It has been suggested that high affinity/avidity interactions are required for the thymic selection of Treg. Here, we investigated the role of CD5, a negative regulator of TCR signaling, in the selection and function of "naturally occurring" CD4(+)CD25(+) Treg (nTreg). Analysis of CD5(-/-) mice showed a significant increase in the percentage and absolute numbers of CD4(+) CD25(+)Foxp3(+) thymocytes and peripheral T lymphocytes, compared with BALB/c mice. Thymi from CD5(-/-) mice showed reduced cellularity due to increased apoptosis, which preferentially affected naïve T cells. To characterize nTreg selection at the molecular level we investigated the phosphorylation of Erk, c-Cbl, PI3K and Akt. CD5(-/-) nTreg showed increased basal levels of p-Erk compared with wild-type nTreg. Interestingly, in response to CD3 plus CD28 costimulation, CD5(-/-) naïve T cells but not CD5(-/-) nTreg showed lower levels of p-Akt. Finally, CD5(-/-) nTreg were thymus-derived and fully functional. We conclude that the enrichment of nTreg observed in the absence of CD5 signaling is due to de novo generation of nTreg and selective reduction of CD4(+)CD25(-) naïve thymocytes. Furthermore, we provide new evidence supporting a potential role of CD5 in thymocyte survival, through a mechanism that may involve the phosphorylation of Akt.
Fast and selective isolation of single cells with unique spatial and morphological traits remains a technical challenge. Here, we address this by establishing high-speed image-enabled cell sorting (ICS), which records multicolor fluorescence images and sorts cells based on measurements from image data at speeds up to 15,000 events per second. We show that ICS quantifies cell morphology and localization of labeled proteins and increases the resolution of cell cycle analyses by separating mitotic stages. We combine ICS with CRISPR-pooled screens to identify regulators of the nuclear factor κB (NF-κB) pathway, enabling the completion of genome-wide image-based screens in about 9 hours of run time. By assessing complex cellular phenotypes, ICS substantially expands the phenotypic space accessible to cell-sorting applications and pooled genetic screening.
Abstract The plethora of chemical, physical, and biological factors that can damage microbial cells has triggered the evolution of sophisticated stress response (SR) mechanisms. While individual SR pathways have been monitored with genetically encoded reporters, sensor concepts for the detection of multimodal effects of stressing conditions in living microorganisms are still lacking. Orthogonally detectable red, green, and blue fluorescent proteins combined in a single vector system, dubbed RGB-S reporter, enable the simultaneous, independent and real-time analysis of the stress response in Escherichia coli to physiological stress, genotoxicity, and cytotoxicity. The sensor system can be read out via conventional fluorescence microscopy or microtiter plate analysis and can also be combined with Fluorescent Activated Cell Sorting (FACS) and subsequent transcriptome analysis. Various stressors, such as the biotechnologically relevant 2-propanol, lead to the activation of one, two or all three SRs, which can have a significant impact on non-stress-related metabolic pathways. Implemented in microfluidic cultivation with confocal fluorescence microscopy imaging, the technology enabled spatiotemporal analysis of live biofilms to discover stratified subpopulations of bacteria with heterogeneous stress responses.
Summary Dishevelled (Dvl) proteins are essential transducers in Wnt signaling pathways, which have been implicated in development, stem cell maintenance, and human diseases such as cancer. Several studies have shown that Dvl proteins form dynamic biomolecular condensates. However, how cellular signals and cell states influence the formation of biomolecular condensates remains poorly understood. Here, we analyzed cells with endogenous Dvl2 condensates using image-based cell sorting in combination with phosphoproteomics and identified protein enrichment for Wnt/PCP signaling and the G2/M cell cycle transition. We then performed an image-based high-throughput screen to identify small molecule kinase inhibitors that affect Dvl2 liquid-liquid phase separation. Strikingly, CK1δ/ε inhibition blocked Dvl2 condensate formation. Its effect on Wnt signaling was modulated in genetic epistasis experiments with loss-of-function alleles of APC, Axin1, and MCC. Our study highlights the interplay between post-translational modifications and condensate dynamics, opening new avenues for research on their role in cellular signaling and disease intervention.
We discovered that the proteostasis modulating transcription factor Nrf1 requires cytosolic de-N-glycosylation by the N-glycanase NGly1 as part of its activation mechanism. Through a covalent small molecule library screen, we discovered an inhibitor of NGly1 that blocks Nrf1 activation in cells and potentiates the activity of proteasome inhibitor cancer drugs. The requirement of NGly1 for Nrf1 activity likely underlies several pathologies associated with a rare hereditary deficiency in NGly1.
Proteasome inhibitors are used to treat blood cancers such as multiple myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs is frequently undermined by acquired resistance. One mechanism of proteasome inhibitor resistance may involve the transcription factor Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1), which responds to proteasome insufficiency or pharmacological inhibition by upregulating proteasome subunit gene expression. This "bounce-back" response is achieved through a unique mechanism. Nrf1 is constitutively translocated into the ER lumen, N-glycosylated, and then targeted for proteasomal degradation via the ER-associated degradation (ERAD) pathway. Proteasome inhibition leads to accumulation of cytosolic Nrf1, which is then processed to form the active transcription factor. Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human PNGase) is essential for Nrf1 activation in response to proteasome inhibition. Chemical or genetic disruption of NGLY1 activity results in the accumulation of misprocessed Nrf1 that is largely excluded from the nucleus. Under these conditions, Nrf1 is inactive in regulating proteasome subunit gene expression in response to proteasome inhibition. Through a small molecule screen, we identified a cell-active NGLY1 inhibitor that disrupts the processing and function of Nrf1. The compound potentiates the cytotoxicity of carfilzomib, a clinically used proteasome inhibitor, against MM and T cell-derived acute lymphoblastic leukemia (T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation and represents a new therapeutic approach for cancers that depend on proteasome homeostasis.
Introduction: Non-Hodgkin lymphoma (NHL) NHL arise in lymph nodes whose normal architecture is variably altered by different NHL subtypes. While follicular lymphoma maintains a germinal center-like growth program, diffuse large B cell lymphomas (DLBCL) is characterized by a diffuse growth pattern. To uncover driving forces of these distinct growth patterns we studied lymph node derived immune cell subsets and stroma cells (SC) which are known to function as tissue organizers through chemokine gradients that enable the compartmentalization of specific immune cell subsets into functionally specialized microdomains (Figure 1). Methods: Here, we utilized a combined single-cell and spatially-resolved mapping approach of lymph node derived stroma cells, lymphoma infiltrating T-cells and malignant B-cells to dissect the pathophysiological mechanisms underlying the gradual loss of tissue organization in indolent and diffusely growing aggressive lymphomas. We validated altered chemokine gradients in large lymphoma patient cohorts and in-vitro SC co-culture lymphocyte co-culture models. Results: First, we characterized and quantified how lymph node resident cells organize into spatially distinct cellular neighborhoods and how these functional units are disturbed and disrupted in diffusely growing lymphomas using ultra-high-plex immunofluorescence (CODEX). To determine the molecular programs underlying loss of tissue organization, we employed holistic single-cell transcriptomic mapping of the lymph node ecosystem, covering major lymph node resident cell types, including rare mesenchymal and endothelial populations. Combined with transcriptomic and outcome data from patient cohorts, this approach revealed that highly specialized mesenchymal cells, which create chemokine gradients in normal lymph nodes, downregulate chemokines responsible for the maintenance of tissue organization and enter a dysfunctional state characterized by inflammatory and fibrotic phenotypes. In silico modelling of intercellular attractions based on receptor ligand expression levels recapitulated lymphoma specific cellular neighborhoods and revealed that DLBCL specific chemokine signatures were sufficient to explain the loss of lymph node organization in DLBCL. In addition to the loss of mesenchymal-derived chemokine gradients, inflammatory immune cells create ectopic sources of chemokines in diffusely growing lymphomas, further disturbing the highly orchestrated chemokine gradients. Ongoing Trial Keywords: Bioinformatics, Computational and Systems Biology, Genomics, Epigenomics, and Other -Omics, Microenvironment No conflicts of interests pertinent to the abstract.
We discovered that the proteostasis modulating transcription factor Nrf1 requires cytosolic de-N-glycosylation by the N-glycanase NGly1 as part of its activation mechanism. Through a covalent small molecule library screen, we discovered an inhibitor of NGly1 that blocks Nrf1 activation in cells and potentiates the activity of proteasome inhibitor cancer drugs. The requirement of NGly1 for Nrf1 activity likely underlies several pathologies associated with a rare hereditary deficiency in NGly1.
