Matrix mechanics regulates epithelial defence against cancer by tuning dynamic localization of filamin

In epithelia, normal cells recognize and extrude out newly emerged transformed cells by competition. This process is the most fundamental epithelial defence against cancer, whose occasional failure promotes oncogenesis. However, little is known about what factors determine the success or failure of this defence. Here we report that mechanical stiffening of extracellular matrix attenuates the epithelial defence against HRasV12-transformed cells. Using photoconversion labelling, protein tracking, and loss-of-function mutations, we attribute this attenuation to stiffening-induced perinuclear sequestration of a cytoskeletal protein, filamin. On soft matrix mimicking healthy epithelium, filamin exists as a dynamically single population, which moves to the normal cell-transformed cell interface to initiate the extrusion of transformed cells. However, on stiff matrix mimicking fibrotic epithelium, filamin redistributes into two dynamically distinct populations, including a new perinuclear pool that cannot move to the cell-cell interface. A matrix stiffness-dependent differential between filamin-Cdc42 and filamin-perinuclear cytoskeleton interaction controls this distinctive filamin localization and hence, determines the success or failure of epithelial defence on soft versus stiff matrix. Together, our study reveals how pathological matrix stiffening leads to a failed epithelial defence at the initial stage of oncogenesis.

Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

Focal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we link the molecular behavior and 3D nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displays free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation, cell spreading and FAs formation. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.

TriTag: an integrative tool to correlate chromatin dynamics and gene expression in living cells

A wealth of single-cell imaging studies have contributed novel insights into chromatin organization and gene regulation. However, a comprehensive understanding of spatiotemporal gene regulation requires developing tools to combine multiple monitoring systems in a single study. Here, we report a versatile tag, termed TriTag, which integrates the functional capabilities of CRISPR-Tag (DNA labeling), MS2 aptamer (RNA imaging) and fluorescent protein (protein tracking). Using this tag, we correlate changes in chromatin dynamics with the progression of endogenous gene expression, by recording both transcriptional bursting and protein production. This strategy allows precise measurements of gene expression at single-allele resolution across the cell cycle or in response to stress. TriTag enables capturing an integrated picture of gene expression, thus providing a powerful tool to study transcriptional heterogeneity and regulation.

Matrix mechanics regulates epithelial defence against cancer by tuning dynamic localization of filamin

In epithelia, normal cells recognize and extrude out newly emerged transformed cells by competition. This process is the most fundamental epithelial defence against cancer, whose occasional failure promotes oncogenesis. However, little is known about what factors determine the success or failure of this defence. Here we report that mechanical stiffening of extracellular matrix attenuates the epithelial defence against activated HRasV12-transformed cells. Using photoconversion labelling, protein tracking, and loss-of-function mutations, we attribute this attenuation to stiffening-induced perinuclear sequestration of a cytoskeletal protein, filamin. On soft matrix mimicking healthy epithelium, filamin exists as a dynamically single population, which moves to the normal cell-transformed cell interface to initiate transformed cell-extrusion. But, on stiff matrix mimicking fibrotic epithelium, filamin redistributes into two dynamically distinct populations, including a new perinuclear pool, which cannot move to the cell-cell interface. A tug-of-war between filamin-Cdc42 and filamin-perinuclear cytoskeleton interactions controls this differential filamin localization and hence, determines the success or failure of epithelial defence on soft versus stiff matrix. Together, our study reveals how pathological matrix stiffening leads to a failed epithelial defence at the initial stage of oncogenesis.

Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

Focal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we linked the molecular behavior and tridimensional nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displayed free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation during cell adhesion and spreading. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.

High-precision protein-tracking with interferometric scattering microscopy

The mobility of proteins and lipids within the cell, sculpted oftentimes by the organisation of the membrane, reveals a great wealth of information on the function and interaction of these molecules as well as the membrane itself. Single particle tracking has proven to be a vital tool to study the mobility of individual molecules and unravel details of their behaviour. Interferometric scattering (iSCAT) microscopy is an emerging technique well suited for visualising the diffusion of gold nanoparticle-labelled membrane proteins to a spatial and temporal resolution beyond the means of traditional fluorescent labels. We discuss the applicability of interferometric single particle tracking (iSPT) microscopy to investigate the minutia in the motion of a protein through measurements visualising the mobility of the epidermal growth factor receptor in various biological scenarios on the live cell.