The Role of Tensin in Cell Migration and Fibronectin Fibrillogenesis
thesisposted on 24.05.2010, 12:44 by Jonathan David Howe
The tensin family of cytoskeletal-associated proteins are implicated in fibronectin fibrillogenesis and integrin-mediated cell migration. There are four members of the human tensin family. Tensin1, 2 and 3 are large proteins (~150-200 kDa) with widespread expression in adult tissues that are thought to couple integrins to the actin cytoskeleton, whereas tensin4 (cten) is a smaller protein (77 kDa) with a restricted expression profile that is not thought to interact with actin. To study the role of tensin in fibronectin fibrillogenesis, which is mediated by fibrillar adhesions, I first characterised the isoform specificities of several anti-tensin antibodies. These antibodies were then used to determine the tensin expression profile of human foreskin fibroblasts (HFFs), which were found to express tensin1, 2 and 3. Unfortunately, most of the antibodies did not work sufficiently well for immunofluorescence microscopy so the sub-cellular localisation of the tensins was largely investigated using GFP-tagged proteins. Tensin1, 2 and 3 localised to both focal and fibrillar adhesions, although relative distribution between these two adhesion types was isoform-specific. Since tensin3 preferentially localised to fibrillar adhesions, I used an siRNA approach to investigate its role in fibronectin fibrillogenesis, and found that tensin3 was required for this process. Although tensins play an important role in cell migration, it is unclear whether they act as positive or negative regulators. Therefore, I used over-expression and siRNAmediated knockdown to clarify the role of tensin1, 2 and 3 in both 2D and 3D migration. Interestingly, I found that modulating tensin expression did not affect the 2D migration of HFF, HEK293, or A2780 ovarian cancer cells. However, all three tensins were required for the 3D migration of A2780 cells in fibronectin-containing microenvironments. Over-expressing Rab25, a small GTPase up-regulated in aggressive ovarian cancers, in A2780 cells promotes invasive migration in 3D microenvironments by recycling and retaining integrin at the cell front (Caswell et al., 2007). Tensin was required for the invasive migration and morphology of Rab25-expressing A2780 cells. However, tensin depletion did not affect global α5 integrin recycling or the retention of photoactivated GFP-α5 integrin at the cell front. Interestingly, Rab25 over-expression was also found to promote tensin-dependent fibronectin fibrillogenesis in A2780 cells, suggesting that tensin promotes 3D migration by facilitating fibronectin fibril remodelling.