In vivo regulation of cell trafficking and the extracellular matrix

University Of Wyoming, Laramie WY

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Abstract

SUMMARY. We use C. elegans to study membrane trafficking and its impact on development and the apical extracellular matrix (aEMC) that surrounds the epidermis. Our work with collaborators centers on two conserved “pathways”, which until recently had been largely overlooked by the membrane trafficking field. Theme 1. We found that conserved NIMA-related kinases NEKL-2/NEK8/9 and NEKL-3/NEK6/7 regulate membrane trafficking and actin dynamics in worms and mammals. Theme 2. We found that membrane-associated SYM-3/FAM102A and SYM-4/WDR44 bind to trafficking factors and likely mediate the transport of bulk lipids at membrane contact sites (MCSs) via the lipid transferase BLTP-2/BLTP2. Both pathways ultimately impact the epidermal aEMC: loss of NEKLs causes defective molting, a cyclic form of aECM (cuticle) remodeling, whereas loss of SYMs leads to mechanical deformation of the embryo due to defects in the pre-cuticle. Many central questions remain regarding the mechanisms by which NEKLs and SYMs carry out their conserved functions. Theme 1 questions include: (i) How does loss of NEKLs/NEKs alter membrane trafficking and actin dynamics? (ii) What are the functions of nekl genetic suppressors and how do they alleviate nekl defects? (iii) How do proteins identified by NEKL–MLT BioID studies physically and functionally interact with NEKLs? (iv) What are the substrates of NEKLs/NEKs and how are they regulated by phosphorylation? Theme 2 questions include: (i) Which MCSs are bridged by the SYMs and BLTP-2? (ii) What roles do the SYMs play in localizing BLTP-2 to MCSs? (iii) What proteins act with the SYMs and BLTP-2 to mediate lipid transport? (iv) How does perturbation of the SYMs and BLTP-2 affect membrane lipid composition and trafficking? We address these questions through molecular, genetic, cell biological, proteomic, modeling, and biochemical approaches and complementary studies in C. elegans and mammalian cell culture to broaden the impact of our work. Collectively, these studies will lead to new mechanistic insights into the functions of conserved but largely uncharacterized regulators of membrane trafficking implicated in cancers, ciliopathies, and neurological disorders. Theme 3. We use the C. elegans syncytial epidermis as a model to understand how cell fusion affects nuclear functions and tissue differentiation. Our findings indicate that fusion is necessary for the normal transition from embryonic to larval development including the production of larval cuticle components. Syncytia are found throughout the animal kingdom in both healthy and diseased tissues, but little is known about the effects of syncytialization on subsumed nuclei. Theme 3 questions include: (i) How does syncytium formation impact nuclear transcriptomes and tissue differentiation? (ii) Are syncytial nuclei heterogenous (specialized) or homogenous? (iii) How are nuclear identities maintained or modified foll fusion? We will address these questions using genetics; omics methods, including snRNAseq; and cell biological analysis with smFISH methods and transcriptional reporters. These studies will expand our knowledge of the roles and properties of multinucleate syncytia, including a role in aECM assembly, which are largely unexplored.