Tubulin Modifications Affect Monolayer Formation and Apical Trafficking in Epithelial Cells

Application for GSD microscopy

December 05, 2012

The development and maintenance of polarized epithelial cells requires the establishment of complicated subcellular machinery. We studied the role of post-translational tubulin modifications within this process. At first, the distribution of detyrosinated microtubules was assessed in MDCK cells via immunofluorescence microscopy. No preferential accumulation of tyrosinated or detyrosinated microtubules could be detected at the apical or basal cell poles in epithelial cell cysts. However, during monolayer formation, the quantities of detyrosinated tubulin increased significantly over time. Arrangements of this tubulin modification along single microtubules were studied in greater detail by GSD microscopy. Here, we observed alternating stretches of detyrosinated and tyrosinated tubulin. When the balance between tyrosinated and detyrosinated tubulin molecules was altered by overexpression of the enzyme tubulin tyrosine ligase, the formation of epithelial monolayers as well as subcellular trafficking events were heavily disturbed.

 

Introduction

Polar epithelial cells can be found on the surface of multi-cellular organisms and can cover cavities throughout the body and serve in this way as protection barrier. They thus regulate the secretion and exchange of different substances. One of the most important issues in the development and maintenance of a polarized epithelium is the specific composition of the apical and the basolateral plasma membrane. Therefore, proteins and lipids are segregated into distinct vesicle populations destined for one of the two membrane domains. The underlying process of vesicle trafficking involves the integrity of cytoskeletal tracks formed by actin microfilaments and microtubules. Especially microtubules are required for long-range transport processes and consequently the integrity of an epithelial cell sheet can be disturbed by alterations in the microtubule composition (Yap et al., 1995). One of the early described post-translational modifications is the tyrosination-detyrosination cycle of tubulin, where a tyrosine residue is added (tyrosination) or removed (detyrosination) from the C-terminus of α-tubulin. Dynamic microtubules are considered to be tyrosinated (tyr-) while the detyrosinated (detyr-) form can be found in stable microtubules inhibiting microtubule disassembly (Peris et al., 2009). In this article we show that an intervention into this balance by overexpression or knockdown of one of the responsible enzymes, the tubulin tyrosine ligase (TTL, which adds tyrosine residues to tubulin), alters the balance between tyr- and detyr-microtubules, which results in the disruption of epithelial monolayer formation and affects the apical protein trafficking.

Distribution of detyrosinated and tyrosinated tubulin in polarized epithelial cells and fluctuations during the polarization process

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).

Fig. 1: (A) MDCK cell cysts grown for 7 d in Matrigel were studied by confocal microscopy. Antibodies directed against detyrosinated (AlexaFluor488) and tyrosinated tubulin (AlexaFluor546) were used for immunostaining. Scale bar, 20 µm. (B) The immunostaining for GSD microscopy was performed with AlexaFluor488 and AlexaFluor647 to label detyr-, tyr- or alpha-tubulin on cells one day after seeding. Scale bars, 5 µm, enlarged sections, 1 µm. (C) MDCK and MDCKTTL-GFP cells (green) together were immunostained 3 days after seeding with specific detyr-tubulin antibodies (AlexaFluor546). Scale bars, 20 µm (D) MDCK and MDCKTTL-GFP cells were seeded at identical densities on coverslips, incubated for one day and fixed for immunostaining with anti-tubulin antibodies (Alexa546). Scale bars, 20 µm. Nuclei are indicated in blue.

Depletion of detyr-tubulin by tubulin tyrosine ligase

To approve the effect of changes in tyr- and detyr-tubulin during the polarization process we impaired the posttranslational modification of tubulin by stably overexpressing the tubulin tyrosine ligase in MDCK cells. These MDCKTTL-GFP cells show a dramatic decrease in detyr-tubulin (Figure 1C). The detyr-tubulin deficient MDCK strain (MDCKTTL-GFP) was then analyzed concerning alterations in cell morphology. One day after plating MDCKTTL-GFP cells revealed a tendency to join into islands (Figure 1D) and prematurely polarize. Further studies also indicated that TTL expression modulates the surface delivery of apical proteins in MDCK cells (Zink et al., in press).

Conclusion

In this study we analyzed the role of post-translational tubulin modifications in the generation and maintenance of polarized epithelial cells. The distribution of detyr- and tyr-tubulin was investigated by super-resolution GSD microscopy in greater detail, so that we were able to confirm observations that were previously based on electron microscopy with a fluorescence microscopic technique.

Comments