Studies on the development and maintenance of epithelial cell surface polarity with monoclonal antibodies.

Abstract

We examined epithelial cell surface polarity in subconfluent and confluent MadinDarby canine kidney (MDCK) cells with monoclonal antibodies directed against plasma membrane glycoproteins of 35,000, 50,000, and 60,000 mol wt. The cell surface distribution of these glycoproteins was studied by immunofluorescence and immunoelectron microscopy. At the ultrastructural level, the electron-dense reaction product localizing all three glycoproteins was determined to be uniformly distributed over the apical and basal cell surfaces of subconfluent MDCK cells as well as on the lateral surfaces between contacted cells; however, after formation of a confluent monolayer, these glycoproteins could only be localized on the basal-lateral plasma membrane. The development of cell surface polarity was followed by assessing glycoprotein distribution with immunofluorescence microscopy at selected time intervals during growth of MDCK cells to form a confluent monolayer . These results were correlated with transepithelial electrical resistance measurements of tight junction permeability and it was determined by immunofluorescence that polarized distributions of cell surface glycoproteins were established just after electrical resistance could be detected, but before the development of maximal resistance . Our observations provide evidence that intact tight junctions are required for the establishment of the apical and basal-lateral plasma membrane domains and that development of epithelial cell surface polarity is a continuous process .The specialized functions of epithelia such as absorption, secretion, and vectorial ion transport are dependent on the existence of two distinct cell surfaces; the apical (or mucosal) domain which is separated from the basal-lateral (or serosal) surface by a circumferential occluding belt of tight junctions (1). These two membrane domains are in contact with different environments and each exhibits unique structural and biochemical components that are responsible for the specialized physiological properties of epithelia .Ultrastructural examination of a variety of epithelial tissues has demonstrated structural polarity with plasma membrane specializations such as microvilli and cilia being confined to the apical cell surface while intercellular junctions are localized to the basal-lateral membrane (1, 2). Structural polarity has also been obseved by freeze-fracture electron microscopy with the intramembrane particle density being higher on the basal-lateral than the apical plasma membrane (3-5). Biochemical studies of purified apical and basal-lateral mem-

The Syntaxin 4 N Terminus Regulates Its Basolateral Targeting by Munc18c-dependent and -independent Mechanisms

“…Rabbit anti-syntaxin 4 (Calbiochem, San Diego, CA), rabbit anti-syntaxin 3 (7), rabbit anti-GFP (Rockland, Gilbertville, PA), mouse anti-GFP (Roche Applied Science), rabbit anti-FLAG, mouse anti-FLAG M2, mouse anti-Na,K-ATPase ␤1 chain, rabbit anti-SNAP23, mouse anti-E-cadherin DECMA (Sigma), mouse anti-E-cadherin, mouse anti-␤1-integrin, mouse syntaxin 4 (BD Biosciences), rat anti-␤1-integrin clone AIIB2 (17), rat anti-ZO-1 clone R40.76 (18), rabbit anti-claudin 2 (Invitrogen), mouse anti-p58 clone 6.23.3 (19), mouse anti-gp135 clone 3F21D8 (20), mouse anti-LAMP2 clone AC17 (21), rabbit anti-occludin (Zymed Laboratories Inc.), goat anti-Scribble, rabbit anti-␤-catenin (Santa Cruz Biotechnology), and rabbit anti-syntaxin 2 and mouse anti-sec6 (StressGen Biotechnologies, Victoria, Canada). The rabbit anti-canine munc18c was raised to a C-terminal peptide of canine munc18c as immunogen (Sigma).…”

Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras.

“…In most epithelial cells, the Na+,K÷-ATPase, for example, is located in the basolateral domain (Louvard, 1980), whereas other proteins, such as the amiloride sensitive Na ÷ channel, are predominantly expressed on the apical surface (Simons and Fuller, 1985;Rodriguez-Boulan and Nelson, 1989). In addition, the restriction of several plasma membrane antigens of unknown function to specific domains has been demonstrated using mAbs (Balcarova-Stiinder et al, 1984;Hertzlinger and Ojakian, 1984). The unique protein and lipid composition is generated by sorting membrane components along specialized transport pathways to their respective domains and preventing the components of the distinct domains from becoming intermixed (Caplan and Matlin, 1989).…”

AP1B sorts basolateral proteins in recycling and biosynthetic routes of MDCK cells

“…Moreover, domain-selective biotinylation, followed by Western blot analysis of polarized (4.5 d) 1B-KD MDCK cells revealed a dramatic redistribution of TfR and ␤1-integrin (Ϸ40%) to the apical surface, whereas other basolateral proteins were either marginally affected (e.g., E-cadherin) or not affected (e.g., the ␤-subunit of Na, K-ATPase). Two apical markers, gp114 (42) and gp135 (43), displayed normal polarity in 1B-KD MDCK cells (Fig. 3).…”