Scavenger receptor endocytosis controls apical membrane morphogenesis in the Drosophila airways
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eLife assessment
In this important study, the authors present evidence for the novel involvement of a scavenger receptor in tubular morphogenesis. Using a convincing set of data, the authors propose that the Drosophila scavenger receptor Emp (homologous to human CD36 ) couples endocytosis of luminal molecules and regulates tube length via controlling Crumbs and Src. This work will be of broad interest to cell and development biologists as well as cancer biologists.
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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.
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eLife assessment
In this important study, the authors present evidence for the novel involvement of a scavenger receptor in tubular morphogenesis. Using a convincing set of data, the authors propose that the Drosophila scavenger receptor Emp (homologous to human CD36 ) couples endocytosis of luminal molecules and regulates tube length via controlling Crumbs and Src. This work will be of broad interest to cell and development biologists as well as cancer biologists.
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Reviewer #1 (Public Review):
In their manuscript entitled "Scavenger receptor endocytosis controls apical membrane morphogenesis in the Drosophila airways," Pinheiro and colleagues identify a requirement for Epithelial membrane protein (Emp), a Drosophila CD36 homolog, in embryonic viability and show that mutant embryos display tracheal tube elongation and gas-filling defects. The authors first generate a null allele of emp. The authors validate gene-specific defects by the transgenic rescue of a deletion allele of emp, and further show partial rescuing activity of human CD36. The authors generate and validate an Emp antibody. In mutant trachea, the authors determine there is defective internalization of Serpentine and Vermiform from the tube lumens. Endocytic defects appear selective as GASP internalization does not appear affected. …
Reviewer #1 (Public Review):
In their manuscript entitled "Scavenger receptor endocytosis controls apical membrane morphogenesis in the Drosophila airways," Pinheiro and colleagues identify a requirement for Epithelial membrane protein (Emp), a Drosophila CD36 homolog, in embryonic viability and show that mutant embryos display tracheal tube elongation and gas-filling defects. The authors first generate a null allele of emp. The authors validate gene-specific defects by the transgenic rescue of a deletion allele of emp, and further show partial rescuing activity of human CD36. The authors generate and validate an Emp antibody. In mutant trachea, the authors determine there is defective internalization of Serpentine and Vermiform from the tube lumens. Endocytic defects appear selective as GASP internalization does not appear affected. Crb is also found to accumulate to higher levels. The affected process is not clathrin-dependent, as disruption of clathrin function blocks endocytosis of GASP but not Serp or Verm. Emp localizes to apical/adherens junction membranes in epithelia. Actin bundles regulate endocytosis and affect Emp internalization as seen by disruption of actin bundles with Ptp mutants or expression of DAAM dominant negative. Some Emp-GFP colocalized with Rab and Rab7 endosomal markers. A fraction of Srp-GFP co-localized with early and late endosomes and that colocalization was decreased in emp mutants. The LDLr domain was identified as responsible for Emp-dependent endocytosis. In Srp-GFP overexpression, Emp and Crb accumulate on the apical membrane; in serp, verm double mutants Emp and Crb levels on the apical membrane are decreased. These data are consistent with ligand clustering driving Emp and Crb apical localization. Overall Emp and Crb protein levels did not change, arguing for a role in subcellular distribution rather than protein stability. In emp; SerpGFP embryos, DT length was decreased relative to SerpGFP alone; authors suggest this implies that SerpGFP increase in length is partially dependent upon Emp.
In emp mutants, DE-Cad and Crb accumulate along longitudinal junctions, whereas only DE-Cad shows increased accumulation at transverse junctions. Western blots indicate no change in overall protein levels for DE-Cad or Crb. alpha-Catenin (adherens junction component) levels were indistinguishable from wt.
MoeGFP distribution in emp embryos is altered compared to wild-type, with a diffuse appearance. The formin, DAAM, accumulates apically in emp mutant embryos as compared to wt.
A yeast 2 hybrid screen revealed a physical interaction with beta-heavy spectrin. Co-IP experiments in S2 cells support this interaction. kst mutants show tube elongation defects suggesting that the two proteins function in the same process. Kst levels were reduced near the apical membrane in emp mutants. Emp localization was not notably altered in kst mutant embryos.
In emp mutants, pSrc levels are higher. Also seen in western blot. Embryos mutants for src have a shorter dorsal trunk. Double mutant embryos (emp; src42A) showed significant suppression of the emp phenotype. Crb and DE-Cad accumulation could be suppressed by the expression of an srcDN transgene.
The authors propose that Emp affects pSrc levels to regulate tube size and possibly other morphogenetic processes.
The manuscript makes excellent use of genetic and cell biological approaches to provide insight into the regulation of tube length during embryonic tracheal development. Many genes and pathways have been implicated in this process and this study begins to make some connections. A weakness of the manuscript is the lack of a molecular mechanism linking Emp to pSrc distribution.
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Reviewer #2 (Public Review):
Pinheiro et al unravel the role of a new scavenger receptor in tubular morphogenesis. To do so they use the Drosophila respiratory network, the tracheal system. Here, the apical extracellular matrix (aECM) and the apical cytoskeleton are essential players in tube length regulation. A few years ago, a feedback mechanism between the aECM and the underlying cells was proposed (Ozturk-Çolak et al., eLife 2016), by which the aECM and the apical F-actin could regulate levels of phosphorylated Src protein to ensure proper tube morphogenesis. However, the connection between the aECM and the cells had not been found. In this manuscript, that Emp, a Drosophila scavenger receptor homologous to human CD36, could fulfill such a role. The authors show that Emp localizes in apical epithelial membranes and shows cargo …
Reviewer #2 (Public Review):
Pinheiro et al unravel the role of a new scavenger receptor in tubular morphogenesis. To do so they use the Drosophila respiratory network, the tracheal system. Here, the apical extracellular matrix (aECM) and the apical cytoskeleton are essential players in tube length regulation. A few years ago, a feedback mechanism between the aECM and the underlying cells was proposed (Ozturk-Çolak et al., eLife 2016), by which the aECM and the apical F-actin could regulate levels of phosphorylated Src protein to ensure proper tube morphogenesis. However, the connection between the aECM and the cells had not been found. In this manuscript, that Emp, a Drosophila scavenger receptor homologous to human CD36, could fulfill such a role. The authors show that Emp localizes in apical epithelial membranes and shows cargo selectivity for LDLr-domain-containing proteins. They show that 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 and over-elongated tracheal tubes with increased levels of the apical proteins Crb, DE-cad and phosphorylated Src (p-Src). Overexpression or loss of the Emp cargo protein Serp leads to abnormal apical accumulations of Emp and perturbations in p-Src levels. They propose a model linking aECM with cell elongation and open new lines of research in downstream signalling effectors.
Strengths:
The finding of a novel receptor involved in the modulation of aECM-cellular homeostasis. A solid genetic and cellular analysis was provided. The implications for a scavenger receptor function during morphogenesis and overall implications in ECM to cell interactions and downstream signalling.Weaknesses:
The authors fail to clearly show the localization of Emp at the apical membrane and its connection to apical actin structures and chitinous aECM. -
Reviewer #3 (Public Review):
Controlling the shape of biological tubes (blood vessels, lungs, etc) is essential for optimizing the traffick of liquid and gas in organisms. Tracheal tubulogenesis of Drosophila embryos is regulated separately in two dimensions, width, and length. Molecules controlling the tracheal tube length function at three levels of location, luminal ECM, plasma membrane mediating cell-apical ECM interaction, and the signaling at the membrane/cell junction. In this paper, Pinheiro et al. reported a novel function of a scavenger receptor family molecule, Emp, which mediates endocytosis of a subset of luminal proteins including chitin deacetylates Serp and Verm that are required for restricting the tube length. It was previously shown that endocytosis and recycling of Serp and Verm maintain the level of luminal chitin …
Reviewer #3 (Public Review):
Controlling the shape of biological tubes (blood vessels, lungs, etc) is essential for optimizing the traffick of liquid and gas in organisms. Tracheal tubulogenesis of Drosophila embryos is regulated separately in two dimensions, width, and length. Molecules controlling the tracheal tube length function at three levels of location, luminal ECM, plasma membrane mediating cell-apical ECM interaction, and the signaling at the membrane/cell junction. In this paper, Pinheiro et al. reported a novel function of a scavenger receptor family molecule, Emp, which mediates endocytosis of a subset of luminal proteins including chitin deacetylates Serp and Verm that are required for restricting the tube length. It was previously shown that endocytosis and recycling of Serp and Verm maintain the level of luminal chitin deacetylates for keeping the property of the apical ECM to restrict the tube length (10.1016/j.celrep.2014.03.066).
This work is novel in two ways. First, Emp was shown to mediate the endocytosis of Serp and Verm by most likely interacting with the LDLr domains of cargo molecules and acting in parallel with the clathrin-mediated endocytosis to clear luminal materials. Second and surprisingly, the Emp-mediate endocytosis is coupled with the widespread alteration of the apical plasma membrane, including reduction of junctional E-cadherin and Crumbs, apical membrane protein DAAM1, and the cortical membrane skeleton component beta heavy spectrin (Kst). The elevation of junctional Crumbs protein in Emp mutants is noteworthy because the authors showed Crumbs was selectively upregulated in the longitudinal cell junctions. This change in Crumbs polarity may be related to the axial over-elongation of the trachea in Emp mutants. Furthermore, the authors showed that Src42A, which was previously shown to promote tube elongation, is also regulated negatively by Emp.
Overall, the information provided in this work supports a model of endocytic coupling of luminal materials and the axial polarity of the tracheal tube. This leads to a new idea distinct (but none-exclusive) from the previously proposed mechanical coupling model (10.1016/j.celrep.2014.03.066) and would advance a fundamental understanding of biological tube shape regulation. One critical point of linking endocytosis to the axial polarity is the selective enrichment of Crumbs to the longitudinal cell boundaries (10.1371/journal.pgen.1007824), which is shown to be enhanced in Emp mutants (Fig. 5D-F). Discussing how the junction-enriched Crmbs contribute to selective axial cell elongation will be desirable to expand the scope of this work. This point is essential, given that the expression of the dominant-active form of Crumbs lacking the extracellular domain (Crumbs-intra) is mislocalized in the cytoplasmic puncta promotes axial cell elongation (Laprise et al., 2010).
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