The general goal of this project is to elucidate the process of membrane differentiation and secretory function in intestinal goblet cells. A goblet cell subclone (18-N2) of the HT29 colon carcinoma line will be used to obtain goblet cell membrane protein preparations. These will serve as immunogens to raise monoclonal antibodies with which we will identify and characterized goblet cell-specific membrane proteins. Selected plasma membrane antigens will serve as markers for commitment to the goblet cell phenotype, and for specific stages of differentiation. Monoclonal and polyclonal antibodies against these antigens will then be applied to normal intestinal mucosa to identify committed crypt cells, and cells at progressive stages of goblet cell differentiation. Similar immunologic methods will be used to identify membrane components of regulated mucin granules, first in 18-N2 cells, and then in normal human and mouse goblet cells. The time of expression of these components during goblet cell differentiation will be determined immunologically, and correlated with initial onset of mucin synthesis and packaging. These membrane markers will be used to elucidate patterns of segregation and sorting of membrane and soluble proteins into polarized regulated and constitutive secretory pathways of goblet cells. Entry of membrane proteins and mucins into these pathways will be directly visualized by EM immunocytochemistry, and the kinetics of transport determined by immunoprecipitation of pulse-radiolabeled antigens secreted by 18-N2 cell monolayers. The ability of 18-N2 goblet cells transfected with foreign genes to sort and package a regulated endocrine cell product (human growth hormone) and a non-regulated plasma cell product (immunoglobulin light chain) will be analyzed before and after differentiation of the regulated secretory pathway. Finally, sorting of these proteins will be followed in goblet cells of transgenic mice in which foreign gene products are expressed in intestinal epithelial cells. together, these studies will provide new information about stage-specific expression and polarized delivery of specific membrane and secretory proteins as goblet cells migrate along the crypt-villus axis. these phenomena are basic to normal intestinal secretory functions, and to maintenance of an effective epithelial barrier.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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General Medicine A Subcommittee 2 (GMA)
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Children's Hospital Boston
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