Scavenger receptor endocytosis controls apical membrane morphogenesis in the Drosophila airways

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.

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 (emp^e3d1, 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).

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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.

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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.

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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 Gasp^FL-mCherry and Gasp^FL+LDLr-mCherry in wild-type and emp mutant embryos. Both constructs were normally cleared form the airways of wild-type embryos. However, btl>GaspFL^FL+LDLr-mCherry, but not btl>Gasp^FL-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 verm^ex245,serp^ex7 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.

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Emp controls tube morphogenesis

Crb and DE-cad trafficking underlies the anisotropic growth of the apical surface and elongation of Drosophila airways (Förster and Luschnig, 2012; Nelson et al., 2012). We first quantified the ratio of longitudinal/transverse cell junction lengths (LCJ/TCJ) in emp mutants and wild-type embryos stained for Crb. The longitudinal junction length and aspect ratio (LCJ/TCJ) of emp mutant tracheal cells significantly increased, while the length of junctions along the transverse tube axis was not affected (Figure 5A–C).

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The selective accumulation of the apical polarity protein Crb at longitudinal junctions relates to tube elongation (Olivares-Castiñeira and Llimargas, 2018). Consistent with this, we detected increased Crb signals along the longitudinal but not transverse junctions in over-elongated tubes of emp mutants (Figure 5D–F). Stainings for the AJ component DE-cad showed increased levels along both longitudinal and transverse junctions in emp mutants (Figure 5D–F). To examine whether the intensity differences may be due to changes in overall protein levels, we analyzed the relative protein levels of Crb and DE-cad in emp mutants by western blots. We observed no significant difference in total protein levels of Crb (Figure 5GI) or DE-cad (Figure 5H, J) in emp mutant embryos compared to wild-type. These results suggest that Crb accumulates in LCJ and DE-cad in LCJs and TCJs in emp mutants presumably due to defects in their endocytic uptake and trafficking. The relative LCJ vs. TCJ intensities (Raw Intensity/Junctional length) of stainings for the AJs component α-CAT (α-Catenin) (Pai et al., 1996) and the SJs protein Dlg (disc large) (Olivares-Castiñeira and Llimargas, 2018; Sharifkhodaei et al., 2019) were indistinguishable between emp and wild-type embryos (Figure 4—figure supplement 1A–D), suggesting that intracellular junction components are intact in the mutants. Similarly, a dextran leakage assay comparing paracellular junction integrity of wild-type, emp, and ATPα mutant embryos showed that emp loss does not affect general SJ integrity (Figure 4—figure supplement 1E). Overall, these results suggest that Emp function regulates the apical membrane levels of Crb and DE-cad without majorly affecting junctional integrity or function.

Emp modulates the apical actin organization

Our analysis until now suggests that Emp is a scavenger receptor involved in the continuous, basal-level trafficking of Serp during tube elongation and in its massive uptake during luminal protein clearance. Since the organization of the transverse actin bundles restrict both tube elongation and the timing of luminal protein clearance, we investigated the apical actin cytoskeleton in emp mutants. We first examined the apical F-actin in DTs of late stage embryos by live imaging using btl>Moe-GFP. At 17.5 h AEL, at the time interval of airway protein clearance, emp mutants showed a more diffuse and continuous apical actin bundle density compared to wild-type embryos (Figure 6A, B and Figure 6—figure supplement 1E). We also detected higher apical accumulation of the dDAAM formin, in emp mutants compared with wild-type embryos by antibody stainings. Stainings for Gasp that visualized the forming taenidial of ECM, did not show detectable defects in chitin deposition in the emp mutants (Figure 6C). These observations suggested that Emp modulates the actin bundle organization at the apical membrane, presumably upon engagement with its luminal cargoes.

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To identify proteins that might provide a direct connection between the cytoskeleton and Emp, we used the cytoplasmic C-terminus of Emp (484–520 aa) as bait in a yeast two hybrid (Y2H) screen at Hybrigenics. This uncovered three potential interacting proteins, CG32506 encoding a RabGAP protein, CG34376 encoding a protein with a predicted Zinc Finger motif and βH-Spec/Kst (Figure 6D). We further characterized the interaction with βH-Spec/Kst because of its established role in the apical cytoskeleton organization in Drosophila epithelial tissues (Thomas and Williams, 1999; Phillips and Thomas, 2006). The α- and β-heavy Spectrin subunits form tetramers, which assemble in two dimensional networks together with actin underneath the apical plasma membranes. Additionally, βH-Spec/Kst is required for the early steps and endocytic trafficking of V-ATPase in the brush border of intestinal epithelial cells (Phillips and Thomas, 2006). We used a knock-in, fusion construct of Venus into the kst locus to detect endogenous, βH-Spec/Kst together with Emp and α-Spectrin in epithelial cells of the trachea and hindgut. As expected Kst-Venus colocalized with Emp apically, whereas α-Spectrin was also detected along the lateral sides (Figure 6E and Figure 6—figure supplement 1D). To confirm the Emp binding to Kst and also map their interaction domains we used immunoprecipitation experiments of tagged proteins in Drosophila S2 cells. We expressed V5-tagged Emp and a series of constructs expressing different fragments of βH-Spec/Kst protein fused to the FLAG epitope. We found that the intracellular C-terminus of Emp co-precipitates with the C-terminal region of βH-Spec/Kst (Figure 6F) consistent with their interaction detected in the Y2H system. Additionally, kst² mutants show similar tracheal over elongation phenotypes with emp suggesting a functional interaction between Emp and Kst (Figure 6—figure supplement 1A, B). To further test this, we analyzed the localization and abundance of tagged βH-Spec/Kst in the airways of emp mutants. The apical levels of βH-Spec/Kst were severely reduced, while Crb staining was increased and staining of an unrelated apical protein Uninflatable remained unaffected in the emp mutants compared to the wild-type (Figure 6G–I). The localization or levels of Emp were not noticeably affected in kst mutants (Figure 6—figure supplement 1C). Together, these results indicate that the C-terminal intracellular domain of Emp binds to apical βH-Spectrin (Kst) and controls the spectrin cytoskeleton presumably by stabilizing βH-Spec/Kst. Additionally, in emp mutants the intensity and distribution of actin bundles is distorted and the levels of the diaphanous-related formin, DAAM are increased.

Emp regulates Src phosphorylation

Src phosphorylation and activation are required for tracheal tube elongation and controls Crb accumulation at the longitudinal cell junctions (Olivares-Castiñeira and Llimargas, 2018), and the Beitel laboratory proposed that Src together with DAAM orient the directed membrane expansion during tube elongation (Nelson et al., 2012). Since emp mutants showed higher Crb accumulation in longitudinal junctions and an overall increase in the apical levels of DAAM, we compared Src phosphorylation (p-Src) levels by immunostainings and by western blot in wild-type, emp mutants, btl>Serp-GFP and emp;btl>Serp-GFP embryos (Figure 7A–C). The specificity of the p-Src⁴¹⁹ antibody was first confirmed by staining of src42A src64B double mutants (Figure 7—figure supplement 1A). Both immunostaining and western blot analysis showed increased p-Src levels in emp mutants compared to wild-type (Figure 7A–C). Interestingly, p-Src levels were also increased in the verm serp double or verm serp heterozygous (Figure 7—figure supplement 1C, D). Mutant embryos and decreased in wild-type embryos overexpressing btl>Serp-GFP (Figure 7B) This decrease by Serp overexpression was partly ameliorated in emp mutants (Figure 7A–C), suggesting that the effect of Serp-GFP overexpression on Src phosphorylation is, at least partly, mediated by Emp on the cell surface. We also detected increased p-Src levels in total protein extract from emp and verm serp mutant embryos (Figure 7B) suggesting that their interaction controls Src phosphorylation in other ectodermal tissues. In agreement with this, neurons of the ventral neve cord in emp mutant embryos also showed increased p-Src levels compared to wild-type (Figure 7—figure supplement 1B). To test if the increase in p-Src levels in emp mutants underlies the apical membrane over-elongation defects, we performed genetic interaction experiments between src42A and emp mutants. As expected, emp mutants showed over-elongated tubes while the src42A^E1 mutants showed short tubes (Nelson et al., 2012). The increase of tube length in emp mutants was significantly suppressed in emp; src42A^E1 embryos, where the levels of Src protein were reduced (Figure 7D, E). Similarly, the increased apical accumulation of Crb and DE-cad in emp mutants was partly restored by overexpression of a Src42A^DN dominant negative form (Src42A^DN) in emp mutant embryos (Figure 7—figure supplement 1E–H). Previous work has shown that changes in chitin synthesis or ECM integrity affects the levels of phosphorylated Src42A (pSrc) at cell junctions (Öztürk-Çolak et al., 2016). Verm and Serp loss also causes p-Src upregulation and over-elongation and we also show that Serp overexpression leads to p-Src downregulation as expected (Figure 7B). Counter intuitively, Serp overexpression also results in tube over-elongation (Wang et al., 2006). Although Src42A is necessary and sufficient for cell shape changes during tracheal tube elongation (Förster and Luschnig, 2012; Nelson et al., 2012), this overexpression paradox could be explained by an indirect and src-independent effect of the overexpressed luminal Serp-GFP on tube elongation.

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Emp, a CD36 homolog, is a selective scavenger receptor required for endocytosis of a subset of luminal proteins. The endocytosis defects in emp mutants become most apparent during the massive endocytosis wave that removes all secreted luminal components just before gas-filling of the airways. Our comparison of the endocytosis requirements of Serp and Gasp together with the LDLr-domain swap experiments defined a subset of Emp cargoes and indicates that the LDLr-domain targets cargoes to Emp through clathrin-independent endocytosis. These selective requirements of Serp and Gasp internalization are consistent with the view that the choice of endocytic route is a cargo-driven process (Mettlen et al., 2018).

The machinery involved in class B scavenger receptor endocytosis have not been characterized in Drosophila, but an important characteristic of all clathrin-independent endocytosis pathways is their dependance on the dynamic control of actin polymerization to distort the plasma membrane for cargo internalization (Mayor et al., 2014). Pioneering biochemical experiments suggested that CD36 receptor clustering is essential for its internalization and signaling in response to multivalent cargo binding. Single molecule tracking of CD36 in primary human macrophages showed that the un-ligated receptor diffuses in linear confinement tracks set by the actin cytoskeleton. These diffusion tracks enable clustering and internalization upon oxLDL-ligand addition (Jaqaman et al., 2011). Similarly, in human endothelial cells the actin cytoskeleton is required for the increase of CD36 clustering upon thrombospondin binding and Fyn, a Src-family kinase, activation (Githaka et al., 2016). These studies argue that CD36 is confined in cytoskeletal tracks and cargo/ligand binding induces its clustering and signaling ability. Similarly, to CD36, Emp function is also tightly connected with the apical cytoskeleton. First, emp activity is confined in apical ‘macro’-domains along the longitudinal tube axis by the transverse, DAAM-dependent actin filaments. Disruption of these filaments induces massive endocytosis and re-localization of Emp. Additionally, the initially punctate Emp distribution along the apical membrane becomes aggregated upon overexpression of luminal Serp, suggesting that LDLr-domains on chitin-binding proteins induce Emp clustering. Apart from the Emp similarities to CD36, our results also reveal an unexpected direct function of Emp in organizing the apical Spectrin and actin cytoskeleton. The distribution of the DAAM-formin, the transverse actin bundle density and the apical accumulation of Kst are all disrupted in emp mutants. Because we showed that the conserved C-terminal intracellular domain of Emp binds directly to Kst, we propose that a central function of Emp, and possibly CD36, is to also directly organize the epithelial cytoskeleton. This notion is supported by the ability of human CD36 to partially rescue the emp mutant phenotypes when overexpressed in the Drosophila airways.

How can a scavenger receptor restrict epithelial tube elongation? In the interval of embryonic stages 13–16, regulated Src and DAAM activities promote tube elongation and the formation and alignment of the transverse actin bundles (Matusek et al., 2006). These bundles inhibit Emp internalization and restrict Serp endocytosis and trafficking to the longitudinal tube axis. We showed that Emp is directly linked to the apical βH-Spectrin cytoskeleton and is required for its assembly. This cytoskeletal organization might indirectly interfere with DAAM localization and presumably with Src42A activation and function as also proposed by Nelson et al., 2012. These data suggest that endocytosis of luminal proteins during tube elongation is controlled by at least two parallel pathways, one enabling Emp-endocytosis along the longitudinal tube axis and one restricting it along the transverse axis. We hypothesize that in the bundle-free membrane domains, Emp associates with Kst and presumably establishes an endocytosis domain, where Serp-mediated Emp clustering leads to internalization (Figure 8A, B). A similar function for Kst enabling endocytosis and trafficking of apical H⁺ V-ATPase has been proposed in the brush border of the larval Drosophila intestine, where its anchoring to the membrane remains unknown. The luminal levels of chitin deacetylases, like Serp, and other chitin modifying proteins are transcriptionally regulated (Yao et al., 2017) and are predicted to control the biophysical properties of the apical ECM (Cui et al., 2016). We infer that luminal Serp bound to chitin generates a multivalent ligand and is continuously recognized and endocytosed by Emp selectively along the longitudinal tube axis. Together with the clustered receptors, apical membrane and ‘passenger’ transmembrane proteins are expected to follow in the Emp endocytic vesicles. The over-elongation defects in emp mutant airways can be explained by the failure to balance elongation induced by Src activation with endocytosis of membrane and transmembrane regulators on the longitudinal tube axis (Figure 8A, B). Src42A phosphorylation levels have been proposed as a feedback mechanism of the aECM to the underlying cells to modulate proper cytoskeletal organization in airways (Öztürk-Çolak et al., 2016). Our work is complementary to this mechanism and further argues that Emp senses and responds to the levels of specific ECM cargoes by initiating apical membrane protein endocytosis and recycling during tube elongation. After protein clearance, Emp and Crb accumulate at the subapical region of the longitudinal junctions, where they could enable cytoskeletal interactions and the modulation of apical membrane tension.

Figure 8

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At the initiation of protein clearance, the transverse bundles are transiently resolved and luminal proteins together with apical transmembrane proteins and membrane are massively internalized and targeted for degradation or recycling to the junctional areas (Figure 8C). The mechanism of Crb recycling in airway cells involves retromer components (Olivares-Castiñeira and Llimargas, 2017) but the selective targeting mechanism to longitudinal junctions is still unknown. The re-localization of Emp to the apical epithelial junctions during airway maturation is accompanied by massive alteration in Src activity, which is not restricted to the tracheal system but is common to several ectodermal organs including neurons. This suggests that scavenger receptor endocytosis has a general, but poorly understood role, in embryonic morphogenesis of epithelial tissues. Our work on Emp provides an entry toward elucidating the roles of scavenger receptor class B in development and pathogenesis.

All data generated or analyzed during this study are included in the manuscript and supporting files. Unprocessed western blots are provided as source data files in zip format.

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Author details

1. Ana Sofia Pinheiro

   Science for Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft

   Contributed equally with

   Vasilios Tsarouhas

   Competing interests

   No competing interests declared

2. Vasilios Tsarouhas

   Science for Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

   Contribution

   Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Ana Sofia Pinheiro

   For correspondence

   Vasilios.Tsarouhas@su.se

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2933-1351

3. Kirsten André Senti

   1. Science for Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden
   2. IMBA – Institute of Molecular Biotechnology, Austrian Academy of Sciences, Vienna, Austria

   Contribution

   Conceptualization, Investigation

   Competing interests

   No competing interests declared

4. Badrul Arefin

   1. Science for Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden
   2. Sahlgrenska Academy, Gothenburg University, Gothenburg, Sweden

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1117-9125

5. Christos Samakovlis

   1. Science for Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden
   2. Cardiopulmonary Institute, Justus Liebig University of Giessen, Giessen, Germany

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   christos.samakovlis@scilifelab.se

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9153-6040

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