observed are indicated in black, and light grey indicates absence of the phenotype, Peer reviewed

All images are projections of confocal sections. (A, B) Overexpressed full-length Reb localises mainly apically in trachea (A) and in salivary glands (B). (C) The simultaneous overexpression of MH2-exp and GFP-kkv does not rescue the absence of extracellular chitin deposition, and it produces intracellular chitin vesicles (pink arrowheads). (D) Endogenous Exp localises mainly apically in trachea, although a bit of the protein can be detected intracellularly. (E) Example of a salivary gland used to quantify the number of GFP-Kkv vesicles in Fig 5I. (E’) A mask to count the vesicles is generated by substracting background and the apical membrane region. Scale bars: 10 μm., Peer reviewed

(A, B) Kkv distribution on the apical surface of a control (A) and an embryo expressing reb in salivary glands (fkhGal4>UAS-reb, B) and zoomed images for Kkv punctae (magenta) on the apical cell area marked by arm (green) of a control (C) and a fkhGal4>UAS-reb (D) embryo. The corresponding observed F function for the control (E) and the fkhGal4>UAS-reb (F) are displayed within and below the reference simulated random distributions (black) and the 95% confidence interval (light gray), respectively, indicating a random spatial pattern for the control and a tendency towards the formation of aggregates for the reb ectopic expression. (G) SDI histogram for the F-Function of the control (blue) and the fkhGal4>UAS-reb (magenta) samples. A significant difference between the frequency distributions for each group of individuals has been observed. (Kolmogorov–Smirnov D = 0.6667, p < 0.01) (H) Frequency distribution histograms for the Nearest Neighbour Distances (NNDs) between Kkv punctae in control (blue) and fkhGal4>UAS-reb samples. The distribution of values between the two groups is found significantly different (Kolmogorov–Smirnov D = 0.1463, p < 0.005). All images are projections of confocal sections, of super-resolution microscopy. The underlying data for quantifications can be found in the S1 Data. (J, K) Western blot using αKkv (J, two different exposure times are shown) or αReb (K) of embryo extracts that were subjected to immunoprecipitation with αKkv or an unrelated antibody (mock). Input correspond to 7.5% of the immunoprecipitated material. The position of MW markers (in kDa) is indicated. Scale bars A, B: 5 μm; C, D: 2 μm., Peer reviewed