Cellular segregation in cocultures is driven by differential adhesion and contractility on distinct timescales.

Cellular sorting and pattern formation are crucial for many biological processes such as development, tissue regeneration, and cancer progression. Prominent physical driving forces for cellular sorting are differential adhesion and contractility. Here, we studied the segregation of epithelial cocultures containing highly contractile, ZO1/2-depleted MDCKII cells (dKD) and their wild-type (WT) counterparts using multiple quantitative, high-throughput methods to monitor their dynamical and mechanical properties. We observe a time-dependent segregation process governed mainly by differential contractility on short (<5 h) and differential adhesion on long (>5 h) timescales. The overly contractile dKD cells exert strong lateral forces on their WT neighbors, thereby apically depleting their surface area. Concomitantly, the tight junction-depleted, contractile cells exhibit weaker cell-cell adhesion and lower traction force. Drug-induced contractility reduction and partial calcium depletion delay the initial segregation but cease to change the final demixed state, rendering differential adhesion the dominant segregation force at longer timescales. This well-controlled model system shows how cell sorting is accomplished through a complex interplay between differential adhesion and contractility and can be explained largely by generic physical driving forces.

Single-cell transcriptomics reveals immune response of intestinal cell types to viral infection.

Human intestinal epithelial cells form a primary barrier protecting us from pathogens, yet only limited knowledge is available about individual contribution of each cell type to mounting an immune response against infection. Here, we developed a framework combining single-cell RNA-Seq and highly multiplex RNA FISH and applied it to human intestinal organoids infected with human astrovirus, a model human enteric virus. We found that interferon controls the infection and that astrovirus infects all major cell types and lineages and induces expression of the cell proliferation marker MKI67. Intriguingly, each intestinal epithelial cell lineage exhibits a unique basal expression of interferon-stimulated genes and, upon astrovirus infection, undergoes an antiviral transcriptional reprogramming by upregulating distinct sets of interferon-stimulated genes. These findings suggest that in the human intestinal epithelium, each cell lineage plays a unique role in resolving virus infection. Our framework is applicable to other organoids and viruses, opening new avenues to unravel roles of individual cell types in viral pathogenesis.

Role of Clathrin Light Chains in Regulating Invadopodia Formation.

One of the most fundamental processes of the cell is the uptake of molecules from the surrounding environment. Clathrin-mediated endocytosis (CME) is the best-described uptake pathway and regulates nutrient uptake, protein and lipid turnover at the plasma membrane (PM), cell signaling, cell motility and cell polarity. The main protein in CME is clathrin, which assembles as a triskelion-looking building block made of three clathrin heavy chains and three clathrin light chains. Compared to clathrin heavy chains (CHCs), the role of the two isoforms of clathrin light chains (CLCA and CLCB) is poorly understood. Here, we confirm that the simultaneous deletion of both CLCA/B causes abnormal actin structures at the ventral PM and we describe them, for the first time, as functional invadopodia rather than disorganized actin-cytoskeleton assembly sites. Their identification is based on the occurrence of common invadopodia markers as well as functional invadopodia activity characterized by an increased local proteolytic activity of the extracellular matrix proteins. We demonstrate that CLCA/B deletion impacts the intracellular trafficking and recovery of the matrix metalloproteinase 14 (MMP14) leading to its accumulation at the plasma membrane and induction of invadopodia formation. Importantly, we show that invadopodia formation can be prevented by depletion of MMP14. As such, we propose that CLCA/B regulate invadopodia formation by regulating MMP14 delivery to the plasma membrane.

Critical Role of Type III Interferon in Controlling SARS-CoV-2 Infection in Human Intestinal Epithelial Cells.

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) is an unprecedented worldwide health problem that requires concerted and global approaches to stop the coronavirus 2019 (COVID-19) pandemic. Although SARS-CoV-2 primarily targets lung epithelium cells, there is growing evidence that the intestinal epithelium is also infected. Here, using both colon-derived cell lines and primary non-transformed colon organoids, we engage in the first comprehensive analysis of the SARS-CoV-2 life cycle in human intestinal epithelial cells (hIECs). Our results demonstrate that hIECs fully support SARS-CoV-2 infection, replication, and production of infectious de novo virus particles. We found that viral infection elicits an extremely robust intrinsic immune response where interferon-mediated responses are efficient at controlling SARS-CoV-2 replication and de novo virus production. Taken together, our data demonstrate that hIECs are a productive site of SARS-CoV-2 replication and suggest that the enteric phase of SARS-CoV-2 may participate in the pathologies observed in COVID-19 patients by contributing to increasing patient viremia and fueling an exacerbated cytokine response.

Asymmetric distribution of TLR3 leads to a polarized immune response in human intestinal epithelial cells.

Intestinal epithelial cells (IECs) act as a physical barrier separating the commensal-containing intestinal tract from the sterile interior. These cells have found a complex balance allowing them to be prepared for pathogen attacks while still tolerating the presence of bacterial or viral stimuli present in the lumen of the gut. Using primary human IECs, we probed the mechanisms that allow for such a tolerance. We discovered that viral infections emanating from the basolateral side of IECs elicit a stronger intrinsic immune response in comparison to lumenal apical infections. We determined that this asymmetric immune response is driven by the clathrin-sorting adaptor AP-1B, which mediates the polarized sorting of Toll-like receptor 3 (TLR3) towards the basolateral side of IECs. Mice and human IECs lacking AP-1B showed an exacerbated immune response following apical stimulation. Together, these results suggest a model where the cellular polarity program plays an integral role in the ability of IECs to partially tolerate apical commensals while remaining fully responsive to invasive basolateral pathogens.

HIV-1 Vpu Antagonizes CD317/Tetherin by Adaptor Protein-1-Mediated Exclusion from Virus Assembly Sites.

UNLABELLED: The host cell restriction factor CD317/tetherin traps virions at the surface of producer cells to prevent their release. The HIV-1 accessory protein Vpu antagonizes this restriction. Vpu reduces the cell surface density of the restriction factor and targets it for degradation; however, these activities are dispensable for enhancing particle release. Instead, Vpu has been suggested to antagonize CD317/tetherin by preventing recycling of internalized CD317/tetherin to the cell surface, blocking anterograde transport of newly synthesized CD317/tetherin, and/or displacing the restriction factor from virus assembly sites at the plasma membrane. At the molecular level, antagonism relies on the physical interaction of Vpu with CD317/tetherin. Recent findings suggested that phosphorylation of a diserine motif enables Vpu to bind to adaptor protein 1 (AP-1) trafficking complexes via two independent interaction motifs and to couple CD317/tetherin to the endocytic machinery. Here, we used a panel of Vpu proteins with specific mutations in individual interaction motifs to define which interactions are required for antagonism of CD317/tetherin. Impairing recycling or anterograde transport of CD317/tetherin to the plasma membrane was insufficient for antagonism. In contrast, excluding CD317/tetherin from HIV-1 assembly sites depended on Vpu motifs for interaction with AP-1 and CD317/tetherin and correlated with antagonism of the particle release restriction. Consistently, interference with AP-1 function or its expression blocked these Vpu activities. Our results define displacement from HIV-1 assembly sites as active principle of CD317/tetherin antagonism by Vpu and support a role of tripartite complexes between Vpu, AP-1, and CD317/tetherin in this process. IMPORTANCE: CD317/tetherin poses an intrinsic barrier to human immunodeficiency virus type 1 (HIV-1) replication in human cells by trapping virus particles at the surface of producer cells and thereby preventing their release. The viral protein Vpu antagonizes this restriction, and molecular interactions with the restriction factor and adaptor protein complex 1 (AP-1) were suggested to mediate this activity. Vpu modulates intracellular trafficking of CD317/tetherin and excludes the restriction factor from HIV-1 assembly sites at the plasma membrane, but the relative contribution of these effects to antagonism remain elusive. Using a panel of Vpu mutants, as well as interference with AP-1 function and expression, we show here that Vpu antagonizes CD317/tetherin by blocking its recruitment to viral assembly sites in an AP-1-dependent manner. These results refine our understanding of the molecular mechanisms of CD317/tetherin antagonism and suggest complexes of Vpu with the restriction factor and AP-1 as targets for potential therapeutic intervention.

Activation of the TCR complex by peptide-MHC and superantigens.

Drug hypersensitivity reactions are immune mediated, with T lymphocytes being stimulated by the drugs via their T-cell antigen receptor (TCR). In the nonpathogenic state, the TCR is activated by foreign peptides presented by major histocompatibility complex molecules (pMHC). Foreign pMHC binds with sufficient affinity to TCRαß and thereby elicits phosphorylation of the cytoplasmic tails of the TCRαß-associated CD3 subunits. The process is called TCR triggering. In this review, we discuss the current models of TCR triggering and which drug properties are crucial for TCR stimulation. The underlying molecular mechanisms mostly include pMHC-induced exposure of the CD3 cytoplasmic tails or alterations of the kinase-phosphatase equilibrium in the vicinity of CD3. In this review, we also discuss triggering of the TCR by small chemical compounds in context of these general mechanisms.