Structural insights in cell-type specific evolution of intra-host diversity by SARS-CoV-2

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants with increased transmissibility and pathology. In addition to this entrenched diversity, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriSΔ variant, originally identified as a viral subpopulation from SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike an eight amino-acid deletion encompassing a furin recognition motif and S1/S2 cleavage site. We elucidate the structure, function and molecular dynamics of this spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Our results reveal long-range allosteric communication between functional domains that differ in the wild-type and the deletion variant and support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host

Vesicle Induced Receptor Sequestration: Mechanisms behind Extracellular Vesicle-Based Protein Signaling

Extracellular vesicles (EVs) are fundamental for proper physiological functioning of multicellular organisms. By shuttling nucleic acids and proteins between cells, EVs regulate a plethora of cellular processes, especially those involved in immune signalling. However, the mechanistic understanding concerning the biophysical principles underlying EV‐based communication is still incomplete. Towards holistic understanding, particular mechanisms explaining why and when cells apply EV‐based communication and how protein‐based signalling is promoted by EV surfaces are sought. Here, the authors study vesicle‐induced receptor sequestration (VIRS) as a universal mechanism augmenting the signalling potency of proteins presented on EV‐membranes. By bottom‐up reconstitution of synthetic EVs, the authors show that immobilization of the receptor ligands FasL and RANK on EV‐like vesicles, increases their signalling potential by more than 100‐fold compared to their soluble forms. Moreover, the authors perform diffusion simulations within immunological synapses to compare receptor activation between soluble and EV‐presented proteins. By this the authors propose vesicle‐triggered local clustering of membrane receptors as the principle structural mechanism underlying EV‐based protein presentation. The authors conclude that EVs act as extracellular templates promoting the local aggregation of membrane receptors at the EV contact site, thereby fostering inter‐protein interactions. The results uncover a potentially universal mechanism explaining the unique structural profit of EV‐based intercellular signalling

Cell Spreading and Focal Adhesion Dynamics Are Regulated by Spacing of Integrin Ligands

Integrin-mediated adhesion is regulated by multiple features of the adhesive surface, including its chemical composition, topography, and physical properties. In this study we investigated integrin lateral clustering, as a mechanism to control integrin functions, by characterizing the effect of nanoscale variations in the spacing between adhesive RGD ligands on cell spreading, migration, and focal adhesion dynamics. For this purpose, we used nanopatterned surfaces, containing RGD-biofunctionalized gold dots, surrounded by passivated gaps. By varying the spacing between the dots, we modulated the clustering of the associated integrins. We show that cell-surface attachment is not sensitive to pattern density, whereas the formation of stable focal adhesions and persistent spreading is. Thus cells plated on a 108-nm-spaced pattern exhibit delayed spreading with repeated protrusion-retraction cycles compared to cells growing on a 58-nm pattern. Cell motility on these surfaces is erratic and nonpersistent, leaving thin membrane tethers bound to the RGD pattern. Dynamic molecular profiling indicated that the adhesion sites formed with the 108-nm pattern undergo rapid turnover and contain reduced levels of zyxin. These findings indicate that a critical RGD density is essential for the establishment of mature and stable integrin adhesions, which, in turn, induce efficient cell spreading and formation of focal adhesions

Cell adhesion and response to synthetic nanopatterned environments by steering receptor clustering and spatial location

During adhesion and spreading, cells form micrometer-sized structures comprising transmembrane and intracellular protein clusters, giving rise to the formation of what is known as focal adhesions. Over the past two decades these structures have been extensively studied to elucidate their organization, assembly, and molecular composition, as well as to determine their functional role. Synthetic materials decorated with biological molecules, such as adhesive peptides, are widely used to induce specific cellular responses dependent on cell adhesion. Here, we focus on how surface patterning of such bioactive materials and organization at the nanoscale level has proven to be a useful strategy for mimicking both physical and chemical cues present in the extracellular space controlling cell adhesion and fate. This strategy for designing synthetic cellular environments makes use of the observation that most cell signaling events are initiated through recruitment and clustering of transmembrane receptors by extracellular-presented signaling molecules. These systems allow for studying protein clustering in cells and characterizing the signaling response induced by, e.g., integrin activation. We review the findings about the regulation of cell adhesion and focal adhesion assembly by micro- and nanopatterns and discuss the possible use of substrate stiffness and patterning in mimicking both physical and chemical cues of the extracellular space