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

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.

Scavenger receptor endocytosis controls apical membrane morphogenesis in the Drosophila airways.

The acquisition of distinct branch sizes and shapes is a central aspect in tubular organ morphogenesis and function. In the Drosophila airway tree, the interplay of apical extracellular matrix (ECM) components with the underlying membrane and cytoskeleton controls tube elongation, but the link between ECM composition with apical membrane morphogenesis and tube size regulation is elusive. Here, we characterized Emp (epithelial membrane protein), a Drosophila CD36 homolog belonging to the scavenger receptor class B protein family. emp mutant embryos fail to internalize the luminal chitin deacetylases Serp and Verm at the final stages of airway maturation and die at hatching with liquid filled airways. Emp localizes in apical epithelial membranes and shows cargo selectivity for LDLr-domain containing proteins. emp mutants also display over elongated tracheal tubes with increased levels of the apical proteins Crb, DE-cad, and phosphorylated Src (p-Src). We show that Emp associates with and organizes the ßH-Spectrin cytoskeleton and is itself confined by apical F-actin bundles. Overexpression or loss of its cargo protein Serp lead to abnormal apical accumulations of Emp and perturbations in p-Src levels. We propose that during morphogenesis, Emp senses and responds to luminal cargo levels by initiating apical membrane endocytosis along the longitudinal tube axis and thereby restricts airway elongation.

WASH activation controls endosomal recycling and EGFR and Hippo signaling during tumor-suppressive cell competition.

Cell competition is a conserved homeostatic mechanism whereby epithelial cells eliminate neighbors with lower fitness. Cell communication at the interface of wild-type "winner" cells and polarity-deficient (scrib-/-) "losers" is established through Sas-mediated Ptp10D activation in polarity-deficient cells. This tumor-suppressive cell competition restrains EGFR and Hippo signaling and enables Eiger-JNK mediated apoptosis in scrib-/- clones. Here, we show that the activation state of the endosomal actin regulator WASH is a central node linking EGFR and Hippo signaling activation. The tyrosine kinase Btk29A and its substrate WASH are required downstream of Ptp10D for "loser" cell elimination. Constitutively active, phosphomimetic WASH is sufficient to induce both EGFR and Yki activation leading to overgrowth. On the mechanistic level we show that Ptp10D is recycled by the WASH/retromer complex, while EGFR is recycled by the WASH/retriever complex. Constitutive WASH activation selectively interferes with retromer function leading to Ptp10D mistargeting while promoting EGFR recycling and signaling activation. Phospho-WASH also activates aberrant Arp2/3 actin polymerization, leading to cytoskeletal imbalance, Yki activation and reduced apoptosis. Selective manipulation of WASH phosphorylation on sorting endosomes may restrict epithelial tumorous growth.

Dietary nitrite extends lifespan and prevents age-related locomotor decline in the fruit fly.

Aging is associated with decreased nitric oxide (NO) bioavailability and signalling. Boosting of a dietary nitrate-nitrite-NO pathway e.g. by ingestion of leafy green vegetables, improves cardiometabolic function, mitochondrial efficiency and reduces oxidative stress in humans and rodents, making dietary nitrate and nitrite an appealing intervention to address age-related disorders. On the other hand, these anions have long been implicated in detrimental health effects of our diet, particularly in formation of carcinogenic nitrosamines. The aim of this study was to assess whether inorganic nitrite affects lifespan in Drosophila melanogaster and investigate possible mechanisms underlying any such effect. In a survival assay, female flies fed a nitrite supplemented diet showed lifespan extension by 9 and 15% with 0.1 and 1 µM nitrite respectively, with no impact of nitrite on reproductive output. Interestingly, nitrite could also protect female flies from age-dependent locomotor decline, indicating a protective effect on healthspan. NO generation from nitrite involved Drosophila commensal bacteria and was indicated by a fluorescent probe as well as direct measurements of NO gas formation with chemiluminescence. Nutrient sensing pathways such as TOR and sirtuins, have been strongly implicated in lifespan extension. In aged flies, nitrite supplementation significantly downregulated dTOR and upregulated dSir2 gene expression. Total triglycerides and glucose were decreased, a described downstream effect of both TOR and sirtuin pathways. In conclusion, we demonstrate that very low doses of dietary nitrite extend lifespan and favour healthspan in female flies. We propose modulation of nutrient sensing pathways as driving mechanisms for such effects.

Yorkie controls tube length and apical barrier integrity during airway development.

Epithelial organ size and shape depend on cell shape changes, cell-matrix communication, and apical membrane growth. The Drosophila melanogaster embryonic tracheal network is an excellent model to study these processes. Here, we show that the transcriptional coactivator of the Hippo pathway, Yorkie (YAP/TAZ in vertebrates), plays distinct roles in the developing Drosophila airways. Yorkie exerts a cytoplasmic function by binding Drosophila Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water tightness of tracheal tubes by transcriptional regulation of the δ-aminolevulinate synthase gene (Alas). We conclude that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality.

WASH phosphorylation balances endosomal versus cortical actin network integrities during epithelial morphogenesis.

Filamentous actin (F-actin) networks facilitate key processes like cell shape control, division, polarization and motility. The dynamic coordination of F-actin networks and its impact on cellular activities are poorly understood. We report an antagonistic relationship between endosomal F-actin assembly and cortical actin bundle integrity during Drosophila airway maturation. Double mutants lacking receptor tyrosine phosphatases (PTP) Ptp10D and Ptp4E, clear luminal proteins and disassemble apical actin bundles prematurely. These defects are counterbalanced by reduction of endosomal trafficking and by mutations affecting the tyrosine kinase Btk29A, and the actin nucleation factor WASH. Btk29A forms protein complexes with Ptp10D and WASH, and Btk29A phosphorylates WASH. This phosphorylation activates endosomal WASH function in flies and mice. In contrast, a phospho-mimetic WASH variant induces endosomal actin accumulation, premature luminal endocytosis and cortical F-actin disassembly. We conclude that PTPs and Btk29A regulate WASH activity to balance the endosomal and cortical F-actin networks during epithelial tube maturation.

Meeting report - Cellular dynamics: membrane-cytoskeleton interface.

The first ever 'Cellular Dynamics' meeting on the membrane-cytoskeleton interface took place in Southbridge, MA on May 21-24, 2017 and was co-organized by Michael Way, Elizabeth Chen, Margaret Gardel and Jennifer Lippincott-Schwarz. Investigators from around the world studying a broad range of related topics shared their insights into the function and regulation of the cytoskeleton and membrane compartments. This provided great opportunities to learn about key questions in various cellular processes, from the basic organization and operation of the cell to higher-order interactions in adhesion, migration, metastasis, division and immune cell interactions in different model organisms. This unique and diverse mix of research interests created a stimulating and educational meeting that will hopefully continue to be a successful meeting for years to come.

Early development of Drosophila embryos requires Smc5/6 function during oogenesis.

Mutations in structural maintenance of chromosomes (Smc) proteins are frequently associated with chromosomal abnormalities commonly observed in developmental disorders. However, the role of Smc proteins in development still remains elusive. To investigate Smc5/6 function during early embryogenesis we examined smc5 and smc6 mutants of the fruit fly Drosophila melanogaster using a combination of reverse genetics and microscopy approaches. Smc5/6 exhibited a maternally contributed function in maintaining chromosome stability during early embryo development, which manifested as female subfertility in its absence. Loss of Smc5/6 caused an arrest and a considerable delay in embryo development accompanied by fragmented nuclei and increased anaphase-bridge formation, respectively. Surprisingly, early embryonic arrest was attributable to the absence of Smc5/6 during oogenesis, which resulted in insufficient repair of pre-meiotic and meiotic DNA double-strand breaks. Thus, our findings contribute to the understanding of Smc proteins in higher eukaryotic development by highlighting a maternal function in chromosome maintenance and a link between oogenesis and early embryogenesis.

Src kinases and ERK activate distinct responses to Stitcher receptor tyrosine kinase signaling during wound healing in Drosophila.

Metazoans have evolved efficient mechanisms for epidermal repair and survival following injury. Several cellular responses and key signaling molecules that are involved in wound healing have been identified in Drosophila, but the coordination of cytoskeletal rearrangements and the activation of gene expression during barrier repair are poorly understood. The Ret-like receptor tyrosine kinase (RTK) Stitcher (Stit, also known as Cad96Ca) regulates both re-epithelialization and transcriptional activation by Grainy head (Grh) to induce restoration of the extracellular barrier. Here, we describe the immediate downstream effectors of Stit signaling in vivo. Drk (Downstream of receptor kinase) and Src family tyrosine kinases bind to the same docking site in the Stit intracellular domain. Drk is required for the full activation of transcriptional responses but is dispensable for re-epithelialization. By contrast, Src family kinases (SFKs) control both the assembly of a contractile actin ring at the wound periphery and Grh-dependent activation of barrier-repair genes. Our analysis identifies distinct pathways mediating injury responses and reveals an RTK-dependent activation mode for Src kinases and their central functions during epidermal wound healing in vivo.

Control of airway tube diameter and integrity by secreted chitin-binding proteins in Drosophila.

The transporting function of many branched tubular networks like our lungs and circulatory system depend on the sizes and shapes of their branches. Understanding the mechanisms of tube size control during organ development may offer new insights into a variety of human pathologies associated with stenoses or cystic dilations in tubular organs. Here, we present the first secreted luminal proteins involved in tube diametric expansion in the Drosophila airways. obst-A and gasp are conserved among insect species and encode secreted proteins with chitin binding domains. We show that the widely used tracheal marker 2A12, recognizes the Gasp protein. Analysis of obst-A and gasp single mutants and obst-A; gasp double mutant shows that both genes are primarily required for airway tube dilation. Similarly, Obst-A and Gasp control epidermal cuticle integrity and larval growth. The assembly of the apical chitinous matrix of the airway tubes is defective in gasp and obst-A mutants. The defects become exaggerated in double mutants indicating that the genes have partially redundant functions in chitin structure modification. The phenotypes in luminal chitin assembly in the airway tubes are accompanied by a corresponding reduction in tube diameter in the mutants. Conversely, overexpression of Obst-A and Gasp causes irregular tube expansion and interferes with tube maturation. Our results suggest that the luminal levels of matrix binding proteins determine the extent of diametric growth. We propose that Obst-A and Gasp organize luminal matrix assembly, which in turn controls the apical shapes of adjacent cells during tube diameter expansion.

The tyrosine kinase Stitcher activates Grainy head and epidermal wound healing in Drosophila.

Epidermal injury initiates a cascade of inflammation, epithelial remodelling and integument repair at wound sites. The regeneration of the extracellular barrier and damaged tissue repair rely on the precise orchestration of epithelial responses triggered by the injury. Grainy head (Grh) transcription factors induce gene expression to crosslink the extracellular barrier in wounded flies and mice. However, the activation mechanisms and functions of Grh factors in re-epithelialization remain unknown. Here we identify stitcher (stit), a new Grh target in Drosophila melanogaster. stit encodes a Ret-family receptor tyrosine kinase required for efficient epidermal wound healing. Live imaging analysis reveals that Stit promotes actin cable assembly during wound re-epithelialization. Stit activation also induces extracellular signal-regulated kinase (ERK) phosphorylation along with the Grh-dependent expression of stit and barrier repair genes at the wound sites. The transcriptional stimulation of stit on injury triggers a positive feedback loop increasing the magnitude of epithelial responses. Thus, Stit activation upon wounding coordinates cytoskeletal rearrangements and the level of Grh-mediated transcriptional wound responses.

Resistance to Melampsora larici-epitea leaf rust in Salix: analyses of quantitative trait loci.

Quantitative resistance of Salix to Melampsora larici-epitea leaf rust was studied in 2 Salix mapping populations. One population was a backcross between a S. schwerinii x S. viminalis hybrid and S. viminalis, and the other was an F2 population between S. viminalis and S. dasyclados. A leaf disc bioassay was used to study the components of quantitative resistance (latent period, uredinia number, and uredinia size) to 3 isolates of the leaf rust. The analysis of quantitative trait loci (QTLs) revealed 9 genomic regions in the backcross population and 7 genomic regions in the F2 population that were important for rust resistance, with QTLs explaining 8-26% of the phenotypic variation. An important genomic region was identified for the backcross population in linkage group 2, where QTLs were identified for all resistance components for 2 of the rust isolates. Four of the QTLs had overlapping mapping intervals, demonstrating a common genetic background for latent period, uredinia diameter, and uredinia number. QTLs specific to some rust isolates and to some resistance components were also found, indicating a combination of common and specific mechanisms involved in the various resistance components. Breeding implications in relation to these findings are discussed.

COPI vesicle transport is a common requirement for tube expansion in Drosophila.

BACKGROUND: Tube expansion defects like stenoses and atresias cause devastating human diseases. Luminal expansion during organogenesis begins to be elucidated in several systems but we still lack a mechanistic view of the process in many organs. The Drosophila tracheal respiratory system provides an amenable model to study tube size regulation. In the trachea, COPII anterograde transport of luminal proteins is required for extracellular matrix assembly and the concurrent tube expansion. PRINCIPAL FINDINGS: We identified and analyzed Drosophila COPI retrograde transport mutants with narrow tracheal tubes. gammaCOP mutants fail to efficiently secrete luminal components and assemble the luminal chitinous matrix during tracheal tube expansion. Likewise, tube extension is defective in salivary glands, where it also coincides with a failure in the luminal deposition and assembly of a distinct, transient intraluminal matrix. Drosophila gammaCOP colocalizes with cis-Golgi markers and in gammaCOP mutant embryos the ER and Golgi structures are severely disrupted. Analysis of gammaCOP and Sar1 double mutants suggests that bidirectional ER-Golgi traffic maintains the ER and Golgi compartments and is required for secretion and assembly of luminal matrixes during tube expansion. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate the function of COPI components in organ morphogenesis and highlight the common role of apical secretion and assembly of transient organotypic matrices in tube expansion. Intraluminal matrices have been detected in the notochord of ascidians and zebrafish COPI mutants show defects in notochord expansion. Thus, the programmed deposition and growth of distinct luminal molds may provide distending forces during tube expansion in diverse organs.

Sequential pulses of apical epithelial secretion and endocytosis drive airway maturation in Drosophila.

The development of air-filled respiratory organs is crucial for survival at birth. We used a combination of live imaging and genetic analysis to dissect respiratory organ maturation in the embryonic Drosophila trachea. We found that tracheal tube maturation entails three precise epithelial transitions. Initially, a secretion burst deposits proteins into the lumen. Solid luminal material is then rapidly cleared from the tubes, and shortly thereafter liquid is removed. To elucidate the cellular mechanisms behind these transitions, we identified gas-filling-deficient mutants showing narrow or protein-clogged tubes. These mutations either disrupt endoplasmatic reticulum-to-Golgi vesicle transport or endocytosis. First, Sar1 is required for protein secretion, luminal matrix assembly, and diametric tube expansion. Subsequently, a sharp pulse of Rab5-dependent endocytic activity rapidly internalizes and clears luminal contents. The coordination of luminal matrix secretion and endocytosis may be a general mechanism in tubular organ morphogenesis and maturation.

The nucleoporin Nup214 sequesters CRM1 at the nuclear rim and modulates NFkappaB activation in Drosophila.

CRM1-mediated protein export is an important determinant of the nuclear accumulation of many gene regulators. Here, we show that the NFkappaB transcription factor Dorsal is a substrate of CRM1 and requires the nucleoporin Nup214 for its nuclear translocation upon signaling. Nup214 bound to CRM1 directly and anchored it to the nuclear envelope. In nup214 mutants CRM1 accumulated in the nucleus and NES-protein export was enhanced. Nup214 formed complexes with Nup88 and CRM1 in vivo and Nup214 protected Nup88 from degradation at the nuclear rim. In turn, Nup88 was sufficient for targeting the complex to the nuclear pores. Overexpression experiments indicated that Nup214 alone attracts a fraction of CRM1 to the nuclear envelope but does not interfere with NES-GFP export. By contrast, overexpression of the Nup214-Nup88 complex trapped CRM1 and Dorsal to cytoplasmic foci and inhibited protein export and immune response activation. We hypothesize that variation in levels of the Nup214-Nup88 complex at the pore changes the amount of NPC-bound CRM1 and influences the relative strength and duration of NFkappaB signaling responses.

Septate-junction-dependent luminal deposition of chitin deacetylases restricts tube elongation in the Drosophila trachea.

The function of tubular epithelial organs like the kidney and lung is critically dependent on the length and diameter of their constituting branches. Genetic analysis of tube size control during Drosophila tracheal development has revealed that epithelial septate junction (SJ) components and the dynamic chitinous luminal matrix coordinate tube growth. However, the underlying molecular mechanisms controlling tube expansion so far remained elusive. Here, we present the analysis of two luminal chitin binding proteins with predicted polysaccharide deacetylase activities (ChLDs). ChLDs are required to assemble the cable-like extracellular matrix (ECM) and restrict tracheal tube elongation. Overexpression of native, but not of mutated, ChLD versions also interferes with the structural integrity of the intraluminal ECM and causes aberrant tube elongation. Whereas ChLD mutants have normal SJ structure and function, the luminal deposition of the ChLD requires intact cellular SJs. This identifies a new molecular function for SJs in the apical secretion of ChLD and positions ChLD downstream of the SJs in tube length control. The deposition of the chitin luminal matrix first promotes and coordinates radial tube expansion. We propose that the subsequent structural modification of chitin by chitin binding deacetylases selectively instructs the termination of tube elongation to the underlying epithelium.

Mapping of quantitative trait loci controlling timing of bud flush in Salix.

Dormancy release is an important phenological stage, which determines plant growth and survival in northern temperate regions. Spring bud flushing was studied in a Salix pedigree (n=82) derived from a cross between the male hybrid clone "Björn" (Salix viminalis x Salix schwerinii) and the female clone "78183" (Salix viminalis). The timing of bud flush was recorded outdoors in two consecutive years (1998, 1999) and indoor in the spring of 1998. Timing of bud flush was found to be under moderately strong genetic control (clonal mean heritabilities ranging from 0.43 to 0.72). Phenotypic correlations between height growth and bud flushing were negative but non-significant (r=0.1-0.3). Using a Salix linkage map composed of 325 AFLP and 38 RFLP markers, six quantitative trait loci (QTLs) and three unmapped marker loci associated with timing of bud flush were detected. Four QTLs were detected in the field experiment while two QTLs and three unmapped marker loci were identified in the indoor experiment. One QTL associated with indoor bud flushing coincided with one of the QTL detected from the field data. Individual QTL explained 6-16% of the phenotypic variance [corrected]. None of the bud flush QTLs coincided with QTLs controlling height growth identified previously in the same pedigree.