Emerging mechanistic understanding of cilia function in cellular signalling.

Primary cilia are solitary, immotile sensory organelles present on most cells in the body that participate broadly in human health, physiology and disease. Cilia generate a unique environment for signal transduction with tight control of protein, lipid and second messenger concentrations within a relatively small compartment, enabling reception, transmission and integration of biological information. In this Review, we discuss how cilia function as signalling hubs in cell-cell communication using three signalling pathways as examples: ciliary G-protein-coupled receptors (GPCRs), the Hedgehog (Hh) pathway and polycystin ion channels. We review how defects in these ciliary signalling pathways lead to a heterogeneous group of conditions known as 'ciliopathies', including metabolic syndromes, birth defects and polycystic kidney disease. Emerging understanding of these pathways' transduction mechanisms reveals common themes between these cilia-based signalling pathways that may apply to other pathways as well. These mechanistic insights reveal how cilia orchestrate normal and pathophysiological signalling outputs broadly throughout human biology.

Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched.

Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.

GRK2 kinases in the primary cilium initiate SMOOTHENED-PKA signaling in the Hedgehog cascade.

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

Smoothened stimulation by membrane sterols drives Hedgehog pathway activity.

Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration1. Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma2. Hedgehog proteins bind to and inhibit the transmembrane cholesterol transporter Patched-1 (PTCH1), which permits activation of the seven-transmembrane transducer Smoothened (SMO) via a mechanism that is poorly understood. Here we report the crystal structure of active mouse SMO bound to both the agonist SAG21k and to an intracellular binding nanobody that stabilizes a physiologically relevant active state. Analogous to other G protein-coupled receptors, the activation of SMO is associated with subtle motions in the extracellular domain, and larger intracellular changes. In contrast to recent models3-5, a cholesterol molecule that is critical for SMO activation is bound deep within the seven-transmembrane pocket. We propose that the inactivation of PTCH1 by Hedgehog allows a transmembrane sterol to access this seven-transmembrane site (potentially through a hydrophobic tunnel), which drives the activation of SMO. These results-combined with signalling studies and molecular dynamics simulations-delineate the structural basis for PTCH1-SMO regulation, and suggest a strategy for overcoming clinical resistance to SMO inhibitors.

Smoothened transduces Hedgehog signals via activity-dependent sequestration of PKA catalytic subunits.

The Hedgehog (Hh) pathway is essential for organ development, homeostasis, and regeneration. Dysfunction of this cascade drives several cancers. To control expression of pathway target genes, the G protein-coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here, we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase (GRK)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking Hh signal transduction at the membrane to GLI transcription in the nucleus. This process is more fundamentally similar between species than prevailing hypotheses suggest. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades in diverse areas of biology.

Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium.

Hedgehog signaling specifies tissue patterning and renewal, and pathway components are commonly mutated in certain malignancies. Although central to ensuring appropriate pathway activity in all Hedgehog-responsive cells, how the transporter-like receptor Patched1 regulates the seven-transmembrane protein Smoothened remains mysterious, partially due to limitations in existing tools and experimental systems. Here we employ direct, real-time, biochemical and physiology-based approaches to monitor Smoothened activity in cellular and in vitro contexts. Patched1-Smoothened coupling is rapid, dynamic, and can be recapitulated without cilium-specific proteins or lipids. By reconstituting purified Smoothened in vitro, we show that cholesterol within the bilayer is sufficient for constitutive Smoothened activation. Cholesterol effects occur independently of the lipid-binding Smoothened extracellular domain, a region that is dispensable for Patched1-Smoothened coupling. Finally, we show that Patched1 specifically requires extracellular Na+ to regulate Smoothened in our assays, raising the possibility that a Na+ gradient provides the energy source for Patched1 catalytic activity. Our work suggests a hypothesis wherein Patched1, chemiosmotically driven by the transmembrane Na+ gradient common to metazoans, regulates Smoothened by shielding its heptahelical domain from cholesterol, or by providing an inhibitor that overrides this cholesterol activation.

Stimuli-Responsive DNA-Linked Nanoparticle Arrays as Programmable Surfaces.

Self- and directed-assembly approaches have enabled precise control over the composition and geometry of 2D and 3D nanoparticle constructs. However, the resulting structures are typically static, providing only a single structural arrangement of the nanoparticle building blocks. In this work, the power of DNA-linked nanoparticle assembly is coupled to a grayscale patterning technique to create programmable surfaces for assembly and thermally activated reorganization of gold nanoparticle arrays. Direct grayscale patterning of DNA monolayers by electron-beam lithography (DNA-EBL) enables the production of surfaces with nanometer-scale control over the density of functional DNA. This enables tuning of the particle-surface interactions with single-nanoparticle resolution and without the need for a physical template as employed in most directed assembly methods. This technique is applied on suspended membrane structures to achieve high-resolution assembly of 2D nanoparticle arrays with highly mutable architectures. Gold nanorods assembled on grayscale-patterned surfaces exhibit temperature-dependent configurations and ordering behavior that result in tunable polarization-dependent optical properties. In addition, spherical gold particles assembled from a bimodal suspension produce arrays with temperature-dependent configurations of small and large particles. These results have important implications for the design and fabrication of reconfigurable nanoparticle arrays for application as structurally tunable optical metasurfaces.

Suppressing Electron Exposure Artifacts: An Electron Scanning Paradigm with Bayesian Machine Learning.

Electron microscopy of biological, polymeric, and other beam-sensitive structures is often hampered by deleterious electron beam interactions. In fact, imaging of such beam-sensitive materials is limited by the allowable radiation dosage rather that capabilities of the microscope itself, which has been compounded by the availability of high brightness electron sources. Reducing dwell times to overcome dose-related artifacts, such as radiolysis and electrostatic charging, is challenging due to the inherently low contrast in imaging of many such materials. These challenges are particularly exacerbated during dynamic time-resolved, fluidic cell imaging, or three-dimensional tomographic reconstruction-all of which undergo additional dosage. Thus, there is a pressing need for the development of techniques to produce high-quality images at ever lower electron doses. In this contribution, we demonstrate direct dose reduction and suppression of beam-induced artifacts through under-sampling pixels, by as much as 80% reduction in dosage, using a commercial scanning electron microscope with an electrostatic beam blanker and a dictionary learning in-painting algorithm. This allows for multiple sparse recoverable images to be acquired at the cost of one fully sampled image. We believe this approach may open new ways to conduct imaging, which otherwise require compromising beam current and/or exposure conditions.

A simple and scalable zebrafish model of Sonic hedgehog medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is stratified into three major subgroups. The Sonic hedgehog (SHH) subgroup represents ∼30% of all MB cases and has significant survival disparity depending upon TP53 status. Here, we describe a zebrafish model of SHH MB using CRISPR to create mutant ptch1, the primary genetic driver of human SHH MB. In these animals, tumors rapidly arise in the cerebellum and resemble human SHH MB by histology and comparative onco-genomics. Similar to human patients, MB tumors with loss of both ptch1 and tp53 have aggressive tumor histology and significantly worse survival outcomes. The simplicity and scalability of the ptch1-crispant MB model makes it highly amenable to CRISPR-based genome-editing screens to identify genes required for SHH MB tumor formation in vivo, and here we identify the gene encoding Grk3 kinase as one such target.

A Simple and Scalable Zebrafish Model of Sonic Hedgehog Medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is stratified into three major subgroups. The Sonic hedgehog (SHH) subgroup represents ~30% of all MB cases and has significant survival disparity depending upon TP53 status. Here, we describe the first zebrafish model of SHH MB using CRISPR to mutate ptch1, the primary genetic driver in human SHH MB. These tumors rapidly arise adjacent to the valvula cerebelli and resemble human SHH MB by histology and comparative genomics. In addition, ptch1-deficient MB tumors with loss of tp53 have aggressive tumor histology and significantly worse survival outcomes, comparable to human patients. The simplicity and scalability of the ptch1 MB model makes it highly amenable to CRISPR-based genome editing screens to identify genes required for SHH MB tumor formation in vivo, and here we identify the grk3 kinase as one such target.

GRK2 Kinases in the Primary Cilium Initiate SMOOTHENED-PKA Signaling in the Hedgehog Cascade.

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO, and the ensuing PKA-C binding and inactivation, are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

Studies of SMOOTHENED Activation in Cell-Free and Reconstituted Systems.

Much of our current understanding of Hedgehog signal transduction derives from studies involving intact cells and organisms. Here we describe the use of cell-free and reconstituted systems to study a key step in Hedgehog signal transduction: the activation of SMOOTHENED by membrane lipids. These methods can be adapted to study other steps in Hedgehog signal transduction, particularly those that occur at the membrane.

Rapid, Direct SMOOTHENED Activity Assays in Live Cells Using cAMP-Based Conformational Sensors.

Communication between PATCHED1 (PTCH1) and SMOOTHENED (SMO) is fundamental to Hedgehog (Hh) signal transduction in development and disease. We describe a real-time cell-based SMO functional assay based on SMO activity-dependent changes in cellular cAMP concentrations. This assay is capable of detecting changes in SMO conformation within minutes of PTCH1 inactivation by Hh ligands. As a result, it expands the range of experimental perturbations that can be used to dissect PTCH1-SMO communication, enabling a deeper mechanistic understanding of a longstanding mystery in Hh signal transduction.

Oxygen-Tolerant RAFT Polymerization Initiated by Living Bacteria.

Living organisms can synthesize a wide range of macromolecules from a small set of natural building blocks, yet there is potential for even greater materials diversity by exploiting biochemical processes to convert unnatural feedstocks into new abiotic polymers. Ultimately, the synthesis of these polymers in situ might aid the coupling of organisms with synthetic matrices, and the generation of biohybrids or engineered living materials. The key step in biohybrid materials preparation is to harness the relevant biological pathways to produce synthetic polymers with predictable molar masses and defined architectures under ambient conditions. Accordingly, we report an aqueous, oxygen-tolerant RAFT polymerization platform based on a modified Fenton reaction, which is initiated by Cupriavidus metallidurans CH34, a bacterial species with iron-reducing capabilities. We show the synthesis of a range of water-soluble polymers under normoxic conditions, with control over the molar mass distribution, and also the production of block copolymer nanoparticles via polymerization-induced self-assembly. Finally, we highlight the benefits of using a bacterial initiation system by recycling the cells for multiple polymerizations. Overall, our method represents a highly versatile approach to producing well-defined polymeric materials within a hybrid natural-synthetic polymerization platform and in engineered living materials with properties beyond those of biotic macromolecules.

A PKA inhibitor motif within SMOOTHENED controls Hedgehog signal transduction.

The Hedgehog (Hh) cascade is central to development, tissue homeostasis and cancer. A pivotal step in Hh signal transduction is the activation of glioma-associated (GLI) transcription factors by the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO). How SMO activates GLI remains unclear. Here we show that SMO uses a decoy substrate sequence to physically block the active site of the cAMP-dependent protein kinase (PKA) catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular and organismal models, we demonstrate that interfering with SMO-PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism uncovered echoes one used by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

TRP channel structural biology: new roles for an old fold.

The capsaicin receptor, TRPV1, contributes to thermal and chemical sensitivity of primary afferent neurons of the pain pathway, but many aspects of its regulation remain elusive. In this issue of Neuron, Lishko et al. describe a high-resolution structure of a TRPV1 domain, providing insight into the molecular basis of channel modulation while revealing new functions for a widely expressed protein interaction fold.

Leveraging the United States Epicenter to Provide Insights on COVID-19 in Patients With Systemic Lupus Erythematosus.

OBJECTIVE: To characterize patients with systemic lupus erythematosus (SLE) affected by coronavirus disease 2019 (COVID-19) and to analyze associations of comorbidities and medications on infection outcomes. METHODS: Patients with SLE and reverse transcriptase-polymerase chain reaction-confirmed COVID-19 were identified through an established New York University lupus cohort, query of 2 hospital systems, and referrals from rheumatologists. Data were prospectively collected via a web-based questionnaire and review of medical records. Data on baseline characteristics were obtained for all patients with COVID-19 to analyze risk factors for hospitalization. Data were also collected on asymptomatic patients and those with COVID-19-like symptoms who tested negative or were not tested. Statistical analyses were limited to confirmed COVID-19-positive patients. RESULTS: A total of 226 SLE patients were included: 41 with confirmed COVID-19, 19 who tested negative for COVID-19, 42 with COVID-19-like symptoms who did not get tested, and 124 who remained asymptomatic without testing. Of the SLE patients with confirmed COVID-19, hospitalization was required in 24 (59%) and intensive care unit-level of care in 4, and 4 died. Hospitalized patients tended to be older, nonwhite, Hispanic, have higher body mas index (BMI), history of nephritis, and at least 1 comorbidity. An exploratory (due to limited sample size) logistic regression analysis identified race, presence of at least 1 comorbidity, and BMI as independent predictors of hospitalization. CONCLUSION: In general, the variables predictive of hospitalization in our SLE patients were similar to those identified in the general population. Further studies are needed to understand additional risk factors for poor COVID-19 outcomes in patients with SLE.

Zebrafish TRPA1 channels are required for chemosensation but not for thermosensation or mechanosensory hair cell function.

Transient receptor potential (TRP) ion channels have been implicated in detecting chemical, thermal, and mechanical stimuli in organisms ranging from mammals to Caenorhabditis elegans. It is well established that TRPA1 detects and mediates behavioral responses to chemical irritants. However, the role of TRPA1 in detecting thermal and mechanical stimuli is controversial. To further clarify the functions of TRPA1 channels in vertebrates, we analyzed their roles in zebrafish. The two zebrafish TRPA1 paralogs are expressed in sensory neurons and are activated by several chemical irritants in vitro. High-throughput behavioral analyses of trpa1a and trpa1b mutant larvae indicate that TRPA1b is necessary for behavioral responses to these chemical irritants. However, TRPA1 paralogs are not required for behavioral responses to temperature changes or for mechanosensory hair cell function in the inner ear or lateral line. These results support a role for zebrafish TRPA1 in chemical but not thermal or mechanical sensing, and establish a high-throughput system to identify genes and small molecules that modulate chemosensation, thermosensation, and mechanosensation.

Stage-Rocked Electron Channeling for Crystal Orientation Mapping.

Microstructural analysis by crystal orientation mapping of bulk functional materials is an essential and routine operation in the engineering of material properties. Far and away the most successfully employed technique, Electron Backscattered Diffraction (EBSD), provides high spatial resolution information at the cost of limited angular resolution and a distorted imaging condition. In this work, we demonstrate a stage-rocked electron channeling approach as a low-cost orientation mapping alternative to EBSD. This is accomplished by automated electron channeling contrast imaging (ECCI) as the microscope stage physically tilts/rotates a sample through a reduced hemisphere of orientations followed by computational reconstruction of electron channeling patterns (ECP). Referred to as Orientation Mapping by Electron Channeling (OMEC), our method offers advantages in terms of local defect analysis, as it combines the advantages of selected area ECP (SACP) and ECCI. We also illustrate dynamic or "adaptive" sampling schemes to increase the throughput of the technique. Finally, we discuss the implications for sample analysis in which large 3D maps of ECCI images can be routinely constructed of challenging crystalline samples. As an electron channeling-based approach to orientation mapping, OMEC may open new routes to characterize crystalline materials with high angular and spatial resolution.

Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next-Generation Sequencing.

This unit describes a technique for generating exome-enriched sequencing libraries using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. Utilizing commercially available kits, we present a low-input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution-targeted selection for exons, followed by sequencing using the Illumina next-generation short-read sequencing platform. © 2017 by John Wiley & Sons, Inc.