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Mechanisms of basement membrane secretion and assembly

基底膜分泌和组装机制

基本信息

批准号：
10352423
负责人：
Sally Horne-Badovinac
金额：
$ 31.92万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10352423
关键词：
Apical Architecture Basement membrane Binding Biochemical Biological Assay Biology Bulla Cell surface Cells Cytoplasm Data Defect Destinations Disease Drosophila genus Dystroglycan ECM receptor Ensure Epithelial Epithelial Cells Extracellular Matrix Genetic Goals Golgi Apparatus Gossypium Heart Homeostasis Image Kidney Diseases Kinesin Lateral Lead Location Logic Lung Mammary gland Mechanics Membrane Protein Traffic Membrane Proteins Molecular Monomeric GTP-Binding Proteins Morphogenesis Motion Motor Neoplasm Metastasis Organ Pathway interactions Play Proteins Research Role Salivary Glands Shapes Signal Transduction Site Skin Stroke Surface System Testing Three-Dimensional Imaging Tissue constructs Tissues Travel Tubular formation Variant Vesicle Vision Work biophysical techniques cell motility density egg imaging approach mechanical force membrane assembly migration network architecture novel programs recruit tool trafficking

项目摘要

PROJECT SUMMARY / ABSTRACT Basement membranes (BM) are specialized extracellular matrices found at the basal surface of all epithelial tissues. These sheet-like protein networks provide mechanical stability to the cells, promote cell-cell and cell- matrix signaling, and act as a physical barrier to metastasis. Moreover, defects in BM assembly lead to skin blistering, vision problems, nephropathy, and stroke. Some aspects of BM assembly are shared by all epithelia. For example, when new BM proteins are made by the epithelial cells, post-Golgi vesicles filled with BM proteins (BM vesicles) must be trafficked exclusively to basal cellular regions for secretion. Other aspects of BM assembly are tissue-specific. For example, local variations in BM composition and density direct the branching of the mammary gland, salivary gland and lung. However, despite their clear importance to epithelial biology, we know remarkably little about how BMs are built. The goals of this proposal are: (1) to identify the molecular logic underlying polarized BM secretion, and (2) to determine how the basic BM assembly program can be modified to create a specialized BM architecture for organ morphogenesis. To this end, we are studying the BM that surrounds the Drosophila egg chamber (the multi-cellular precursor to the egg). The epithelial cells that form the egg chamber’s outer layer (follicle cells) secrete their own BM that is easily visualized on the egg chamber’s surface. We and others previously used this system to identify two small GTPases, Rab10 and Rab8, that act as master regulators of polarized BM secretion. However, how these Rabs direct the transport of BM vesicles from their point of origin at the Golgi to the basal- most region of the cell where the BM needs to be assembled is unknown. We have identified two kinesins that, when depleted, cause BM proteins to be mis-secreted. In Aim 1, we will test the hypothesis that Rab10 and/or Rab8 recruit the kinesins to BM vesicles for directed transport. This work will identify fundamental mechanisms that determine where and how a BM is built and identify new guiding principles for polarized membrane traffic. The BM we study also has a specialized architecture that allows it to act as a “molecular corset” to elongate the egg chamber. The follicle cells collectively migrate along their BM, which remains stationary. We showed that this motion synergizes with new BM secretion to build an oriented array of fibrils into the planar BM that form the heart of the molecular corset. This work has now led us to hypothesize that there may be two BM secretion pathways, a Rab10-dependent pathway that builds the planar BM and a Rab8-dependent pathway that builds the fibrils. Aim 2 will test this hypothesis. Our supporting data further suggest that BM proteins that exit the cell via the fibril-forming pathway must be placed under tension by the migrating cells for linear fibrils to form. Aim 3 will test this hypothesis. By studying these two tissue-specific mechanisms for BM formation, we will identify general principles that cells can use to build a specialized BM architecture for organ morphogenesis and identify a new role for mechanical forces in BM assembly.

项目概要/摘要基底膜 (BM) 是一种特殊的细胞外基质，存在于所有上皮细胞的基底表面组织。这些片状蛋白质网络为细胞提供机械稳定性，促进细胞-细胞和细胞- 基质信号传导，并充当转移的物理屏障。此外，BM组装缺陷会导致皮肤起泡、视力问题、肾病和中风。 BM 组装的某些方面是所有上皮细胞共有的。例如，当上皮细胞产生新的 BM 蛋白时，高尔基体后囊泡充满 BM 蛋白质（BM 囊泡）必须专门运输到基底细胞区域进行分泌。其他方面 BM 组装是组织特异性的。例如，BM 成分和密度的局部变化直接影响乳腺、唾液腺和肺的分支。然而，尽管它们对上皮细胞具有明显的重要性生物学方面，我们对 BM 的构建方式知之甚少。该提案的目标是： (1) 确定极化 BM 分泌的分子逻辑，以及 (2) 确定基本 BM 组装程序如何可以进行修改以创建用于器官形态发生的专门 BM 架构。为此，我们正在研究围绕果蝇卵室（多细胞卵的前体）。形成卵室外层的上皮细胞（卵泡细胞）分泌其自己的 BM，在卵室表面很容易看到。我们和其他人之前使用这个系统鉴定出两种小 GTPases，Rab10 和 Rab8，它们充当极化 BM 分泌的主要调节因子。然而，这些 Rabs 如何引导 BM 囊泡从高尔基体的起源点运输到基底层？细胞中需要组装 BM 的大部分区域是未知的。我们已经鉴定出两种驱动蛋白，当耗尽时，会导致 BM 蛋白错误分泌。在目标 1 中，我们将检验 Rab10 和/或 Rab8 将驱动蛋白募集到 BM 囊泡中进行定向运输。这项工作将确定基本机制确定 BM 的建造地点和方式，并确定极化膜交通的新指导原则。我们研究的 BM 还具有专门的架构，使其能够充当“分子紧身衣” 延长蛋室。毛囊细胞沿着其BM集体迁移，BM保持静止。我们研究表明，这种运动与新的 BM 分泌协同作用，将原纤维定向阵列构建到平面中 BM 构成分子紧身胸衣的核心。这项工作现在使我们假设可能有两种 BM 分泌途径，构建平面 BM 的 Rab10 依赖性途径和 Rab8 依赖性途径构建原纤维的途径。目标 2 将检验这一假设。我们的支持数据进一步表明 BM 通过原纤维形成途径离开细胞的蛋白质必须受到迁移细胞的张力形成线性原纤维。目标 3 将检验这一假设。通过研究 BM 的这两种组织特异性机制形成后，我们将确定细胞可以用来为器官构建专门的 BM 架构的一般原则形态发生并确定机械力在 BM 组装中的新作用。