Deletion of the cytoplasmic domain of N-cadherin reduces, but does not eliminate, traction force-transmission

https://doi.org/10.1016/j.bbrc.2016.08.173Get rights and content

Highlights

  • N-cadherin mediated traction force analysis of HGF-transformed MDCK cells.

  • The assembly of N-cadherin puncta requires the cytoplasmic domain of N-cadherin.

  • Loss of N-cadherin cytoplasmic domain reduces, but does not eliminate, traction.

Abstract

Collective migration of epithelial cells is an integral part of embryonic development, wound healing, tissue renewal and carcinoma invasion. While previous studies have focused on cell-extracellular matrix adhesion as a site of migration-driving, traction force-transmission, cadherin mediated cell-cell adhesion is also capable of force-transmission. Using a soft elastomer coated with purified N-cadherin as a substrate and a Hepatocyte Growth Factor-treated, transformed MDCK epithelial cell line as a model system, we quantified traction transmitted by N-cadherin-mediated contacts. On a substrate coated with purified extracellular domain of N-cadherin, cell surface N-cadherin proteins arranged into puncta. N-cadherin mutants (either the cytoplasmic deletion or actin-binding domain chimera), however, failed to assemble into puncta, suggesting the assembly of focal adhesion like puncta requires the cytoplasmic domain of N-cadherin. Furthermore, the cytoplasmic domain deleted N-cadherin expressing cells exerted lower traction stress than the full-length or the actin binding domain chimeric N-cadherin. Our data demonstrate that N-cadherin junctions exert significant traction stress that requires the cytoplasmic domain of N-cadherin, but the loss of the cytoplasmic domain does not completely eliminate traction force transmission.

Introduction

A unique feature of epithelial cell migration is their ability to move together as a cohesive unit. Collective cell migration is an essential feature in both physiological and pathological processes [1], [2]. Previous studies have focused on migration-driving, force-transmission through cell-extracellular matrix adhesion, yet, physical contacts between neighboring cells can also serve as a site of traction force-transmission. Recent studies uncovered an important role of molecular linkers between the cadherin complex and the actin cytoskeleton. Similar to the integrin complex at focal adhesions, the cadherin complex is regulated by force-sensitive proteins thought to control traction force necessary for collective cell migration [3].

Collective cell migration depends on adhesive properties at the sites of cell-extracellular matrix and cell-cell adhesion. For example, Madin-Darby canine kidney (MDCK) epithelial cells with abundant junctional proteins migrate as collective units on a 2D surface [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. The addition of Hepatocyte Growth Factor (HGF), however, induces MDCK cells to rapidly adapt a mesenchymal phenotype and migrate as single cells on a 2D surface [6], [17], [18], [19], [20]. These data suggest that traction force onto cell-extracellular matrix is sufficient to move away from neighboring cells and disrupt cell-cell contacts. In contrast, HGF-treated MDCK cells collectively migrate while maintaining cell-cell contacts in a 3D matrix [17], [21], [22], [23]. This is in part due to the soft 3D matrix preventing efficient traction force transmission to the matrix, thus limiting cells from moving away from each other. Instead, HGF-treated MDCK cells maintain cell-cell contacts while invading a 3D matrix.

Interestingly, HGF-treated MDCK cells migrate more efficiently as a cell cluster, which requires N-cadherin adhesion proteins [17], [24]. Unlike brief exposure of HGF to MDCK cells, prolonged exposure of HGF to MDCK cells induces complete Epithelial-to-Mesenchymal Transition (EMT), which decreases the level of epithelial markers (e.g., E-cadherin) and increases the level of mesenchymal markers (e.g., N-cadherin) [24]. In the absence of the full-length N-cadherin, the transformed MDCK cells do not migrate and remain single cells in a 3D matrix, while in the absence of the extracellular domain of N-cadherin, the cells migrate but as single cells [24]. Therefore, the adhesive activity of the N-cadherin extracellular domain is required for 3D collective migration of the transformed MDCK cells. One possible explanation for the efficient collective cell migration is N-cadherin's ability to transmit traction forces along neighboring cells which propels individual cells along the cell cluster. Using the transformed MDCK cells and traction force sensing substrates, we sought to analyze traction forces transmitted via N-cadherin junctions.

Section snippets

Cell culture and reagents

MDCK epithelial cells were treated with HGF to induce EMT [24]. As a result, these transformed cells down-regulated E-cadherin and up-regulated N-cadherin as the primary cell-cell adhesion protein. The cells expressing mutant cadherins were generated by transfecting both shRNA against canine N-cadherin and tandem dimer Tomato tagged, shRNA resistant mouse N-cadherin. The detail characterization of these N-cadherin mutant expressing cells were described previously [24]. The actin filaments were

Cell spreading on a purified N-cadherin coated surface

Traction force generated by the actin-myosin network is transmitted via adhesion receptors. Transformed MDCK epithelial cells express both N-cadherin and fibronectin receptors [24]. To specify the adhesion molecules responsible for traction force transmission, we coated an elastomer surface with fibronectin or the extracellular domain of N-cadherin, and plated the transformed epithelial cells in the presence or absence of calcium. Homophilic, trans-interactions of cadherins strongly depend on

Discussion

N-cadherin is often up-regulated in aggressive cancers, and N-cadherin mediated cell-cell adhesion may play a role in the progression of cancers [29], [30]. Therefore, the regulation of N-cadherin-mediated cell-cell adhesion and traction force transmission is likely to be critical for understanding how cancer cells invade and metastasize. In our study, N-cadherin mediated junctions exert traction stress in the order of 100 Pa (Fig. 2, Fig. 4A). This is consistent with previous studies using

Acknowledgements

We thank Dr. Kent Leach (University of California Davis) for the use of the Discovery HR2 Hybrid rheometer. This work was supported by NIH EUREKA GM094798, UC Davis Bridge Fund, NIH R03 EB021636 and NSF 1562095 (all to SY).

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