Regulating the emergence of leaders is a central aspect of collective cell migration, but the underlying mechanisms remain ambiguous. Here we show that the selective emergence of leader cells at the epithelial wound-margin depends on the dynamics of the follower cells and is spatially limited by the length-scale of collective force transduction. Owing to the dynamic heterogeneity of the monolayer, cells behind the prospective leaders manifest locally increased traction and monolayer stresses much before these leaders display any phenotypic traits. Followers, in turn, pull on the future leaders to elect them to their fate. Once formed, the territory of a leader can extend only to the length up-to which forces are correlated, which is similar to the length up-to which leader cells can transmit forces. These findings provide mechanobiological insight into the hierarchy in cell collectives during epithelial wound healing.

In this manuscript, we introduce a simple, off-the-shelf approach for the on-demand creation of giant unilamellar vesicles (GUVs) or multicompartment synthetic cell model systems in a high-throughput manner. To achieve this, we use microfluidics to encapsulate small unilamellar vesicles in block-copolymer surfactant-stabilized water-in-oil droplets. By tuning the charge of the inner droplet interface, adsorption of lipids can be either inhibited, leading to multicompartment systems, or induced, leading to the formation of droplet-stabilized GUVs. To control the charge density, we formed droplets using different molar ratios of an uncharged PEG-based fluorosurfactant and a negatively-charged PFPE carboxylic acid fluorosurfactant (Krytox). We systematically studied the transition from a multicompartment system to 3D-supported lipid bilayers as a function of lipid charge and Krytox concentration using confocal fluorescence microscopy, cryo-scanning electron microscopy and interfacial tension measurements. Moreover, we demonstrate a simple method to release GUVs from the surfactant shell and the oil phase into a physiological buffer -providing a remarkably high-yield approach for GUV formation. This widely applicable microfluidics-based technology will increase the scope of GUVs as adaptable cell-like compartments in bottom-up synthetic biology applications and beyond.

The extracellular environment is a complex medium in which cells secrete and consume metabolites. Molecular gradients are thereby created near cells, triggering various biological and physiological responses. However, investigating these molecular gradients remains challenging because the current tools are ill-suited and provide poor temporal and special resolution while also being destructive. Herein, we report the development and application of a machine learning approach in combination with a surface-enhanced Raman spectroscopy (SERS) nanoprobe to measure simultaneously the gradients of at least eight metabolites in vitro near different cell lines. We found significant increase in the secretion or consumption of lactate, glucose, ATP, glutamine, and urea within 20 μm from the cells surface compared to the bulk. We also observed that cancerous cells (HeLa) compared to fibroblasts (REF52) have a greater glycolytic rate, as is expected for this phenotype. Endothelial (HUVEC) and HeLa cells exhibited significant increase in extracellular ATP compared to the control, shining light on the implication of extracellular ATP within the cancer local environment. Machine-learningdriven SERS optophysiology is generally applicable to metabolites involved in cellular processes, providing a general platform on which to study cell biology.

Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments. In contrast to higher eukaryotic cells with their large filamentous actin structures, apicomplexan parasites typically contain only short and highly dynamic microfilaments. In apicomplexans, profilin appears to be the main monomer-sequestering protein. Compared to classical profilins, apicomplexan profilins contain an additional arm-like β-hairpin motif, which we show here to be critically involved in actin binding. Through comparative analysis using two profilin mutants, we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites, the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes. Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility. Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites.

Spatial molecular patterning enables the regulation of adhesion receptor clustering and can thus play a pivotal role in multiple biological activities such as cell adhesion, viability, proliferation, and differentiation. A wide range of nanopatterned, adhesive interfaces have been designed to decipher the essence of molecular-scale interactions between cells and the adhesive interface. Although an interligand spacing of less than 70 nm is a proven prerequisite for the formation of stable focal adhesions, there is a paucity of data concerning how cells behave on substrates featuring heterogeneous adhesiveness. In this study, a stretchable hydrogel functionalized with a quasi-hexagonally arranged nanoarray was stretched along one direction, resulting in ligands periodically arranged in a pattern resembling a centered rectangular lattice with an interligand spacing smaller than 70 nm in one direction and greater than 70 nm in the orthogonal direction. This substrate was utilized to modulate interligand spacing and investigate cell adhesion and migration. An interligand spacing larger than 70 nm-even in just one direction-prevented the establishment of stable focal adhesions. The stretched interface promoted dynamic remodeling at cell contacts, resulting in higher cellular mobility. Our nanopatterned stretchable hydrogel permits reversible control over cell adhesion and migration on nanopatterned ligand interfaces.

Success in the bottom‐up assembly of synthetic cells will depend on strategies for the division of protocellular compartments. Here, we describe the controlled division of phase‐separated giant unilamellar lipid vesicles (GUVs). We derive an analytical model based on the vesicle geometry, which makes four quantitative predictions that we verify experimentally. We find that the osmolarity ratio required for division is 2 , independent of the GUV size, while asymmetric division happens at lower osmolarity ratios. Remarkably, we show that a suitable osmolarity change can be triggered by water evaporation, enzymatic decomposition of sucrose or light‐triggered uncaging of CMNB‐fluorescein. The latter provides full spatiotemporal control, such that a target GUV undergoes division whereas the surrounding GUVs remain unaffected. Finally, we grow phase‐separated vesicles from single‐phased vesicles by targeted fusion of the opposite lipid type with programmable DNA tags to enable subsequent division cycles.

Extracellular biophysical cues have a profound influence on a wide range of cell behaviors, including growth, motility, differentiation, apoptosis, gene expression, adhesion, and signal transduction. Cells not only respond to definitively mechanical cues from the extracellular matrix (ECM) but can also sometimes alter the mechanical properties of the matrix and hence influence subsequent matrix-based cues in both physiological and pathological processes. Interactions between cells and materials in vitro can modify cell phenotype and ECM structure, whether intentionally or inadvertently. Interactions between cell and matrix mechanics in vivo are of particular importance in a wide variety of disorders, including cancer, central nervous system injury, fibrotic diseases, and myocardial infarction. Both the in vitro and in vivo effects of this coupling between mechanics and biology hold important implications for clinical applications.

Adoptive cell therapy (ACT) has shown very promising results as treatment for cancer in a few clinical trials, such as the complete remissions of otherwise terminal leukemia patients. Nevertheless, the introduction of ACT into clinics requires overcoming not only medical but also technical challenges, such as the ex vivo expansion of large amounts of specific T-cells. Nanostructured surfaces represent a novel T-cell stimulation technique that enables us to fine-tune the density and orientation of activating molecules presented to the cells. In this work, we studied the influence of integrin-mediated cell-adhesion on T-cell activation, proliferation, and differentiation using nanostructured surfaces, which provide a well-defined system at the nanoscale compared with standard cultures. Specifically, we synthesized a polymeric polyethylene glycol (PEG) hydrogel cross-linked with two fibronectin-derived peptides, cyclic Arg-Gly-Asp (cRGD) and cyclic Leu-Asp-Val (cLDV), that are known to activate different integrins. Moreover, the hydrogels were decorated with a quasi-hexagonal array of gold nanoparticles (AuNPs) functionalized with the activating antibody CD3 to initiate T-cell activation. Both cLDV and cRGD hydrogels showed higher T-cell activation (CD69 expression and IL-2 secretion) than nonfunctionalized PEG hydrogels. However, only the cRGD hydrogels clearly supported proliferation giving a higher proportion of cells with memory (CD4CD45RO) than naı̈ve (CD4CD45RA) phenotypes when interparticle distances smaller than 150 nm were used. Thus, T-cell proliferation can be enhanced by the activation of integrins through the RGD sequence.