The association of microtubules with tight junctions is promoted by cingulin phosphorylation by AMPK

J Cell Biol. 2013 Nov 25;203(4):605-14. doi: 10.1083/jcb.201304194.

Abstract

Epithelial cells characteristically have noncentrosomal microtubules that are arranged in the apicobasal direction. In this paper, we examined cell sheets formed by an epithelial (Eph4) cell line by structure illumination microscopy and found a previously not clearly described planar apical network of noncentrosomal microtubules (MTs) in which the sides of the MT bundles were associated with tight junctions (TJs). In a gel overlay assay with taxol-stabilized MTs, cingulin showed strong binding to MTs, and a domain analysis showed that this binding occurred through cingulin's N-terminal region. The association of planar apical MTs with TJs was compromised by cingulin knockdown (KD) or the expression of dephosphomimetic mutants of cingulin at its adenosine monophosphate-activated protein kinase (AMPK) target sites, whereas phosphorylation at these sites facilitated cingulin-tubulin binding. In addition, although wild-type colonies formed spheres in 3D culture, the cingulin KD cells had anisotropic shapes. These findings collectively suggest that the regulated cingulin-MT association has a specific role in TJ-related epithelial morphogenesis that is sensitive to metabolic homeostasis-related AMPK activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases / metabolism*
  • Amino Acid Sequence
  • Animals
  • Calcium / metabolism
  • Cell Polarity
  • Centrosome / metabolism
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism
  • Epithelium / growth & development
  • Epithelium / metabolism
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Antibody Technique
  • Gene Knockdown Techniques
  • HEK293 Cells
  • Humans
  • Intercellular Junctions / metabolism
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism*
  • Mice
  • Microtubules / metabolism*
  • Models, Biological
  • Molecular Sequence Data
  • Morphogenesis
  • Phosphorylation
  • Phosphoserine / metabolism
  • Protein Structure, Tertiary
  • Sus scrofa
  • Tight Junctions / metabolism*
  • Tubulin / metabolism

Substances

  • Cgn protein, mouse
  • Membrane Proteins
  • Tubulin
  • Phosphoserine
  • AMP-Activated Protein Kinases
  • Calcium