Molecular mechanisms defining the size of biological tubes in vivo

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1. BACKGROUND AND HYPOTHESIS. Epithelial tubes sustain gas, nutrient, and waste exchange. The precise size of epithelial tubes defines luminal fluid and gas dynamics, which are crucial physiological parameters in many organs. Our goal is to delineate the molecular mechanisms specifying the size of multicellular epithelial tubes by using Drosophila embryonic dorsal trunks as model system. Apical secretion of matrix-modifying enzymes in the lumen of dorsal trunks modulates the mechanical properties of the luminal matrix to restrain tube elongation. The surface area of the apical membrane of dorsal trunk cells also defines tube size. We have demonstrated that the protein Crb promotes apical membrane growth and elongation of dorsal trunks independently of the luminal extracellular matrix pathway. We recently showed that Rac1 acts as a central regulator of tube-size specification by promoting both apical secretion of matrix-modifying enzymes, and endocytosis and degradation of Crb. Loss of Rac1 function thus results in over-elongation of dorsal trunks. Identification of the regulators and effectors of Rac1 in this context is an outstanding puzzle to be solved in delineating the mechanisms regulating epithelial tube size. Our hypothesis is that defining the protein network of Rac1 specifically in epithelial tubes in vivo will contribute to defining the molecular mechanisms sustaining tube-size regulation. 2. AIMS AND EXPERIMENTAL SCHEME. 2.1 Unraveling the protein network of Rac1 in epithelial tubes using proximity-biotin labeling coupled to mass spectrometry (MS) (TurboID-MS). We established transgenic fly lines allowing inducible expression of TurboID-FLAG-Rac1, or the control fusion proteins TurboID-FLAG-GFP and TurboID-FLAG-GFPCAAX, in embryonic dorsal trunks. Biotinylated proteins will be purified and then identified by MS. Background and non-specific interactions will be removed using the SAINT algorithm, and Rac1-associated proteins will be classified into functional categories using DAVID. 2.2 Identification and characterization of novel epithelial tube-size regulators. We will use Drosophila genetics to couple our MS data with phenotypic analysis of dorsal trunk length and diameter. Thus, we will efficiently identify novel regulators of epithelial tube-size among the Rac1-associated proteins. We will then explore their mechanism of action and their functional relationship with Rac1 using in vivo approaches and biochemistry. 3. CONCLUSIONS. Our work will provide molecular data contributing to a comprehensive understanding of epithelial tube-size regulation, which is crucial for the morphogenesis and survival of animals. We will also reveal novel regulators and effectors of Rac1. This will have a strong impact beyond our field of research, as Rac1 signalling modulates a vast array of cellular functions. Our work will thus solve fundamental issues in cell and developmental biology, and will be of broad interest in biological sciences.