Scavenger receptor endocytosis controls apical membrane morphogenesis in the Drosophila airways

The acquisition of distinct branch sizes and shapes is a central aspect in tubular organ morphogenesis and function. In the Drosophila airway tree, the interplay of apical extracellular matrix (ECM) components with the underlying membrane and cytoskeleton controls tube elongation, but the link between ECM composition with apical membrane morphogenesis and tube size regulation is elusive. Here, we characterized Emp (epithelial membrane protein), a Drosophila CD36 homolog belonging to the scavenger receptor class B protein family. emp mutant embryos fail to internalize the luminal chitin deacetylases Serp and Verm at the final stages of airway maturation and die at hatching with liquid filled airways. Emp localizes in apical epithelial membranes and shows cargo selectivity for LDLr-domain containing proteins. emp mutants also display over elongated tracheal tubes with increased levels of the apical proteins Crb, DE-cad, and phosphorylated Src (p-Src). We show that Emp associates with and organizes the βH-Spectrin cytoskeleton and is itself confined by apical F-actin bundles. Overexpression or loss of its cargo protein Serp lead to abnormal apical accumulations of Emp and perturbations in p-Src levels. We propose that during morphogenesis, Emp senses and responds to luminal cargo levels by initiating apical membrane endocytosis along the longitudinal tube axis and thereby restricts airway elongation.


224
proteins. We stained clathrin (chc 1 ) mutants for Serp, Verm and Gasp and analyzed 225 them at late embryonic stages, when luminal clearance is completed in wild-type 226 embryos. We found that Serp and Verm were cleared, whereas Gasp clearance 227 was selectively impaired in chc 1 mutants ( Figure 1F). This indicates that Emp is 228 involved in a selective, clathrin-independent endocytosis pathway internalizing 229

261
To further elucidate Emp functions, we generated an antibody against its extracellular        LDLr reporter was cleared from the lumen as efficient as the full-length Serp-GFP but 365 the CBD-GFP fusion was retained in the tracheal lumen of 20% of wild-type embryos. 366 Interestingly both the Serp-GFP and LDLr-GFP were strongly retained in the lumen of 367 emp mutants. These results suggest that the LDLr domain of Serp, targets GFP to 368 Emp-mediated internalization ( Figure 3E, F). CBD-GFP clearance failed in 40% of the 369 emp mutants suggesting that this cargo is also internalized by alternative receptors. 370 To further test if the addition of the LDLr-domain is sufficient to target an unrelated 371 protein for Emp-mediated uptake, we fused the Serp LDLr-domain to the Gasp-372 mCherry protein, which does not require emp for its luminal clearance. As with the 373

Serp-based constructs we analyzed the clearance of Gasp FL -mCherry and 374
Gasp FL+LDLr -mCherry in wild-type and emp mutant embryos ( Figure 3D). Both 375 constructs were normally cleared form the airways of wild-type embryos. However, ChtB2 ChtB2                  Emp apically, whereas α-spectrin was also detected along the lateral sides. To confirm 582 the Emp binding to Kst and also map their interaction domains we used 583 immunoprecipitation experiments of tagged proteins in Drosophila S2 cells. We 584 expressed V5-tagged Emp and a series of constructs expressing different fragments 585 of βH-Spec/Kst protein fused to the FLAG epitope. We found that the intracellular C-586 terminus of Emp co-precipitates with the C-terminal region of βH-Spec/Kst ( Figure 6E) 587 consistent with their interaction detected in the yeast-two-hybrid system. Additionally This zip archive contains the raw unedited western-blots shown in Figure 6E.

607
Uninflatable remained unaffected in the emp mutants compared to the wild-type 608       The emp e3d1 null mutant was generated by FLP-FRT site-directed recombination using 780 two piggyBac elements (PBacWH#021071 and PBacRB#e0441541  imaged with an Airy-scan-equipped confocal microscope system (Zeiss LSM 800, Carl 848 Zeiss) using a Plan-Apo 63x/1.40 DIC oil immersion objective. 849 Yeast two-hybrid screen 850 The screen was carried out by Hybrigenics using a prey library constructed from RNA 851 of embryos that were 0-24-h old. A fragment encoding the C-terminus domain of Emp 852 (amino acids 484-520) was inserted into the pB27 vector (N-LexA-bait-C fusion) and 853 was used to screen 167 million clones. Embryos dechorionated in bleach, hand-sorted for GFP expression, collected in 300 857 uL TRizol LS Reagent and stored at −80°C until further use. For RNA extraction, the 858 embryos were homogenized in TRIzol LS Reagent using a 1.5 mL tube pestle and the reverse transcribed using High-Capacity RNA to cDNA kit (4387406 ThermoFisher). 863 The cDNA products were subsequently diluted 1:5 and 2 μl were used as a template 864 in each qPCR reaction. qPCR was performed using iTaq Universal SYBR Green 865 Supermix (Biorad). Generation of specific PCR products was confirmed by melting-866 curve analysis. Ct values for all genes were normalized to the levels of Rp49. For data 867 analysis, the delta-delta Ct values was applied. The sequences of primers used are 868 provided in Supplementary Table 2. an Airy-scan-equipped confocal microscope system (Zeiss LSM 800, Carl Zeiss) was 878 used. Z-stacks (0.16-0.2-μm step size) were taken every 15 min over a 2-4-h period 879 using a Plan-Apo 63x/1.40 DIC oil or a C-Apochromat ×63/1.2 NA water objective 880 (Zeiss). Raw data were processed with the Airy-scan processing tool available on the 881 Zen Black software version 2.3 (Carl Zeiss). Images were converted to tiff format using 882 the Zen Black or ImageJ/Fiji software. 883

Morphometric analysis 884
Tube length measurements were conducted in embryos stained for the luminal 885