Generating and maintaining polarity is fundamental to multicellular life and a prerequisite for spatial diversity and morphogenesis. Epithelial membrane biogenesis requires distinct polarizing cues from: (a) outside the cell (e.g. the matrix), (b) insie the cell (e.g. through directional trafficking), and (c) the plasma membrane and its junctions (e.g by plasma-membrane associated polarity determinants such as the partitioning-defective PAR s). Many of these highly conserved polarity cues have been identified, but their integration during the complex process of tissue morphogenesis is not well understood. We have conducted genome-wide unbiased as well as targeted RNAi-based screens in the transparent roundworm C. elegans, engineered with fluorescently-labeled apical membranes, to explore polarized membranes biogenesis in simple multi- and unicellular tubes at single-cell resolution. Here, tubular organogenesis involves coincident apical domain and lumen morphogenesis, permitting polarity analysis in a 3D in vivo setting during morphogenesis. These screens identified, among other novel tube phenotypes, an intriguing intestinal polarity and multiple-ectopic-lumen phenotype, where all apical molecules tested, including the apical polarity complex component PAR-6 were found displaced to the basolateral membrane. Among other molecules, the loss of several unrelated lipid -biosynthetic enzymes, trafficking-related genes, as well as actin-modulating genes were found to cause this phenotype. Subsequent analyses identified membrane sphingolipids (SLs) as well as clathrin/AP-1 as trafficking molecules required for epical sorting and lumen positioning, a novel in vivo function for these proposed raft components and an unexpected trafficking route for this classical post-Golgi vesicle coat and its adaptor. Here, we will characterize this new vesicular sorting pathways for apical polarity and lumen morphogenesis by functionally, genetically, morphologically and biochemically characterizing some of the other molecules suggested by these screens are to participate in polarity regulation and lumen formation, and explore their relationship to SLs, clathrin/AP-1, each other and classical polarity determinants such as the PARs in this function. Specifically, we will assess the role of the actin cytoskeleton in apical sorting via three identified actin modulators (Aim1);analyze the relationship of PAR-6 and CDC-42 to SLs, clathrin/AP-1 and these actin modulators (Aim2);and delineate the components and routes of this new apical sorting pathway by integrating multiple trafficking-related genes also identified as involved in apical membrane biogenesis by our multi-and unicellular tubulogenesis screens (Aim3). Given the fundamental nature of polarity and the high structural and functional conservation of all molecules examined, the results of these studies should be directly relevant to human epithelial polarity and the related human diseases, two examples being cancer( associated with loss of polarity) and diseases of internal organs (all composed of, and functioning through, polarized tubular epithelia).

Public Health Relevance

We propose to analyze polarity and apical membrane modeling (lumen formation) in tubular epithelia, using the simple transparent roundworm C. elegans as a model organism, with a track record of discovering basic biological principles that also operate in humans. Our analysis should contribute to the understanding of polarity, a basic requirement for all multicellular organisms, and the diseases connected to its loss (such as cancer). It should also contribute to our understanding of diseases of internal organs (all composed of polarized tubular epithelia), specifically of developmental diseases such as the fatal orphan Microvillus Inclusion Disease, where additional lumens form inside intestinal cells, a defect also found in the C. elegans mutants we are studying.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Development - 1 Study Section (DEV1)
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Dunsmore, Sarah
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Massachusetts General Hospital
United States
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