Tubulin Modifications Affect Monolayer Formation and Apical Trafficking in Epithelial Cells

To analyze the apical and basolateral transport processes in polarized cells it was necessary to check the arrangement of different tubulin modifications by using immunofluorescence microscopy. In MDCK cell cysts (grown in Matrigel) detyrosinated and tyrosinated microtubules were equally distributed along the apical-basolateral axis (Figure 1A). Neither detyr- nor tyr-tubulin preferentially concentrates at one of the two cell poles. The distribution of detyr- and tyr-tubulin on single microtubules was then analyzed by high-resolution GSD microscopy (Leica SR GSD Leica Microsystems, Wetzlar, Germany) of MDCK cells. Because this technique requires relatively flat objects for optimal results, the MDCK wild type cells were grown on coverslips for only one day. After a five-minute fixation with ice- cold methanol the cells were blocked for 1 hour with 1 % milk powder in PBS++ (PBS with 1 mM CaCl2 and 1 mM MgCl2). The immunostaining was performed by using anti-alpha tubulin, anti-detyr-tubulin or anti-tyr-tubulin primary antibodies for 2 hours, which were then labeled with Alexa488- or Alexa647-coupled secondary antibodies for 1 hour. The coverslips were sealed onto the cavity of a microscope slide in 10 mM MEA (β-Mercaptoethylamine) in PBS and by using Twinsil® in a ratio of 1:1. As depicted in Figure 1B, alpha tubulin antibodies were evenly distributed along the microtubule. If specific antibodies for tyr- or detyr-tubulin were used, short detyr-tubulin sections of up to 1 µm length were disrupted by longer segments of tyr-tubulin, which was also concentrated at the tubulus ends. So detyr- and tyr- tubulin were arranged like a pearl cord along microtubules. Similar observations based on electron microscopy have been described before (Geuens et al., 1986).

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