Sonic Hedgehog activates prostaglandin signaling to stabilize primary cilium length.

Sonic Hedgehog (SHH) is a driver of embryonic patterning that, when corrupted, triggers developmental disorders and cancers. SHH effector responses are organized through primary cilia (PC) that grow and retract with the cell cycle and in response to extracellular cues. Disruption of PC homeostasis corrupts SHH regulation, placing significant pressure on the pathway to maintain ciliary fitness. Mechanisms by which ciliary robustness is ensured in SHH-stimulated cells are not yet known. Herein, we reveal a crosstalk circuit induced by SHH activation of Phospholipase A2α that drives ciliary E-type prostanoid receptor 4 (EP4) signaling to ensure PC function and stabilize ciliary length. We demonstrate that blockade of SHH-EP4 crosstalk destabilizes PC cyclic AMP (cAMP) equilibrium, slows ciliary transport, reduces ciliary length, and attenuates SHH pathway induction. Accordingly, Ep4-/- mice display shortened neuroepithelial PC and altered SHH-dependent neuronal cell fate specification. Thus, SHH initiates coordination between distinct ciliary receptors to maintain PC function and length homeostasis for robust downstream signaling.

Unswitched memory B cell deficiency in children with sickle cell disease and response to pneumococcal polysaccharide vaccine.

Early mortality in sickle cell disease (SCD) is attributed to increased infections due to loss of splenic function. Marginal zone B cells are important for initial opsonization of pathogens and can be absent in spleen histopathology in SCD. The frequency of unswitched memory B cells (UMBC), the circulating correlate of marginal zone B cells, reflects the immunologic function of the spleen. We hypothesized that asplenia in SCD is associated with alterations in the peripheral blood lymphocyte population and explored whether UMBC deficiency was associated with a clinical phenotype. We analyzed B cell subsets and clinical history for 238 children with SCD and 63 controls. The median proportion of UMBCs was lower in children with SCD compared with controls (4.7% vs. 6.6%, p < .001). Naïve B cells were higher in SCD compared with controls (80.6 vs. 76.3%, respectively, p = .02). UMBC frequency declined by 3.4% per year increase in age in SCD (95% CI: 2%, 4.7%, p < .001), but not in controls. A majority of children in all cohorts had an IgM concentration in the normal range for age and there were no differences between groups (p = .13). Subjects developed titers adequate for long-term protection to fewer serotypes in the polysaccharide vaccine than controls (14.7 vs. 19.4, p < .001). In this cohort, bacteremia was rare and specific clinical complications were not associated with UMBC proportion. In summary, UMBC deficiency occurs in SCD and is associated with age. Future studies should investigate B cell subsets prospectively and identify the mechanism of B cell loss in the spleen.

SHP2 promotes sarcoidosis severity by inhibiting SKP2-targeted ubiquitination of TBET in CD8+ T cells.

Sarcoidosis is an interstitial lung disease (ILD) characterized by interferon-γ (IFN-γ) and T-box expressed in T cells (TBET) dysregulation. Although one-third of patients progress from granulomatous inflammation to severe lung damage, the molecular mechanisms underlying this process remain unclear. Here, we found that pharmacological inhibition of phosphorylated SH2-containing protein tyrosine phosphatase-2 (pSHP2), a facilitator of aberrant IFN-γ abundance, decreased large granuloma formation and macrophage infiltration in the lungs of mice with sarcoidosis-like disease. Positive treatment outcomes were dependent on the effective enhancement of TBET ubiquitination within CD8+ T cells. Mechanistically, we identified a posttranslational modification pathway in which the E3 F-box protein S-phase kinase-associated protein 2 (SKP2) targets TBET for ubiquitination in T cells under normal conditions. However, this pathway was disrupted by aberrant pSHP2 signaling in CD8+ T cells from patients with progressive pulmonary sarcoidosis and end-stage disease. Ex vivo inhibition of pSHP2 in CD8+ T cells from patients with end-stage sarcoidosis enhanced TBET ubiquitination and suppressed IFN-γ and collagen synthesis. Therefore, these studies provided new mechanistic insights into the SHP2-dependent posttranslational regulation of TBET and identified SHP2 inhibition as a potential therapeutic intervention against severe sarcoidosis. Furthermore, these studies also suggest that the small-molecule SHP2 inhibitor SHP099 might be used as a therapeutic measure against human diseases linked to TBET or ubiquitination.

Caspase-2 is essential for proliferation and self-renewal of nucleophosmin-mutated acute myeloid leukemia.

Mutation in nucleophosmin (NPM1) causes relocalization of this normally nucleolar protein to the cytoplasm ( NPM1c+ ). Despite NPM1 mutation being the most common driver mutation in cytogenetically normal adult acute myeloid leukemia (AML), the mechanisms of NPM1c+-induced leukemogenesis remain unclear. Caspase-2 is a pro-apoptotic protein activated by NPM1 in the nucleolus. Here, we show that caspase-2 is also activated by NPM1c+ in the cytoplasm, and DNA damage-induced apoptosis is caspase-2-dependent in NPM1c+ AML but not in NPM1wt cells. Strikingly, in NPM1c+ cells, loss of caspase-2 results in profound cell cycle arrest, differentiation, and down-regulation of stem cell pathways that regulate pluripotency including impairment in the AKT/mTORC1 and Wnt signaling pathways. In contrast, there were minimal differences in proliferation, differentiation, or the transcriptional profile of NPM1wt cells with and without caspase-2. Together, these results show that caspase-2 is essential for proliferation and self-renewal of AML cells that have mutated NPM1. This study demonstrates that caspase-2 is a major effector of NPM1c+ function and may even be a druggable target to treat NPM1c+ AML and prevent relapse.

A murine model of hnRNPH2-related neurodevelopmental disorder reveals a mechanism for genetic compensation by Hnrnph1.

Mutations in HNRNPH2 cause an X-linked neurodevelopmental disorder with features that include developmental delay, motor function deficits, and seizures. More than 90% of patients with hnRNPH2 have a missense mutation within or adjacent to the nuclear localization signal (NLS) of hnRNPH2. Here, we report that hnRNPH2 NLS mutations caused reduced interaction with the nuclear transport receptor Kapß2 and resulted in modest cytoplasmic accumulation of hnRNPH2. We generated 2 knockin mouse models with human-equivalent mutations in Hnrnph2 as well as Hnrnph2-KO mice. Knockin mice recapitulated clinical features of the human disorder, including reduced survival in male mice, impaired motor and cognitive functions, and increased susceptibility to audiogenic seizures. In contrast, 2 independent lines of Hnrnph2-KO mice showed no detectable phenotypes. Notably, KO mice had upregulated expression of Hnrnph1, a paralog of Hnrnph2, whereas knockin mice failed to upregulate Hnrnph1. Thus, genetic compensation by Hnrnph1 may counteract the loss of hnRNPH2. These findings suggest that HNRNPH2-related disorder may be driven by a toxic gain of function or a complex loss of HNRNPH2 function with impaired compensation by HNRNPH1. The knockin mice described here are an important resource for preclinical studies to assess the therapeutic benefit of gene replacement or knockdown of mutant hnRNPH2.

The Efficiency of p27 Cleavage during In Vitro Respiratory Syncytial Virus (RSV) Infection Is Cell Line and RSV Subtype Dependent.

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. IMPORTANCE 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.

Caspase-2 regulates S-phase cell cycle events to protect from DNA damage accumulation independent of apoptosis.

In addition to its classical role in apoptosis, accumulating evidence suggests that caspase-2 has non-apoptotic functions, including regulation of cell division. Loss of caspase-2 is known to increase proliferation rates but how caspase-2 is regulating this process is currently unclear. We show that caspase-2 is activated in dividing cells in G1-phase of the cell cycle. In the absence of caspase-2, cells exhibit numerous S-phase defects including delayed exit from S-phase, defects in repair of chromosomal aberrations during S-phase, and increased DNA damage following S-phase arrest. In addition, caspase-2-deficient cells have a higher frequency of stalled replication forks, decreased DNA fiber length, and impeded progression of DNA replication tracts. This indicates that caspase-2 protects from replication stress and promotes replication fork protection to maintain genomic stability. These functions are independent of the pro-apoptotic function of caspase-2 because blocking caspase-2-induced cell death had no effect on cell division, DNA damage-induced cell cycle arrest, or DNA damage. Thus, our data supports a model where caspase-2 regulates cell cycle and DNA repair events to protect from the accumulation of DNA damage independently of its pro-apoptotic function.

Effect of sex chromosomes versus hormones in neonatal lung injury.

The main mechanisms underlying sexually dimorphic outcomes in neonatal lung injury are unknown. We tested the hypothesis that hormone- or sex chromosome-mediated mechanisms interact with hyperoxia exposure to impact injury and repair in the neonatal lung. To distinguish sex differences caused by gonadal hormones versus sex chromosome complement (XX versus XY), we used the Four Core Genotypes (FCG) mice and exposed them to hyperoxia (95% FiO2, P1-P4: saccular stage) or room air. This model generates XX and XY mice that each have either testes (with Sry, XXM, or XYM) or ovaries (without Sry, XXF, or XYF). Lung alveolarization and vascular development were more severely impacted in XYM and XYF compared with XXF and XXM mice. Cell cycle-related pathways were enriched in the gonadal or chromosomal females, while muscle-related pathways were enriched in the gonadal males, and immune-response-related pathways were enriched in chromosomal males. Female gene signatures showed a negative correlation with human patients who developed bronchopulmonary dysplasia (BPD) or needed oxygen therapy at 28 days. These results demonstrate that chromosomal sex - and not gonadal sex - impacted the response to neonatal hyperoxia exposure. The female sex chromosomal complement was protective and could mediate sex-specific differences in the neonatal lung injury.

A versatile oblique plane microscope for large-scale and high-resolution imaging of subcellular dynamics.

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.

HEM1 deficiency disrupts mTORC2 and F-actin control in inherited immunodysregulatory disease.

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 NCKAP1L, 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.

The cell biologist's guide to super-resolution microscopy.

Fluorescence microscopy has become a ubiquitous method to observe the location of specific molecular components within cells. However, the resolution of light microscopy is limited by the laws of diffraction to a few hundred nanometers, blurring most cellular details. Over the last two decades, several techniques - grouped under the 'super-resolution microscopy' moniker - have been designed to bypass this limitation, revealing the cellular organization down to the nanoscale. The number and variety of these techniques have steadily increased, to the point that it has become difficult for cell biologists and seasoned microscopists alike to identify the specific technique best suited to their needs. Available techniques include image processing strategies that generate super-resolved images, optical imaging schemes that overcome the diffraction limit and sample manipulations that expand the size of the biological sample. In this Cell Science at a Glance article and the accompanying poster, we provide key pointers to help users navigate through the various super-resolution methods by briefly summarizing the principles behind each technique, highlighting both critical strengths and weaknesses, as well as providing example images.

Human signal transducer and activator of transcription 5b (STAT5b) mutation causes dysregulated human natural killer cell maturation and impaired lytic function.

BACKGROUND: 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. OBJECTIVE: In this study we characterized the NK cell defect in STAT5b-deficient human NK cells, as well as Stat5b-/- mice. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function.

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 CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 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.

Heterozygous Truncating Variants in POMP Escape Nonsense-Mediated Decay and Cause a Unique Immune Dysregulatory Syndrome.

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.

Nanoscale Dynamism of Actin Enables Secretory Function in Cytolytic Cells.

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.

Ruxolitinib partially reverses functional natural killer cell deficiency in patients with signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations.

BACKGROUND: Natural killer (NK) cells are critical innate effector cells whose development is dependent on the Janus kinase-signal transducer and activator of transcription (STAT) pathway. NK cell deficiency can result in severe or refractory viral infections. Patients with STAT1 gain-of-function (GOF) mutations have increased viral susceptibility. OBJECTIVE: We sought to investigate NK cell function in patients with STAT1 GOF mutations. METHODS: NK cell phenotype and function were determined in 16 patients with STAT1 GOF mutations. NK cell lines expressing patients' mutations were generated with clustered regularly interspaced short palindromic repeats (CRISPR-Cas9)-mediated gene editing. NK cells from patients with STAT1 GOF mutations were treated in vitro with ruxolitinib. RESULTS: Peripheral blood NK cells from patients with STAT1 GOF mutations had impaired terminal maturation. Specifically, patients with STAT1 GOF mutations have immature CD56dim NK cells with decreased expression of CD16, perforin, CD57, and impaired cytolytic function. STAT1 phosphorylation was increased, but STAT5 was aberrantly phosphorylated in response to IL-2 stimulation. Upstream inhibition of STAT1 signaling with the small-molecule Janus kinase 1/2 inhibitor ruxolitinib in vitro and in vivo restored perforin expression in CD56dim NK cells and partially restored NK cell cytotoxic function. CONCLUSIONS: Properly regulated STAT1 signaling is critical for NK cell maturation and function. Modulation of increased STAT1 phosphorylation with ruxolitinib is an important option for therapeutic intervention in patients with STAT1 GOF mutations.

FiloQuant reveals increased filopodia density during breast cancer progression.

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.

Quantitative Imaging Approaches to Study the CAR Immunological Synapse.

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.