Dynamic microtubules regulate the local concentration of E-cadherin at cell-cell contacts

Samantha J. Stehbens 2 Andrew D. Paterson 1 2 Matthew S. Crampton 2 Annette M. Shewan 2 Charles Ferguson 0 2 Anna Akhmanova 3 Robert G. Parton 0 2 Alpha S. Yap ) 2 0 Centre for Microscopy and Microanalysis, The University of Queensland , St Lucia, Brisbane, Queensland 4072 , Australia 1 School for Biomedical Science 2 Division of Molecular Cell Biology, Institute for Molecular Bioscience 3 MGC Department of Cell Biology and Genetics, Erasmus Medical Center , 3000 DR Rotterdam , The Netherlands - In contrast to the well-established relationship between cadherins and the actin cytoskeleton, the potential link between cadherins and microtubules (MTs) has been less extensively investigated. We now identify a pool of MTs that extend radially into cell-cell contacts and are inhibited by manoeuvres that block the dynamic activity of MT e plus-ends (e.g. in the presence of low concentrations of c n nocodazole and following expression of a CLIP-170 ice mutant). Blocking dynamic MTs perturbed the ability S of cells to concentrate and accumulate E-cadherin at ll cell-cell contacts, as assessed both by quantitative C immunofluorescence microscopy and fluorescence recovery e f after photobleaching (FRAP) analysis, but did not affect lo either transport of E-cadherin to the plasma membrane or na the amount of E-cadherin expressed at the cell surface. This ru indicated that dynamic MTs allow cells to concentrate Eo J Introduction Classical cadherin adhesion molecules function as membranespanning macromolecular complexes. The binding properties of the cadherin ectodomains support surface adhesion and cellcell recognition, whereas the cytoplasmic tails can interact with a range of proteins that couple cadherins to cell signalling pathways and to the cytoskeleton (Perez-Moreno et al., 2003). Importantly, cadherin-based cell-cell contacts are dynamic adhesive structures (Adams et al., 1998). Even the apparently commonplace observation that cadherins accumulate at cellcell contacts reflects the ongoing interplay between local adhesive remodelling (Gumbiner, 2000), cytoskeletal association (Adams and Nelson, 1998; Shewan et al., 2005) and the trafficking and turnover of cadherins to and from the plasma membrane (Bryant and Stow, 2004). Such interplay, in turn, probably arises from dynamic interactions between cadherins and the cytoskeleton that are coordinated at the cell cortex by membrane-local cell signalling (Gumbiner, 2000; Perez-Moreno et al., 2003; Yap and Kovacs, 2003). The microtubule (MT) cytoskeleton is a major determinant of cortical dynamics in a wide range of circumstances and MTs can interact with the cortices of animal cells in a variety of ways. One particularly striking interaction involves MT plus (+)-ends, which are commonly oriented towards the cell cadherin at cell-cell contacts by regulating the regional distribution of E-cadherin once it reaches the cell surface. Importantly, dynamic MTs were necessary for myosin II to accumulate and be activated at cadherin adhesive contacts, a mechanism that supports the focal accumulation of E-cadherin. We propose that this population of MTs represents a novel form of cadherin-MT cooperation, where cadherin adhesions recruit dynamic MTs that, in turn, support the local concentration of cadherin molecules by regulating myosin II activity at cell-cell contacts. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/119/9/1801/DC1 periphery (Akhmanova and Hoogenraad, 2005; Gundersen et al., 2004). Dynamic instability allows these (+)-ends to grow outwards and potentially explore peripheral structures (Howard and Hyman, 2003), including integrin-based focal adhesions (Small and Kaverina, 2003), as well as regions of the free cell surface (Gundersen et al., 2004). Moreover, there is increasing evidence that such interactions with MT(+)-ends affect cellular processes at the cortex, including the dynamics of integrin adhesion complexes and actin cytoskeletal activity (Rodriguez et al., 2003). MT(+)-ends might exert these effects by facilitating vesicular transport to the cell surface (Watanabe et al., 2005), as well as by delivering regulatory molecules to the cortex (Rodriguez et al., 2003). In contrast to the well-established connection between cadherins and the actin cytoskeleton (Adams and Nelson, 1998), less is known about the potential relationship between cadherins and MTs. MTs are often identified in close proximity to cadherin contacts, where they are reported to organise in a variety of patterns that include running parallel to the lateral cell surface in polarised Madin-Darby canine kidney (MDCK) cells (Bacallao et al., 1989) and extending towards contacts in myoblasts (Mary et al., 2002). Furthermore, several recent studies have suggested molecular mechanisms that can link cadherins to MTs (Kaufmann et al., 1999). Thus, -catenin is reported to bind the MT-based motor dynein (Ligon et al., 2001), N-cadherin can form a complex with the KIF3 kinesin (Teng et al., 2005), and p120 catenin might interact both with conventional kinesin (Chen et al., 2003) and directly with MTs themselves (Yanagisawa et al., 2004). Without yet providing a comprehensive picture, these findings suggest the potential for cadherins to associate physically with MTs. There is also emerging evidence for functional interaction between cadherins and MTs. Cell-cell contact in lung epithelial cells was reported to stabilise the dynamic behaviour of MT(+)-ends (Waterman-Storer et al., 2000), whereas Ncadherin adhesions could stabilise MT minus-ends in cytoplasts (Chausovsky et al., 2000). Therefore, cadherinbased adhesion can regulate MT dynamics. By contrast, MT integrity might also affect cadherins, as depolymerisation of MTs disrupted the morphology and organisation of E-cadherinbased contacts in thyroid monolayers (Waterman-Storer et al., 2000; Yap et al., 1995), whereas low concentrations of nocodazole that affect MT(+)-end dynamics perturbed the distribution of catenins at contacts between newt lung epithelial cells (Waterman-Storer et al., 2000). One way in which MTs might influence cell-cell contacts is through kinesin-based transport of cadherin-containing vesicles, which have been implicated in both the maintenance and reassembly of cell-cell adhesions (Chen et al., 2003; Mary et al., 2002). Together, these disparate observations suggest the capacity for cadherin contacts to interact with the MT cytoskeleton, without yet providing a detailed understanding of what this could entail. We sought to pursue this possibility in this study. We identified a subpopulation of MTs that radiate into Ecadherin-based cell-cell contacts with their (+)-ends oriented towards cadherin adhesions. Importantly, the integrity of dynamic MTs was necessary both for cells to accumulate Ecadherin locally and to activate myosin II at cell-cell contacts, thus revealing a novel role for these MTs in controlling the regional distribution o (...truncated)