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• W4387495197 abstract "Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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. Editor's evaluation In this important work, the authors convincingly show that the Drosophila scavenger receptor Emp (homologous to human CD36) senses and responds to the levels of its cargo apical ECM proteins and triggers the initiation of apical endocytosis, thereby regulating tube length via controlling Crumbs and Src. This work will be of broad interest to cell and development biologists as well as cancer biologists. https://doi.org/10.7554/eLife.84974.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The tube shapes in transporting organs like kidney, lung, and vascular system are precisely controlled to ensure optimal fluid flow and thereby, function. Failure in normal tube size acquisition leads to cystic, stenotic, or winding tubes. The Drosophila respiratory network, the trachea, provides a well-characterized system for the genetic dissection of tubular organ maturation. Like mammalian lungs, the trachea undergoes a precisely timed series of maturation events to convert the nascent branches into functional airways. First, a transient secretion burst of luminal proteins (Tsarouhas et al., 2007; Jayaram et al., 2008; Förster et al., 2010) initiates diametric tube expansion. Luminal proteins assemble into a chitinous central rod and into a cuticular apical extracellular matrix (aECM, taenidia), lining the apical membrane. After 10 hr, luminal material becomes rapidly cleared from the tubes by massive endocytosis involving several endocytic pathways. Finally, a liquid clearance pulse converts the tubes into functional airways (Tsarouhas et al., 2007). Genetic studies suggested an instructive role of luminal chitin and proteins in tube growth coordination and termination. Mutations affecting chitin biosynthesis (kkv) or matrix assembly (knk, gasp) show irregular tube shapes, diametric expansion and tube maturation defects (Moussian et al., 2006; Tiklová et al., 2013; Öztürk-Çolak et al., 2016). Tube elongation is continuous during tracheal development and its termination requires chitin biosynthesis and the secreted chitin deacetylases vermiform (Verm) and serpentine (Serp) (Beitel and Krasnow, 2000; Luschnig et al., 2006; Wang et al., 2006; Öztürk-Çolak et al., 2016). These luminal proteins presumably modify the structure and physical properties of the aECM and thereby restrict tube elongation. In addition to the luminal matrix pathway, components involved in the assembly of basolateral septate junctions (SJs) also restrict tube elongation, through the regulation of the subcellular localization of Crumbs (Crb), a transmembrane protein that promotes expansion of the tracheal cell apical surface and tube elongation (Laprise et al., 2010). More recently, the conserved non-receptor tyrosine kinase, Src oncogene at 42A (Src42A) was found to promote axial elongation by controlling the apical cytoskeleton and apical cell (Förster and Luschnig, 2012; Nelson et al., 2012; Öztürk-Çolak et al., 2016; Olivares-Castiñeira and Llimargas, 2018). Additionally, Yorkie (Yki) and several components of the Hippo pathway control tube elongation, along with transcription factors like Blimp-1 and Grainy head (Grh) (Hemphälä et al., 2003; Robbins et al., 2014; Öztürk-Çolak et al., 2016; McSharry and Beitel, 2019; Skouloudaki et al., 2019). An appealing model suggests that the interaction between apical membrane and aECM elasticity may influence apical cytoskeletal organization and thereby control tube shapes (Dong et al., 2014). Although ECM integrity and the apical cytoskeleton appear crucial in tube length regulation, it is unknown how ECM signals are perceived by the airway cells to regulate their shapes during tube maturation. Scavenger receptors comprise a superfamily of cell surface membrane proteins that bind and internalize modified lipoproteins and various other types of ligands. Cluster of differentiation 36 (CD36) belongs to class B scavenger receptor family, which includes scavenger receptor B1 (SRB1) and lysosomal integral membrane protein 2 (LIMP2). CD36 is expressed on the surface of many cell types including epithelial, endothelial cells, and macrophages. Disruption of CD36 function in mice can lead to inflammation, atherosclerosis, metabolic disorders, tumor growth, and metastasis (Chen et al., 2008; Pascual et al., 2017; Wang et al., 2020). CD36 has several cargoes, including long-chain fatty acids, oxidized LDL (ox-LDL), oxidized phospholipids and thrombospondin-1 (TSP-1) (Githaka et al., 2016; Deng et al., 2022). In vitro imaging studies of macrophages and endothelial cells propose that CD36 clustering at the cell surface upon engagement of multivalent ligands and in conjunction with the cortical cytoskeleton triggers signal transduction and receptor–ligand complex endocytosis (Jaqaman et al., 2011; Githaka et al., 2016). The activity of several signaling effectors, including the Src family kinases, Fyn, Yes (Thorne et al., 2006; Zani et al., 2015) and the mitogen-activated kinases, Jun-kinase (JNK) 1 and 2 (Rahaman et al., 2006) can be regulated by CD36. The Drosophila genome includes a family of 14 CD36-like genes, with distinct tissue-specific expression patterns. The genetic analysis of a few members in this class B scavenger receptor family implicated them in phagocytosis, immune responses, and photoreceptor function (Philips et al., 2005; Stuart et al., 2005; Voolstra et al., 2006). The Drosophila epithelial membrane protein (Emp) shows the highest similarity with CD36 and is selectively expressed in embryonic epithelial tissues (Hart and Wilcox, 1993). Here, we show that Emp is a selective receptor for internalization, endosomal targeting, and tracheal luminal clearance of proteins with LDLr-domains. emp mutants display over elongated tracheal tubes with increased levels of junctional Crb, DE-cad, and phospho-Src. Reduction of Src42A in emp mutants, rescues the tube elongation phenotype indicating that Emp modulates junctional p-Src42A levels to control apical membrane expansion and tube length. The organization of the beta-heavy spectrin (βH-Spectrin) cytoskeletal network is compromised in emp mutants. Emp binds to βH-Spectrin directly, suggesting that it provides a direct link between ECM, apical membrane, and cytoskeleton during tube maturation process. Re-expression of human CD36 in emp mutants can ameliorate the mutant tube phenotypes suggesting conserved functions of Emp. Results Emp is a selective scavenger receptor required for tube elongation and luminal protein clearance emp (or CG2727 in Flybase) encodes a class-B scavenger receptor expressed in embryonic ectodermal epithelial tissues including the tracheal system (Hart and Wilcox, 1993). To elucidate the developmental functions of emp in the airways, a deletion mutant (empe3d1, referred as emp mutant hereafter) was generated using the FLP/FRT recombinase system (Parks et al., 2004; Figure 1—figure supplement 1A). PCR mapping of genomic DNA identified a 4.8-kb deletion encompassing exon 2 in the CG2727 locus. Both immunofluorescence and western blots using a polyclonal antiserum against recombinant Emp (see Methods) (Figure 1—figure supplement 1B), failed to detect Emp protein in emp mutants (Figure 1—figure supplement 1C, D). Similarly, quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) of RNA extracted from late embryos showed a strong reduction of emp RNA in the mutants (Figure 1—figure supplement 1E). emp homozygous or Df(2R)BSC608/emp mutants were embryonic lethal with few escapers surviving to first instar larvae. This embryonic lethality could be rescued by the re-expression of a transgenic emp construct using the ectodermal driver 69BGal4. This suggests that the deletion generates a strong loss-of function mutation in emp and does not affect any neighboring genes required for embryo viability. To examine a potential role of Emp in airway maturation, we visualized the tracheal tubes during embryonic development in wild-type and emp mutants. emp embryos at stage 16 showed a 30% over-elongation of the dorsal trunk (DT) compared to the wild-type (Figure 1A), but showed no defects in tube diameter (Figure 1—figure supplement 1F). emp mutants also failed to fill their airways with gas at hatching (Figure 1H, Figure 1—figure supplement 1I). Both phenotypes could be rescued by re-expression of emp in tracheal cells of the emp mutants (Figure 1A, B, H, Figure 1—figure supplement 1Ic). These data indicate that Emp is required for normal tube elongation and gas-filling during embryonic development. The survival of emp mutants overexpressing emp in the airways was limited to larval stages, suggesting that the tracheal-specific re-expression of emp is not sufficient for larval or adult survival. This suggests additional roles for Emp in other tissues. The human homolog of Emp, CD36 shares the overall protein architecture and 30% of amino acid identity with Emp (Figure 1—figure supplement 1G, H). We generated a transgenic line expressing the coding sequence of human CD36 and drove its expression in fly airways to test if Emp and CD36 have conserved functions. We found that both the tracheal length and gas-filling defects in emp mutants were partially reversed by tracheal CD36 overexpression, arguing for a conserved function of CD36 (Figure 1A, B, H, Figure 1—figure supplement 1Id). Figure 1 with 1 supplement see all Download asset Open asset empe3d1 mutants show over-elongation of the tracheal tubes and severe luminal clearance defect of Serp. (A) Images showing the dorsal trunk (DT) of wild-type, empe3d1, empe3d1;btl>Emp, empe3d1;btl>CD36 embryos stained for the luminal marker Serp. (B) Graph showing the tracheal DT length in wild-type embryos (n = 16), empe3d1 mutants (n = 17), empe3d1;btl>Emp (n = 19) and empe3d1;btl>CD36 (n = 15) embryos. (C) Confocal images showing the DT of wild-type and empe3d1 mutant at late stage 17 embryos, stained for the endogenous luminal proteins Serp, Verm, and Gasp. (D) Confocal images showing the DT of live btl>GaspFL-mCherry (magenta), btl>VermFL-GFP and btl>SerpFL-GFP (green) in wild-type, and empe3d1 mutant at 20.0 h AEL. (E) Plots showing the percentage of embryos with completion of luminal clearance in btl>VermFL-GFP (green, n = 59), empe3d1;btl>VermFL-GFP (green, n = 37), btl>SerpFL-GFP (green, n = 58), empe3d1;btl>SerpFL-GFP (green, n = 15), btl>GaspFL-mCherry (magenta, n = 28) and empe3d1;btl>GaspFL-mCherry (magenta, n = 18). Representative confocal images showing the tracheal DT of wild-type (F) and chc1 (G) mutant embryos, stained for the endogenous luminal markers Serp (magenta), Verm (yellow), and Gasp (green) before and after luminal clearance, stage 16 and late stage 17 (n ≥ 8, for each genotype were analyzed). (H) Bar graph shows the percentage of embryos that fill with gas in wild-type (n = 177), empe3d1 (n = 182), empe3d1; btl>Emp (n = 119), empe3d1;btl>Emp-GFP (n = 178), and empe3d1; btl>CD36 (n = 70) embryos. Error bars denote standard error of the mean (SEM), p > 0.05 not significant (ns), **p < 0.005, ***p < 0.0005, and ***p < 0.0001 (unpaired two-tailed t-tests). Scale bars, (A) 50 μm, (C, D) 10 μm, and (F) 5 μm. To establish whether the failure of tracheal gas-filling originates from a defect in luminal protein clearance, we stained wild-type and emp mutants for the luminal proteins Serp, Verm, and Gasp. These secretory proteins were internalized from the lumen by late stage 17 in wild-type embryos. Serp and Verm, but not Gasp were selectively retained in the emp DT airways (Figure 1C) suggesting a role of Emp in the internalization of a subset of luminal proteins. To confirm the protein clearance phenotypes, we generated emp mutants carrying btl>Serp-GFP or btl>Verm-GFP or btl>Gasp-mCherry transgenes and analyzed them by live imaging from 17 h to 21 h after egg laying (AEL) (Figure 1D, E). The three reporters were normally secreted into the lumen of both emp mutants and wild-type embryos and at 19 hr they were cleared from the tubes of wild-type embryos. In emp mutants Gasp-mCherry was also cleared from the lumen but the Serp-GFP and Verm-GFP reporters remained inside the tube. This suggests that Emp acts as a selective endocytosis receptor during luminal protein clearance. The unperturbed clearance of Gasp-Cherry suggested differential requirements for the internalization of luminal proteins. clathrin (chc1) null mutants show defects in luminal ANF-GFP clearance (Tsarouhas et al., 2007) and tube length (Behr et al., 2007). We stained chc1 mutants for Serp, Verm, and Gasp and analyzed them at late embryonic stages, when luminal clearance is completed in wild-type embryos. This analysis showed that Serp and Verm were cleared, whereas Gasp clearance was selectively impaired in chc1 mutants (Figure 1F, G). This indicates that Emp is involved in a selective, clathrin-independent endocytosis pathway internalizing Verm and Serp. Gasp endocytosis is clathrin dependent and presumably involves an unidentified surface receptor. These genetic experiments suggest that Emp controls tracheal tube elongation and luminal clearance of chitin deacetylases presumably by mediating their endocytosis. Dynamic subcellular localization of Emp during tracheal development To further elucidate Emp functions, we generated an antibody against its extracellular domain (Figure 1—figure supplement 1B) and determined its subcellular localization by co-staining for the previously characterized apical membrane proteins, Crb and Ptp10D, markers of adherens junctions (DE-cad, pY) and septate epithelial junctions (Disc Large/Dlg, Coracle/Cora). During tracheal branch elongation (stage 14–16), wild-type embryos showed an Emp enrichment in epithelial apical membranes and in subapical cytoplasmic puncta (Figure 2A, B). Similar to the tracheal cells, Emp showed apical localization, initially diffuse in dots in subapical regions and progressively more defined at the apical cortical region in the epidermis, hindgut and tracheal terminal branches of stage 15–16 wild-type embryos (Figure 2—figure supplement 1A, B and Figure 2—figure supplement 2). During late stage 16 to early stage 17, Emp localization became predominantly restricted in the junctional subapical region of tracheal, epidermal, and intestinal cells, where it colocalized with Crb, DEcad-GFP, and Phospho-Src (Figure 2A, C and Figure 2—figure supplement 1A–C). The Emp signal showed only weak colocalization with the SJ markers Coracle, Mtf, Dlg, and with the lateral cytoskeleton marked by α-Spectrin (Figure 2—figure supplement 1A–C). The massive uptake of luminal material correlates with the disassembly of apical actin bundles running along the transverse tube axis. The diaphanous-like formin, DAAM and the type III receptor tyrosine phosphatases, Ptp4E and Ptp10D, control the organization of F-actin bundles running along the perpendicular tube axis in the Drosophila airways (Matusek et al., 2006; Tsarouhas et al., 2019). Mutations in Ptp10D4E (Ptp10D and Ptp4E) or expression of a dominant negative form of DAAM (btl>C-DAAM) disrupt the transverse actin bundle arrays and prematurely initiate luminal protein clearance. Similarly, Latrunculin A (Lat-A) injection in embryos destroys the actin bundles and leads to luminal protein uptake. These experiments had suggested that the transverse F-actin bundles restrict the endocytic uptake of luminal cargoes (Tsarouhas et al., 2019). We analyzed wild-type embryos over-expressing the GFP-tagged actin-binding domain of moesin (btl>moe-GFP) in tracheal cells stained for Emp and GFP. This showed low colocalization (r2 = 0.218, n = 5) suggesting distinct apical membrane domains of Emp and actin bundles (GFP) (Figure 2—figure supplement 3A–D). We infer that Emp is predominantly localized in F-actin bundle-free membrane regions to promote the endocytosis and recycling of selected luminal cargos along the longitudinal tube axis. Next, we tested whether the localization of Emp may be altered in Ptp10D4E mutants and in embryos overexpressing the dominant negative C-DAAM construct in the airways. Embryos of both genotypes showed premature translocation of Emp to the airway cell junctions compared to wild-type (Figure 2D, E). This suggests, that similar to luminal protein uptake, the relocation of Emp to the apical junctional region can be induced by the premature disassembly of the actin bundles. Additionally, we analyzed the localization and intensity of Emp and Crb protein stainings along the apical membrane in wild-type and btl>C-DAAM embryos. DAAM inactivation increased the intensity of apical Emp punctate accumulations compared to wild-type (Figure 2F, G), further arguing that the transverse actin bundles restrict Emp localization at the apical membrane. Figure 2 with 3 supplements see all Download asset Open asset Dynamic apical distribution of Emp during tube maturation. (A) Confocal images showing dorsal trunk (DT) projections, from stage 14, 15, and 17 embryos, stained for Emp and Crb (Crumbs). (B) Confocal images of the DT in wild-type embryos stained for Emp and Ptp10D. Inset denotes a region of the apical membrane (arrowheads) with Emp and Ptp10D co-localization. (C) Confocal images of embryonic gut cells stained for Emp, p-Src, α-Spectrin (α-Spec), and 4′,6-diamidino-2-phenylindole ( DAPI) showing the subcellular localization of Emp in stage 17. (D) DT projections, from stage 15, 16, and 17 of wild-type and Ptp4E10D (4E10D) stained for Emp. (E) Confocal image-projections of the DT cortex (depth: 5.2 μm) in wild-type and btl>C-DAAM/+ (stage early-16) embryos stained for Emp and Crb. Emp is prematurely enriched at the AJs in btl>C-DAAM/+, but not in wild-type embryos (arrowheads). Images in insets are single longitudinal sections depicting the luminal borders of the tubes. Note: btl>C-DAAM/+, displays irregular tubes with ellipsoid dilations. (F) Confocal images showing the tracheal DT of wild-type and btl>C-DAAM/+ embryos stained for Emp and Crb. Insets show magnified regions of (F), indicated by the white rectangles. The apical or basal sides of the tracheal cells are indicated. (G) Bar plot showing the relative intensity of apical Emp puncta at DT (Tr8) in wild-type (n = 168 puncta, 5 embryos) and btl>C-DAAM (n = 237 puncta, 6 embryos). Statistically significant shown in p-values, ***p < 0.0005 (unpaired two-tailed t-tests). Scale bars, 5 μm (A, E, F) and 10 μm (B, C, D). To test if a subset of the apical Emp puncta may correspond to endocytic vesicles, we analyzed the localization of Emp relatively to several YFP-tagged Rab GTPases (YRab), expressed at endogenous levels. Co-staining for Emp and GFP (Dunst et al., 2015) showed an overlap with YRab5 (early endosomes) and YRab7 (late endosomes) with the Emp positive cytoplasmic puncta. We also detected weaker colocalization with YRab11 (recycling endosomes), (Figure 3A). Live imaging of embryos expressing btl>Emp-GFP in the time interval of luminal protein clearance (early stage 17) showed an increase of Emp intracellular puncta compared to stage 15 or late stage 17 embryos (Figure 2—figure supplement 1D). Overall, these experiments suggest that the localization of Emp in the apical membrane and endocytic vesicles is dynamic and influenced by actin bundle integrity. The timing of the final, steady-state accumulation of Emp is controlled by PTP signaling. Figure 3 with 1 supplement see all Download asset Open asset The endosomal localization of Serp is strongly reduced in empe3d1 mutants. (A) Confocal images of tracheal dorsal trunk (DT) of wild-type embryos expressing endogenous tagged YFP-Rab (knock-in) proteins, YRab5, YRab7, and YRab11 stained with Emp, Serp, and GFP. Insets show zoomed cross section views of the DT (y–z plane) of Emp co-localization with YRab5, YRab7, and YRab11, as indicated with red arrowheads. (B) Confocal images showing the tracheal DT, stained for Serp and endogenous YRab5, YRab7, and YRab11 in empe3d1 mutant. (C) Scatter plots representing the co-localization between the YRabs and Serp in wild-type and in emp3ed1 mutants calculated by Pearson correlation coefficient (r2). (D) Schematic representation of Serp and Gasp domain organization. The following abbreviations are used: SP, signal peptide (blue); LDLr, low-density lipoprotein receptor (black); ChtB, chitin-binding domain (black); GFP (green); Cht BD2, chitin-binding domain (black); and mCherry (magenta). btl>SerpFL-GFP represents the full length of Serp, btl>SerpLDLr-GFP represents the LDLr-domain of Serp, btl>SerpCBD-GFP expresses ChtB domain of Serp, btl>GaspFL-mCherry represents the full-length Gasp protein and btl>GaspLDLr-mCherry represents the full-length Gasp protein with addition of the LDLr-domain. (E) Confocal images showing the DT of live btl>SerpFL-GFP, btl>SerpLDL-GFP, btl>SerpCBD-GFP (green) embryos before (18.0 h AEL) and after (20.0 h AEL) luminal protein clearance. btl>SerpCBD-GFP embryos show incomplete luminal GFP clearance compared to btl>SerpFL-GFP or btl>SerpLDLr-GFP. (F) Plots showing the percentage of embryos with completion of luminal clearance in btl>SerpFL-GFP (green, n = 58); empe3d1;btl>SerpFL-GFP (green, n = 15); btl>SerpLDLr-GFP (green, n = 3 2); empe3d1;btl>SerpLDLr-GFP (green, n = 26); btl>SerpCBD-GFP (green, n = 45); empe3d1; btl>SerpCBD-GFP (green, n = 28); btl>GaspFL-LDLr-mCherry (magenta, n = 57); and empe3d1;btl>GaspFL-LDLr-mCherry (magenta, n = 56), from at least five independent experiments. The median (horizontal line) is shown in the plots with the data range from 25th to 75th percentile. Error bars denote standard error of the mean (SEM), *p < 0.05, ** p < 0.01, and ***p < 0.0005 (unpaired two-tailed t-tests). Scale bars, 5 μm (A, B) and 10 μm (E). Serp internalization and endosomal targeting requires Emp The luminal retention of Serp in emp mutants and the partial localization of Emp with endosomal markers led us to examine if Emp mediates the endosomal uptake and subsequent trafficking of luminal Serp. We co-stained for endogenous YFP-tagged endosomal markers and Serp in wild-type and emp mutant embryos. This analysis showed that intracellular Serp puncta co-stained for the early endosomal marker Rab5 (Figure 3A–C; arrows, r2 = 0.29) and late endosomal marker, Rab7 (Figure 3A–C; arrows, r2 = 0.27) in wild-type embryos. In the emp mutants, the colocalization of Serp with both early and late endocytic markers was significantly decreased (Figure 3B, C), suggesting that Emp mediates Serp internalization and endosomal vesicle targeting. In addition, the number of intracellular Serp puncta was reduced in emp mutant embryos compared to wild-type (Figure 3—figure supplement 1A), whereas the total number of GFP puncta corresponding to early and late endosomes remained unchanged (Figure 3—figure supplement 1B). The large size of YRab7 vesicles appeared reduced in emp mutants, presumably due to impaired apical internalization and targeting of cargoes to late endosomes. Because Emp also shares homology with the mammalian lysosomal integral membrane protein-2 (LIMP-2) (Hart and Wilcox, 1993), we cannot exclude an additional function of Emp in endo-lysosomal integrity and trafficking. Taken together, these results suggest that Emp functions as a receptor for Serp internalization and endosomal targeting. To further investigate Emp cargo specificity, we tested the luminal clearance of GFP constructs, tagged with different domains of Serp in wild-type embryos and emp mutants (Figure 3D). We used GFP constructs fused to either Serp-full-length or to the Serp-LDLr-domain (low-density lipoprotein receptor-domain) or to the Serp-CBD-domain (chitin-binding domain) (Luschnig et al., 2006; Wang et al., 2006) and examined their luminal secretion and clearance. The constructs were expressed and similarly secreted into the tracheal tubes of wild-type and emp mutant embryos. The Serp-LDLr reporter was cleared from the lumen efficiently as the full-length Serp-GFP but the CBD-GFP fusion was retained in the tracheal lumen of 20% of wild-type embryos. Interestingly, both the Serp-GFP and LDLr-GFP were retained in the lumen of emp mutants. These results suggest that the LDLr-domain of Serp, targets GFP to Emp-mediated internalization. CBD-GFP clearance failed in only 40% of the emp mutants suggesting that this cargo is also internalized by additional unknown receptors (Figure 3E, F). To further test if the addition of the LDLr-domain is sufficient to target an unrelated protein for Emp-mediated uptake, we fused the Serp LDLr-domain to the Gasp-mCherry protein, which does not require emp for its luminal clearance. As with the Serp-based constructs we analyzed the clearance of GaspFL-mCherry and GaspFL+LDLr-mCherry in wild-type and emp mutant embryos. Both constructs were normally cleared form the airways of wild-type embryos. However, btl>GaspFLFL+LDLr-mCherry, but not btl>GaspFL-mCherry, was retained in the airways of emp embryos (Figure 3F). These data suggest that the LDLr-domain can confer cargo specificity for Emp-mediated internalization. Loss of function verm serp mutants or overexpression of Serp-GFP leads to over elongation of the tracheal tubes (Luschnig et al., 2006; Wang et al., 2006). We thus examined the effects of Serp and Verm on the levels and localization of Emp. btl>Serp-GFP overexpressing embryos showed increased punctate accumulations of Emp and Crb at the apical cell surface compared to wild-type (Figure 4A). Conversely, in vermex245,serpex7 double mutants, we detected more diffuse punctate cytoplasmic accumulations for both Emp and Crb compared to wild-type (Figure 4A, B). To investigate whether the changes in intensity and localization of Emp and Crb puncta upon Serp-GFP overexpression or verm and serp deletion were due to changes in protein synthesis or stability we performed western blots of mutant and wild-type embryos. The total protein levels of Crb and Emp did not change in the mutants suggesting that the levels of luminal Serp control the punctate accumulation of Emp and Crb at the apical membrane (Figure 4D–F). As expected, overexpression of Emp in the airways (btl>Emp) increased the overall levels of cytoplasmic Emp, without a major influence on the accumulation of Crb (Figure 4A–C). Overall, these data suggest that the levels of luminal cargo control the punctate accumulation of Emp and Crb at the apical membrane. btl >Serp-GFP overexpression also causes DT tube over-elongation. This phenotype is similar to the loss-of-function phenotype of verm serp double mutants (Wang et al., 2006) and argues that deviations of the normal luminal Serp levels, too low or too high, can similarly cause tube over-elongation. Measurements from the Tr5 to Tr10 showed reduced DT elongation in emp;btl>Serp-GFP embryos compared to btl>Serp-GFP, suggesting that Serp-GFP overexpression control the length of the Drosophila airways, at least partially, through Emp (Figure 4G). Overall, these results suggest that Emp acts as a scavenger receptor for LDLr-domain proteins, such as Serp, thereby facilitating their internalization through clathrin-independent endocytosis. The levels of luminal Serp-GFP influence the localized apical membrane accumulation of Emp and Crb and interfere with tube elongation. Figure 4 with 1 supplement see all Download asset Open asset Serp-GFP overexpression induces apical Emp accumulations and tracheal over-elongation. (A) Confocal images stained for Emp and Crb, in wild-type, btl>Serp-GFP, vermex245,serpex7 mutant, and btl>Emp embryos. Inset shows zoomed view of Emp and Crb signals. The arrows indicate the accumulation of Emp and Crb in yz plane. Bar plots showing total fluorescence intensities of apical enriched Emp (B) or Crb (C) in wild-type (n = 15), btl>Serp-GFP (n = 12), vermex245,serpex7 (n = 10) mutant, and btl>Emp (n = 7) embryos. (D) Representative western blot from protein lysates of wild-type, empe3d1, btl>Serp-GFP, empe3d1;btl>Serp-GFP and vermex245,serpex7 mutants, blotted for Emp and α-Tubulin. (E) and (F) show the quantification of protein levels of Emp and Crb, respectively, based on four independent western blot e" @default.
• W4387495197 created "2023-10-11" @default.
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• W4387495197 date "2023-02-02" @default.
• W4387495197 modified "2023-10-12" @default.
• W4387495197 title "Decision letter: Scavenger receptor endocytosis controls apical membrane morphogenesis in the Drosophila airways" @default.
• W4387495197 doi "https://doi.org/10.7554/elife.84974.sa1" @default.
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