ERK drives protrusion WAVEs

During cell migration, formation of cell protrusions involves Arp2/3 binding to the WAVE2 regulatory complex (WRC) to mediate actin nucleation. ERK is required for motility but its precise function remains unclear. Mendoza et al. now show that one of the roles of ERK (extracellular signal-regulated kinase) in cell migration is to regulate protrusion initiation by promoting WRC activation (Mol. Cell 41, 661–671; 2011).

Visualization of cell protrusion formation revealed that ERK activity was necessary, and depended on the WRC, which co-localised with active ERK at cell protrusion edges. In contrast to previous reports, the authors found that ERK directly phosphorylates multiple sites in the WAVE2 and Abi1 components of the WRC. Inhibition of WAVE2 phosphorylation led to unregulated binding to Arp2/3 and actin, whereas inhibition of Abi1 phosphorylation suppressed these interactions, uncovering distinct modes of WRC regulation by ERK. In live cells, WRC mutants defective for ERK phosphorylation blocked cell protrusions, whereas their phosphomimetic counterparts had the converse effect. Notably, mutation of the Abi1 sites also affected cell migration, showing that Abi1 phosphorylation by ERK is essential for WRC activity in this context.

The authors propose that ERK phosphorylation contributes to allosteric activation of the WRC, which allows Arp2/3 and actin binding, leading to F-actin assembly that drives cell protrusion during migration. AIZ

Aurora B regulates mDia3 in mitosis

mDia3, a Diaphanous-related formin, localizes to kinetochores and is involved in metaphase chromosome alignment, but exactly how mDia3 regulates this process is unclear. Cheng et al. now report that mDia3 directly binds and stabilizes kinetochore microtubules, in a manner regulated by Aurora B phosphorylation (Dev. Cell 20, 342–352; 2011).

RNAi knockdown of mDia3 reduced chromosome alignment and stability of kinetochore microtubules in vitro, whereas co-expression of RNAi-resistant, actin nucleation-deficient mDia3 rescued these defects, showing that the actin nucleation activity of mDia3 is not required for its ability to stabilize microtubules. How then does mDia3 regulate microtubule stability?

mDia3 directly associates with the microbutule-binding proteins EB1 and APC, and a mDia3 mutant deficient in EB1 binding induced misalignment of chromosomes in the metaphase plate, suggesting the involvement of EB1. Interestingly, Aurora B kinase — known to destabilize kinetochore–microtubule interactions — was found to phosphorylate four sites in mDia3, and phosphorylation of at least one of these disrupted microtubule-binding and kinetochore-stabilizing functions of mDia3. Intriguingly, nonphosphorylatable and phosphomimetic mDia3 mutants could bind EB1, but displayed defective chromosome alignment.

It will be interesting to know how this role of mDia3 is coordinated with other kinetochore-associated proteins and whether Aurora B phosphorylation is involved in their functional integration. IO

Cancer cells: stick or Twist?

During metastatic cancer, cancerous cells spread into surrounding tissue or around the body. To do this, the cells use proteases to break through the extracellular matrix (ECM), but the molecular mechanisms that trigger matrix degradation are poorly characterized.

Eckert et al. (Cancer Cell, 19, 372–386; 2011) report that the transcription factor Twist1, previously shown to have a role in metastasis, is necessary and sufficient to break down ECM in mouse mammary tumour cells and human mammary epithelial cells. Twist1 expression resulted in more cells forming invadopodia, which harbour proteases for ECM degradation. Twist1 expression induced and activated the growth factor receptor PDGFRα, which activated Src kinase to phosphorylate invadopodia components, promoting invadopodia formation.

Twist1 also regulates the epithelial−mesenchymal transition (EMT), a process that results in loss of cell adhesion and increased cell mobility and is involved in embryogenesis. Other EMT-inducing factors are involved in metastasis, and Eckert et al. show that certain EMT-inducing signals can also promote invadopodia formation and ECM breakdown, which is mediated by Twist1 and PDGFRα.

Finally, the authors demonstrate that induction of invadopodia and PDGFRα is required for Twist1 to promote metastasis in mice. Furthermore, Twist1 and PDGFRα expression was correlated in human breast cancer samples, and survival of patients was negatively correlated with this expression. GD

Fly signalling networks

Although synthetic genetic interactions have been revealed using deletion strains in yeast and bacteria, this type of analysis is more challenging in multicellular organisms. Boutros and colleagues now demonstrate that synthetic interaction networks can be mapped in Drosophila S2 cells using RNAi (Nat. Methods 8, 341–346; 2011)

In preliminary experiments, the authors validated known interactions between members of the Ras–MAPK signalling pathway using two dsRNAs per gene. The authors employed previously described methods to analyse cellular features (such as cell number, nuclear size and Hoechst staining intensity) with automated microscopy. The result of pair-wise depletion of genes could be predicted by modelling and a deviation from this phenotype then indicated a positive or negative interaction.

They went on to study pair-wise interactions between 93 genes involved in signal transduction in Drosophila. 634 interactions were identified, with roughly equal numbers of positive and negative ones. Assigning genes to known signalling pathways using computational methods confirmed known interactions, such as positive and negative regulators of Ras–MAPK signalling, but also identified new interactions. For example, the Cka gene, encoding a known JNK pathway scaffolding protein, was found to interact with the Ras–MAPK pathway, and biochemical analysis showed that it exists in a complex with the Raf activator PP2A.

This analysis opens the way for large-scale interaction studies in other organisms. CKR

Written by Gary Dorken, Iley Ozerlat, Christina Karlsson Rosenthal & Alexia-IIeana Zaromytidou