Asier Jayo

Fascin: a key regulator of cytoskeletal dynamics.

Fascin is a 55 kDa actin-bundling protein and is an important regulatory element in the maintenance and stability of parallel bundles of filamentous actin in a variety of cellular contexts. Regulation of fascin function is under the control of a number of different signalling pathways that act in concert to spatially regulate the actin-binding properties of this protein. The ability of fascin to bind and bundle actin plays a central role in the regulation of cell adhesion, migration and invasion. Fascin has received considerable attention recently as an emerging key prognostic marker of metastatic disease. Studies are now underway to better understand the precise regulation of this protein in the context of tumour progression and to investigate fascin as a potential therapeutic target for a number of forms of cancer.

The first World Cell Race

Summary Motility is a common property of animal cells. Cell motility is required for embryogenesis [1], tissue morphogenesis [2] and the immune response [3] but is also involved in disease processes, such as metastasis of cancer cells [4]. Analysis of cell migration in native tissue in vivo has yet to be fully explored, but motility can be relatively easily studied in vitro in isolated cells. Recent evidence suggests that cells plated in vitro on thin lines of adhesive proteins printed onto culture dishes can recapitulate many features of in vivo migration on collagen fibers [5,6]. However, even with controlled in vitro measurements, the characteristics of motility are diverse and are dependent on the cell type, origin and external cues. One objective of the first World Cell Race was to perform a large-scale comparison of motility across many different adherent cell types under standardized conditions. To achieve a diverse selection, we enlisted the help of many international laboratories, who submitted cells for analysis. The large-scale analysis, made feasible by this competition-oriented collaboration, demonstrated that higher cell speed correlates with the persistence of movement in the same direction irrespective of cell origin.

Fascin promotes filopodia formation independent of its role in actin bundling

Mutation of a critical residue of fascin eliminates the protein’s actin-bundling activity but maintains its positive role in filopodia formation

Fascin Regulates Nuclear Movement and Deformation in Migrating Cells

Summary Fascin is an F-actin-bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells, and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers. Here, we identified the nuclear envelope protein nesprin-2 as a binding partner for fascin in a range of cell types in vitro and in vivo. Nesprin-2 interacts with fascin through a direct, F-actin-independent interaction, and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery. Moreover, disrupting the interaction between fascin and nesprin-2 C-terminal domain leads to specific defects in F-actin coupling to the nuclear envelope, nuclear movement, and the ability of cells to deform their nucleus to invade through confined spaces. Together, our results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion.

A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability

BackgroundFascin-1 is an actin crosslinking protein that is important for the assembly of cell protrusions in neurons, skeletal and smooth muscle, fibroblasts, and dendritic cells. Although absent from most normal adult epithelia, fascin-1 is upregulated in many human carcinomas, and is associated with poor prognosis because of its promotion of carcinoma cell migration, invasion, and metastasis. Rac and Cdc42 small guanine triphosphatases have been identified as upstream regulators of the association of fascin-1 with actin, but the possible role of Rho has remained obscure. Additionally, experiments have been hampered by the inability to measure the fascin-1/actin interaction directly in intact cells. We investigated the hypothesis that fascin-1 is a functional target of Rho in normal and carcinoma cells, using experimental approaches that included a novel fluorescence resonance energy transfer (FRET)/fluorescence lifetime imaging (FLIM) method to measure the interaction of fascin-1 with actin.ResultsRho activity modulates the interaction of fascin-1 with actin, as detected by a novel FRET method, in skeletal myoblasts and human colon carcinoma cells. Mechanistically, Rho regulation depends on Rho kinase activity, is independent of the status of myosin II activity, and is not mediated by promotion of the fascin/PKC complex. The p-Lin-11/Isl-1/Mec-3 kinases (LIMK), LIMK1 and LIMK2, act downstream of Rho kinases as novel binding partners of fascin-1, and this complex regulates the stability of filopodia.ConclusionsWe have identified a novel activity of Rho in promoting a complex between fascin-1 and LIMK1/2 that modulates the interaction of fascin-1 with actin. These data provide new mechanistic insight into the intracellular coordination of contractile and protrusive actin-based structures. During the course of the study, we developed a novel FRET method for analysis of the fascin-1/actin interaction, with potential general applicability for analyzing the activities of actin-binding proteins in intact cells.

An open access microfluidic device for the study of the physical limits of cancer cell deformation during migration in confined environments

Graphical abstract

A direct interaction between fascin and microtubules contributes to adhesion dynamics and cell migration

ABSTRACT Fascin is an actin-binding and bundling protein that is highly upregulated in most epithelial cancers. Fascin promotes cell migration and adhesion dynamics in vitro and tumour cell metastasis in vivo. However, potential non-actin bundling roles for fascin remain unknown. Here, we show for the first time that fascin can directly interact with the microtubule cytoskeleton and that this does not depend upon fascin-actin bundling. Microtubule binding contributes to fascin-dependent control of focal adhesion dynamics and cell migration speed. We also show that fascin forms a complex with focal adhesion kinase (FAK, also known as PTK2) and Src, and that this signalling pathway lies downstream of fascin–microtubule association in the control of adhesion stability. These findings shed light on new non actin-dependent roles for fascin and might have implications for the design of therapies to target fascin in metastatic disease. Summary: Fascin associates directly with microtubules independently of F-actin binding, and this contributes to microtubule dynamics, adhesion assembly and cell migration.

Prostaglandins regulate nuclear localization of Fascin and its function in nucleolar architecture

Fascin, a conserved actin-bundling protein, is not only cytoplasmic but also localizes to the nucleus and nuclear periphery in both Drosophila and mammalian cell contexts. In Drosophila, prostaglandin signaling regulates this localization. In addition, Fascin plays a critical role in nucleolar architecture in both Drosophila and mammalian cells.

Local dimensionality determines imaging speed in localization microscopy

Localization microscopy allows biological samples to be imaged at a length scale of tens of nanometres. Live-cell super-resolution imaging is rare, as it is generally assumed to be too slow for dynamic samples. The speed of data acquisition can be optimized by tuning the density of activated fluorophores in each time frame. Here, we show that the maximum achievable imaging speed for a particular structure varies by orders of magnitude, depending on the sample dimensionality (that is, whether the sample is more like a point, a strand or an extended structure such as a focal adhesion). If too high an excitation density is used, we demonstrate that the analysis undergoes silent failure, resulting in reconstruction artefacts. We are releasing a tool to allow users to identify areas of the image in which the activation density was too high and correct for them, in both live- and fixed-cell experiments.

Imaging of cell adhesion events in 3D matrix environments.

Cell adhesion plays an essential role in development and homeostasis, but is also a key regulator of many diseases such as cancer and immune dysfunction. Numerous studies over the past three decades have revealed a wealth of information detailing signalling molecules required for cell adhesion to two-dimensional surfaces. However, in vivo many cells are completely surrounded by matrix and this will very likely influence the size, composition and dynamics of adhesive structures. The study of adhesion in cells within three-dimensional environments is still in its infancy, thus the role and regulation of adhesions in these complex environments remains unclear. The recent development of new experimental models coupled with significant advances in cell imaging approaches have provided platforms for researchers to begin to dissect adhesion signalling in cells in 3D matrices. Here we summarise the recent insights in cell adhesion formation and regulation in 3D model systems and the imaging approaches used to analyse these events.