Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells.

Abstract

Abstract. We characterized the three-dimensional organization of microtubules in the human intestinal epithelial cell line Caco-2 by laser scanning confocal microscopy. Microtubules formed a dense network ",,4-/zm thick parallel to the cell surface in the apical pole and a loose network 1-/zm thick in the basal pole. Between the apical and the basal bundles, microtubules run parallel to the major cell axis, concentrated in the vicinity of the lateral membrane. Colchicine treatment for 4 h depolymerized 99.4 % of microtubular tubulin. Metabolic pulse chase, in combination with domain-selective biotinylation, immune and streptavidin precipitation was used to study the role of microtubules in the sorting and targeting of four apical and one basolateral markers. Apical proteins have been recently shown to use both direct and transcytotic (via the basolateral membrane) routes to the apical surface of Caco-2 cells. Colchicine treatment slowed down the transport to the cell surface of apical and basolateral proteins, but the effect on the apical proteins was much more drastic and affected both direct and indirect pathways. The final effect of microtubular disruption on the distribution of apical proteins depended on the degree of steady-state polarization of the individual markers in control cells. Aminopeptidase N (APN) and sucrase-isomaltase (SI), which normally reach a highly polarized distribution (110 and 75 times higher on the apical than on the basolateral side) were still relatively polarized (9 times) after colchicine treatment. The decrease in the polarity of APN and SI was mostly due to an increase in the residual basolateral expression (10% of control total surface expression) since 80% of the newly synthesized APN was still transported, although at a slower rate, to the apical surface in the absence of microtubules. Alkaline phosphatase and dipeptidylpeptidase IV, which normally reach only low levels of apical polarity (four times and six times after 20 h chase, nine times and eight times at steady state) did not polarize at all in the presence of colchicine due to slower delivery to the apical surface and increased residence time in the basolateral surface. Colchicinetreated cells displayed an ectopic localization of microvilli or other apical markers in the basolateral surface and large intracellular vacuoles. Polarized secretion into apical and basolateral media was also affected by microtubular disruption. Thus, an intact microtubular network facilitates apical protein transport to the cell surface of Caco-2 cells via direct and indirect routes; this role appears to be crucial for the final polarity of some apical plasma membrane proteins but only an enhancement factor for others.PITHELIAL cells characteristically display two plasma membrane domains, apical and basolateral, with different protein and lipid compositions, separated by tight junctions (for reviews, see references 55, 63). Both the cytoplasmic and submembrane cytoskeletons are also asymmetrically distributed. The polarized organization of ...

Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites.

“…Upon reestablishing secretory flow from the ER to the Golgi in this manner, transport from dispersed Golgi sites to the cell surface, as shown by occurs efficiently, but is no longer directed to specific regions of the cell surface. This interpretation offers a reasonable explanation to the conflicting data regarding the role of microtubules in secretion, where some studies have shown no effect of microtubule disruption Van De Moortele et al, 1993), while others have found significant effects (Redman et al, 1978;Boyd et al, 1982;Breitfield et al, 1990;Gilbert et al, 1991;Rennison et al, 1992). It also supports studies of Saraste and Svensson (1991) that suggested that ER-to-Golgi transport intermediates use microtubules to translocate into the Golgi region.…”

Nezha/CAMSAP3 and CAMSAP2 cooperate in epithelial-specific organization of noncentrosomal microtubules

“…In vivo, however, cells are tightly packed together, epithelial cells are apicobasally polarized, and microtubule minus ends are oriented toward the apical membrane (11,17,18). Therefore, it will be interesting to determine whether CAMSAPs can also regulate the orientation of microtubules in polarized epithelial cells; such studies are actually under way.…”

θ Isoform of Protein Kinase C Alters Barrier Function in Intestinal Epithelium through Modulation of Distinct Claudin Isotypes: A Novel Mechanism for Regulation of Permeability

“…In addition, Caco-2 cells closely resemble normal intestinal cells in that they express intestinal hydrolases such as sucrase-isomaltase and alkaline phosphatase. Furthermore, these cells are similar to native intestinal epithelial cells in that they have receptors for prostaglandins, growth factors, vasoactive intestinal peptide, low-density lipoprotein, insulin, and specific substrates such as dipeptides, fructose, glucose, hexoses, and vitamin B 12 (Gilbert et al, 1991;Meunier et al, 1995;Banan et al, 1998b). Accordingly, this cell line provides a suitable in vitro model for our studies.…”

“…Caco-2 cells were obtained from American Type Culture Collection (Manassas, VA) at passage 15. This widely used colonic cell line was chosen for our studies because they form monolayers that morphologically resemble intestinal cells, with defined apical brush borders and tight junctions, and a highly organized claudin-ring network upon differentiation (Gilbert et al, 1991;Meunier et al, 1995;Banan et al, 1998b). Caco-2 cells are a transformed cell line, and monolayers of tumor cells may respond differently than do nontransformed cells, including enterocytes in native tissue.…”