In vivo regulation of the extracellular matrix

摘要动物是由活细胞和三维分子网络组成的， 细胞外基质（ECM）。除了其结构和保护功能外，ECM是一种重要的 细胞组织、分化、形态发生和生理学的调节因子。以前的ECM研究 主要集中在基底膜上，即与极化细胞基底表面接触的ECM。少得多 已知存在于上皮、间皮和内皮管腔内的顶端ECM（aECM）， 在表皮细胞的表面。最近的研究表明，aECM参与了细胞形状、组织结构和细胞周期的控制。 形态发生和管形成，导致对aECM对发育和 疾病目前，对aECM的调控知之甚少，包括控制其表达的途径。 沉积组织和重塑拟议的研究将通过调查aECM来解决这些差距 在两种不同的情况下进行监管：C。（1）胚胎形态发生;（2）幼虫蜕皮。这些 最近，不同的研究方向发现，细胞内的运输因子在细胞内的运输中起着重要的作用。 在这两个阶段的aECM调节中起关键作用。在胚胎发生的情况下，两个保守的，但以前 SYM-3/FAM 102 A和SYM-4/WDR 44这两种未表征的蛋白质使新生表皮能够抵抗 由于生物力学的力量而变形。目前的数据表明，SYMs与多种内吞因子， 包括RAB-11，以控制贩运和aECM完整性。在幼虫蜕皮的情况下， 蛋白激酶NEK家族，NEKL-2/NEK 8/9和NEKL-3/NEK 6/7，在每次蜕皮时都是必需的， 角质层的重塑，一种来源于表皮的aECM。目前的数据表明，NEKL调节 与AP 2密切相关的运输，网格蛋白包被囊泡的核心成分，并通过控制 内吞肌动蛋白未来对SYM和NEKLs的研究将结合联合收割机遗传学、细胞生物学、生物化学和生物化学， 以组学为基础的方法了解其在贩运中的具体功能并将这些活动与影响联系起来 在aECM上为了扩大影响，分析将纳入哺乳动物细胞培养系统，作为当前数据 表明NEKL和SYM功能是保守的。除了阐明ECM生物学，这些研究 将描述顶端运输的机制，这是知之甚少，并有很大不同， 在非极化或基底外侧膜的内吞作用。关于NEKL的工作还将涉及以下方面的作用： 内吞机制的调节组分中的磷酸化，这被认为是普遍的， 基本上没有特征。此外，尽管绝大多数贩运研究都使用体外 在细胞培养系统中，NEKL和SYM的工作将利用研究细胞内贩运的能力。 一个完整的发育中的有机体最后，拟议的研究将深入了解贩运，信号， 以及线虫蜕皮中的ECM重塑，这是一个与人类生物学和健康相关的未充分研究的过程。 总的来说，这项工作将影响细胞内运输，ECM生物学，信号传导和发展领域。

Summary. Animals are composed of living cells and the 3-diminsional network of molecules that surrounds them, the extracellular matrix (ECM). In addition to its structural and protective functions, the ECM is an important regulator of cell organization, differentiation, morphogenesis, and physiology. Previous ECM studies have focused largely on basement membranes, the ECM that contacts the basal surface of polarized cells. Much less is known about the apical ECM (aECM), which resides within epithelial, mesothelial, and endothelial lumens and on the surface of epidermal cells. Recent studies have implicated the aECM in the control of cell shape, tissue morphogenesis, and tube formation, leading to a new appreciation of aECM impacts on development and disease. At present, little is known about the regulation of the aECM, including the pathways that control its deposition, organization, and remodeling. The proposed studies will address these gaps by investigating aECM regulation in two distinct contexts: C. elegans (1) embryonic morphogenesis and (2) larval molting. These separate lines of investigation recently converged with the discovery that intracellular trafficking factors play a crucial role in aECM regulation at both stages. In the case of embryogenesis, two conserved but previously uncharacterized proteins, SYM-3/FAM102A and SYM-4/WDR44, enable the nascent epidermis to resist deformation by biomechanical forces. Current data suggest that SYMs partner with multiple endocytic factors, including RAB-11, to control trafficking and aECM integrity. In the case of larval molting, conserved members of the NEK family of protein kinases, NEKL-2/NEK8/9 and NEKL-3/NEK6/7, are required at each molt to facilitate remodeling of the cuticle, an aECM derived from the epidermis. Current data indicate that NEKLs regulate trafficking in close association with AP2, a core component of clathrin-coated vesicles, and through the control of endocytic actin. Future studies on SYMs and NEKLs will combine genetics, cell biological, biochemical, and omics-based approaches to understand their specific functions in trafficking and to link these activities to effects on the aECM. To broaden impact, analyses will incorporate mammalian cell culture systems, as current data indicate that NEKL and SYM functions are conserved. Beyond elucidating aECM biology, these investigations will characterize mechanisms of apical trafficking, which is poorly understood and differs substantially from endocytosis at non-polarized or basolateral membranes. Work on NEKLs will also address the role of phosphorylation in regulating components of the endocytic machinery, which is thought to be pervasive but remains largely uncharacterized. Moreover, whereas the vast majority of trafficking studies have used in vitro cell culture systems, work on the NEKLs and SYMs will take advantage of the ability to study trafficking within an intact developing organism. Finally, proposed studies will yield insights into the roles of trafficking, signaling, and ECM remodeling in nematode molting, an understudied process with relevance to human biology and health. Collectively, this work will impact the fields of intracellular trafficking, ECM biology, signaling, and development.