The proteolysis of ZP proteins is essential to control cell membrane structure and integrity of developing tracheal tubes in Drosophila

Tube networks are essential for organisms transporting liquids, gases, or cells across bodies. Endothelial and epithelial cells generate such networks with strict hierarchical order and precise tube dimensions as a prerequisite for proper tube network functioning (Ochoa-Espinosa and Affolter, 2012; Potente and Mäkinen, 2017). Defective cell shapes and tube dimensions result in severe syndromes such as chronic obstructive pulmonary diseases (384 million people in 2010) with high global mortality as well as tube dysfunctions like aortic aneurysms (152,000 deaths worldwide) in blood vessel systems and polycystic kidney diseases (1 in 1000 people) (Adeloye et al., 2015; Barnes et al., 2015; Harris and Torres, 2009; Quintana and Taylor, 2019; Zhai et al., 2021). One fundamental question regarding the formation of functional tube systems is how cells balance forces at the cell membranes emerging during organ growth and the simultaneous maintenance of the tubular network integrity. Drosophila melanogaster embryos form a tracheal system, an excellent model for studying molecular mechanisms controlling cell expansion in combination with tube elongation.

The development of tracheal tubes is subject to precise genetic control. Genes that control cell polarity, junction formation at the lateral membranes, cytoskeletal organization, and intracellular trafficking of tube size determinants support apical cell membrane expansion (Behr et al., 2003; Dong et al., 2014; Förster and Luschnig, 2012; Laprise et al., 2010; McSharry and Beitel, 2019; Nelson et al., 2012; Olivares-Castiñeira and Llimargas, 2017; Skouloudaki et al., 2019; Syed et al., 2012; Tonning et al., 2005; Tsarouhas et al., 2007). In contrast, genes controlling the meshwork of chitinous apical extracellular matrix (aECM) formation restrict excessive cell expansion to prevent tube overexpansion (Luschnig et al., 2006; Moussian et al., 2006; Öztürk-Çolak et al., 2016; Petkau et al., 2012; Tiklová et al., 2013; Wang et al., 2006). Subsequently, genetically controlled mechanisms establish tracheal airway clearance, aeration, and tube stabilization (Behr et al., 2007; Behr and Riedel, 2020; Drees et al., 2019; Stümpges and Behr, 2011; Tsarouhas et al., 2007; Tsarouhas et al., 2019).

Previous studies revealed that axial and radial forces affect tracheal tube elongation. The apical membrane grows axially, pulling on the associated aECM until the aECM’s elastic resistance balances the elongation force throughout the tubes (Dong and Hayashi, 2015). Given the association between cell membranes and aECM, ongoing tube expansion and luminal shear stresses inevitably lead to problematic membrane tension. Once forces are out of equilibrium, tracheal tubes show curvy appearance. Similarly, also blood vessels can appear unstable, twist, kink, and buckle. High stresses even lead to vascular damage and aneurysm rupture (Dong et al., 2014; Han et al., 2013). However, it is not known how cells manage to integrate the axial forces to stabilize the cell membrane and aECM.

Zona pellucida (ZP) domain proteins are critical components of apical cell membranes and aECM (Plaza et al., 2010) and assemble into extracellular fibrillar polymers (Jovine et al., 2005; Litscher and Wassarman, 2020). For example, Uromodulin plays a role in chronic kidney diseases and hypertension (Rampoldi et al., 2011). Secreted Uromodulin requires proteolysis at the apical cell membrane for shedding and polymerization within the tube lumen (Brunati et al., 2015). Similarly, the ZP domain protein Piopio (Pio) (Figure 1—figure supplement 1A) is secreted into the tracheal tubes of Drosophila embryos (Grieder et al., 2008; Massarwa et al., 2009). Pio restricts the elongation of autocellular junctions (Jaźwińska et al., 2003). Further, Pio is involved in relocating microtubule organizing center components γ-TuRC (γ-tubulin and Grips, gamma-tubulin ring proteins). This requires Spastin-mediated release from the centrosome and Pio-mediated γ-TuRC anchoring in the apical membrane (Brodu et al., 2010). However, while Pio is a transmembrane protein, it was detected in the tube lumen (Jaźwińska et al., 2003), but the release mechanism remains unknown. A promising candidate for Pio proteolysis is Notopleural (Np), the functional homolog of the human Matriptase. It is a type II single-transmembrane serine protease in tracheal and lung epithelia and is capable of ectodomain shedding (Bugge et al., 2009). Initial in vitro studies prove that Np cleaves the Pio ZP domain (Drees et al., 2019). The elastic luminal matrix is essential for the integrity of the tubular network. During tube elongation, the matrix balances elongation forces in the anterior-posterior direction (Dong et al., 2014). Here, we show that the ZP domain proteins Pio and Dumpy and the protease Np respond to mechanical stresses when tracheal tubes elongate to ensure normal membrane-aECM morphology.

Tracheal tube lumen expansion requires mechanical stress regulation at apical cell membranes and attached aECM. This involves the proteolytic processing of proteins that set local membrane-matrix linkages. Thus, the membrane microenvironment exhibits critical roles in regulating tube and network functionality.

ZP domain proteins organize protective aECM in the kidney, tectorial inner ear, and ZP (Jovine et al., 2005; Litscher and Wassarman, 2020), as well as in Drosophila epidermis, tendon cells, and appendages (Bökel et al., 2005; Plaza et al., 2010; Ray et al., 2015). Drosophila ZP domain proteins link the aECM to actin and polarity complexes in epithelial cells (Fernandes et al., 2010). Dumpy establishes force-resistant filaments for anchoring tendon cells to the pupal cuticle (Chu and Hayashi, 2021).

We identify that ZP protein-mediated microenvironmental changes increase the flexibility of membrane-matrix association, resulting from the activity of ZP domain proteins (Figures 1 and 7). Shear stress stimulates the activity of membrane-anchored proteases (Kang et al., 2015) and potentially also Np since we did not observe the misdistribution of the tracheal cytoskeleton when blisters arise. The dynamic membrane-matrix association control is based on our findings that loss of Np prevents Pio ectodomain shedding at the apical cell membrane resulting in immobile localization of Pio at the membrane and Dpy localization within the matrix (Figure 6). Direct interaction and overlapping subcellular localization at the cell surface showed that both proteins form a ZP matrix that potentially attaches membrane and ECM (Figure 5; Figure 5—figure supplement 1). Deregulation of Pio shedding blocks ZP matrix rearrangement and release of membrane-matrix linkages under tube expansion and subsequent shear stress. This destabilizes the microenvironment of membranes, causing blister formation at the membrane due to ongoing membrane expansion (Figure 7). Additionally, Pio could be part of a force-sensing signal transduction system destabilizing the membrane and matrix. Our observation that the membrane deformations are maintained in Np mutant embryos supports our postulated Np function to redistribute and deregulate membrane-matrix associations in stage 16 embryos when tracheal tube length expands. In contrast, Np overexpression potentially uncouples the Pio-Dpy ZP matrix membrane linkages resulting very likely in unbalanced forces causing sinusoidal tubes (Figure 8).

The membrane defects observed in both Pio and Np mutants indicate errors in the coupling of the membrane matrix due to the involvement of Pio (Figures 1 and 7). In pio mutants, gaps appear between the deformed membrane and the apical matrix (Figure 1B–D). These changes in apical cell membrane shape are consistent with increased cell and tube elongation in pio mutant embryos because the matrix is uncoupled from the membrane in such mutants (see model Figure 9). In contrast to pio mutants, the large membrane bulges in Np mutants affect the membrane and the apical matrix (Figure 7). Since apical Pio is not cleaved in Np mutants (Figure 6D), the matrix is not uncoupled from the membrane as in pio mutant embryos but is likely more intensely coupled, which leads to tearing of the matrix axially along the membrane bulges (Figures 7 and 9; Figure 7—figure supplement 1), when the tube expands in length. If apical Pio detachment reduces coupling between the matrix and apical membrane, then it is likely that Np mutant embryos may exhibit a reduced tube length phenotype. In Np mutant embryos, average tracheal dorsal trunk length tends to be reduced compared to wt embryos (Figure 8B), suggesting that Pio shedding is critical in controlling tracheal tube lumen length.

Figure 9

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The btl-Gal4-driven Np expression mimics the endogenous Np from stage 11 onward in all tracheal cells throughout embryogenesis (Drees et al., 2019), suggesting that Np is not expressed at a wrong time point. However, the ratio between Np and Pio is essential. We assume that tracheal Np overexpression increases Pio shedding in stage 16 embryos (Figure 8—figure supplement 1), resulting in a pio loss-of-function-like phenotype. Thus, the tube length overexpansion upon Np overexpression indicates that Pio cleavage is required for tube length control.

Is Pio ectodomain shedding in response to tension? We did not measure tension directly. However, the developmental profile of mechanical tension during tracheal tube length elongation in stage 16 embryos (Dong et al., 2014) is consistent with the profile of Pio shedding. Np cleaves apical Pio during stage 16 when tube length expands (Figures 5 and 6). In contrast, Pio shedding decreases sharply at early stage 17 when tube elongation is completed (Figures 5A and 6A). Our model, therefore, predicts that loss of Pio or increased Pio secretion at stage 16 may reduce the coupling of the membrane matrix so that increased tracheal tube elongation is maintained until the end of stage 16, which is found in pio mutants and upon Np overexpression (Figure 8A and B). Unknown proteases may likely be involved in Pio processing since cleaved mCherry::Pio is also detectable in inactive NpS990A cells. Previously we identified a mutation at the Pio ZP domain (R196A) resistant to NP cleavage in cell culture experiments (Drees et al., 2019). Establishing a corresponding mutant fly line would be essential in determining whether the observed phenotype resembles the phenotype of the Np mutant embryos. In addition, unknown mechanisms, such as distinct membrane connections during development and emerging links to the developing cuticle, may also influence tension at the apical membrane during tube length control.

Indeed, the anti-Pio antibody, which detects all different Pio variants, showed a punctuate Pio pattern overlapping with the apical cell membrane markers Crb and Uif at the dorsal trunk cells of stage 16 embryos (Figure 2; Figure 2—figure supplement 1, Figure 2—figure supplement 2). Additionally, Pio antibody also revealed early tracheal expression from embryonic stage 11 onward, and due to Pio function in narrow dorsal and ventral branches, strong luminal Pio antibody staining is detectable from early stage 14 until stage 17, when airway protein clearance removes luminal contents. In the pio^5m and pio^17c mutants, Pio stainings were strongly reduced although some puncta were still detectable in the trachea (Figure 1—figure supplement 1G and H). Similarly, Pio antibody staining is intracellular in the trachea of stage 11 pio^2R-16 point mutation embryos (Jaźwińska et al., 2003). Interestingly, also dpy mutants showed strongly reduced and intracellular Pio antibody staining (Figure 5—figure supplement 3E).

We generated mCherry::Pio as a tool for in vivo Pio expression and localization pattern analysis during tube lumen length expansion. The mCherry::Pio resembled the Pio antibody expression pattern from early tracheal development onward. However, luminal mCherry::Pio enrichment occurs specifically during stage 16, when tubes expand. The stage 16 embryos showed mCherry::Pio puncta accumulating apically in dorsal trunk cells. Moreover, mCherry::Pio puncta partially overlapped with Dpy::YFP and chitin at the taenidial folds, forming at apical cell membranes. Supported by several observations, such as antibody staining, video monitoring, FRAP experiments, and western blot studies (Figure 5; Figure 8—figure supplement 1; Figure 5—videos 1 and 2; Figure 6—videos 1; 2), these findings indicate that Pio may play a significant role at the apical cell membrane and matrix in dorsal trunk cells of stage 16 embryos.

Furthermore, we show that Np mediates Pio ZP domain cleavage for luminal release of the short Pio variant during ongoing tube length expansion. The luminal cleaved mCherry::Pio is enriched at the end of stage 16 and finally internalized by the subsequent airway clearance process during stage 17 after tube length expansion (Figures 5 and 6). Such rapid luminal Pio internalization is consistent with a sharp pulse of endocytosis rapidly internalizing the luminal contents during stage 17 (Tsarouhas et al., 2007). Wurst is required to mediate the internalization of proteins in the airways (Behr et al., 2007; Stümpges and Behr, 2011). In consistence, during stage 17, luminal Pio antibody staining fades in control embryos but not in Wurst deficient embryos.

Nevertheless, Pio and its endocytosis depend on its interaction with the chitin matrix and the Np-mediated cleavage. In stage 16 wurst and mega mutant embryos, we detect Pio antibody staining at the chitin cable, suggesting that Pio is cleaved and released into the dorsal trunk tube lumen. Also, the Cht2 overexpression did not prevent the luminal release of Pio. However, reduced wurst, mega function, and Cht2 overexpression caused an enrichment of punctuate Pio staining at the apical cell membrane and matrix (Figures 1 and 2). Although the three proteins are involved in different subcellular requirements, they all contribute to the determination of tube size by affecting either the apical cell membrane or the formation of a well-structured apical extracellular chitin matrix, indicating that changes at the apical cell membrane and matrix in stage 16 embryos affect the Pio pattern at the membrane. It also shows that local Pio linkages at the cell membrane and matrix are still cleaved by the Np function for luminal Pio release, which explains why those mutant embryos do not show pio mutant-like membrane deformations and Np-mutant-like bulges. This is in line with our observations that tracheal Pio overexpression cannot cause tube size defects as the Np function is sufficient to organize local Pio linkages at the membrane and matrix. Therefore, it is unlikely that tracheal tube length defects in wurst and mega mutants as well as in Cht2 misexpression embryos are caused by the apical Pio density enrichment.

Nevertheless, oversized tube length due to the misregulation of the apical cell membrane and adjacent chitin matrix may cause changes to local Pio set linkages and the need for Np-mediated cleavage. Strikingly, we observe a lack of Pio release in Np mutants. This shows that Pio density at the membrane versus lumen depends predominantly on Np function. The molecular mechanisms that coordinate the Np-mediated Pio cleavage are unknown and will be necessary for understanding how tubes resist forces that impact cell membranes and matrices. On the other hand, Pio is required for the extracellular secretion of its interaction partner Dpy (Figure 5—figure supplement 2). At the same time, Dpy is needed for Pio localization at the cell membrane and its distribution into the tube lumen (Figure 5—figure supplement 3). Consistently, in vivo, mCherry::Pio, and Dpy::eYFP localization patterns overlap at the apical cell surface and within the tube lumen (Figure 5—figure supplement 1). These observations support our model that Pio and Dpy interact at the cell surface where Np mediates Pio cleavage to support luminal Pio release by the large and stretchable matrix protein Dpy (Figure 9).

Taenidial organization prevents the collapse of the tracheal tube. Therefore, cortical (apical) actin organizes into parallel-running bundles that proceed to the onset of cuticle secretion and correspond precisely to the cuticle’s taenidial folds (Matusek et al., 2006; Öztürk-Çolak et al., 2016). Mutant larvae of the F-actin nucleator formin DAAM show mosaic taenidial fold patterns, indicating a failure of alignment with each other and along the tracheal tubes (Matusek et al., 2006). In contrast, pio mutant dorsal tracheal trunks contained increased ring spacing (Figure 3A). Fusion cells are narrow doughnut-shaped cells where actin accumulates into a spotted pattern. Formins, such as diaphanous, are essential in organizing the actin cytoskeleton. However, we do not observe dorsal trunk tube fusion defects as found in the presence of the activated diaphanous.

On the other hand, ectopic expression of DAAM in fusion cells induces changes in apical actin organization but does not cause any phenotypic effects (Matusek et al., 2006). DAAM is associated with the tyrosine kinase Src42A (Nelson et al., 2012), which orients membrane growth in the axial tube dimension (Förster and Luschnig, 2012). The Src42 overexpression elongates tracheal tubes due to flattened axially elongated dorsal trunk cells and AJ remodeling. Although flattened cells and tube overexpansion are similar in pio mutant embryos, we did not observe a mislocalization of AJ components, as found upon constitutive Src42 activation (Förster and Luschnig, 2012). Instead, we detected an unusual stretched appearance of AJs at the fusion cells of pio mutant dorsal trunks (Figure 4B and C), which to our knowledge, has not been observed before and may play a role in regulating axial taenidial fold spacing and tube elongation.

Self-organizing physical principles govern the regular spacing pattern of the tracheal taenidial folds (Hannezo et al., 2015). The actomyosin cortex and increased actin activity before and turnover at stage 16 drive the regular pattern formation. However, the cell cortex and actomyosin are in frictional contact with a rigid apical ECM. The Src42A mutant embryos contain shortened tube length but increased taenidial fold period pattern due to decreased friction. In contrast, the chitinase synthase mutant kkv¹ has tube dilation defects and no regular but an aberrant pearling pattern caused by zero fiction (Hannezo et al., 2015).

In contrast, pio mutant embryos do not contain tube dilation defects or shortened tubes but increased tube length (Figures 1 and 8; Figure 1—figure supplement 1). Furthermore, our cbp and antibody stainings reveal the presence of a luminal chitin cable and a solid aECM structure in pio mutant stage 16 embryos (Figure 8; Figure 1—figure supplement 1; Figure 3—figure supplement 2). In addition, apical actin enrichment in tracheal cells of pio mutant embryos appeared wt-like. Nonetheless, pio mutant embryos show an increased taenidial fold period compared with wt, indicating a decreased friction. Thus, we propose that the lack of Pio reduces friction. Reasons might be subtle defects of actomyosin constriction or chitin matrix, which we have not detected in the pio mutant tracheal cells. Further reasons for lower friction might also be the loss of Pio set local linkages between apical cortex and aECM in stage 16 embryos, which are modified by Np, as proposed in our model (Figure 9).

Heterozygous and homozygous pio mutant embryos generally do not show tubal collapse. However, the loss of Pio and accompanying lack of Dpy secretion in stage 17 pio mutant embryos led to the loss of a Pio/Dpy matrix, impacting the late embryonic maturation and differentiation of a normal chitin matrix at the apical cell surface. TEM images reveal reduced dense chitin matrix material at taenidial folds and misarranged taenidial fold pattern (Figure 1; Figure 2—figure supplement 1), suggesting impaired taenidial function prevents tube lumen from collapsing after tube protein clearance. Wurst knockdown and mutant embryos do not show general tube collapse, but luminal chitin fiber organization is disturbed in stage 17 embryos (Behr et al., 2007). Therefore, transheterozygous wurst;pio mutant embryos may combine both defects and suffer from maturation deficits of the chitin/ZP matrix at the apical cell surface and within the tube lumen, which finally causes a high number of embryos with incomplete gas-filling due to tube collapse. These maturation deficits are even more dramatic in the wurst;pio double mutants, which show no gas-filling.

Our studies on human Matriptase provide evidence for a mechanistic conservation of ZP domain protein as a substrate for ectodomain shedding (Figure 8). The upregulation of Matriptase activity and increased TGFβ receptor density affect human and mouse model idiopathic pulmonary fibrosis cells on pulmonary fibrogenesis (Bardou et al., 2016; Naik et al., 2012). Furthermore, the human Matriptase induces the release of proinflammatory cytokines in endothelial cells, which contribute to atherosclerosis and probably also to abdominal aortic aneurysms (Seitz et al., 2007). The membrane bulges arising in our Drosophila model during tracheal tube elongation upon Np loss of function showed analogy to the appearance of artery aneurysms. Bulges with varying phenotypic expression in different organs can lead to aortic rupture due to fragile artery walls or degeneration of layers in responses to stimuli, such as shear stresses (Kubo et al., 2015). Indeed, aneurysms development is forced by alterations in the ECM (Yoon et al., 1999) and are characterized by extensive ECM fragmentation caused by shedding of membrane-bound proteins (Antalis et al., 2016; Quintana and Taylor, 2019; Yoon et al., 1999; Zhong and Khalil, 2019).

We identified a dynamic control of matrix proteolysis, very likely enabling fast and site-specific uncoupling of membrane-matrix linkages when tubes expand. Such a scenario has not yet been studied in angiogenesis. It may represent a new starting point for genetic studies to decipher the putative roles of ZP domain proteins and Matriptase in clinically relevant syndromes, including the formation of aneurysms caused by membrane deformation and defects in size determination of airways and vessels.

All data confocal raw data used for this this study are either included in the manuscript as supporting files or available from the research data repository of the saxon universities, Open Access Repository and Archive OpARA, https://doi.org//10.25532/OPARA-240.

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Author details

1. Leonard Drees

   Research Group Molecular Organogenesis, Department of Molecular Developmental Biology, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany

   Contribution

   Conceptualization, Data curation, Supervision, Validation, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3191-8709

2. Susi Schneider

   Cell biology, Institute for Biology, Leipzig University, Leipzig, Germany

   Contribution

   Validation, Investigation, Visualization

   Competing interests

   No competing interests declared

3. Dietmar Riedel

   Facility for electron microscopy, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany

   Contribution

   Validation, Investigation, Visualization

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2970-6894

4. Reinhard Schuh

   Research Group Molecular Organogenesis, Department of Molecular Developmental Biology, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

5. Matthias Behr

   Cell biology, Institute for Biology, Leipzig University, Leipzig, Germany

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   matthias.behr@uni-leipzig.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9869-1257

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