Mechanisms of basement membrane secretion and assembly

项目总结/摘要 基底膜（BM）是在所有上皮细胞的基底表面发现的特化细胞外基质。 组织中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，并作为转移的物理屏障。此外，BM组装中的缺陷导致皮肤 水泡、视力问题、肾病和中风。BM组装的某些方面由所有上皮细胞共享。 例如，当上皮细胞产生新的BM蛋白时，充满BM的高尔基体后囊泡 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 BM组装是组织特异性的。例如，BM成分和密度的局部变化指导了 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， 在生物学上，我们对BM是如何构建的知之甚少。本建议的目标是：（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 可以被修饰以产生用于器官形态发生的专门的BM结构。 为此，我们正在研究果蝇卵室周围的BM（多细胞 蛋的前身）。形成卵腔外层的上皮细胞（卵泡细胞）分泌它们的 在卵室的表面上很容易看到自己的BM。我们和其他人以前使用这个系统， 鉴定了两种小GTP酶Rab 10和Rab 8，它们作为极化BM分泌的主要调节物。 然而，这些Rabs如何指导BM囊泡从高尔基体的起源点运输到基底膜， 需要组装BM的单元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，导致BM蛋白被错误分泌。在目标1中，我们将检验Rab 10和/或 Rab 8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 这决定了在哪里以及如何构建BM，并为极化膜交通确定了新的指导原则。 我们研究的BM也有一个特殊的结构，使它能够作为一个“分子紧身胸衣”， 拉长卵室毛囊细胞集体沿着它们的BM迁移，BM保持静止。我们 表明这种运动与新的BM分泌协同作用，以在平面内建立定向的原纤维阵列， 构成了分子紧身衣的核心。这项工作现在使我们假设可能有两个 BM分泌途径，一种Rab 10依赖性途径，建立平面BM和Rab 8依赖性途径， 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 通过原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， 形成线状原纤维。目标3将检验这一假设。通过研究这两种组织特异性机制， 形成，我们将确定细胞可以用来建立器官的专门BM架构的一般原则， 形态发生，并确定BM组装中机械力的新作用。

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.