Tropomyosin Promotes Lamellipodial Persistence by Collaborating with Arp2/3 at the Leading Edge

At the leading edge of migrating cells, protrusion of the lamellipodium is driven by Arp2/3-mediated polymerization of actin filaments [1]. This dense, branched actin network is promoted and stabilized by cortactin [2, 3]. In order to drive filament turnover, Arp2/3 networks are remodeled by proteins such as GMF, which blocks the actin-Arp2/3 interaction [4, 5], and coronin 1B, which acts by directing SSH1L to the lamellipodium where it activates the actin-severing protein cofilin [6, 7]. 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-stabilizing protein tropomyosin (Tpm) can bind [8]. The presence of Tpm in lamellipodia, however, is disputed in the literature [9-19]. Here, we report that the Tpm isoforms 1.8/9 are enriched in the lamellipodium of fibroblasts as detected with a novel isoform-specific monoclonal antibody. RNAi-mediated silencing of Tpm1.8/9 led to an increase of Arp2/3 accumulation at the cell periphery and a decrease in the persistence of lamellipodia and cell motility, a phenotype consistent with cortactin- and coronin 1B-deficient cells [2, 7]. In the absence of coronin 1B or cofilin, Tpm1.8/9 protein levels are reduced while, conversely, inhibition of Arp2/3 with CK666 leads to an increase in Tpm1.8/9 protein. These findings establish a novel regulatory mechanism within the lamellipodium whereby Tpm collaborates with Arp2/3 to promote lamellipodial-based cell migration

The regulation of cell motility by tropomyosin

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

Heterotypic contact inhibition of locomotion can drive cell sorting between epithelial and mesenchymal cell populations

Interactions between different cell types can induce distinct contact inhibition of locomotion (CIL) responses that are hypothesised to control population-wide behaviours during embryogenesis. However, our understanding of the signals that lead to cell-type specific repulsion and the precise capacity of heterotypic CIL responses to drive emergent behaviours is lacking. Using a new model of heterotypic CIL, we show that fibrosarcoma cells, but not fibroblasts, are actively repelled by epithelial cells in culture. We show that knocking down EphB2 or ERK in fibrosarcoma cells specifically leads to disruption of the repulsion phase of CIL in response to interactions with epithelial cells. We also examine the population-wide effects when these various cell combinations are allowed to interact in culture. Unlike fibroblasts, fibrosarcoma cells completely segregate from epithelial cells and inhibiting their distinct CIL response by knocking down EphB2 or ERK family proteins also disrupts this emergent sorting behaviour. These data suggest that heterotypic CIL responses, in conjunction with processes such as differential adhesion, may aid the sorting of cell populations

βIII-Tubulin Structural Domains Regulate Mitochondrial Network Architecture in an Isotype-Specific Manner

βIII-tubulin is a neuronal microtubule protein that is aberrantly expressed in epithelial cancers. The microtubule network is implicated in regulating the architecture and dynamics of the mitochondrial network, although the isotype-specific role for β-tubulin proteins that constitute this microtubule network remains unclear. High-resolution electron microscopy revealed that manipulation of βIII-tubulin expression levels impacts the volume and shape of mitochondria. Analysis of the structural domains of the protein identifies that the C-terminal tail of βIII-tubulin, which distinguishes this protein from other β-tubulin isotypes, significantly contributes to the isotype-specific effects of βIII-tubulin on mitochondrial architecture. Mass spectrometry analysis of protein–protein interactions with β-tubulin isotypes identifies that βIII-tubulin specifically interacts with regulators of mitochondrial dynamics that may mediate these functional effects. Advanced quantitative dynamic lattice light sheet imaging of the mitochondrial network reveals that βIII-tubulin promotes a more dynamic and extended reticular mitochondrial network, and regulates mitochondrial volume. A regulatory role for the βIII-tubulin C-terminal tail in mitochondrial network dynamics and architecture has widespread implications for the maintenance of mitochondrial homeostasis in health and disease