In vivo regulation of the extracellular matrix

总结。动物是由活细胞和围绕在其周围的三维分子网络组成的 它们是细胞外基质(ECM)。除了其结构和保护功能外，ECM还是一个重要的 细胞组织、分化、形态发生和生理的调节者。之前的ECM研究已经 主要集中在基底膜，即接触极化细胞基面的细胞外基质。少得多 已知顶端ECM(AECM)，它存在于上皮腔、间皮腔和内皮腔内，并且 在表皮细胞的表面。最近的研究表明，AECM在控制细胞形状、组织 形态发生和管状形成，导致对AECM对发育和 疾病。目前，人们对AECM的调控知之甚少，包括控制其 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 在两个不同的环境中调节：线虫(1)胚胎形态发生和(2)幼虫蜕皮。这些 最近，不同的调查路线汇聚在一起，发现细胞内转运因子在 在这两个阶段的AECM监管中发挥关键作用。在胚胎发生的情况下，有两个保守的 未知的蛋白质，SYM-3/FAM102A和SYM-4/WDR44，使新生表皮能够抵抗 生物机械力造成的变形。目前的数据表明，Syms与多种内吞因子合作， 包括RAB-11，以控制贩运和AECM完整性。在幼虫蜕皮的情况下， NEK家族的蛋白激酶NEKL-2/NEK8/9和NEKL-3/NEK6/7在每次蜕皮时都是必需的，以促进 角质层的重塑，这是一种来自表皮的AECM。目前的数据表明，NEKLS调节 与笼蛋白包裹的囊泡的核心成分AP2密切相关的贩运，并通过控制 内吞肌动蛋白。未来对Syms和NEKL的研究将结合遗传学、细胞生物学、生化和 以组学为基础的方法，以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 说明NEKL和SYM功能是保守的。除了阐明AECM生物学，这些研究 将描述心尖部运输的机制，这一机制知之甚少，与 非极化或基侧膜的内吞作用。关于NEKL的工作也将涉及到 在调节内吞机制的组件中的磷酸化，被认为是普遍存在的，但 在很大程度上仍然没有特征。此外，尽管绝大多数的人口贩运研究都是在体外使用 细胞培养系统，关于NEKL和SYMS的工作将利用研究内部贩运的能力 一个完整的发育中的有机体。最后，拟议的研究将对人口贩运、信号传递、 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被充分研究的过程。 总的来说，这项工作将影响细胞内贩运、细胞外基质生物学、信号和开发领域。

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.