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Thursday, August 23, 2012
Hongjie Zhang, PhD.; Division of Pediatric Hematology-Oncology, Mass General Hospital for Children; Instructor of Pediatrics, Harvard Medical School
Clathrin and intestinal polarity: Growth regulation and the generation and maintenance of epithelial polarity are two basic cellular mechanisms whose disruption/deregulation are important causes of human diseases, such as cancer and inherited defects of organ development. For instance, microvillus inclusion disease (MVID) is an inherited intestinal failure syndrome of infancy, characterized clinically by watery diarrhea and malabsorption from birth, and ultrastructurally by shortening or absence of apical microvilli and the appearance of ectopic lumens inside the cytoplasm of the enterocytes, with microvilli pointing inwards (microvillus inclusions). The precise etiology of MVID remains unclear, but the similarity of this phenotype with the vacuolar apical compartment in epithelial cell lines has led to the proposition that the pathogenesis of MVID includes defects in apical-membrane-trafficking-dependent epithelial polarity regulation. Recently, the disease has been linked to mutations in the motor protein MYO5B and the GTPase RAB8, molecules that play a role in actin-based protein and organelle transport. However, the questions how these genes contribute to the development of the disease and how MVID specific complex developmental morphogenesis defects occur are not easy to address in the complex vertebrate system, and not at all in humans.
Here in Dr. Verena Gobel’s lab, we found that C. elegans, the soil worm, is emerging as an effective tool for analyzing the pathogenesis of human diseases. The lab is interested in identifying genes involved in epithelial polarity and growth regulation. Worms have several experimental advantages, including their rapid life cycle, genetically tractable character, genome-wide resources, accessibility to embryonic analyses and the large number of individuals that can be generated, which make them ideal for sophisticated genetic screens, and for the analysis of complex multigenic disorders.
We have performed such a genetic screen to identify genes involved in epithelial polarity, tubulogenesis and growth regulation. This screen identified many genes involved in polarity (potentially cancer-related genes) that were also involved in organ morphogenesis, as well as many intriguing phenotypes mimicking human congenital organ diseases (for example, intestinal and kidney diseases). The C. elegans intestine is comprised of twenty large epithelial cells that are mostly positioned as bilaterally symmetrical pairs to form a single-layered epithelial tube. Polarized intestinal cells contain a distinct apical membrane facing the central lumen and basolateral surfaces contacting adjacent cells and the extracellular matrix. The C. elegans “kidney” – the excretory canals -- is built from one cell that expands into an H-shaped canal system.
After completion of the screen, we began to investigate an intriguing intestinal phenotype: the transformation of a contiguous central, into multiple ectopic, intestinal lumens. This phenotype turned out to be very closely reminiscent of human intestinal defects observed in MVID and related syndromes from both a phenotypic and mechanistic point of view. First, detailed analysis indicated that multiple-lumen formation is caused by a conversion of apicobasal polarity in the intestinal epithelium, with displacement of many apical membrane components to the lateral membrane and/or cytoplasm (Figure 1A). Second, ultrastructural analysis by transmission electron microscopy (TEM) revealed MVID like morphological defects: microvilli and the terminal web were lost from the apical membrane and acquired at the lateral ectopic lumenal membrane (Figure 1B). Third, in animals with severe defects, we observed early polarity defects together with cytoplasmic lumenal membrane inclusions (Figure 1C). Fourth, the polarity defect appeared to be caused by a vesicular trafficking-dependent mechanism by which apicobasal domains are maintained through directionally targeting submembraneous, transmembraneous and polarity components (Figure 1D).
Our studies identified a couple of evolutionarily conserved components underlying this MVID-like phenotype. As an example, we found a critical role of glycosphingolipids (GSLs)1, as presumed components of lipid rafts, in the apical trafficking process and the determination of apical membrane domains for the first time in vivo. The identification of several classical trafficking molecules -- CHC-1 (the clathrin heavy chain ortholog) and its two AP-1 adaptor subunits -- provided an even tighter connection of this MVID-like phenotype with a trafficking defect2. Clathrin/AP-1, as one major type of post-Golgi vesicle coats, was previously thought to be restricted to endocytic and basolateral membrane-directed routes in epithelia. Our subsequent genetic and functional studies suggested that clathrin/AP1, together with GSLs, contribute to MVID by their novel apical sorting role, for instance through their concurrent recruitment on GSL-rich endomembranes at the Golgi (Figure 1E).
There are several research avenues that have evolved out of our studies. In a continuous effort to understand the molecular mechanism of epithelial polarity establishment and maintenance, we are investigating the functions of other genes identified in our unbiased screen and implicated to be essential for this process. I am also planning to perform a suppressor screen to identify genes or compounds whose knocking down might suppress the polarity and organogenesis phenotype. This hopefully will enable us to understand the pathogenesis of cancer and congenital organ diseases further and provide a molecular basis for discovering novel treatment strategies for these conditions and related syndromes.
Figure Legend: C. elegans is a genetically tractable model for Microvillus Inclusion Disease. A. A genome-wide RNAi tubulogenesis screen identified a novel polarity and ecotopic lumen phenotype with displacement of apical markers to the basolateral membranes and subsequent ectopic lumen formation. B. TEM micrographs of cross sections through the C. elegans intestine, showing brushborder defects, microvillar atrophy and inclusions in mutant versus wild-type animals. C. Severe depletion of clathrin/AP-1 results in cytoplasmic inclusions in the early C. elegans embryonic intestine, in addition to basolateral ERM-1::GFP displacement. D. The biogenesis, localization and association of CHC-1- and GSL-rich vesicles require AP-1. E. Our working model: GSLs recruit clathrin via AP-1at the Golgi to direct apical trafficking, thereby regulating apical polarity and lumen formation during C. elegans tubulogenesis.
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