Thursday, April 25, 2024
4/25/2024
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.
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