Understanding How Invadosomes Breach Basement Membrane In Vivo

描述（由申请人提供）：基底膜是一种薄、致密、片状的细胞外基质，环绕大多数组织，为上皮细胞和内皮细胞提供结构支持。了解特化侵袭细胞如何跨越基底膜对人类健康具有深远的重要性：基底膜迁移是许多发育过程中细胞分散的基础，是白细胞免疫监视所必需的，细胞侵袭行为的不适当表现是转移性癌症发展的基础。富含f -肌动蛋白的细胞膜相关结构，称为浸润体，三十多年前在正常侵袭性细胞类型和转移性癌细胞系中被发现。侵入体被认为介导细胞通过基底膜屏障侵入的能力。由于在体内检测细胞侵入行为的困难，侵入体仅在体外研究，其中细胞培养条件不概括天然环境信号或基质条件。因此，体内浸润体的相关性、调控和潜在功能仍然是我们对细胞侵袭行为理解中最关键的空白之一。秀丽隐杆线虫的锚定细胞入侵是一种简单的、高度定型的细胞通过基底膜入侵的体内模型，它独特地结合了单细胞视觉和遗传分析。通过活细胞成像方法的发展，我们的团队最近发现了锚定细胞内破坏基底膜的动态侵入体结构。我们发现当一个侵入体穿透基底膜时，新的侵入体停止形成，并从浸润的侵入体中形成一个单一的侵入性突起。通过对基底膜成分的光学高亮显示，我们发现这种突起将基底膜推到一边，为入侵清除了道路。本应用程序的目的是揭示这些侵入体如何用于破坏基膜的机制细节。我们建议的研究将活细胞成像与遗传和分子分析相结合，以确定：(1)调节锚细胞侵袭的关键通路如何协调调节基底膜侵入前和侵入过程中侵入体的形成、动力学和功能；(2)网蛋白信号在选择单一侵入体进行基底膜侵入中的作用；(3)肌动蛋白调节因子Ena/VASP在侵入体侵入后促进锚细胞-基底膜接触点基底膜间隙扩张的作用。总之，这项工作将确定细胞入侵的新机制，并阐明它们如何共同作用以破坏基膜。该项目与NIH的使命相关，因为它将导致特定的治疗策略来控制转移性癌症等疾病的侵袭性行为。

DESCRIPTION (provided by applicant): Basement membrane is a thin, dense, sheet-like extracellular matrix that encircles most tissues and provides structural support for epithelial and endothelial cells. Understanding how specialized invasive cells cross basement membrane is of profound importance to human health: Basement membrane transmigration underlies the dispersal of cells in many developmental processes, is required in leukocytes for immune surveillance and inappropriate manifestation of cell-invasive behavior underpins the development of metastatic cancers. F-actin-rich, cell membrane associated structures, termed invadosomes, were identified over three decades ago in normally invasive cell types and metastatic cancer cell lines. Invadosomes are thought to mediate the ability of cells to invade through basement membrane barriers. Owing to the difficulty of examining cell-invasive behavior in vivo, invadosomes have only been studied in vitro, where cell culture conditions do not recapitulate native environmental signals or matrix conditions. As a result, the relevance, regulation and potential functions of invadosomes in vivo remain one of the most critical gaps in our understanding of cell- invasive behavior. Anchor cell invasion in C. elegans is a simple, highly stereotyped in vivo model of cell invasion through basement membrane that uniquely combines single-cell visual and genetic analysis. Through the development of live-cell imaging approaches, our group has recently identified dynamic invadosome structures within the anchor cell that breach the basement membrane. We find that when one invadosome penetrates the BM, new invadosome formation ceases, and a single invasive protrusion matures from the infiltrated invadosome. Using optical highlighting of basement membrane components, we have found that this protrusion pushes the basement membrane aside to clear a path for invasion. The goal of this application is to uncover the mechanistic details of how these invadosomes are used to breach basement membrane. Our proposed research will combine live-cell imaging with genetic and molecular analysis to determine: (1) how key pathways that regulate anchor cell invasion coordinately regulate the formation, dynamics and function of invadosomes prior to and during BM penetration, (2) the role of netrin signaling in selecting a single invadosome for basement membrane invasion, (3) the function of the actin regulator Ena/VASP in promoting basement membrane gap expansion at the anchor cell-basement membrane contact points after invadosome penetration. Together, this work will identify new mechanisms underlying cell invasion and elucidate how they function together to breach basement membrane. This project is relevant to NIH's mission because it will lead to specific therapeutic strategies to control invasive behavior in diseases such as metastatic cancer. PUBLIC HEALTH RELEVANCE: Cell invasion through basement membrane barriers occurs during the dispersal of cells in many normal physiological processes. In human diseases such as metastastic cancer, cell invasion is one of the most clinically relevant yet least understood aspects of cancer progression. The proposed research will investigate the mechanisms that control the subcellular structures that allow invasive cells to penetrate through basement membrane barriers. This work will reveal new molecular targets and strategies to develop more effective and specific treatments to control cell invasive behavior in important human diseases such as cancer.