Effects of microtubule disruption on endocytosis, membrane recycling and polarized distribution of Aquaporin-1 and gp330 in proximal tubule cells

Microtubules are critically involved in membrane traffic and maintenance of epithelial cell polarity. In this study we examined the effect of microtubule disruption by colchicine on 1) the subcellular organization of the apical endocytic apparatus, 2) apical endocytosis, and 3) subcellular distribution of gp330 and Aquaporin-1 water channels in renal proximal tubule cells. Rats were treated in vivo with colchicine for 5 h before fixation of the kidneys, and sections of proximal tubules were studied using electron microscopy, morphometry and immunocytochemistry. In proximal tubule cells from colchicine-treated animals, virtually no endocytic invaginations are present, indicating that colchicine blocks the formation of endocytic invaginations. Furthermore, no large endocytic vacuoles are present, also consistent with a block of endocytosis. This was confirmed by functional studies revealing a colchicine-induced arrest in apical endocytosis of peroxidase. There is a marked reduction in dense apical tubules (the exocytic vehicle for membrane recycling) but an extensive accumulation of small vesicles, suggesting a disruption in membrane recycling. This disruption may be primary or secondary to the block of endocytosis. Immunofluorescence and immunoelectron microscopy reveal extensive colchicine-induced changes in the subcellular distribution of gp330 and Aquaporin-1. The localization of gp330 is normally restricted to the apical endocytic apparatus. However, after colchicine treatment gp330 is localized to numerous vesicles distributed throughout the entire cytoplasm, as previously shown. Also Aquaporin-1 water channels, which are normally almost exclusively present in the plasma membranes, are redistributed to small vesicles in addition to the plasma membrane localization after colchicine treatment. In summary, the present study suggests that cytoplasmic microtubules are critically involved in 1) formation and stabilization of endocytic invaginations, 2) formation of large endocytic vacuoles, 3) apical endocytosis, 4) maintaining the polarized distribution of vesicles in the apical part of the cell, and 5) maintaining the polarized organization of gp330 in the apical endocytic apparatus, and maintaining Aquaporin-1 water channels in the plasma membranes.