Unraveling intestinal lumen formation in Caenorhabditis elegans

Most organs within our body are comprised of tubular structures essential for nutrient and waste transportation. Examples include blood vessels through which our blood flows, the intestines where we digest food, and airways through which air reaches our lungs. A crucial aspect of tube formation lies in the creation of a lumen, the central hollow space within the tube, which is orchestrated by a complex interplay of molecular players and cellular mechanisms. In this thesis, we employ cutting-edge genome engineering techniques and extensive microscopy analyses to delve into the molecular underpinnings of lumen formation, using the Caenorhabditis elegans intestine as a model system. Specifically, we focus on elucidating the roles of ERM proteins, NHERF proteins, and Ste20-like kinases in modulating the actin cytoskeleton to orchestrate lumen formation. ERM‑1, the sole C. elegans ERM ortholog, is essential for lumen formation, and disruptions in its membrane binding domain lead to widening of the intestinal lumen accompanied by constrictions. Furthermore, our research uncovers the nuanced regulatory role of ERM‑1's phosphorylation state at the C‑terminal T544 residue, which intricately modulates its stability and apical recruitment. In addition, ERM-1 activity involves actin binding modulated by this C-terminal phosphorylation and NRFL-1, with the latter being recruited to the microvilli by ERM-1, suggesting a vital role in organizing the apical domain. Moreover, our research identifies the GCK‑4 kinase as a novel regulator of lumen formation, localized at the tips of microvilli. Depletion of GCK‑4 results in a widened lumen, distorted adherent junctions, and alterations in the actin network, similar to defects observed in erm‑1 mutants. Interestingly, unlike its Ste20-like kinase counterparts, GCK‑4 is not a ERM C-terminal kinase, thereby contributing to lumen formation in a unique and yet-to-be-discovered manner. In addition to unraveling the molecular intricacies of lumen formation, we also improved the auxin-inducible degradation (AID) system in C. elegans by integrating a novel mIAA7 degron. This enhancement demonstrates significant efficacy in inducing protein degradation across various proteins and tissues compared to the conventional degron, thereby facilitating the study of specific proteins involved in lumen formation with enhanced precision and efficiency. By elucidating the intricate mechanisms underlying lumen formation through the lens of the C. elegans model organism, this work contributes to our understanding of fundamental biological processes and holds potential implications for the treatment of tube-related disorders.