Mechanisms of basement membrane secretion and assembly

项目摘要/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面 纸巾。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，并充当转移的物理屏障。此外，BM组装中的缺陷会导致皮肤 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所共有的。 例如，当上皮细胞制造新的BM蛋白时，高尔基体后的囊泡充满BM 蛋白质(BM囊泡)必须专门运输到基底细胞区域进行分泌。的其他方面 骨髓的组装是组织特异性的。例如，BM组成和密度的局部变化将引导 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，我们对大气层管理系统是如何构建的知之甚少。这项建议的目标是：(1)确定 BM分泌极化的分子逻辑，以及(2)确定基本BM组装程序 可以被修改以创建用于器官形态发生的专门的BM架构。 为此，我们正在研究围绕果蝇卵室(多细胞)的BM 卵子的前体)。形成卵室外层的上皮细胞(滤泡细胞)分泌它们的 自己的BM很容易在卵室的表面上看到。我们和其他人以前使用这个系统来 识别两个小的GTP酶，Rab10和Rab8，它们是极化BM分泌的主要调节者。 然而，这些RAB如何引导BM囊泡从它们在高尔基体的起始点运输到基底- BM需要组装的电池的大部分区域都是未知的。我们已经确定了两种激动素， 当耗尽时，会导致BM蛋白分泌错误。在目标1中，我们将检验假设Rab10和/或 Rab8将激动素招募到BM囊泡进行定向运输。这项工作将确定基本的机制 这决定了BM在哪里以及如何建造，并确定了极化膜交通的新指导原则。 我们研究的BM也有一个特殊的体系结构，使它能够充当 拉长卵室。毛囊细胞沿着它们的BM集体迁移，BM保持不动。我们 显示这一运动与新的BM分泌协同作用，将定向的纤维阵列构建到平面中 BM构成了分子胸衣的心脏。这项工作现在让我们假设可能有两个 BM分泌途径，依赖Rab10构建平面BM的途径和依赖Rab8的途径 形成纤维的途径。《目标2》将检验这一假设。我们的支持数据进一步表明，BM 通过纤维形成途径离开细胞的蛋白质必须被迁移的细胞置于张力下，以便 形成线状原纤维。《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，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。

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.