# A surfactant lipid layer of endosomal membranes facilitates airway gas filling in Drosophila

Vasilios Tsarouhas, Dan Liu, Georgia Tsikala, Ylva Engström, Maura Strigini, Christos Samakovlis

► To cite this version:

Vasilios Tsarouhas, Dan Liu, Georgia Tsikala, Ylva Engström, Maura Strigini, et al. A surfactant lipid layer of endosomal membranes facilitates airway gas filling in Drosophila. Current Biology, 2023, 33 (23), pp.5132-5146.e5. 10.1016/j.cub.2023.10.058. hal-04966992

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# Current Biology

## A surfactant lipid layer of endosomal membranes facilitates airway gas filling in *Drosophila*

**Graphical abstract**

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**Authors**

Vasilios Tsarouhas, Dan Liu,
Georgia Tsikala, Ylva Engström,
Maura Strigini, Christos Samakovlis

**Correspondence**

vasilios.tsarouhas@su.se (V.T.),
christos.samakovlis@scilifelab.se (C.S.)

**In brief**

Tsarouhas et al. unveil a surfactant film of exosomal-derived lipids in the embryonic *Drosophila* airways. Just before gas filling, distinct but coordinated secretion events of MVB vesicles and lysosomes generate a lipid layer that facilitates gas filling. The presence of a lipid coating argues that gas filling in insects occurs by cavitation.

**Highlights**

* Mature fly airways assemble a film of exosomal lipids in the air-liquid interface

* Coordinated secretion of MVB vesicles and lysosomes synergize in the film formation

* Secreted acid sphingomyelinase converts exosomal membrane vesicles to a lipid film

* The film is necessary for gas filling of the airways and animal survival

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## Article

# A surfactant lipid layer of endosomal membranes facilitates airway gas filling in *Drosophila*

Vasilios Tsarouhas,<fnref n="1" />,<fnref n="2" />,* Dan Liu,<fnref n="1" /> Georgia Tsikala,<fnref n="1" />,<fnref n="4" /> Ylva Engström,<fnref n="1" /> Maura Strigini,<fnref n="4" />,<fnref n="5" />,<fnref n="6" /> and Christos Samakovlis<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,<fnref n="6" />,*

## SUMMARY

The mechanisms underlying the construction of an air-liquid interface in respiratory organs remain elusive. Here, we use live imaging and genetic analysis to describe the morphogenetic events generating an extracellular lipid lining of the *Drosophila* airways required for their gas filing and animal survival. We show that sequential Rab39/Syx1A/Syt1-mediated secretion of lysosomal acid sphingomyelinase (*Drosophila* ASM [dASM]) and Rab11/35/Syx1A/Rop-dependent exosomal secretion provides distinct components for lipid film assembly. Tracheal inactivation of Rab11 or Rab35 or loss of Rop results in intracellular accumulation of exosomal, multi-vesicular body (MVB)-derived vesicles. On the other hand, loss of dASM or Rab39 causes luminal bubble-like accumulations of exosomal membranes and liquid retention in the airways. Inactivation of the exosomal secretion in *dASM* mutants counteracts this phenotype, arguing that the exosomal secretion provides the lipid vesicles and that secreted lysosomal dASM organizes them into a continuous film. Our results reveal the coordinated functions of extracellular vesicle and lysosomal secretions in generating a lipid layer crucial for airway gas filling and survival.

## INTRODUCTION

The embryonic respiratory networks in vertebrates and insects are filled with liquid and are rapidly cleared at birth or hatching to ensure normal respiratory function. Failure of newborns to effectively clear the fetal lung fluid can lead to bronchopulmonary dysplasia (BPD) and respiratory distress syndrome (RDS). Small airways and alveolar surfaces in the lungs are covered by a film of phospholipids and proteins termed surfactant.<fnref n="1" /> This film counteracts surface tension and optimizes air flow not only in mammalian alveoli during the respiratory cycle but also in more rigid lungs lacking alveoli, similar to the respiratory tubes of birds.<fnref n="2" /> In type II alveolar epithelial cells (AT2), lipids are packed in specialized lysosome-related vesicles, the lamellar bodies. Although several proteins have been implicated in surfactant film assembly, the cellular processes and regulatory mechanisms generating the air-liquid interface in respiratory organs are poorly defined.<fnref n="3" /> Following fusion with multi-vesicular bodies (MVBs), lamellar lipids are released into the lumen and become organized into a lipid film covering the air-liquid interface in the terminal airways and alveoli.<fnref n="4" /> Liquid clearance in the lung is initiated by the activity of amiloride-sensitive epithelial Na⁺ channels (ENaC). Mice lacking the ENaC α-subunit die at birth with respiratory failure. Similar to mammalian lungs, the embryonic respiratory network of *Drosophila* undergoes a precisely timed series of maturation events converting the nascent tubes into air-filled,

functional airways. The fly airways consist of a single epithelial cell layer, surrounded by a lamina on its basal surface and a specialized cuticular lining on its apical surface. The cuticular layer is perforated by nanopores<fnref n="5" /> that provide communication channels between the epithelial cells and the lumen. The apical extracellular matrix (aECM) is organized in three distinct layers, consisting of proteins, chitin, and lipids.<fnref n="4" />,<fnref n="6" />,⁷ The outermost layer of lipids and waxes is thought to prevent the entry of liquid and epithelial cell desiccation after larval hatching.⁷⁻⁹ Just before larval hatching, a gas bubble is generated in one of the main branches and spreads throughout the entire network within 10 min. The surface hydrophobicity is expected to contribute to the generation of gas by reducing the tensile strength of the liquid inside the trachea.¹⁰ The gene *waterproof* (*wat*), encoding a *Drosophila* fatty acyl-coenzyme A (CoA) reductase, is required for embryonic airway gas filling and reduces very long fatty acids to alcohols, presumably by participating in wax biosynthesis.¹¹ Although a surfactant film has been proposed to participate in airway liquid clearance, its role and the mechanisms of its generation remain unknown. At larval molts, airway liquid clearance is initiated by the neuro-endocrine system via the induction of calcium oscillations and activation of ENaCs in the airway epithelium,¹² illustrating the systemic control of the gas filling process.

Conventional lysosomes or lysosome-related organelles (LROs) can undergo regulated secretion in several cell types including polarized epithelial cells,¹³,¹⁴ melanocytes, mast cells,

<footnote n="1">¹Stockholm University, Department of Molecular Biosciences, The Wenner-Gren Institute, 10691 Stockholm, Sweden</footnote>

<footnote n="2">²Science for Life Laboratory, SciLifeLab, 171 65 Stockholm, Sweden</footnote>

<footnote n="3">³ECCPS, Justus Liebig University of Giessen, 35390 Giessen, Germany</footnote>

<footnote n="4">⁴IMBB, 70013 Heraklion, Crete, Greece</footnote>

<footnote n="5">⁵Present address: Université Jean Monnet Saint-Etienne, 42100 Saint-Étienne, France</footnote>

<footnote n="6">⁶Lead contact</footnote>

<footnote>*Correspondence: vasilios.tsarouhas@su.se (V.T.), christos.samakovlis@scilifelab.se (C.S.)</footnote>

<footnote>https://doi.org/10.1016/j.cub.2023.10.058</footnote>
platelets, cytotoxic T cells, and osteoclasts.¹⁵ Membrane fusion is facilitated by vesicle SNAREs (v-SNAREs) on lysosomes and the target SNAREs (t-SNAREs) at the plasma membrane (PM).¹³ Ca²⁺ triggers lysosomal exocytosis to promote PM repair, neurite outgrowth, and ECM remodeling. Acid sphingomyelinase (ASM) catalyzes the conversion of sphingomyelin (SM), a component of membranes, into ceramide and phosphocholine. Secretion of the lysosomal ASM enables lipid modifications in the outer leaflet of the PM and induces endocytosis and PM repair.¹⁶ Similarly, the release of lysosomal cysteine proteases, cathepsins, participate in PM repair and ECM remodeling.¹⁷,¹⁸ MVBs can fuse with the PM and extrude micro-vesicles (exosomes with diameters of 30–100 nm) originating from the inward budding of MVB membrane.¹⁹ Exosomal vesicles have emerged as carriers of cytosolic and membrane proteins, lipids, and nucleic acids into the extracellular space and recipient cells.²⁰,²¹ Exosomal secretion utilizes the endosomal-sorting complexes required for transport (ESCRT) machinery,²²,²³ but there are also ESCRT-independent secretion mechanisms,²⁴ reflecting the heterogeneity of intraluminal MVB vesicles and their differential regulation. Several Rabs,²³,²⁵,²⁶ v-SNARE and t-SNARE proteins,²⁷⁻²⁹ have been implicated in exosome release. Both Ca²⁺-triggered³⁰,³¹ and calcium-independent factors²⁷ can induce exosome secretion. In *Drosophila*, the SNARE proteins YKT6, Syntaxin 1A (STX1 in mammals), and Rab11 control exosome-mediated secretion of Wingless or Evenness interrupted (Evi) via an ESCRT-dependent mechanism.³²⁻³⁵ Extracellular vesicles from MVBs are secreted by a Ca²⁺-triggered mechanisms during branch fusion in the *Drosophila* airway network.³⁶

Here, we identified a membrane-film enriched in exosomal markers in the gas-exchange surface of airways just before gas filling. We show that coordinated Rab11/Rab35/Syx1A/Rop-dependent exosome secretion and Rab39/Syx1A/Syt1-dependent lysosomal secretion are required for the formation of this layer. Fusion of both MVBs and lysosomes with the PM requires Syntaxin-1. Loss of lysosomal *Drosophila* ASM (dASM) or Rab39 causes exosomal membrane accumulations in the lumen and failure in airway gas filling. Similarly, interference with exosomal secretion leads to liquid retention in the airways. The aberrant lipid accumulations in *dASM* mutants are eliminated by airway-specific inactivation of the exosomal secretion, suggesting that the secretion of exosomal lipids is independent of the lysosomal secretion of dASM. Our data identify a cellular mechanism coordinating exosomal and lysosomal secretion for the generation of an extracellular lipid film crucial for airway liquid clearance and animal respiration.

## RESULTS

### Drosophila ASM is essential for gas filling

Developmentally regulated endocytosis and degradation of luminal material is a central step in the morphogenesis and maturation of the *Drosophila* respiratory system.³⁷ We followed the expression of the lysosomal marker, lysosome-associated membrane protein-1 tagged with GFP (LAMP1-GFP), during airway maturation and detected a progressive increase in apical lysosomes and an overall decrease in total lysosome number before gas filling (Figures S1A–S1F). Similarly, stained wild-

type embryos for the endogenous marker Arl8 (Arf-like protein 8) showed increased apical lysosomes at late stage 17 (Figure S1G). This localization switch during organ morphogenesis suggests that lysosomal function is regulated and presumably required for the late steps of maturation. To investigate this further, we focused on the gene CG3376 gene, which encodes a predicted dASM (Figure S1H). Whole-mount RNA *in situ* hybridization showed *dASM* expression in the developing tracheal tree (Figure S1I) from embryonic stage 14 and onward. dASM expression was also found in the epidermis, salivary glands, hindgut, esophagus, and hemocytes. We generated deletion mutants by imprecise P-element excision of a transposon inserted 126 bp upstream of the predicted dASM transcription start site (Figure S1J). PCR and DNA sequencing mapped a deletion in the *dASM* M1.M2 excision allele removing the whole transcription unit of *dASM* and part of the next gene, GC13577. Analysis of mutant embryos showed that the airways of *dASM* mutants (92%, n = 98) retained liquid at 21 h AEL, whereas in wild-type embryos, they were filled with gas (Figures 1A and 1B). Live imaging of the membrane-associated markers, *myr-RFP* and *CAAX-mCherry*, revealed prominent bubble-like accumulations of lipids inside the respiratory tubes of *dASM* mutants at the end of embryogenesis (Figures 1C–1F). We also detected these membranous structures together with remnants of the luminal matrix in the dorsal trunk (DT) tubes of *dASM* mutants by transmission electron microscopy (TEM) (Figures 1G and 1H). The luminal accumulations and gas-filling defects of the *dASM* mutants were rescued by tracheal re-expression of dASM, indicating that dASM function is required in the tracheal cells for gas filling (Figures 1A and 1B). Immunostaining of wild-type embryos with an anti-dASM antibody-detected intracellular punctate structures throughout the cytoplasm from stage 14 and enriched apical localization at stage 16 (Figures S1K–K'', S1L, and S1L'). Stainings for dASM and GFP in embryos expressing endogenously tagged Rab GTPases (YRab), lysosomal LAMP1-YFP<sup>CT1</sup>,³⁸ or btl/>LAMP1-GFP showed overlap of the dASM puncta with YRab7, YRab39, and LAMP1 (Figures S1N and S1O), suggesting that dASM is predominantly localized in lysosomes until late stage 16. Surprisingly, we observed dASM inside the tracheal tubes at early stage 17 (Figures S1K''' and L–L'), suggesting that the majority of dASM becomes luminal just before gas filling. This luminal localization pattern in the airways was not observed in other ectodermal organs including the hindgut tube, which is also covered by a cuticular layer (Figure S1M), suggesting that the lysosomal dASM secretion is selectively induced in the airways. Similarly, to dASM, two independent GFP fusions of the putative lysosomal cysteine proteinase Cathepsin-F were detected inside the tracheal lumen at stage 17, arguing that lysosomal enzymes are secreted into the airways at the end of embryogenesis (Figures S1P and S1Q). Taken together, our data indicate that lysosomal dASM and Cathepsin-F become predominantly extracellular during the last steps of airway maturation. The lipid membranous structures in the liquid-filled tubes of *dASM* mutants further suggest a crucial role of lysosomal proteases and dASM in luminal gas filling and lipid modification (Figure 1A). Next, we generated transgenic strains carrying either the human *SMPD1* (SM phosphodiesterase 1 or *hASM*) or the *dASM* ORFs in the same chromosomal position and asked whether airway expression of
<figure><img src="image_5.png" /><figcaption>**Figure 1.** Loss of *dASM* shows intra-luminal lipid accumulations and liquid clearance in the airways</figcaption></figure>

(A) Wide-field images of a wild-type and *dASM*, *dASM; btl > dASM*, and *dASM; btl > hASM* mutant embryo 21 h AEL. *dASM* mutants show severe gas-filling defects.

(B) Scatterplot showing the percentage of wild-type (n = 137), *dASM* (n = 178), *dASM; btl > dASM* (n = 140), and *dASM; btl > hASM* (n = 48) embryos with completion of gas filling. Each data point represents an average of a single embryos collection. Means values are calculated by at least six repeated experiments. Error bars are SEM. ***p < 0.001 (unpaired two tailed t tests).

(C–F) Confocal sections showing the tracheal dorsal trunk (DT) of living wild-type (C and E) and *dASM* mutant (D and F) embryos expressing *bt1 > ANF-GFP* (green) and *myr-RFP* (C and D) or *CAAX-mCherry* (magenta) (E and F) in airways. Embryos were recorded simultaneously. Time points are hours after egg laying (AEL). *dASM* mutant embryos show strong luminal accumulations of the membrane markers.

(G and H) Transmission electron micrographs of wild-type (G) and *dASM* (H) dorsal trunks at the end of embryogenesis. *dASM* mutants accumulate amorphous lipids and electrodense material in the lumen.

(I) Merged fluorescent and wide-field images of living *dASM*, *dASM; Abd-B GAL4*, *dASM; Abd-B GAL4 UAS-dASM*, and *Abd-B-Gal4* mutant embryos expressing the membrane marker CD8-GFP (green).

(J) Plots showing the % of DT filled with gas in *dASM* (n = 25), *dASM; Abd-B > CD8-GFP* (n = 22), *dASM; Abd-B > CD8-GFP UAS-dASM* (n = 31), and *Abd-B > CD8-GFP* (n = 18) embryos. ***p < 0.001 (unpaired two tailed t tests). Error bars show SEM. Scale bars, 10 µm (A–D, K, and G); 2 µm (E and F); 50 µm (I). See also Figure S1.

*SMPD1/hASM* could substitute for *dASM* in the mutants. Re-expression of the *hASM* or the *dASM* transgenes in *dASM* mutants improved the liquid clearance phenotypes of these mutants

(Figure 1B). Human ASM activity stabilizes the lysosomal limiting membrane.³⁹ Consistently, *dASM* is also required for lysosomal integrity in flies (Figures S1R–S1V), as *dASM* mutants showed
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reduced numbers, size, and intensity of LAMP1-YFP<sup>CPT1</sup> (Figures S1R–S1U) and Arl8-labeled (Figure S1V) structures. To more precisely address the potential role of luminal dASM in the gas filling process, we re-expressed dASM in parasegment-13 of *dASM* mutants using the Abdominal B-Gal4 driver strain (*Abd-B-Gal4::CD8-GFP*).<sup>40</sup> This activates the upstream activating sequence (UAS) transgenes only in the posterior metameres (Tr9 and Tr10) of the airway network and in posterior spiracles (Figures 1I and 1J). However, we observed striking restoration of gas filling, not only in the Tr9 and Tr10 branches but also in the rest of the tracheal network, which is not expressing dASM (Figures 1I and 1J). This indicates that secreted dASM spreads in the lumen and is sufficient to induce liquid clearance in the entire tracheal network. We thus focused on the function of secreted dASM.

## An apical extracellular film of exosomal membranes covers the airways during gas filling

To better understand the luminal function of dASM, we addressed the origin of luminal lipid bubbles in *dASM* mutants. Upon the onset of liquid clearance, we analyzed the tracheal air-liquid interface of wild-type and *dASM* embryos by expansion microscopy and a prolonged (4 days) fixation protocol to permeabilize the cuticle<sup>41-43</sup> (see also STAR Methods). Stainings for the lipid raft-associated protein flotilin-2 (FLOT-2) revealed an apical extracellular lipid layer at late stage 17 (Figures 2A-2C). The labeling of the chitin deacetylase Serpentine (Serp) shows that this layer covers the upper-most part of the aECM, presumably the envelope. To confirm the lipid composition of this layer, we injected wild-type and *dASM* mutant embryos (at stage 10) with the fluorescent lipophilic-dye (FM1-43), which visualizes plasma and vesicular membranes.<sup>44</sup> Before gas filling at late stage 17, FM1-43 labeled two structures, the apical extracellular layer and the PM in wild-type embryos. By contrast, in *dASM* mutant embryos, labeling was detected on only one layer, presumably the PM and in intraluminal bubble-like accumulations (Figures S2A-S2B). This further supports the existence of an extracellular apical layer composed of lipids. Stainings for various transmembrane apical proteins (Uif, Sas, and Ptp10D) only revealed one layer and did not show any noticeable change in *dASM* mutants (Figures S2D-S2F and S2J). Because flotillins

are enriched in exosomal membranes and have been used as protein markers to quantify exosomal release,<sup>24</sup> we examined whether secreted intraluminal vesicles (ILVs) from MVBs contribute with lipids to the formation of the extracellular lipid layer. The human tetraspanin CD63 is enriched on ILVs of MVBs, which are secreted as exosomes through the fusion of MVBs with the PM.<sup>45,46</sup> Live imaging of tracheal cells expressing a human CD63-GFP transgene<sup>47</sup> showed an apical enrichment during luminal protein clearance at 19 h after egg laying (AEL) and colocalized with CAAX-mCherry in the apical membrane (Figures S2G–S2G"). Strikingly, 37 (± 8) min before the initiation of gas filling (19.0–19.5 h AEL), an auxiliary CD63-GFP + layer appeared extracellularly, which was 0.489 ± 0.067 µm apart from the apical membrane (Figures 2D, 2H, and S2H–S2H"). In *btl/>CD63-GFP* embryos, the CD63-GFP + extracellular layer was co-labeled by FLOT-2 (Figure S2C). Stainings from stage 16 to early 17 embryos (16–19 h AEL) showed that the intracellular dots of CD63-GFP co-localized with the MVB/exosomal proteins, ALIX (ALG 2-interacting protein X), hrs (hepatocyte growth factor receptor substrate), and TSG101 (tumor susceptibility gene 101) but not with lysosomal dASM (Figure S2I). This suggests that the extracellular lipid cover derives from MVB vesicles and not from lysosomes. In *dASM* mutants, the CD63-GFP<sup>+</sup> extracellular lipid layer was disrupted, and instead, CD63-GFP accumulated together with CAAX-mCherry in bubble-like structures in the liquid-filled lumen (Figures 2D-2G). Stainings for the *Drosophila* ALIX and TSG101 proteins also showed stronger luminal accumulation in *dASM* mutants compared with wild type (Figures S2J–S2K). Re-expression of both dASM and CD36-GFP only in para-segment 13 (Tr9 and Tr10 by Abd-B-Gal4) in *dASM* mutants, strongly reduced the spread luminal accumulations of CD36-GFP observed in *dASM* mutants lacking the UAS-dASM transgene. In the embryos expressing both UAS constructs, CD36 staining was aligned to the Crb-marked PM even in the remote central metamere (Tr6), where the transgenes are not activated by Abd-B Gal4 (Figures 2I-2J'). Collectively, the results suggest that the luminal lipids derive from exocytosed MVB vesicles, which are converted into a lipid surface by secreted dASM. The gas-filling defects of *dASM* mutants suggest that this lipid film and liquid clearance are functionally linked.

## Figure 2. dASM is essential for the formation of an extracellular CD63<sup>+</sup> layer in the airways

(A–C) Airy-scan confocal sections showing the tracheal DT of wild-type embryos stained for FLOT-2 (green) and Serp (magenta). Embryos were fixed, immunolabeled, and expanded before imaging.

(A'–C') Images depict zoomed areas of airways indicated by the rectangular frames in (A)–(C).

(D–E') Airy-scan confocal sections showing the DT of living wild-type embryos expressing the exosomal marker *btl/>CD63-GFP* (green) and the membrane marker CAAX-mCherry (magenta).

(D' and C') Images depict zoomed areas of the airways indicated by the rectangular frames.

(F and G) Plots showing the average fluorescence intensity profile (a.u.) of CD63-GFP (green) and CAAX-mCherry (magenta) in wild-type (n = 5) (F) and *dASM* (n = 6) mutants (G), along the length of 2 µm lines of the apical ECM (D and E).

(H) Scheme of extracellular CD63-GFP<sup>+</sup> lipid-layer detection during *Drosophila* airway maturation. The x axis depicts developmental time AEL (hours) with the corresponding embryonic stages. The airway maturation steps: luminal protein endocytosis and liquid clearance/gas filling are indicated. The lipid layer is transient and cannot be detected after the gas filling (dot line).

(I and J) Confocal sections showing the tracheal dorsal trunk (DT) of *dASM;Abd-B>CD63-GFP* (I) and *dASM;Abd-B>CD63-GFP* UAS-*dASM* (J) embryos stained for the apical membrane marker Crb (magenta) and GFP (green).

(I' and J') Images depict zoomed areas of airways indicated by the rectangular frames in (I) and (J).

Re-expression of dASM in para-segment 13 (Tr9 and Tr10) under the control of *Abd-B-Gal4* in *dASM* mutants, promotes re-formation of the CD63-GFP<sup>+</sup> extracellular layer (arrow heads in J') in remote areas (Tr6) of the DT that are not expressing dASM. The CD36-GFP layer appears interrupted because only exosomal membranes deriving from Tr9 and Tr10 are labeled in this experiment.

Scale bars, 10 µm. See also Figure S2.
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## Lysosomal secretion of dASM relies on the t-SNARE Syx1A and the Rab39 GTPase

To better understand luminal dASM secretion, we investigated its genetic requirements. In *Drosophila*, the *t*-SNARE Syntaxin 1A (Syx1A) enables secretory vesicle fusion in neurons and epidermal cells<sup>48–50</sup> and is also required for airway gas filling (Figure 3L). We stained wild-type and syx1A-mutant embryos for dASM at stage 17 and found a strong reduction of luminal dASM in the mutants. Instead, we observed cytoplasmic dASM accumulation in clusters just underneath the apical membrane, labeled by the transmembrane protein Crumbs (Crb) (Figure 3A). Similarly, loss of syx1A strongly reduced the levels of Cathepsin-F in the lumen (Cts-F-GFP<sup>KM0156</sup> trap line) at stage 17 (Figures S3A and S3C), but the secretion of other luminal matrix proteins was unaltered (Figure S3B). These data argue for a selective requirement of Syx1A for lysosomal targeting and fusion to the apical membrane. Next, we investigated whether components of the classical secretory machinery may also contribute to the luminal secretion of dASM, as it has been reported for the mammalian secretory form of ASM (S-ASM). We analyzed mutants of the small GTPase Sar1 (a component of the coat protein complex II), which promotes endoplasmic reticulum (ER) to Golgi transport, mutants for Sec61β, a member of the translocon complex in the ER, and mutants for CrebA, which activates secretory gene expression in epithelial tissues.<sup>51</sup> These mutants showed strong gas-filling phenotypes<sup>52,53</sup> but intact luminal dASM accumulation (Figures S3D and S3E), indicating that the luminal secretion of dASM primarily relies on Syx1A-mediated lysosomal secretion.<sup>52</sup> To test whether the block in luminal deposition of lysosomal proteins reflects a more general defect of lysosomal fusion with the apical membrane, we recorded living wild-type and syx1A embryos for the lysosomal LAMP1-GFP marker. syx1A embryos showed abundant accumulation of lysosomal structures just underneath the apical membrane (Figure 3B). This argues that Syx1A is selectively required for the docking of lysosomes to the apical membrane and releasing

lysosomal enzymes into the lumen. To further test whether the apical dASM accumulations in the mutants represent lysosomes, we injected syx1A embryos with the hydrophobic Lysotracker dye that labels LAMP1-GFP-positive puncta in tracheal cells (Figure S3F). Lysotracker-injected syx1A mutants showed a strong apical accumulation of GFP and Lysotracker (Figure 3C), indicating that syx1A mediates apical secretion of conventional lysosomes. The LAMP1-GFP puncta in syx1A mutants were also dASM positive (Figure 3D). Time-lapse recordings in syx1A embryos showed that apical LAMP1-GFP signals appear at stage 17 (18 h 19' ± 38') (Figure 3M). The apical accumulation continued until the onset of liquid clearance correlating with the apical lysosomal staining seen in wild-type embryos (Figures S1A–S1G). These data suggest that the developmentally controlled migration of lysosomes occurs normally in the syx1A mutants, but their fusion with the apical PM fails. Because intracellular dASM colocalizes with Rab39 (Figure S1O), we tested whether the transport of lysosomal vesicles to the apical membrane requires Rab39 activity. Overexpression of Rab39<sup>DN</sup> in the airways reduced the luminal secretion of dASM and disturbed airway gas filling in 57% of the Rab39<sup>DN</sup> embryos (n = 116) (Figures 3J–3L). We conclude that Rab39 is involved in the transport of lysosomes to the apical membrane, where Syx1A facilitates membrane fusion and dASM release.

## Ca²⁺-activated v-SNARE synaprotagmin-1 controls lysosomal secretion of dASM

As Ca²⁺ signaling is crucial for the control of lysosomal exocytosis,<sup>54–56</sup> we hypothesized that Ca²⁺ signals may also be involved in lysosomal secretion in the fly airways. Live imaging of embryos expressing the calcium sensor GCAMP5G-GFP in the trachea showed a pronounced increase in cytoplasmic Ca²⁺, preceding protein clearance and gas filling (Video S1). To identify additional components of lysosomal secretion, we analyzed mutant alleles affecting calcium-binding proteins of the conserved SNARE exocytosis complex. This included

<caption>**Figure 3.** Lysosomal secretion of dASM relies on the *t*-SNARE Syntaxin 1A, the *v*-SNARE protein Syt-1 and Rab39</caption>

(A) Confocal sections of the DT of wild-type and syx1A embryos stained with the anti-Crb (green), anti-dASM (magenta), and DAPI (blue). Arrowheads depict apical accumulations of dASM in syx1A mutants.
(B) Confocal sections of the DT in wild-type and syx1A mutant embryos expressing the lysosomal marker LAMP1-GFP (btl>LAMP1-GFP), co-stained for Ppt10D (magenta), GFP (green), and DAPI (blue). syx1A mutant embryos showed accumulation of LAMP1-GFP. Inset depicts YZ cross-sections of the DT.
(C) Confocal sections of the tracheal DT in live wild-type and syx1A mutant embryos expressing the luminal protein ANF-GFP (btl>ANF-GFP) at 20 h AEL, pre-loaded with lysotracker at stage 14. Arrows showing apical accumulation of acidic cellular compartments in syx1A mutant embryos.
(D) Confocal sections of the DT in wild-type and syx1A mutant embryos expressing the lysosomal protein LAMP1-GFP (btl>LAMP1-GFP), co-stained for dASM (magenta), GFP (green), and DAPI (blue). LAMP1-GFP accumulation largely overlapped with dASM staining in syx1A mutant embryos (arrowheads).
(E and F) Confocal sections showing the DT of wild-type (E) and syt1<sup>K05909</sup> (F) embryos co-stained for Crb (green) and dASM (magenta). The luminal dASM-signal was reduced in syt1<sup>K05909</sup> mutants.
(G) The DT in wild-type living embryos expressing the Syt1-GFP at 18 and 20 h AEL. Arrowheads denote the apical enrichment of Syt-1- GFP at 20 h AEL (just before the gas filling).
(H) Confocal sections of the DT in syx1A living embryos expressing the Syt1-GFP at 20 h AEL. Arrowheads denote an apical punctuate-accumulation of Syt-1-GFP at 20 h AEL.
(I) Confocal sections of the DT in wild-type embryos expressing the Syt1-GFP stained for dASM (magenta), GFP (green), and DAPI (blue). Arrowheads depict the co-localization of dASM in Syt1-GFP⁺ vesicles.
(J) Confocal images of the tracheal DT of wild-type and btl>Rab39<sup>DN</sup> mutant embryos co-stained with anti-Crb (green), anti-dASM (magenta), and DAPI (blue).
(K) Plots showing the ratio of luminal/intracellular dASM fluorescence intensities in wild-type (n = 6), or syx1A (n = 9) or syt1<sup>K05909</sup> (n = 8) or btl>Rab39<sup>DN</sup> (n = 12).
(L) Plots showing the % of embryos with completed gas filling in wild-type (n = 86), syx1A (n = 137), btl>Rab7<sup>DN</sup> (n = 231), syt1<sup>K05909</sup> (n = 157), and btl>Rab39<sup>DN</sup> (n = 231) embryos. Data collected from 6 to 8 independent experiments. The boxplots (K and L) show the median (horizontal line), and the data range from 25th to 75th percentiles. The bars depict maximum and minimum values. **p < 0.01, ***p < 0.0001. Unpaired two tailed t tests were performed in relation to the wild-type (control) values.
(M) Schematics of the lysosomal secretion of dASM and CtsF in relation to the time of the maturation process. The x axis depicts the time of embryo development AEL (hours). The time points of luminal protein clearance and liquid clearance/gas filling are indicated. Scale bars, 10 µm. See also Figure S3, Tables S1 and S2, and Video S1.
<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /></figure>

<figure><img src="image_51.png" /><figcaption>**Figure 4.** *Rab11*, *Rab35*, *Rop*G27, and *syx1A* mutations accumulate intracellular CD63⁺ vesicles and fail to form the extracellular-lipid layer

(A) Air-scan confocal frames of the DT of living wild-type, Rab11DN and Rab35DN1/2 embryos expressing CD63-GFP (green) and CAAX-mCherry (magenta). Rab11DN and Rab35DN1/2 embryos accumulate intracellular CD63-GFP⁺ vesicles. The indicated time point is hours AEL.

(B) Confocal frames of (A) showing only the CD63-GFP marker (gray).

(B–B'') Zoomed view of airway regions in (B) indicated by the rectangular frames. Rab11DN (B'') and Rab35DN1/2 (B'') mutant embryos fail to form the CD63⁺ extracellular lipid layer.</figcaption></figure>

(legend continued on next page)
mutants for synaptotagmin-1 (syt-1), synaptotagmin-like (btsz), Unc-13-4/staccato (stac), c-synaptobrevin (c-syb), and of Rop, encoding a conserved syntaxin-1-binding protein (Munc18), involved in synaptic exocytosis (Table S1). Alleles of all of these mutants displayed liquid clearance defects ranging from 22% to 98% (Table S2), indicating their requirement in airway maturation. Next, we analyzed whether the luminal secretion of dASM is affected in mutant embryos. We found a significant reduction in luminal dASM fluorescence only in the syt-1 mutant (Figures 3F, S3G, and S3H), implying that Syt-1 is essential for the exocytosis of dASM. To further test this, we expressed the eGFP-tagged Syt1 transgene (Syt-1-GFP), a widely used secretory vesicle marker, in the airways.⁵⁷,⁵⁸ In wild-type embryos, Syt1-GFP was enriched apically just before gas filling and labeled both the PM and a small number of vesicles in airway cells (Figure 3G and 3M). Interestingly, loss of Syx1A resulted in prominent cytoplasmic accumulations of the Syt-1-GFP in areas underneath the apical membrane (Figure 3H). We also detected strong colocalization of Syt1-GFP and dASM puncta (Figure 3I), suggesting that Syt1 marks the lysosomal vesicles targeting Syx1a on the PM. Overall, our data suggest that Rab39 transports lysosomes to the apical membrane, where Syt1, Syx1A, and a Ca²⁺ pulse trigger membrane fusion leading to dASM and Cathepsin-F release.

## Rab11/Syx1A exosomal secretion contributes to the extracellular lipid layer

The enrichment of FLOT-2 and CD63-GFP in the extra-cellular lipid layer led us to hypothesize that MVBs fuse with the apical membrane and release vesicles into the airway lumen. To further test this hypothesis, we examined the effects of airway-specific inactivation of Rab11, Rab27, and Rab35 on the formation of the extra-cellular lipid layer. These GTPases are known to regulate exosome secretion,⁵⁹ and we expressed dominant-negative (DN) constructs targeting these Rabs⁶⁰ in the airways and then monitored gas filling and intracellular CD63-GFP accumulation by live imaging. Rab11DN¹; btl>CD63-GFP and Rab35DN¹/²; btl>CD63-GFP, but not btl>Rab27DN¹ or Rab27DN² btl>CD63-GFP embryos, showed CD63-GFP intracellular accumulations, a defective luminal lipid coat and gas-filling defects (Figures 4A-4D and S4A-S4D). We conclude that MVBs marked by Rab11 and Rab35 are targeted to the apical membrane where they fuse and release ILVs for the extracellular lipid-film formation. The liquid retention in the airways of btl >Rab35DN and btl >Rab11DN DN mutants further indicates the requirement of the extracellular lipid layer for gas filling.

To identify additional components of the MVB exocytosis pathway, we analyzed the CD63-GFP marker in previously characterized mutants affecting vesicle exocytosis (Table S1). We found increased intracellular CD63⁺ puncta and a reduced extracellular CD63⁺ layer in RopG27 or RopG27/Df(3L)GN4 mutants (Figures 4E-4G; Table S2). This suggests that Rop is selectively involved in the exosomal secretory pathway, presumably tethering MVBs to the membrane and facilitating ILV release. Similarly, inactivation of ept—the *Drosophila* TSG101 homolog encoding a component of the ESCRT-I complex, affecting the biogenesis of the ILVs in MVBs—resulted in intracellular CD63-GFP accumulation and liquid clearance defects (Figures S4E-S4G). During vesicle fusion in mammalian cells, Rop/Sec1-Munc18 proteins regulate the assembly and activity of trans-SNARE complexes by interacting with Syntaxin.⁶¹,⁶² Thus, we examined the role of Syntaxin-1A in the formation of the extracellular lipid layer, since Syntaxin-1A also regulates the exosome-mediated secretion of Wingless in *Drosophila*.³³,³⁵ syx1A mutant embryos failed to form the apical extracellular lipid layer and instead showed enriched CD63-GFP⁺ signal at the apical membrane during the onset of liquid clearance (Figure 4H). This indicates that Syx1A is essential for both lysosomal secretion and the fusion of MVBs with the apical membrane to release CD63-GFP⁺ vesicles into the lumen. None of the intracellular components of exosomal vesicle biogenesis and transport showed any defects in dASM secretion (Figures 4I, S3G, and S4F; Table S2), suggesting that the cytoplasmic targeting machineries of lysosomal and exosomal secretion are independent but converge on Syt1A at the apical membrane.

## Exosomal secretion follows a parallel pathway to the lysosomal secretion

To further test the convergence of lysosomal and exosomal delivery routes, we examined mutants affecting dASM secretion, lysosomal biogenesis, and endosome-lysosomes fusion. Mutations in *rab39*, *syt1*, and *vps18/dor* (*Deep orange*), a subunit of the HOPS complex, showed luminal bubble-like membrane accumulation of CAAX-mCherry, comparable to the dASM mutants, whereas CAAX-mCherry localization was unaffected in *syx1A* and *RobG27* mutants (Figures 5A-5C). The extracellular CD63-GFP⁺ layer was present but significantly reduced in *syt1*K05909 and *btl>*Rab39DN (Figures S5A-S5D). These results suggest that the disruption of lysosomal biogenesis and transport does not affect the secretion of exosomal membranes but affects the assembly of the extracellular lipid layer. Next,

<footnote>(C) Bar graph depicting the average number of intracellular CD63⁺ vesicles of the DT (Tr7 and Tr8) in wild type (n = 49), Rab27DN (n = 43), Rab11DN (n = 41), Rab35DN¹ (n = 44), Rab35DN¹/² (n = 32), and syt1K05909 (n = 39) embryos. **, *** indicate statistical significance p < 0.01, p < 0.001, respectively, in comparison with wild type (unpaired two tailed t tests).</footnote>

<footnote>(D) Plots showing the % of embryos with gas filling completed in wild-type, (n = 109), Rab11DN, (n = 152), Rab27DN (n = 124), and Rab35DN¹/² (n = 167) embryos. The boxplots show the median (horizontal line) and the range from 25th to 75th percentiles. The bars depict maximum and minimum values. *, *** denote statistical significance p < 0.02, p < 0.001, respectively, calculated by unpaired two tailed t tests.</footnote>

<footnote>(E) Air-scan confocal frames showing the DT of living wild-type and RopG27 embryos expressing CD63-GFP (green) in airways. RopG27 mutant embryos accumulate intracellular CD63-GFP⁺ vesicles. The indicated time point represents hours AEL.</footnote>

<footnote>(F and G) Zoomed view of apical airway regions from (E) indicated by the rectangular frames.</footnote>

<footnote>(H) The DT of living wild-type and Syx1A embryos expressing btl>CD63-GFP (green) and actin RFP (magenta). Syx1 mutant embryos accumulate intracellular CD63⁺ vesicles. The time point represents hours AEL.</footnote>

<footnote>(I) Representative confocal sections showing the tracheal DT of stage 17 wild-type, Rab11DN, and Rab35DN¹/² mutant embryos stained for dASM (magenta), Crb (green), and DAPI (blue).</footnote>

<footnote>Scale bars, 10 µm. See also Figure S4.</footnote>
<figure><img src="image_52.png" /><figcaption>**Figure 5. Mutations in Rab39, syt1, and vps18/dor⁸, but not in syx1A or Rop, cause luminal membrane accumulations**</figcaption></figure>

(A) Confocal frames showing the DT of living wild-type, *dASM*, *btl>Rab39DN*, *dor⁸*, *syx1A*, and *RopG27* embryos expressing the membrane marker CAAX-mCherry (gray) in airways. Luminal lipids accumulated in *btl>Rab39DN* and *dor⁸* mutant embryos but not in *RopG27* and *syx1A*. The time points are hours AEL.

(B) Confocal frames of the DT of living *dASM* and *syt1K05909* mutant embryos expressing CAAX-mCherry (gray) in airways. Arrowheads depict luminal accumulations of CAAX-mCherry.

(C) Plots showing the percentage of luminal area covered by bubble-like structures in wild-type, (n = 7), *dASM* (n = 11), and *syt1K05909* (n = 12), embryos at 20 h AEL. Tubes collapsed in *syt1K05909* mutants during gas filling, and the phenotypic assessment of luminal bubble-like structures was not possible at the later stages of development. **, *** denotes statistical significance p < 0.01, p < 0.001, respectively. Mann-Whitney nonparametric test. Error bars are SEM. Scale bars, 10 µm. See also Figure S5.

we expressed *btl>CAAX-mCherry* in *dASM;Rab11DN1*, *dASM; Rab35DN1*, *dASM;Rab39DN*, and *dASM; vps18/dor* double mutants and quantified the intra-luminal CAAX-mCherry lipid accumulation. Interference with Rab11 or Rab35, but not with Rab39 or vps18/dor, in the *dASM* mutants strongly reduced the intra-luminal membrane accumulation of CAAX-mCherry (Figures 6A and 6B). These results further argue for two parallel apical exocytosis pathways. The Rab11/Rab35/Rop/Syx1A pathway mediates exosome secretion, and the Rab39/Syt1/Syx1A/pathway facilitates lysosomal secretion. These pathways are coordinately regulated to provide the structural components and enzymes for the generation of a continuous lipid sheet and facilitate airway gas filling (Figure 6C).

DISCUSSION

Our work revealed the origins and cellular mechanisms generating an extracellular lipid film at the air-liquid interface of the *Drosophila* airways, where it is required for airway gas filling and larval survival. Intriguingly, the lipid surface coating is a phylogenetically conserved property of air-liquid interfaces in many internal organs ranging from the *Drosophila* respiratory system to the swim bladder in fish and the lungs of birds and mammals. In both the swim bladder⁶³ and respiratory tubes of flies, this lipid film is required for gas filling. A lipid surfactant-like film has been predicted to contribute to the formation of the initial gas bubble in the fly airways by cavitation, where it
<figure><img src="image_53.png" /></figure>

<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /></figure>

would reduce the tensile strength of the liquid to the point where the pressure gradient in the tubes exceeds the tensile strength of the liquid.<sup>10,64</sup> The extracellular lipid film may also serve to concentrate protons, provided by the lysosomal secretion to efficiently dehydrate bicarbonate, liberate CO<sub>2</sub>, and fill the airways with gas.<sup>10</sup> In mammals with specialized alveolar structures, the surfactant film is further modified with additional surfactant proteins, which alter its structure and further reduce surface tension. Surfactant membranes in AT2 cells are stored in lysosomal-related lamellar bodies, which fuse with the apical membrane by an unknown mechanism and then release the lipid membranes into the lumen. The targeting of lamellar bodies and the mechanisms converting the exocytosed membranes into a continuous surfactant film remain unknown. We show that the construction of the surfactant coat in flies depends on MVB-derived exosomes and the coordinated secretion of lysosomes. Both

<caption>**Figure 6. Disruption of Rab11 and Rab35 but not of Rab39 or vps18/dor<sup>B</sup> in dASM mutants reduced the intra-luminal membrane accumulations**</caption>

(A) Confocal sections of the tracheal DT in living wild-type, *dASM*, *dASM; btl>Rab11<sup>DN</sup>*, *dASM; btl>-Rab35<sup>DN1</sup>*), *dASM; btl>-Rab39<sup>DN</sup>*), and *dASM; dor<sup>B</sup>* mutant embryos expressing the membrane marker *btl>-CAAX-mCherry* (magenta). Scale bars, 10 µm.

(B) Quantitation graph showing the percentage of luminal area covered by bubble-like structures in *dASM* (*n* = 53), *dASM+btl>Rab11<sup>DN1</sup>* (*n* = 58), *dASM+btl>Rab27<sup>DN1</sup>* (*n* = 29), *dASM+btl>Rab35<sup>DN2</sup>* (*n* = 27), *dASM+btl>Rab39<sup>DN</sup>* (*n* = 11), *dASM;dor<sup>B</sup>* (*n* = 18), and *dASM;Syx1A* (*n* = 7). \*\*\* denote statistical significance *p* < 0.01, *p* < 0.001, respectively, calculated by unpaired two tailed t tests.

(C) Schematic model. Rab11/Rab35/Rop/Syx1A-regulated MVB exocytosis promotes the secretion of exosomes into the lumen. Syx1A/Rab39-mediated lysosomal secretion releases dASM (magenta dots) and presumably other enzymes (yellow dots) to modify exosomal membrane-derived lipids and forms an extracellular lipid layer, presumably converted to parts of the envelope. Our work reveals that coordinated exosomal and lysosomal secretion mechanisms generate a film covering the aECM and ensure normal gas filling.

pathways employ distinct Rabs. Exosome secretion is dependent on Rab11 and Rab35 but not on Rab27<sup>65</sup> or its effector Stac/Munc13-4.<sup>66</sup> In turn, lysosomal dASM secretion requires Rab39. This argues for distinct transport pathways and presumably discrete sets of Rab regulators on the membrane microdomains driving the secretion of the two organelles. The *t*-SNARE Syx1A is a crucial factor for targeting both dASM<sup>+</sup> lysosomes and MVBs to the apical membrane. The Syx1A-dependent luminal secretion of Cathepsin-F suggests that other lysosomal enzymes in addition to dASM are released extracellularly, presumably to modify the luminal

contents or extracellular matrix.<sup>67,68</sup> A future task will be to uncover the upstream regulatory mechanisms initiating the apical migration of lysosomes and MVBs at the end stages of respiratory tube maturation. The involvement of the calcium-activated v-SNARE Syt1 in the lysosomal release of dASM, the timely activation of the Ca<sup>2+</sup>-reporter in the airway cells, and the clustering of lysosomes in Syx1A mutants suggest a Ca<sup>2+</sup>-dependent fusion mechanism of pre-docked lysosomes to the apical membrane. Previous work reported that Ca<sup>2+</sup> spikes induce conformational changes in the mammalian calcium sensor Synaptotagmin VII on lysosomal membranes to facilitate exocytosis.<sup>69</sup> Whether dASM exocytosis is regulated by local or systemic intracellular Ca<sup>2+</sup> signals needs further investigation. Local Ca<sup>2+</sup> sparks can activate Stac/Munc13-4, presumably via its conserved Ca<sup>2+</sup> binding residues, to control the secretion of lysosomal-related vesicles during branch fusion in the
*Drosophila* airways.⁷⁰ We found that intraluminal MVB vesicles marked by CD63-GFP⁺ provide the structural lipids for construction of the extracellular lipid layer. In AT2 cells, lamellar bodies have been found to be encapsulated by the same membrane as MVBs, suggesting that they may derive from MVBs.⁷¹ We found that exosome secretion from MVBs requires Rop. We speculate that an unknown v-SNARE targets MVBs to Rop/Syx1A sites at the apical membrane and facilitates ILV release.

Our work identified a new role for ASM in the developing airways. In contrast to neutral sphingomyelinase,²⁴ loss of dASM did not affect the multi-vesicular endosome biogenesis or release but was crucial for lysosomal integrity and the conversion of exosomal membranes into a film. Lysosomal ASM is also secreted from epithelial cells for membrane repair upon wounding.¹⁶ ASM-deficient cells from Niemann-Pick type A (NPA) patients undergo normal lysosomal secretion after wounding but are defective in PM repair.¹⁶ The extracellular function of dASM in *Drosophila* airways is supported by the following: (1) the concurrent luminal accumulation of dASM with the formation of extracellular lipid film, (2) the bubble-like accumulations of membranes and the aberrant extracellular lipid coat in *dASM* mutants, and (3) the sufficiency of local dASM expression in Tr9 and 10 to rescue the gas filling in the entire respiratory network in the *dASM* mutants. However, the biochemical composition of the secreted membrane vesicles or the anticipated modifications exerted by dASM to convert them into a film remain unknown. Mammalian exosomes are enriched in SM, the substrate for ASM, in the outer leaflet of the lipid bilayer,⁷² raising the possibility that extracellular ASM can modify exosomal membranes in mammals. *Drosophila* lacks SM but instead synthesizes the SM analog, phosphoethanolamine ceramide (PECer).⁷³,⁷⁴ The partial but statistically significant rescue of the gas-filling phenotype in *dASM* mutants by re-expression of the human *SMPD1/hASM* in the *Drosophila* airways suggests that human ASM can accept PECer as a substrate. *SMPD1/hASM* is mutated in patients with Niemann-Pick Disease,⁷⁵⁻⁷⁸ and ASM-deficient mice suffer from alveolar lipo-proteinosis in early adulthood.⁷⁹ In this context, it will be interesting to test whether secreted ASM is detected in lamellar bodies or the lung air-liquid interface of mammalian lungs and whether its extracellular function contributes to the assembly of the surfactant film.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* **KEY RESOURCES TABLE**

* **RESOURCE AVAILABILITY**

* Lead Contact

* Materials Availability

* Data and code availability

* **EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS**

* *Drosophila*

* **METHOD DETAILS**

* *Drosophila* genetics

* Molecular Biology

* Fixation and Immunostaining

* Injections of FM1-43 and Lysotracker dyes and imaging

* Live Imaging

* Expansion microscopy of *Drosophila* embryos

* **QUANTIFICATIONS AND STATISTICAL ANALYSIS**

* Quantifications of tissue dynamics

* Statistics

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.cub.2023.10.058.

## ACKNOWLEDGMENTS

We thank the fly community that isolated, characterized, or distributed mutants or antibodies. We specially thank Clive Wilson, Ernesto Sánchez-Herrero, Suzanne Eaton, Stefan Luschnig, Jean-Paul Vincent, Thomas L. Schwarz, the Bloomington Drosophila Stock Center, Kyoto Drosophila Stock Center, the Drosophila Genomics Resource Center (DGRC; Indiana), and the Developmental Studies Hybridoma Bank (DSHB; Iowa) for fly strains, clones, and antibodies. Special thanks to Flybase for the Drosophila genomic resources. We thank the Stockholm University Imaging Facility (IFSU) and the BioVis platform of Uppsala University. We thank Kirsten Senti for his scientific feedback in the project. This work was funded by the Swedish Research Council and the Swedish Cancer Society to C.S.; by the Magn. Bergvalls stiftelse and O. E. och Edla Johanssons vetenskapliga stiftelse to V.T.; and by the German Research Foundation (DFG), grant KFO309 (project number 284237345) to C.S. This paper is dedicated to the memory of our colleague, G.T., who sadly passed away on October 14, 2021.

## AUTHOR CONTRIBUTIONS

V.T. conceived the project, planned and executed most of the experiments, analyzed the data, and wrote the paper. D.L. generated and analyzed the human ASM and dASM transgenic flies. G.T. planned and executed in situ experiments, fly genetics, and analyzed data. Y.E. suggested experiments and commented on the manuscript. M.S. generated the ASM mutant and hASM, dASM transgenic flies, and commented on the manuscript. C.S. conceived the project, proposed experiments, analyzed data, and wrote the paper.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## INCLUSION AND DIVERSITY

We support inclusive, diverse, and equitable conduct of research.

Received: January 3, 2022

Revised: September 14, 2023

Accepted: October 26, 2023

Published: November 21, 2023

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Antibodies</td></tr><tr><td>Guinea pig anti-dASM</td><td>This paper</td><td>NA</td></tr><tr><td>rat anti-DE-Cad</td><td>Developmental Studies Hybridoma Bank (DSHB)</td><td>Cat#DCAD2, RRID:AB_528120</td></tr><tr><td>mouse anti-Crb</td><td>DSHB</td><td>Cat#Cq4, RRID:AB_528181</td></tr><tr><td>mouse anti-Coracle</td><td>DSHB</td><td>Cat#C615.16, RRID:AB_1161644</td></tr><tr><td>rabbit anti-GFP</td><td>Thermo Fisher Scientific</td><td>Cat#A-11122, RRID:AB_221569</td></tr><tr><td>chicken anti-GFP</td><td>Thermo Fisher Scientific</td><td>Cat #PA1-9533, RRID:AB_1074893</td></tr><tr><td>Guinea pig anti-Verm</td><td>Wang et al.<sup>80</sup></td><td>NA</td></tr><tr><td>Guinea pig anti-Gasp</td><td>Tiklová et al.<sup>81</sup></td><td>NA</td></tr><tr><td>rabbit anti-Uif</td><td>Zhang and Ward<sup>82</sup></td><td>NA</td></tr><tr><td>mouse anti-Syntaxin</td><td>DSHB</td><td>Cat#8C3, RRID:AB_528484</td></tr><tr><td>rabbit anti-TSG101</td><td>Sigma-Aldrich</td><td>Cat#HPA006161, RRID:AB_1080408</td></tr><tr><td>mouse anti-Flotilin-2</td><td>BD Biosciences</td><td>Cat#610383,RRID:AB_397766</td></tr><tr><td>rabbit anti-Arl8</td><td>DSHB</td><td>Cat#Arl8, RRID:AB_2618258</td></tr><tr><td>mouse anti-Ptp10D</td><td>DSHB</td><td>Cat#8B22F5, RRID:AB_528443</td></tr><tr><td>Cy3</td><td>Jackson Immunochemicals</td><td>Variable host and target species</td></tr><tr><td>Cy5</td><td>Jackson Immunochemicals</td><td>Variable host and target species</td></tr><tr><td>Alexa Fluor-488</td><td>Jackson Immunochemicals</td><td>Variable host and target species</td></tr><tr><td>Alexa Fluor-568</td><td>Jackson Immunochemicals</td><td>Variable host and target species</td></tr><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>LysoTracker Red<sup>DND-99</sup></td><td>Invitrogen/Thermo Fisher Scientific</td><td>Cat#L7528</td></tr><tr><td>Halocarbon oil (#700)</td><td>Sigma-Aldrich</td><td>Cat#H8898</td></tr><tr><td>VECTASHIELD PLUS Antifade Mounting Medium</td><td>BioNordika</td><td>VEH-1900-10</td></tr><tr><td>Methyl salicylate</td><td># 155665</td><td>MP Biochemicals</td></tr><tr><td>FM1-43 probe</td><td># HY-D1434</td><td>MCE</td></tr><tr><td>DAPI</td><td>Thermo Fischer Scientific</td><td>Cat#D1306</td></tr><tr><td>Heptane</td><td>Sigma-Aldrich</td><td>Cat#H2198</td></tr><tr><td>Triton X-100</td><td>Thermo Fisher Scientific</td><td>Cat#BP151-500</td></tr><tr><td>Formaldehyde 37% Solution</td><td>Sigma-Aldrich</td><td>Cat#1.03999</td></tr><tr><td>Sodium acrylate</td><td>Sigma-Aldrich</td><td>Cat#408220</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>Cat#S9888</td></tr><tr><td>30% Acrylamide/Bis Solution, 29:1</td><td>Bio-Rad</td><td>Cat#1610156</td></tr><tr><td>N,N'-Methylenebisacrylamide</td><td>Sigma-Aldrich</td><td>Cat#7279</td></tr><tr><td>TEMED (tetramethylethylenediamine)</td><td>Bio-Rad</td><td>Cat#1610800</td></tr><tr><td>4-Hydroxy-TEMPO</td><td>Sigma-Aldrich</td><td>Cat#176141</td></tr><tr><td>Ammonium persulfate (APS)</td><td>Thermo Fischer Scientific</td><td>Cat#17874</td></tr><tr><td>Proteinase K</td><td>Thermo Fischer Scientific</td><td>Cat#EO0492</td></tr><tr><td>EDTA</td><td>Thermo Fischer Scientific</td><td>Cat#15575020</td></tr><tr><td>Tris-HCl</td><td>Life Technologies</td><td>Cat#AM9855</td></tr><tr><td>Guanidine-HCl</td><td>Sigma-Aldrich</td><td>Cat#G3272</td></tr><tr><td>Tris-base</td><td>MP Biomedicals</td><td>Cat#819638</td></tr><tr><td>Tetramethylethylenediamine (TEMED)</td><td>Thermo Fischer Scientific</td><td>Cat#17919</td></tr><tr><td>Albumin bovine serum (BSA)</td><td>Sigma-Aldrich</td><td>Cat#A2153</td></tr><tr><td>Dimethyl sulfoxide, anhydrous (DMSO)</td><td>Sigma-Aldrich</td><td>Cat# 276855</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Experimental models: Organisms/strains</td></tr><tr><td>w<sup>1118</sup></td><td>Bloomington Drosophila Stock Center (BDSC), Indiana University</td><td>BDSC: 3605</td></tr><tr><td>P-element P(XP) CG3376<sup>d09724</sup></td><td>BDRC</td><td>BDSC: 19318</td></tr><tr><td>dASM</td><td>This work</td><td>NA</td></tr><tr><td>dor<sup>8</sup></td><td>BDSC</td><td>BDSC: 28</td></tr><tr><td>FRT80::ept<sup>2</sup>/TM3Sb</td><td>Kenneth H. Moberg/Emory University, Atlanta, GA, USA</td><td>NA</td></tr><tr><td>Df(3L)Exel9002</td><td>BDSC</td><td>BDSC: 7937</td></tr><tr><td>Df(3L)Exel9004</td><td>BDSC</td><td>BDSC: 7935</td></tr><tr><td>hrs (hrs<sup>D28</sup>)</td><td>BDSC</td><td>BDSC: 56816</td></tr><tr><td>syx1A, (Delta<sup>229</sup>)</td><td>BDSC</td><td>BDSC: 4379</td></tr><tr><td>Df(1)Exel6227</td><td>BDSC</td><td>BDSC: 7704</td></tr><tr><td>Df(1)BSC648</td><td>BDSC</td><td>BDSC: 25420</td></tr><tr><td>Df(3L)GN24</td><td>BDSC</td><td>BDSC: 3686</td></tr><tr><td>btl-GAL4</td><td>Shiga et al.<sup>83</sup></td><td>NA</td></tr><tr><td>UAS-Rab11<sup>DN</sup> (DN1)</td><td>Donald F. Ready Lab.<sup>84</sup></td><td>NA</td></tr><tr><td>UAS-Rab11<sup>DN</sup> (DN2)</td><td>BDSC</td><td>BDSC: 23261</td></tr><tr><td>UAS-Rab27<sup>DN</sup> (DN1)</td><td>BDSC</td><td>BDSC: 9810</td></tr><tr><td>UAS-Rab27<sup>DN</sup> (DN2)</td><td>BDSC</td><td>BDSC: 23267</td></tr><tr><td>UAS-Rab35<sup>DN</sup> (DN1)</td><td>BDSC</td><td>BDSC: 9819</td></tr><tr><td>UAS-Rab35<sup>DN</sup> (DN2)</td><td>BDSC</td><td>BDSC: 9820</td></tr><tr><td>UAS-Rab39<sup>DN</sup></td><td>BDSC</td><td>BDSC: 9824</td></tr><tr><td>YFP-Rab1</td><td>BDSC</td><td>BDSC: 62539</td></tr><tr><td>YFP-Rab4</td><td>BDSC</td><td>BDSC: 62542</td></tr><tr><td>YFP-Rab5</td><td>BDSC</td><td>BDSC: 62543</td></tr><tr><td>YFP-Rab7</td><td>BDSC</td><td>BDSC: 62545</td></tr><tr><td>YFP-Rab11</td><td>BDSC</td><td>BDSC: 62549</td></tr><tr><td>YFP-Rab18</td><td>BDSC</td><td>BDSC: 62551</td></tr><tr><td>YFP-Rab21</td><td>BDSC</td><td>BDSC: 62553</td></tr><tr><td>YFP-Rab27</td><td>BDSC</td><td>BDSC: 62556</td></tr><tr><td>YFP-Rab35</td><td>BDSC</td><td>BDSC: 62559</td></tr><tr><td>YFP-Rab39</td><td>BDSC</td><td>BDSC: 62560</td></tr><tr><td>UAS-CD36-GFP (human)</td><td>Clive Wilson Lab /S. Eaton<sup>47</sup></td><td>NA</td></tr><tr><td>UAS-CAAX-mCherry</td><td>BDSC</td><td>BDSC: 59021</td></tr><tr><td>UAS-ANF-GFP</td><td>Tsarouhas et al.<sup>52</sup></td><td>NA</td></tr><tr><td>UAS-ANF-mCherry</td><td>Tsarouhas et al.<sup>37</sup></td><td>NA</td></tr><tr><td>UAS-myr-RFP</td><td>BDSC</td><td>BDSC: 7119</td></tr><tr><td>UAS-GFP-Rab7</td><td>BDSC</td><td>BDSC: 42706</td></tr><tr><td>GCaMP5G-GFP</td><td>BDSC (and Loren L. Looger)</td><td>BDSC: 42038</td></tr><tr><td>UAS-CD8-GFP</td><td>BDSC</td><td>BDSC: 5130</td></tr><tr><td>YFP- LAMP1<sup>CPTI</sup></td><td>Kyoto Fly Stock Center</td><td>BDSC: 115240</td></tr><tr><td>UAS- GFP LAMP1 (Lamp1<sup>EY09768</sup>)</td><td>BDSC</td><td>BDSC: 20051</td></tr><tr><td>UAS- GFP LAMP1</td><td>Helmut Krämer<sup>85</sup></td><td>NA</td></tr><tr><td>UAS-dASM</td><td>This work</td><td>NA</td></tr><tr><td>UAS-hASM</td><td>This work</td><td>NA</td></tr><tr><td>Abd-B-Gal4</td><td>Ernesto Sánchez-Herrero<sup>40</sup></td><td>NA</td></tr><tr><td>Sas-GFP</td><td>VDRC<sup>86</sup></td><td>BDSC: 318129</td></tr><tr><td>Syt1-GFP</td><td>BDSC</td><td>BDSC: 6925</td></tr></tbody></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>sar1<sup>151</sup></td><td>Tsarouhas et al.<sup>52</sup></td><td>NA</td></tr><tr><td>γ-Cop<sup>938</sup></td><td>Jayaram et al.<sup>53</sup></td><td>NA</td></tr><tr><td>Creb-A</td><td>Deborah Andrew, Johns Hopkins<br>Univ. Baltimore, USA</td><td>#CrebA<sup>wR23</sup></td></tr><tr><td>sec61β</td><td>Jayaram et al.<sup>53</sup></td><td>NA</td></tr><tr><td>CtsF<sup>32769</sup></td><td>BDSC</td><td>BDSC: 51538</td></tr><tr><td>CtsF<sup>KM0156</sup></td><td>Kyoto Stock Center</td><td>NA</td></tr><tr><td>syb<sup>25-77</sup></td><td>Thomas L. Schwarz Lab<sup>87</sup></td><td>NA</td></tr><tr><td>syb<sup>144</sup></td><td>Thomas L. Schwarz Lab<sup>87</sup></td><td>NA</td></tr><tr><td>Rop<sup>G27</sup></td><td>BDSC</td><td>BDSC: 4381</td></tr><tr><td>Rop<sup>A3</sup></td><td>BDSC</td><td>BDSC: 39670</td></tr><tr><td>btsz<sup>J 5-2</sup></td><td>BDSC</td><td>BDSC: 8664</td></tr><tr><td>btsz<sup>k13-4</sup></td><td>BDSC</td><td>BDSC: 8665</td></tr><tr><td>stac<sup>3B20</sup></td><td>Stefan Luschnig Lab<sup>70</sup></td><td>NA</td></tr><tr><td>syt1<sup>T77</sup></td><td>BDSC</td><td>BDSC: 4377</td></tr><tr><td>syt1<sup>KG5909</sup></td><td>BDSC</td><td>BDSC: 10586</td></tr><tr><td>syt1<sup>N6</sup></td><td>BDSC</td><td>BDSC: 3910</td></tr><tr><td>FM7c</td><td>BDSC</td><td>BDSC: 23229</td></tr></tbody></table>

Recombinant DNA

<table><tr><td>pcDNA3</td><td>Thermo Fisher Scientific</td><td>Cat#V79020</td></tr><tr><td>pJFRC-MUH</td><td>addgene</td><td>Cat#26213</td></tr><tr><td>pATH11</td><td>Addgene</td><td>Cat#37699</td></tr><tr><td>pUAST-CG3376</td><td>This study</td><td>N/A</td></tr></table>

Software and algorithms

<table><tr><td>ImageJ</td><td>National Institute of Health, USA</td><td>https://imagej.nih.gov/ij/</td></tr><tr><td>ZEN</td><td>Carl Zeiss</td><td>https://www.zeiss.com/corporate/int/home.html</td></tr><tr><td>Signal P 4.1</td><td>Petersen et al.<sup>88</sup></td><td>https://services.healthtech.dtu.dk/service.php?SignalP-4.1</td></tr><tr><td>Python scripts</td><td>Python 3.10.4</td><td>https://www.python.org</td></tr><tr><td>Colab</td><td>Google</td><td>https://colab.research.google.com/?utm_source=scs-index</td></tr><tr><td>Affinity Designer 1.10.4</td><td>Serif (Europe) Ltd</td><td>https://affinity.store/en-us/</td></tr><tr><td>GraphPad Prism</td><td>GraphPad</td><td>https://www.graphpad.com/scientific-software/prism/</td></tr></table>

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Christos Samakovlis (christos.samakovlis@su.se).

Materials Availability

Fly-lines, plasmids and antibodies generated in this study are available upon request from the lead contact.

Data and code availability

* All original data reported in this paper will be shared by the lead contact upon request.

* This paper does not report original code.

* Any additional information required to re-analyze the data reported in this paper is available from the lead contact upon request.
# EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

## Drosophila

Flies were grown on standard cornmeal/yeast food at 25°C, and experiments were performed at 25° unless otherwise specified. *Drosophila melanogaster* stocks used in this study and associated references are listed in the [key resources table](#).

# METHOD DETAILS

## *Drosophila* genetics

The CG3376 null mutants M1M2 were generated by imprecise excision of a P-element P(XP) CG3376<sup>d09724</sup> which is inserted 126 bp upstream of the CG3376 transcription start site. w<sup>1118</sup> was used as the wild-type (wt) strain. Crosses for transgenic expression using *btl*-GAL4 driver were performed at 25 °C. Details for the source of these genotypes are in the [key resources table](#). We used dominant negative constructs to inactivate *Rabs* in the embryo because several of the *Rab* gene products are maternally provided in the eggs and their long-lasting persistence skews the phenotypic analysis of loss of function alleles or RNAi inhibition in the embryo. In all experiments CyO and TM3 balancer strains carrying GFP transgenes were used to identify embryos with the desired genotypes.

## Molecular Biology

cDNA encoding for human ASM (SMPD1 gene) and *Drosophila* dASM (CG3376-A isoform) initially cloned into pcDNA3 and then to the pJFRC-MUH vector (Addgene, plasmid #26213) to generate the UAS-hASM and UAS-dASM transgenic flies used in Figure 1B. We injected a pUAST-CG3376 plasmid into flies containing the golden cDNA for CG3376-A (DGRC) and several transgenic lines were generated according to standard protocols. Two independent lines, one on the second (attp40 landing site) and one in the third chromosome (attp2 landing site) were used for the rescue experiment. cDNA encoding for human ASM (SMPD1 gene) and *Drosophila* dASM (CG3376-A isoform) initially cloned into pcDNA3 and then to the pJFRC-MUH fly vector for the generation of UAS-hASM and UAS-dASM transgenic flies used in Figure 1B. The polyclonal antibody (anti-dASM) generated against a protein corresponding to amino acids 180-471 of dASM (CG3376-A). Protein was expressed in the pATH-11 vector system, purified from inclusion bodies and injected to guinea-pigs (Eurogentec). Digoxigenin-labeled RNA probes were transcribed from gold cDNA plasmid LD42214 (DGRC) and hybridized to embryos according to Lehmann and Tautz.<sup>89</sup>

## Fixation and Immunostaining

Embryos were bleached, dechorionated and fixed for 20 minutes in 4% formaldehyde saturated heptane. Stained embryos were mounted in Vectashield plus antifade mounting medium as described in ³⁷. For anti-dASM stainings, embryos were dechorionated by hand, subjected to dehydration treatment at the end of the fixation and mounted in methyl salicylate.⁹⁰ For stainings at the end of embryogenesis (stage late 17) we follow a fixation protocol from Manning and Doe⁴¹ with the following modifications during second fixation: 30% volume of heptane, incubation of 4 days in -20°C with 1-day agitation and no Pro-K treatment. Secondary antibodies conjugated to Cy3 or Cy5 or Alexa Fluor-488 and Alexa Fluor -568 were diluted as recommended by the manufacturers.

## Injections of FM1-43 and Lysotracker dyes and imaging

Dechorionated embryos were placed on cover slips coated by a heptane/glue mix and then covered with halocarbon oil. FM1-43 and lysotracker dyes were diluted in 6% dimethyl sulfoxide (DMSO) in deionized H₂O. Concentrations of 50 µM of the FM1-43 or 15 µM of Lysotracker were injected, using a microinjection system (FemtoJet, Eppendorf) coupled to an inverted fluorescence microscope (Cell Observer, Zeiss). Injections of FM1-43 were performed in perivitelline membrane at stage 10. For Lysotracker, we injected the dye into the hemocoel of stage-13 to stage-14 embryos, which lack the paracellular barrier function provided by fully matured septate junctions. At the 19.00 AEL, coverslips with injected embryos were placed in an airy-scan-equipped confocal microscope system (Zeiss LSM 800, Carl Zeiss) for imaging using a 63 × Plan-APOCHROMAT /1.4 M27 oil-immersion objective.

## Live Imaging

Embryos were dechorionated for 2-3 min in 7% sodium hypochlorite solution and mounted into a slide with a gas permeable membrane as described in ⁴¹. For wide-field live imaging, embryos were imaged with a CCD camera (AxioCam 702, Carl Zeiss) attached to an AxioImagerZ1 (Zeiss) microscope by using either a 20x/0.75NA Plan-APOCHROMAT or a 63x/1.3 NA C- APOCHROMAT water-immersion objective (Carl Zeiss). Individual Z-stacks with a step size of 1.5–2.0 µm were taken every 5–6 min over a 2–6-h period. To measure the developmental time of the formation of CD63-GFP extracellular layer and Syt1-GFP apical enrichment, embryos were collected on yeasted grape juice agar plates every 15 min and aged at 25°C. To quantify the CAAX-mCherry⁺ luminal accumulation, embryos were left to develop at least until 21 AEL. For confocal imaging, embryos were imaged with a laser-scanning confocal microscope (LSM780, Carl Zeiss) using a 63 × Plan-APOCHROMAT /1.4 M27 Oil-immersion objective. For high-resolution confocal imaging (Imaging panels in figures: S1A-G, 2A-E, 2I-J', S2, 3E-J, S3F, 4A-B'''', E-I, S4, 5, S5A-B, 6A), an airy-scan-equipped confocal microscope system (Zeiss LSM 800, Carl Zeiss) with a 63 × Plan-APOCHROMAT /1.4 M27 oil-immersion objective was used. Raw data were processed with the airy-scan processing tool of the Zen Black software version 2.3 (Carl Zeiss). Images were converted to tiff format using ImageJ/Fiji software ([http://rsb.info.nih.gov/ij/](http://rsb.info.nih.gov/ij/)).
Expansion microscopy of *Drosophila* embryos

Following immunostaining, embryos were mounted on #1.5 cover glass of a multi-well plate and incubated in monomer solution (2 M NaCl, 8.6 % sodium acrylate, 2.5% (wt/vol) acrylamide, 0.15% (wt/vol) N,N'-methylenebis-acrylamide in phosphate-buffered saline (PBS) for 1 h, at 4° C. The incubated solution was replaced by fresh and added 0.01% of 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-hydroxy-TEMPO), 0.2% of tetramethylethylenediamine (TEMED) and 0.2% of ammonium persulfate (APS). Embryos were then incubated within a desiccator for 3 h at 37°C. After gelation, samples were trimmed manually under a stereoscope to reduce large embryo-free areas in the gel and rinsed with PBS twice for 5 min. Embryos then digested in 8 units/ml proteinase K solution in digestion buffer (25 mM Tris pH 8.0, 1 mM EDTA, 0.5% Triton X-100, 0.8 M Guanidine HCl) for 1 h at 37°C. Then, samples removed from the digestion solution, washed for 2 x 5 min in proteinase K diluting buffer and allowed to expand overnight in a large excess of deionized water. After expansion, the gel was mounted on a lysine-coated cover glass for imaging. Details for the source of all the above reagents are listed in the key resources table.

QUANTIFICATIONS AND STATISTICAL ANALYSIS

Quantifications of tissue dynamics

Confocal micrographs were transformed into the “binary” images followed by the Fiji/ImageJ plugin “Segmentation Editor” for auto-
matic calculation and image threshold setting. Luminal bubble-like structures were manually selected using "polygon selections".
The % of luminal area covered by bubbles was calculated by the following formula: (bubble area/total lumen area) x 100. Intracellular
puncta, in 2D images were selected and counted using the “Analyze Particles” command in ImageJ (http://imagej.nih.gov/ij/, 1997-
2011). Minimum particle size was set to 0.21 pixels² to exclude background noise. Alternatively, images were segmented with Cell-
pose from Python script⁹¹ using Colab and fed to the ImageJ plugin LabelsToROIs⁹² for quantitation. Values in each embryo derived
from the average of dots in 3-5 sections per Z-stack. Data were extracted in an excel file format for further statistical analysis. The
fluorescent intensity on CD63-GFP⁺ apical and extracellular membranes (Figure S5C) was measured with ImageJ by manually draw-
ing a 5-pixel line with the "Freehand Line tool", over each membrane layer. Background signals of 5-pixel line, were subtracted from
the intensities. The relative fluorescence intensity was calculated as the intensity ratio of the two CD63-GFP layers (apical intra/extra-
cellular). Mean fluorescent intensity within these ROIs was measured in Fiji. The ImageJ FIJI Plot Profile plugin was used to generate
the GFP or RFP intensity profile plots shown in Figures 2F, 2G, and S2G, H. A line of 2 µm in length was drawn starting at a fixed point-
distance (0.5 µm) in the cytoplasm and towards to the apical membrane (CAAX-mCherry) along the axis where fluorescent intensity
values were needed to be quantified. 2 µm long line was sufficient to cover the fluorescence intensity signals of both membrane layers
(green or magenta). Line plots were generated with Graphpad Prism 8 by plotting distance values (µm) against fluorescent intensity
values (a.u.) for each channel in a single plot. The position of the signal peptide domain was predicted by the software PrediSi (http://
www.predisi.de) and confirmed by the SignalP 4.1 program.⁸⁸

Statistics

Error bars are standard error of means ± S.E.M. Graphs with box-plots show the median (horizontal line) and the range from 25th to 75th percentile. The bars depict maximum and minimum values. *p*-values were calculated through the *t*-test for unpaired variables if the data passed normality test (Shapiro-Wilk test) or the Mann-Whitney nonparametric test if the distribution was not normal. The *n* values of all the quantifications are provided in the figures or figure legends. Statistical tests were performed on Graphpad Prism 8.