Blebs promote cell survival by assembling oncogenic signalling hubs.

Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signalling pathways, without which cells undergo anoikis-a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cells often lose firm attachment to surrounding tissue2,3. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs4-11. Bleb function has been thoroughly investigated in the context of amoeboid migration, but it has been examined far less in other scenarios12. Here we show by three-dimensional imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signalling hubs that confer anoikis resistance. Specifically, in melanoma cells, blebbing generates plasma membrane contours that recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signalling hubs activate ERK and PI3K-well-established promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells it causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils a morphological requirement for mutant NRAS to operate as an effective oncoprotein. Furthermore, whereas some BRAF-mutated melanoma cells do not rely on this survival pathway in a basal state, inhibition of BRAF and MEK strongly sensitizes them to both bleb and septin inhibition. Moreover, fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harbouring oncogenic mutations. Thus, blebs are potent signalling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.

Fine-grained, nonlinear registration of live cell movies reveals spatiotemporal organization of diffuse molecular processes.

We present an application of nonlinear image registration to align in microscopy time lapse sequences for every frame the cell outline and interior with the outline and interior of the same cell in a reference frame. The registration relies on a subcellular fiducial marker, a cell motion mask, and a topological regularization that enforces diffeomorphism on the registration without significant loss of granularity. This allows spatiotemporal analysis of extremely noisy and diffuse molecular processes across the entire cell. We validate the registration method for different fiducial markers by measuring the intensity differences between predicted and original time lapse sequences of Actin cytoskeleton images and by uncovering zones of spatially organized GEF- and GTPase signaling dynamics visualized by FRET-based activity biosensors in MDA-MB-231 cells. We then demonstrate applications of the registration method in conjunction with stochastic time-series analysis. We describe distinct zones of locally coherent dynamics of the cytoplasmic protein Profilin in U2OS cells. Further analysis of the Profilin dynamics revealed strong relationships with Actin cytoskeleton reorganization during cell symmetry-breaking and polarization. This study thus provides a framework for extracting information to explore functional interactions between cell morphodynamics, protein distributions, and signaling in cells undergoing continuous shape changes. Matlab code implementing the proposed registration method is available at https://github.com/DanuserLab/Mask-Regularized-Diffeomorphic-Cell-Registration.

Granger-causal inference of the lamellipodial actin regulator hierarchy by live cell imaging without perturbation.

Many cell regulatory systems implicate nonlinearity and redundancy among components. The regulatory network governing lamellipodial and lamellar actin structures is prototypical of such a system, containing tens of actin-nucleating and -modulating molecules with functional overlap and feedback loops. Due to instantaneous and long-term compensation, phenotyping the system response to perturbation provides limited information on the roles the targeted component plays in the unperturbed system. Accordingly, how individual actin regulators contribute to lamellipodial dynamics remains ambiguous. Here, we present a perturbation-free reconstruction of cause-effect relations among actin regulators by applying Granger-causal inference to constitutive image fluctuations that indicate regulator recruitment as a proxy for activity. Our analysis identifies distinct zones of actin regulator activation and of causal effects on filament assembly and delineates actin-dependent and actin-independent regulator roles in controlling edge motion. We propose that edge motion is driven by assembly of two independently operating actin filament systems.

Actin-Membrane Release Initiates Cell Protrusions.

Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin's affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion.