Abstract

The speed of cell migration on 2-dimensional (2D) surfaces is determined by the rate of assembly and disassembly of clustered integrin receptors known as focal adhesions. Different modes of cell migration that have been described in 3D environments are distinguished by their dependence on integrin-mediated interactions with the extra-cellular matrix. In particular, the mesenchymal invasion mode is the most dependent on focal adhesion dynamics. The focal adhesion protein NEDD9 is a key signalling intermediary in mesenchymal cell migration, however whether NEDD9 plays a role in regulating focal adhesion dynamics has not previously been reported. As NEDD9 effects on 2D migration speed appear to depend on the cell type examined, in the present study we have used mouse embryo fibroblasts (MEFs) from mice in which the NEDD9 gene has been depleted (NEDD9 −/− MEFs). This allows comparison with effects of other focal adhesion proteins that have previously been demonstrated using MEFs. We show that focal adhesion disassembly rates are increased in the absence of NEDD9 expression and this is correlated with increased paxillin phosphorylation at focal adhesions. NEDD9−/− MEFs have increased rates of migration on 2D surfaces, but conversely, migration of these cells is significantly reduced in 3D collagen gels. Importantly we show that myosin light chain kinase is activated in 3D in the absence of NEDD9 and is conversely inhibited in 2D cultures. Measurement of adhesion strength reveals that NEDD9−/− MEFs have decreased adhesion to fibronectin, despite upregulated α5β1 fibronectin receptor expression. We find that β1 integrin activation is significantly suppressed in the NEDD9−/−, suggesting that in the absence of NEDD9 there is decreased integrin receptor activation. Collectively our data suggest that NEDD9 may promote 3D cell migration by slowing focal adhesion disassembly, promoting integrin receptor activation and increasing adhesion force to the ECM.

Highlights

  • Cell adhesion is a prime determinant of the rate of cell migration speed on 2D surfaces [1]

  • NEDD9 suppresses focal adhesion disassembly To address whether NEDD9 plays an active role in regulating focal adhesion turnover we used mouse embryo fibroblasts (MEFs) expressing endogenous wild-type NEDD9 (WT) and NEDD92/2 MEFs (Figure 1A)

  • We confirmed that there was no difference in the peak YFP-paxillin fluorescence intensity at focal adhesions in the WT versus NEDD9 2/2 MEFs (Figure S1) differences in adhesion disassembly rates were not due to differences in exogenous YFP-paxillin expression between the cell lines

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Summary

Introduction

Cell adhesion is a prime determinant of the rate of cell migration speed on 2D surfaces [1]. In addition to the contribution that the concentration of extra-cellular matrix plays in cell speed, a role for focal adhesion dynamics has emerged. Clusters of integrin receptors are bound on the external surface to extra-cellular matrix ligands and are associated with filaments of polymerized actin in the cytoplasm. The resulting tensile force generated at the focal adhesions in turn governs integrin activation and cell motility. The rate of focal adhesion assembly and disassembly contributes to 2D cell migration speed [2,3,4,5,6]

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