A lipid and sugar-rich apical (luminal) extracellular matrix (aECM) shapes and protects the narrowest tubes in our bodies. For example, lung surfactant helps narrow airways remain open, and the endothelial glycocalyx protects capillaries in the vascular system. Damage to such luminal matrices can cause tube collapse or leakage, contributing to disease. Despite the importance of luminal aECM, we have only a rudimentary understanding of its multi-layered organization, how it is assembled, and how appropriate lipid content is ensured. Specialized lysosome-related organelles (LROs) store, secrete and recycle aECM lipids, but mechanisms controlling lipid transport to and between LROs and aECM are poorly defined. It is also unclear how dietary and metabolic changes affect aECM lipids, and how this might contribute to effects on tube integrity. Unfortunately, the fragile nature of the luminal aECM makes it difficult to visualize and study in most systems. We will take advantage of the transparent model organism C. elegans to study opposing roles of two conserved families of lipid transporters - lipocalins and Scavenger Receptor B (SCARB) proteins - in aECM organization and tube protection. Both live imaging and electron microscopy allow us to visualize luminal aECM in unprecedented detail. Forward and reverse genetic approaches allow us to identify and manipulate relevant glycolproteins and lipids in the matrix. Molecular similarities between the C. elegans pre-cuticular aECM and the mammalian glycocalyx, and between C. elegans and mammalian LROs, suggest that this system will reveal broadly relevant principles of aECM trafficking and assembly.
Aim 1 will test the hypothesis that two lipocalins act sequentially to transport phosphatidylcholine (PC) to LROs and aECM. We will use imaging, genetic, and biochemical approaches to identify lipocalins' cellular and aECM compartments, partners and cargoes, test metabolic influences on aECM, and tie lipocalins to specific steps of lipid trafficking and aECM assembly.
Aim 2 will test the hypothesis that a SCARB related to human LIMP-2 (lysosomal integral membrane protein 2) opposes lipocalins via effects on PC metabolism in LROs. We will combine imaging and genetic approaches to identify relevant SCAV-2 compartments and cargos, and test if elevated PC levels can explain how SCAV-2 loss suppresses lipocalin mutant lethality. We will also clone other suppressor loci to identify SCAV-2 partners. Together, these Aims will reveal basic principles of luminal aECM assembly and organization, and elucidate the functions of, and relationships between, specific lipid transporter families that have previously been studied for separate roles in human biology and disease.
Collapse of narrow tubes contributes to lung, kidney and heart disease, and to stroke, blindness and age-related dementia. These studies investigate how a sugar- and fat-rich lining is built inside these tubes, and how this lining helps protect the tubes from collapse. Understanding these mechanisms will help in the design of therapies to prevent or treat tube-related disease.
