The Hrs/Stam Complex Acts as a Positive and Negative Regulator of RTK Signaling during Drosophila Development

BACKGROUND: Endocytosis is a key regulatory step of diverse signalling pathways, including receptor tyrosine kinase (RTK) signalling. Hrs and Stam constitute the ESCRT-0 complex that controls the initial selection of ubiquitinated proteins, which will subsequently be degraded in lysosomes. It has been well established ex vivo and during Drosophila embryogenesis that Hrs promotes EGFR down regulation. We have recently isolated the first mutations of stam in flies and shown that Stam is required for air sac morphogenesis, a larval respiratory structure whose formation critically depends on finely tuned levels of FGFR activity. This suggest that Stam, putatively within the ESCRT-0 complex, modulates FGF signalling, a possibility that has not been examined in Drosophila yet. PRINCIPAL FINDINGS: Here, we assessed the role of the Hrs/Stam complex in the regulation of signalling activity during Drosophila development. We show that stam and hrs are required for efficient FGFR signalling in the tracheal system, both during cell migration in the air sac primordium and during the formation of fine cytoplasmic extensions in terminal cells. We find that stam and hrs mutant cells display altered FGFR/Btl localisation, likely contributing to impaired signalling levels. Electron microscopy analyses indicate that endosome maturation is impaired at distinct steps by hrs and stam mutations. These somewhat unexpected results prompted us to further explore the function of stam and hrs in EGFR signalling. We show that while stam and hrs together downregulate EGFR signalling in the embryo, they are required for full activation of EGFR signalling during wing development. CONCLUSIONS/SIGNIFICANCE: Our study shows that the ESCRT-0 complex differentially regulates RTK signalling, either positively or negatively depending on tissues and developmental stages, further highlighting the importance of endocytosis in modulating signalling pathways during development

Changes in SARS-CoV-2 viral load and mortality during the initial wave of the pandemic in New York City

Funding: This work was partially supported by the National Center for Advancing Translational Sciences of the National Institutes of Health (UL1 TR0023484 to Julianne Imperato-McGinley) and the National Institute of Allergy and Infectious Diseases (UM1 AI069470 to M.E.S).Public health interventions such as social distancing and mask wearing decrease the incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but it is unclear whether they decrease the viral load of infected patients and whether changes in viral load impact mortality from coronavirus disease 2019 (COVID-19). We evaluated 6923 patients with COVID-19 at six New York City hospitals from March 15-May 14, 2020, corresponding with the implementation of public health interventions in March. We assessed changes in cycle threshold (CT) values from reverse transcription-polymerase chain reaction tests and in-hospital mortality and modeled the impact of viral load on mortality. Mean CT values increased between March and May, with the proportion of patients with high viral load decreasing from 47.7% to 7.8%. In-hospital mortality increased from 14.9% in March to 28.4% in early April, and then decreased to 8.7% by May. Patients with high viral loads had increased mortality compared to those with low viral loads (adjusted odds ratio 2.34). If viral load had not declined, an estimated 69 additional deaths would have occurred (5.8% higher mortality). SARS-CoV-2 viral load steadily declined among hospitalized patients in the setting of public health interventions, and this correlated with decreases in mortality.Peer reviewe

Cellular and molecular mechanisms underlying the formation of biological tubes

Biological tubes are integral components of many organs. Based on their cellular organization, tubes can be divided into three types: multicellular, unicellular, and intracellular. The mechanisms by which these tubes form during development vary significantly, in many cases even for those sharing a similar final architecture. Here, we present recent advances in studying cellular and molecular aspects of tubulogenesis in different organisms

A Genetic Mosaic Analysis With a Repressible Cell Marker Screen to Identify Genes Involved in Tracheal Cell Migration During Drosophila Air Sac Morphogenesis

Branching morphogenesis of the Drosophila tracheal system relies on the fibroblast growth factor receptor (FGFR) signaling pathway. The Drosophila FGF ligand Branchless (Bnl) and the FGFR Breathless (Btl/FGFR) are required for cell migration during the establishment of the interconnected network of tracheal tubes. However, due to an important maternal contribution of members of the FGFR pathway in the oocyte, a thorough genetic dissection of the role of components of the FGFR signaling cascade in tracheal cell migration is impossible in the embryo. To bypass this shortcoming, we studied tracheal cell migration in the dorsal air sac primordium, a structure that forms during late larval development. Using a mosaic analysis with a repressible cell marker (MARCM) clone approach in mosaic animals, combined with an ethyl methanesulfonate (EMS)-mutagenesis screen of the left arm of the second chromosome, we identified novel genes implicated in cell migration. We screened 1123 mutagenized lines and identified 47 lines displaying tracheal cell migration defects in the air sac primordium. Using complementation analyses based on lethality, mutations in 20 of these lines were genetically mapped to specific genomic areas. Three of the mutants were mapped to either the Mhc or the stam complementation groups. Further experiments confirmed that these genes are required for cell migration in the tracheal air sac primordium

<i>argos</i> expression is strongly reduced in <i>stam</i>, <i>hrs</i> and <i>hrs, stam</i> mutant cells in pupal wings.

<p><i>argos</i> expression was analysed in <i>wild type</i> (A), <i>stam^2L2896</i> (B) and <i>hrs, stam</i> (C) MARCM clones visualized with mCD8-GFP (green) in pupal wings (24–30 hrs APF) using a <i>argos</i>-<i>LacZ</i> enhancer trap line. White dotted squares (in A–C) indicate the position of close-up pictures. White doted lines (in A′–C′) indicated the position of the clones in the close-up pictures. The <i>argos</i> staining (red) is absent in stam and <i>hrs, stam</i> mutant cells.</p

<i>stam</i> is required to properly localise FGFR/Btl and fully activate FGFR signalling.

<p>A–C. Localisation of Btl in wild type and mutant tracheal cells. High magnification pictures of wild type (A), <i>stam</i> (B) and <i>hrs</i> (C) MARCM mutant cells in the ASP. Scale bar equals 15 µm. Mutant cells were visualised <i>via</i> the expression of <i>UAS-btl-GFP</i> (green). The tracheal cells were visualised with RFP-moesin (red). White arrow indicates the presence of Btl at the cell membrane in wild type cells while yellow arrows indicate Btl as dotted structures. The dotted structures are dramatically enlarged in <i>stam</i> and <i>hrs</i> mutant cells as compared to <i>wild type</i> cells. D–F. <i>pointed</i> expression in wild type and <i>stam</i> mutant tracheal cells. Scale bars: 15 µm. <i>pointed</i> expression is restricted to the distal part, the tip, of a wild type ASP (D–D″). In a <i>stam</i> mutant clone located at the proximal part of the ASP, <i>pointed</i> expression is unchanged (E–E″). When the <i>stam</i> clone is positioned close to or at the distal tip of the ASP: <i>pointed</i> expression is lost (F–F″). Dotted lines showed the position of the <i>stam</i> mutant cells in the ASP (E′, F′). Arrows indicate the distal tip of the ASP (E″, F″).</p

<i>stam</i> and <i>hrs</i> are required for the efficient formation of fine cytoplasmic extensions in tracheal terminal cells and interact with FGFR signalling.

<p>Confocal micrographs of MARCM wild type and mutant dorsal terminal cells. Scale bar: 50 µm. The clones are visualised using UAS-mCD8-GFP. FRT40A line was used as a control (A). MARCM clones were induced for <i>stam</i> (B), <i>hrs</i> (C), <i>hrs, stam</i> (D). (E–F). The FRT40A and <i>stam</i> MARCM terminal cells with altered FGFR signalling. <i>bnl+/−</i> corresponds to a larvae heterozygous for <i>bnl</i>. Branch points (visualised in pink) were counted for mutant clonal cells for each genotype. The average number of branch points is given for each genotype.</p

Number of branching points of <i>hrs</i> and <i>stam</i> MARCM terminal cell clones in different FGFR signalling activity backgrounds.

<p>Number of branching points of <i>hrs</i> and <i>stam</i> MARCM terminal cell clones in different FGFR signalling activity backgrounds.</p

<i>stam</i> and <i>hrs</i> are required for tracheal cell migration in the air sac primordium.

<p>A. Schematic representation of the anterior part of a <i>Drosophila</i> third instar larva. The air sac primordium (ASP) (red) buds from the transverse connective branch (in grey) and is attached to the wing imaginal disc (orange). The tracheal system is drawn in grey and imaginal discs other than the wing disc are colored in yellow. B. Model for the formation of the air sac primordium during larval development. Tracheal cells divide and migrate during ASP formation. Migration occurs under the control of the FGFR signalling pathway. Tracheal cells at the distal tip of the primordium are extending filapodia in the direction of the FGF ligand source (blue). Double arrow indicates the position of ASP distal tip. C. Migration behaviour of <i>wild type</i>, <i>stam</i>, <i>hrs</i> and <i>stam hrs</i> mutant cells. Confocal micrographs of the ASP of a <i>Drosophila</i> third instar larva are shown. All tracheal cells are labelled in red (RFP-moesin) and MARCM clones are labelled in green (mCD8-GFP). The <i>FRT40A</i> chromosome was used as a wild-type control. MARCM clones were induced for <i>stam</i>, <i>hrs</i> and <i>stam</i>, <i>hrs</i>. Scale bar: 15 µm. White double arrows indicate the position of ASP distal tip. Percentages of distal clones are indicated for each genotype tested. Note the strong effect of mutations in <i>hrs</i> and <i>hrs, stam</i> on cell migration. For each genotype, more than 20 clones were scored.</p