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，包围大多数组织并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穿过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。三十多年前，在正常侵袭性细胞类型和转移性癌细胞系中发现了富含F-肌动蛋白的细胞膜相关结构，称为侵袭体。侵入体被认为介导细胞通过基底膜屏障侵入的能力。由于在体内检查细胞侵袭行为的困难，侵袭体仅在体外进行了研究，其中细胞培养条件不重现天然环境信号或基质条件。因此，侵袭体在体内的相关性、调节和潜在功能仍然是我们理解细胞侵袭行为的最关键的空白之一。C.锚细胞的侵袭elegans是一种简单的、高度定型的细胞通过基底膜侵入的体内模型，其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，我们的小组最近已经确定了动态侵入体内的锚细胞破坏基底膜的结构。我们发现，当一个侵入体穿透BM时，新的侵入体形成停止，并且单个侵入性突起从浸润的侵入体成熟。利用基底膜成分的光学突出，我们发现该突起将基底膜推到一边以清除入侵路径。该应用的目标是揭示这些侵入体如何用于破坏基底膜的机制细节。我们提议的研究将联合收割机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途径如何在BM穿透之前和期间协调调节侵袭体的形成、动力学和功能，（2）netrin信号传导在选择单个侵袭体用于基底膜侵袭中的作用，（3）肌动蛋白调节剂Ena/VASP在侵袭体穿透后促进锚细胞-基底膜接触点基底膜间隙扩大的作用。总之，这项工作将确定潜在的细胞入侵的新机制，并阐明它们如何共同发挥作用，以打破基底膜。该项目与NIH的使命相关，因为它将导致特定的治疗策略，以控制转移性癌症等疾病的侵袭行为。 公共卫生相关性：在许多正常的生理过程中，细胞通过基底膜屏障的侵入发生在细胞扩散期间。在人类疾病如乳腺癌中，细胞侵袭是癌症进展中最临床相关但最不了解的方面之一。拟议的研究将调查控制亚细胞结构的机制，这些结构允许入侵细胞穿透基底膜屏障。这项工作将揭示新的分子靶点和策略，以开发更有效和特异性的治疗方法，以控制癌症等重要人类疾病的细胞侵袭行为。

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.