Molecular mechanisms defining the size of biological tubes in vivo

1.研究背景和研究假设。上皮细胞维持气体、营养物质和废物的交换。上皮管的精确大小决定了腔内液体和气体动力学，这是许多器官的关键生理参数。我们的目标是以果蝇胚胎背部干为模型系统，描述决定多细胞上皮管大小的分子机制。背部主干管腔中基质修饰酶的顶端分泌调节管腔基质的机械性能，以抑制管子的伸长。背干细胞顶膜的表面积也决定了管的大小。我们已经证明，CRB蛋白促进顶膜的生长和背干的延长，不依赖于腔外基质途径。我们最近发现，rac1通过促进基质修饰酶的顶端分泌，以及CRB的内吞和降解，发挥着管子大小规范的中心调节作用。因此，rac1功能的丧失会导致背部主干过度伸长。在这一背景下，识别rac1的调节因子和效应器是描述上皮管大小调节机制的一个突出的难题。我们的假设是，在活体上皮管中明确rac1的蛋白质网络将有助于确定维持管子大小调节的分子机制。2.实验目标和实验方案。2.1使用邻近生物素标记与质谱仪(TurboID-MS)联用技术(TurboID-MS)解开上皮管中的rac1蛋白网络。我们建立了可诱导TurboID-FLAG-rac1或对照融合蛋白TurboID-FLAG-GFP和TurboID-FLAG-GFPCAAX在胚胎背干中诱导表达的转基因果蝇系。生物素化的蛋白质将被提纯，然后用MS鉴定背景，非特异的相互作用将被使用SAINT算法去除，而rac1相关的蛋白质将使用David被分类为功能类别。2.2新型上皮管大小调节剂的鉴定和表征。我们将利用果蝇遗传学将我们的MS数据与背干长度和直径的表型分析相结合。因此，我们将有效地在rac1相关蛋白中识别新的上皮管大小调节因子。然后，我们将使用体内方法和生物化学来探索它们的作用机制以及它们与rac1的功能关系。3.结论。我们的工作将提供有助于全面了解上皮管大小调节的分子数据，这对动物的形态发生和生存至关重要。我们还将揭示rac1的新的调节因子和效应器。这将在我们的研究领域之外产生强大的影响，因为rac1信号调制了大量的细胞功能。因此，我们的工作将解决细胞和发育生物学中的基本问题，并将在生物科学中引起广泛的兴趣。

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.