Microtubule Network is Necessary to Direct and Maintain The Apical Localization of Slo1 Channels in Epithelial Cells

Abstract

The cytoskeleton plays a key role in different cellular processes such as cell motility, muscle contraction, mitosis and maintenance of cell shape. In polarized cells, microtubules are involved mainly in the apical targeting of proteins. In MDCK cells, Slo1 channels are mainly expressed at the apical surface. Similarly, transiently transfected MDCK cells target Slo1 channels to their apical surface. To study the role of the microtubules on apical localization of Slo1 channels, we used a viral construct of Slo1 attached to EGFP (Slo1-EGFP) to induce the expression of Slo1. After 72 hrs of infection, cells were either treated with vehicle (control) or with 33 μM nocodazole to disrupt the microtubules. Cells were fixed and immunolabeled with anti α-tubulin to visualize the microtubule network. Using high-resolution confocal microscopy, we found that in control cells the microtubules are localized towards the apical surface, as Slo1. After nocodazole treatment, the microtubule network is completely disrupted and Slo1-EGFP is observed in both apical and basolateral surfaces. To further understand the mechanisms of Slo1 targeting in MDCK cells, we blocked protein synthesis with 1 μg/ml cycloheximide simultaneously with microtubule disruption. In these cells, Slo1-EGFP expression, although still localized at both surfaces, was less prominent at the basolateral surface than without cycloheximide. This result indicates that a part of Slo1-EGFP localized at the basolateral surface after microtubule disruption is redirected from the apical surface and a part of these proteins are newly synthesized. Thus, our results show that in MDCK cells the microtubule network plays an important role in Slo1 apical localization and that normal Slo1 traffic is likely transcytotic first reaching the basolateral surface, then traveling to the apical surface via the microtubule network. Supported by NIH.