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Cellular unjamming is the collective fluidization of cell motion and has been linked to many biological processes, including development, wound repair, and tumor growth. In tumor growth, the uncontrolled proliferation of cancer cells in a confined space generates mechanical compressive stress. However, because multiple cellular and molecular mechanisms may be operating simultaneously, the role of compressive stress in unjamming transitions during cancer progression remains unknown. Here, we investigate which mechanism dominates in a dense, mechanically stressed monolayer. We find that long-term mechanical compression triggers cell arrest in benign epithelial cells and enhances cancer cell migration in transitions correlated with cell shape, leading us to examine the contributions of cell–cell adhesion and substrate traction in unjamming transitions. We show that cadherin-mediated cell–cell adhesion regulates differential cellular responses to compressive stress and is an important driver of unjamming in stressed monolayers. Importantly, compressive stress does not induce the epithelial–mesenchymal transition in unjammed cells. Furthermore, traction force microscopy reveals the attenuation of traction stresses in compressed cells within the bulk monolayer regardless of cell type and motility. As traction within the bulk monolayer decreases with compressive pressure, cancer cells at the leading edge of the cell layer exhibit sustained traction under compression. Together, strengthened intercellular adhesion and attenuation of traction forces within the bulk cell sheet under compression lead to fluidization of the cell layer and may impact collective cell motion in tumor development and breast cancer progression. 

The proteins that make up the actin cytoskeleton can self-assemble into a variety of structures. In vitro experiments and coarse-grained simulations have shown that the actin crosslinking proteins α-actinin and fascin segregate into distinct domains in single actin bundles with a molecular size-dependent competition-based mechanism. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that physical confinement can cause these proteins to form much more complex structures, including rings and asters at GUV peripheries and centers; the prevalence of different structures depends on GUV size. Strikingly, we found that α-actinin and fascin self-sort into separate domains in the aster structures with actin bundles whose apparent stiffness depends on the ratio of the relative concentrations of α-actinin and fascin. The observed boundary-imposed effect on protein sorting may be a general mechanism for creating emergent structures in biopolymer networks with multiple crosslinkers.

 

The cytoskeleton of a cell controls all the aspects of cell shape changes and motility from its physiological functions for survival to reproduction to death. The structure and dynamics of the cytoskeletal components: actin, microtubules, intermediate filaments, and septins – recently regarded as the fourth member of the cytoskeleton family – are conserved during evolution. Such conserved and effective control over the mechanics of the cell makes the cytoskeletal components great candidates for in vitro reconstitution and bottom-up synthetic biology studies. Here, we review the recent efforts in reconstitution of the cytoskeleton in and on membrane-enclosed biomimetic systems and argue that co-reconstitution and synergistic interplay between cytoskeletal filaments might be indispensable for efficient mechanical functionality of active minimal cells. Further, mechanical equilibrium in adherent eukaryotic cells is achieved by the formation of integrin-based focal contacts with extracellular matrix (ECM) and the transmission of stresses generated by actomyosin contraction to ECM. Therefore, a minimal mimic of such balance of forces and quasi-static kinetics of the cell by bottom-up reconstitution requires a careful construction of contractile machineries and their link with adhesive contacts. In this review, in addition to cytoskeletal crosstalk, we provide a perspective on reconstruction of cell mechanical equilibrium by reconstitution of cortical actomyosin networks in lipid membrane vesicles adhered on compliant substrates and also discuss future perspectives of this active research area. 

Engineering synthetic interfaces between membranes has potential applications in designing non‐native cellular communication pathways and creating synthetic tissues. Here, InterSpy is introduced as a synthetic biology tool consisting of a heterodimeric protein engineered to form and maintain membrane–membrane interfaces between apposing synthetic as well as cell membranes through the SpyTag/SpyCatcher interaction. The inclusion of split fluorescent protein fragments in InterSpy allows tracking of the formation of a membrane–membrane interface and reconstitution of functional fluorescent protein in the space between apposing membranes. First, InterSpy is demonstrated by testing split protein designs using a mammalian cell‐free expression (CFE) system. By utilizing co‐translational helix insertion, cell‐free synthesized InterSpy fragments are incorporated into the membrane of liposomes and supported lipid bilayers with the desired topology. Functional reconstitution of split fluorescent protein between the membranes is strictly dependent on SpyTag/SpyCatcher. Finally, InterSpy is demonstrated in mammalian cells by detecting fluorescence reconstitution of split protein at the membrane–membrane interface between two cells each expressing a component of InterSpy. InterSpy demonstrates the power of CFE systems in the functional reconstitution of synthetic membrane interfaces via proximity‐inducing proteins. This technology may also prove useful where cell‐cell contacts and communication are recreated in a controlled manner using minimal components.