Small molecule pinocytosis and clathrin-dependent endocytosis at the intestinal brush border: Two separate pathways into the enterocyte.

Pinocytosis at the small intestinal brush border was studied in postweaned porcine cultured mucosal explants, using the fluorescent polar probes Alexa hydrazide (AH, MW 570), Texas red dextran (TRD, MW ~ 3000), and Cascade blue dextran (CBD, MW ~ 10,000). Within 1 h, AH appeared in a string of subapical punctae in enterocytes, indicative of an ongoing constitutive pinocytosis. By comparison, TRD was taken up less efficiently into the same compartment, and no intracellular labeling of CBD was detectable, indicating that only small molecules are pinocytosed from the postweaned gut lumen. AH remained in the terminal web region in EEA-1-positive endosomes ("TWEEs") for at least 2 h, implying that the pinocytic uptake does not proceed towards a transcytic pathway. Like AH, cholera toxin B subunit (CTB) was readily internalized, but the two probes appeared in completely non-overlapping subapical compartments, indicating the existence of two different uptake mechanisms operating simultaneously at the brush border. CTB is internalized by clathrin-dependent receptor mediated endocytosis, but surprisingly the toxin also caused a rapid disappearance from the apical cell surface of two major brush border enzymes, alkaline phosphatase and aminopeptidase N, demonstrating the disruptive effect of this pathway. By immunofluorescence, caveolin-1 was hardly detectable in enterocytes, arguing against a caveolae-mediated uptake of AH, whereas the pinocytosis/phagocytosis inhibitors dimethyl amiloride and cytochalasin D both arrested AH uptake. We propose that the constitutive pinocytic mechanism visualized by AH contributes to maintenance of membrane homeostasis and to enrich the contents of lipid raft constituents at the brush border.

Caveolae in fibroblast-like synoviocytes: static structures associated with vimentin-based intermediate filaments.

The fibroblast-like synoviocyte is a CD13-positive cell-type containing numerous caveolae, both single and interconnected clusters. In unstimulated cells, all single caveolae at the cell surface and the majority of those localized deeper into the cytoplasm were freely accessible from the medium, as judged from electron microscopy of synoviocytes exposed to the membrane impermeable marker Ruthenium Red. Caveolar internalization could be induced by a CD13 antibody or by cholera toxin B subunit (CTB). Thus, in experiments using sequential labeling with Alexa 488- and 594-conjugated CTB, about 50% of CTB-positive caveolae were internalized by 5 min of chase, and these remained inaccessible from the cell surface for periods up to 24 h. No colocalization with an endosomal marker, EEA1, or Lysotracker was observed, indicating that internalized caveolae clusters represent a static compartment. Vimentin was identified as the most abundant protein in detergent resistant membranes (DRM's), and by immunogold electron microscopy caveolae were seen in intimate contact with intermediate-size filaments. These observations indicate that vimentin-based filaments are responsible for the spatio-temporal fixation of caveolae clusters. RECK, a glycosylphosphatidylinositol-anchored protein acting as a negative regulator of cell surface metalloproteinases, was also localized to the caveolae clusters. We propose that these clusters function as static reservoirs of specialized lipid raft domains where proteins involved in cell-cell interactions, such as CD13, can be sequestered by binding to RECK in a regulatory manner.