Endocytic trafficking from the small intestinal brush border probed with FM dye.
The small intestinal brush border functions as the body's main portal for uptake of dietary nutrients and simultaneously acts as the largest permeability barrier against pathogens. To enable this, the digestive enzymes of the brush border are organized in lipid raft microdomains stabilized by cross-linking galectins and intelectin, but little is known about the dynamic properties of this highly specialized membrane. Here, we probed the endocytic membrane trafficking from the brush border of organ-cultured pig intestinal mucosal explants by use of a fixable, lipophilic FM dye. The fluorescent dye readily incorporated into the brush border, and by 15 min faint but distinct punctae were detectable approximately 1 microm beneath the brush border, indicative of a constitutive endocytosis. The punctae represented a subpopulation of early endosomes confined to the actomyosin-rich terminal web region, and their number and intensity increased by 1 h, but trafficking further into the enterocyte was not observed except in immature epithelial cells of the crypts. A powerful ligand for receptor-mediated endocytosis, cholera toxin B subunit, increased apical endocytosis and caused membrane trafficking to proceed to compartments localized deeper into the cytoplasm of the enterocytes. Two major raft-associated brush border enzymes, alkaline phosphatase and aminopeptidase N, were excluded from endocytosis. We propose that the terminal web cytoskeleton, by inhibiting traffic from apical early endosomes further into the cell, contributes to the overall permeability barrier of the gut.
Hansen
Gert H
GH
Department of Cellular and Molecular Medicine, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.
Rasmussen
Karina
K
Niels-Christiansen
Lise-Lotte
LL
Danielsen
E Michael
EM
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D018826:CD13 Antigens / Q000378:metabolism; D002463:Cell Membrane Permeability; D002772:Cholera Toxin / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D020895:Enterocytes / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D007583:Jejunum / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008871:Microvilli / Q000378:metabolism; D011726:Pyridinium Compounds / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D013552:Swine; D046509:Tissue Culture Techniques; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism*
C092350:FM 4-64; C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D002772:Cholera Toxin; D000469:Alkaline Phosphatase; D018826:CD13 Antigens