Mechanisms of basement membrane secretion and assembly

项目摘要/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.