Defective filopodia formation is linked to pathologies such as cancer, wherein actively protruding filopodia, at the invasive front, accompany cancer cell dissemination. Despite wide biological ...significance, delineating filopodia function in complex systems remains challenging and is particularly hindered by lack of compatible methods to quantify filopodia properties. Here, we present FiloQuant, a freely available ImageJ plugin, to detect filopodia-like protrusions in both fixed- and live-cell microscopy data. We demonstrate that FiloQuant can extract quantifiable information, including protrusion dynamics, density, and length, from multiple cell types and in a range of microenvironments. In cellular models of breast ductal carcinoma in situ, we reveal a link between filopodia formation at the cell-matrix interface, in collectively invading cells and 3D tumor spheroids, and the in vitro invasive capacity of the carcinoma. Finally, using intravital microscopy, we observe that tumor spheroids display filopodia in vivo, supporting a potential role for these protrusions during tumorigenesis.
Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having ...overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo
mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation.
mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.
The proteasome processes proteins to facilitate immune recognition and host defense. When inherently defective, it can lead to aberrant immunity resulting in a dysregulated response that can cause ...autoimmunity and/or autoinflammation. Biallelic or digenic loss-of-function variants in some of the proteasome subunits have been described as causing a primary immunodeficiency disease that manifests as a severe dysregulatory syndrome: chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE). Proteasome maturation protein (POMP) is a chaperone for proteasome assembly and is critical for the incorporation of catalytic subunits into the proteasome. Here, we characterize and describe POMP-related autoinflammation and immune dysregulation disease (PRAID) discovered in two unrelated individuals with a unique constellation of early-onset combined immunodeficiency, inflammatory neutrophilic dermatosis, and autoimmunity. We also begin to delineate a complex genetic mechanism whereby de novo heterozygous frameshift variants in the penultimate exon of POMP escape nonsense-mediated mRNA decay (NMD) and result in a truncated protein that perturbs proteasome assembly by a dominant-negative mechanism. To our knowledge, this mechanism has not been reported in any primary immunodeficiencies, autoinflammatory syndromes, or autoimmune diseases. Here, we define a unique hypo- and hyper-immune phenotype and report an immune dysregulation syndrome caused by frameshift mutations that escape NMD.
We present an oblique plane microscope (OPM) that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high ...numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of lattice light-sheet microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.
Natural killer (NK) cells are innate immune effectors that lyse virally infected and tumorigenic cells through the formation of an immunological synapse. Actin remodeling at the lytic immunological ...synapse is a critical requirement for multiple facets of cytotoxic function. Activating receptor and integrin signaling leads to the regulated turnover and remodeling of actin, which is required for adhesion, sustained receptor signaling, and ultimately exocytosis. NK cells undergo lytic granule exocytosis in hypodense regions of a pervasive actin network. Although these requirements have been well demonstrated, neither the dynamic regulation of synaptic actin nor its specific function, however, has been determined at a nanoscale level. Here, live-cell super-resolution microscopy demonstrates nanoscale filamentous actin dynamism in NK cell lytic granule secretion. Following cell spreading, the overall content of the branched actin network at an immune synapse is stable over time and contains branched actin fibers and discrete actin foci. Similar actin architecture is generated in cytolytic T cells, although the timescale differs from that of NK cells. Individual filament displacement leads to stochastic clearance formation and disappearance, which are independent of lytic granule positioning. Actin dynamism is dependent upon branched network formation mediated by Arp2/3 and contractility generated by myosin IIA. Importantly, the use of small-molecule inhibitors demonstrates that actin dynamism is ultimately needed for granule secretion. Thus, we describe a requirement for nanoscale actin fiber rearrangement in generating the complex actin architecture that enables lytic granule secretion.
•F-actin forms a permissive network at the immune synapse of CTLs and NK cells•The actin mesh of the mature IS of an NK cell is formed by Arp2/3 branching activity•Nanoscale actin dynamism is required to allow degranulation•Nanoscale dynamism is a new opportunity for regulation of cytotoxic function
Natural killer cells eliminate target cells via the release of lytic granules into the immune synapse cleft. Degranulation occurs through size-permissive clearances within the cortical actin mesh of NK cells. This Arp2/3-mediated cytoskeleton undergoes constant nanoscale dynamism required to create clearances and achieve cytotoxic function.
Patients with signal transducer and activator of transcription 5b (STAT5b) deficiency have impairment in T-cell homeostasis and natural killer (NK) cells which leads to autoimmunity, recurrent ...infections, and combined immune deficiency.
In this study we characterized the NK cell defect in STAT5b-deficient human NK cells, as well as Stat5b−/− mice.
We used multiparametric flow cytometry, functional NK cell assays, microscopy, and a Stat5b−/− mouse model to elucidate the effect of impaired and/or absent STAT5b on NK cell development and function.
This alteration generated a nonfunctional CD56bright NK cell subset characterized by low cytokine production. The CD56dim NK cell subset had decreased expression of perforin and CD16 and a greater frequency of cells expressing markers of immature NK cells. We observed low NK cell numbers and impaired NK cell maturation, suggesting that STAT5b is involved in terminal NK cell maturation in Stat5b−/− mice. Furthermore, human STAT5b-deficient NK cells had low cytolytic capacity, and fixed-cell microscopy showed poor convergence of lytic granules. This was accompanied by decreased expression of costimulatory and activating receptors. Interestingly, granule convergence and cytolytic function were restored after IL-2 stimulation.
Our results show that in addition to the impaired terminal maturation of NK cells, human STAT5b mutation leads to impairments in early activation events in NK cell lytic synapse formation. Our data provide further insight into NK cell defects caused by STAT5b deficiency.
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Immunodeficiency often coincides with hyperactive immune disorders such as autoimmunity, lymphoproliferation, or atopy, but this coincidence is rarely understood on a molecular level. We describe ...five patients from four families with immunodeficiency coupled with atopy, lymphoproliferation, and cytokine overproduction harboring mutations in
, which encodes the hematopoietic-specific HEM1 protein. These mutations cause the loss of the HEM1 protein and the WAVE regulatory complex (WRC) or disrupt binding to the WRC regulator, Arf1, thereby impairing actin polymerization, synapse formation, and immune cell migration. Diminished cortical actin networks caused by WRC loss led to uncontrolled cytokine release and immune hyperresponsiveness. HEM1 loss also blocked mechanistic target of rapamycin complex 2 (mTORC2)-dependent AKT phosphorylation, T cell proliferation, and selected effector functions, leading to immunodeficiency. Thus, the evolutionarily conserved HEM1 protein simultaneously regulates filamentous actin (F-actin) and mTORC2 signaling to achieve equipoise in immune responses.
Respiratory syncytial virus (RSV) fusion protein (F) is highly conserved between subtypes A and B (RSV/A and RSV/B). To become fully active, F precursor undergoes enzymatic cleavage to yield F1 and ...F2 subunits and releases a 27-amino-acid peptide (p27). Virus-cell fusion occurs when RSV F undergoes a conformational change from pre-F to post-F. Previous data show that p27 is detected on RSV F, but questions remain regarding if and how p27 affects the conformation of mature RSV F. Monoclonal antibodies against p27, site Ø (pre-F specific), and site II were used to monitor RSV F conformation by enzyme-linked immunosorbent assay (ELISA) and imaging flow cytometry. Pre-F to post-F conformational change was induced by a temperature stress test. We found that p27 cleavage efficiency was lower on sucrose-purified RSV/A (spRSV/A) than on spRSV/B. In addition, cleavage of RSV F was cell line dependent: HEp-2 cells had higher retention of p27 than did A549 cells when infected with RSV. Higher levels of p27 were also found on RSV/A-infected cells than on RSV/B-infected cells. We observed that RSV/A F with higher p27 levels could better sustain the pre-F conformation during the temperature stress challenge in both spRSV- and RSV-infected cell lines. Our findings suggest that despite F sequence similarity, p27 of RSV subtypes was cleaved with different efficiencies, which were also dependent on the cell lines used for infection. Importantly, the presence of p27 was associated with greater stability of the pre-F conformation, supporting the possibility that RSV has more than one mechanism for fusion to the host cell.
RSV fusion protein (F) plays an important role in entry and viral fusion to the host cell. The F undergoes proteolytic cleavages releasing a 27-amino-acid peptide (p27) to become fully functional. The role of p27 in viral entry and the function of the partially cleaved F containing p27 has been overlooked. p27 is thought to destabilize the F trimers, and thus, there is need for a fully cleaved F. In this study, we detected p27 on purified RSV virions and on the surface of virus-infected HEp-2 and A549 cells for circulating RSV strains of both subtypes. Higher levels of partially cleaved F containing p27 better sustained the pre-F conformation during the temperature stress challenge. Our findings highlight that the cleavage efficiency of p27 is different between RSV subtypes and among cell lines and that the presence of p27 contributes to the stability of the pre-F conformation.
The lytic immunological synapse (IS) is a discrete structural entity formed after the ligation of specific activating receptors that leads to the destruction of a cancerous cell. The formation of an ...effector cell IS in cytotoxic T lymphocytes or natural killer cells is a hierarchical and stepwise rearrangement of structural and signaling components and targeted release of the contents of lytic granules. While recent advances in the generation and testing of cytotoxic lymphocytes expressing chimeric antigen receptors (CARs) has demonstrated their efficacy in the targeted lysis of tumor targets, the contribution and dynamics of IS components have not yet been extensively investigated in the context of engineered CAR cells. Understanding the biology of the CAR IS will be a powerful approach to efficiently guide the engineering of new CARs and help identify mechanistic problems in existing CARs. Here, we review the formation of the lytic IS and describe quantitative imaging-based measurements using multiple microscopy techniques at a single cell level that can be used in conjunction with established population-based assays to provide insight into the important cytotoxic function of CAR cells. The inclusion of this approach in the pipeline of CAR product design could be a novel and valuable innovation for the field.
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Evaluation of the CAR immune synapse provides avenues for rational CAR design so as to offset failure of CAR products. Mukherjee et al. review quantitative imaging approaches to study the CAR immunological synapse.
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