The regulation of cell motility by tropomyosin

Abstract

Metastasis accounts for over 90% of cancer related mortality, it is therefore of interest to further develop an understanding of the way in which cells migrate. At the leading edge of migrating cells, protrusion of the lamellipodium is driven by Arp2/3-mediated polymerisation of actin filaments. This dense, branched actin network is promoted and stabilised by cortactin. In order to drive filament turnover, Arp2/3 networks are remodelled by proteins such as GMF which blocks the actin-Arp2/3 interaction, and coronin 1B which acts by directing SSH1L to the lamellipodium where it activates the actin severing protein cofilin. It has been shown in vitro that cofilin-mediated severing of Arp2/3 actin networks results in the generation of new pointed ends to which the actin-stabilising protein tropomyosin (Tpm) can bind. The presence of Tpm in lamellipodia however has been controversial with studies reporting the absence of Tpms from lamellipodia, and others reporting their presence at or near lamellipodia. These opposing observations are partly due to the lack of appropriate reagents to detect the Tpms. This thesis reports that the Tpm isoforms 1.8/1.9 are enriched in the lamellipodium of mouse fibroblasts as detected with a novel, isoform-specific monoclonal antibody. RNAi-mediated silencing of Tpm1.8/1.9 led to an increase in Arp2/3 accumulation at the cell periphery paralleled by a reduction in cell speed and the persistence of lamellipodia, a phenotype consistent with coronin 1B-deficient cells. In the absence of coronin 1B or cofilin, Tpm1.8/1.9 protein levels are reduced while conversely, inhibition of Arp2/3 with CK666 led to an increase in Tpm1.8/1.9 protein. The findings presented in this thesis establish a novel regulatory mechanism within the lamellipodium whereby Tpm collaborates with Arp2/3 to promote lamellipodial persistence and cell motility. This study also provides a solution to the controversy found in the literature and serves as a broader paradigm by which to understand how cells can create and utilise multiple actin filament populations to achieve a singular biological outcome.