Abstract
BackgroundEpithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion.ResultsCalcium depletion resulted in disruption and internalization of epithelial TJs and AJs along with reorganization of perijunctional F-actin into contractile rings. Microtubules reorganized into dense plaques positioned inside such F-actin rings. Depolymerization of microtubules with nocodazole prevented junctional disassembly and F-actin ring formation. Stabilization of microtubules with either docetaxel or pacitaxel blocked contraction of F-actin rings and attenuated internalization of junctional proteins into a subapical cytosolic compartment. Likewise, pharmacological inhibition of microtubule motors, kinesins, prevented contraction of F-actin rings and attenuated disassembly of apical junctions. Kinesin-1 was enriched at the AJC in cultured epithelial cells and it also accumulated at epithelial cell-cell contacts in normal human colonic mucosa. Furthermore, immunoprecipitation experiments demonstrated association of kinesin-1 with the E-cadherin-catenin complex.ConclusionOur data suggest that microtubules play a role in disassembly of the AJC during calcium depletion by regulating formation of contractile F-actin rings and internalization of AJ/TJ proteins.
Highlights
Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity
In fully polarized SK-CO-15 cells cultured at normal concentration of extracellular calcium, AJ proteins E-cadherin and β-catenin and TJ proteins occludin and ZO-1 are localized at areas of cell-cell junctions producing a characteristic 'chicken wire' pattern
AJC in calcium-depleted SK-CO-15 cells is dependent on microtubule integrity, we examined this process in cells where microtubules were depolymerized with nocodazole
Summary
Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. Intercellular junctions are a characteristic morphological feature of differentiated epithelial cell monolayers They represent several types of multiprotein complexes assembled at distinct positions within the lateral plasma membrane in areas of cell-cell contacts. The integrity and barrier properties of epithelial cell monolayers are ensured by transmembrane TJ and AJ proteins that are engaged in trans-interactions with their partners on the opposing plasma membrane [2,5,6]. Such transmembrane components of TJs include occludin, members of the claudin family, and immunoglobulin-like proteins junctional adhesion molecule (JAM)-A and coxsackie adenovirus receptor [7,8]. One of the important functions of junctional cytosolic plaques is to provide a link between transmembrane TJ/AJ proteins and the cortical cytosketon [11] allowing efficient transduction of signals from intercellular junctions to the cell interior as well as "inside out signaling" from cytosolic compartments to intercellular contacts [1,12]
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