G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.

The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.

Publisher Correction: G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Role for myosin-V motor proteins in the selective delivery of Kv channel isoforms to the membrane surface of cardiac myocytes.

RATIONALE: Kv1.5 (KCNA5) mediates the ultra-rapid delayed rectifier current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation, Kv1.5 has emerged as a promising target for the treatment of atrial fibrillation. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known. OBJECTIVE: To investigate the role of the unconventional myosin-V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5. METHODS AND RESULTS: Western blot analysis showed MYO5A and MYO5B expression in the heart, whereas disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and short hairpin RNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and connexin-43 but not potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, whereas MYO5B regulated postendocytic recycling. Overexpression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling. CONCLUSIONS: MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current and, consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.

ISG15/GRAIL1/CD3 axis influences survival of patients with esophageal adenocarcinoma.

Immunosuppression is a common feature of esophageal adenocarcinoma (EAC) and has been linked to poor overall survival (OS). We hypothesized that upstream factors might negatively influence CD3 levels and T cell activity, thus promoting immunosuppression and worse survival. We used clinical data and patient samples of those who progressed from Barrett's to dysplasia to EAC, investigated gene (RNA-Seq) and protein (tissue microarray) expression, and performed cell biology studies to delineate a pathway impacting CD3 protein stability that might influence EAC outcome. We showed that the loss of both CD3-ε expression and CD3+ T cell number correlated with worse OS in EAC. The gene related to anergy in lymphocytes isoform 1 (GRAIL1), which is the prominent isoform in EACs, degraded (ε, γ, δ) CD3s and inactivated T cells. In contrast, isoform 2 (GRAIL2), which is reduced in EACs, stabilized CD3s. Further, GRAIL1-mediated CD3 degradation was facilitated by interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein. Consequently, the overexpression of a ligase-dead GRAIL1, ISG15 knockdown, or the overexpression of a conjugation-defective ISG15-leucine-arginine-glycine-glycine mutant could increase CD3 levels. Together, we identified an ISG15/GRAIL1/mutant p53 amplification loop negatively influencing CD3 levels and T cell activity, thus promoting immunosuppression in EAC.

Role of IL4 and GMCSF in Predicting Survival in Esophageal Cancer.

BACKGROUND: Esophageal cancer (EC) originates in the setting of chronic inflammation. Although previous studies have sought to understand the role of inflammatory signaling in EC, the effect of these immunologic changes on patient outcomes remains understudied. This study's objective was to identify relationships between cytokine levels and prognosis in a mixed cohort of esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC) patients. STUDY DESIGN: A total of 37 serum cytokines were profiled at the time of resection using multiplex ELISA in 47 patients (42 esophageal adenocarcinoma, 5 esophageal squamous cell carcinoma). Cytokine levels were median-binarized and assessed using Cox regression models. Findings were validated at the RNA level using The Cancer Genome Atlas EC cohort (81 esophageal adenocarcinoma, 81 esophageal squamous cell carcinoma). RESULTS: Univariable analysis revealed high serum interleukin 4 (IL4) and granulocyte-macrophage colony-stimulating factor (GMCSF) were negatively associated with overall survival (p = 0.046, p = 0.040). Multivariable analysis determined both high serum IL4 or high serum GMCSF were negatively associated with survival independent of important clinical factors (hazard ratio [HR] 7.55, p < 0.001; HR 5.24, p = 0.001). These findings were validated at the RNA level in The Cancer Genome Atlas EC cohort, where multivariable analysis identified high IL4 expression, high CSF2 expression (encodes GMCSF), and advanced pathologic stage as independent negative predictors of survival when controlled for clinical factors (HR 2.35, p = 0.012; HR 1.97, p = 0.040). CONCLUSIONS: These results show that high IL4/GMCSF levels are negatively associated with survival in EC. These relationships are independent of pathologic stage and are identified across modalities, histologic subtypes, and the presence/absence of neoadjuvant therapy.

Factors affecting timing of surgery following neoadjuvant chemoradiation for esophageal cancer.

Background: Neoadjuvant chemoradiation with esophagectomy is standard management for locally advanced esophageal cancer. Studies have shown that surgical timing following chemoradiation is important for minimizing postoperative complications, however in practice timing is often variable and delayed. Although postoperative impact of surgical timing has been studied, less is known about factors associated with delays. Materials and methods: A retrospective review was performed for 96 patients with esophageal cancer who underwent chemoradiation then esophagectomy between 2018 and 2020 at a single institution. Univariable and stepwise multivariable analyses were used to assess association between social (demographics, insurance) and clinical variables (pre-operative weight, comorbidities, prior cardiothoracic surgery, smoking history, disease staging) with time to surgery (≤8 weeks "on-time" vs. >8 weeks "delayed"). Results: Fifty-one patients underwent esophagectomy within 8 weeks of chemoradiation; 45 had a delayed operation. Univariate analysis showed the following characteristics were significantly different between on-time and delayed groups: weight loss within 3 months of surgery (3.9 ± 5.1 kg vs. 1.5 ± 3.6 kg; P = 0.009), prior cardiovascular disease (29% vs. 49%; P = 0.05), prior cardiothoracic surgery (4% vs. 22%; P = 0.01), history of ever smoked (69% vs. 87%; P = 0.04), absent nodal metastasis on pathology (57% vs. 82%; P = 0.008). Multivariate analysis demonstrated that prior cardiothoracic surgery (OR 8.924, 95%CI 1.67-47.60; P = 0.01) and absent nodal metastasis (OR 4.186, 95%CI 1.50-11.72; P = 0.006) were associated with delayed surgery. Conclusions: Delayed esophagectomy following chemoradiotherapy is associated with prior cardiothoracic surgery and absent nodal metastasis. Further investigations should focus on understanding how these factors contribute to delays to guide treatment planning and mitigate sources of outcome disparities.

Single molecule imaging reveals differences in microtubule track selection between Kinesin motors.

Cells generate diverse microtubule populations by polymerization of a common alpha/beta-tubulin building block. How microtubule associated proteins translate microtubule heterogeneity into specific cellular functions is not clear. We evaluated the ability of kinesin motors involved in vesicle transport to read microtubule heterogeneity by using single molecule imaging in live cells. We show that individual Kinesin-1 motors move preferentially on a subset of microtubules in COS cells, identified as the stable microtubules marked by post-translational modifications. In contrast, individual Kinesin-2 (KIF17) and Kinesin-3 (KIF1A) motors do not select subsets of microtubules. Surprisingly, KIF17 and KIF1A motors that overtake the plus ends of growing microtubules do not fall off but rather track with the growing tip. Selection of microtubule tracks restricts Kinesin-1 transport of VSVG vesicles to stable microtubules in COS cells whereas KIF17 transport of Kv1.5 vesicles is not restricted to specific microtubules in HL-1 myocytes. These results indicate that kinesin families can be distinguished by their ability to recognize microtubule heterogeneity. Furthermore, this property enables kinesin motors to segregate membrane trafficking events between stable and dynamic microtubule populations.

Antiarrhythmic drug-induced internalization of the atrial-specific k+ channel kv1.5.

Conventional antiarrhythmic drugs target the ion permeability of channels, but increasing evidence suggests that functional ion channel density can also be modified pharmacologically. Kv1.5 mediates the ultrarapid potassium current (I(Kur)) that controls atrial action potential duration. Given the atrial-specific expression of Kv1.5 and its alterations in human atrial fibrillation, significant effort has been made to identify novel channel blockers. In this study, treatment of HL-1 atrial myocytes expressing Kv1.5-GFP with the class I antiarrhythmic agent quinidine resulted in a dose- and temperature-dependent internalization of Kv1.5, concomitant with channel block. This quinidine-induced channel internalization was confirmed in acutely dissociated neonatal myocytes. Channel internalization was subunit-dependent, activity-independent, stereospecific, and blocked by pharmacological disruption of the endocytic machinery. Pore block and channel internalization partially overlap in the structural requirements for drug binding. Surprisingly, quinidine-induced endocytosis was calcium-dependent and therefore unrecognized by previous biophysical studies focused on isolating channel-drug interactions. Importantly, whereas acute quinidine-induced internalization was reversible, chronic treatment led to channel degradation. Together, these data reveal a novel mechanism of antiarrhythmic drug action and highlight the possibility for new agents that selectively modulate the stability of channel protein in the membrane as an approach for treating cardiac arrhythmias.

Immune determinants of Barrett's progression to esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) develops from Barrett's esophagus (BE), a chronic inflammatory state that can progress through a series of transformative dysplastic states before tumor development. While molecular and genetic changes of EAC tumors have been studied, immune microenvironment changes during Barrett's progression to EAC remain poorly understood. In this study, we identify potential immunologic changes that can occur during BE-to-EAC progression. RNA sequencing (RNA-Seq) analysis on tissue samples from EAC patients undergoing surgical resection demonstrated that a subset of chemokines and cytokines, most notably IL6 and CXCL8, increased during BE progression to EAC. xCell deconvolution analysis investigating immune cell population changes demonstrated that the largest changes in expression during BE progression occurred in M2 macrophages, pro-B cells, and eosinophils. Multiplex immunohistochemical staining of tissue microarrays showed increased immune cell populations during Barrett's progression to high-grade dysplasia. In contrast, EAC tumor sections were relatively immune poor, with a rise in PD-L1 expression and loss of CD8+ T cells. These data demonstrate that the EAC microenvironment is characterized by poor cytotoxic effector cell infiltration and increased immune inhibitory signaling. These findings suggest an immunosuppressive microenvironment, highlighting the need for further studies to explore immune modulatory therapy in EAC.

Ciliary targeting of olfactory CNG channels requires the CNGB1b subunit and the kinesin-2 motor protein, KIF17.

Nonmotile cilia on olfactory sensory neurons (OSNs) compartmentalize signaling molecules, including odorant receptors and cyclic nucleotide-gated (CNG) channels, allowing for efficient, spatially confined responses to sensory stimuli . Little is known about the mechanisms of the ciliary targeting of olfactory CNG channels, composed of three subunits: CNGA2, CNGA4, and CNGB1b . Recent reports suggest that subunit composition of the retinal CNG channel influences localization, leading to disease . However, the mechanistic role of subunits in properly targeting native olfactory CNG channels remains unclear. Here, we show that heteromeric assembly with CNGB1b, containing a critical carboxy-terminal motif (RVxP), is required for ciliary trafficking of olfactory CNG channels. Movement of proteins within the cilia is governed by intraflagellar transport (IFT), a process that facilitates bidirectional movement of cargo along microtubules. Work in C. elegans has established that heterotrimeric and homodimeric kinesin-2 family members play a critical role in anterograde transport . In mammalian systems, the heterotrimeric KIF3a/KIF3b/KAP-3 complex plays a clear role in IFT; however, no role has been established for KIF17, the mammalian homolog of OSM-3 . Here, we demonstrate that KIF17 is required for olfactory CNG channel targeting, providing novel insights into mechanisms of mammalian ciliary transport.

Olfactory cilia: linking sensory cilia function and human disease.

The olfactory system gives us an awareness of our immediate environment by allowing us to detect airborne stimuli. The components necessary for detection of these odorants are compartmentalized in the cilia of olfactory sensory neurons. Cilia are microtubule-based organelles, which can be found projecting from the surface of almost any mammalian cell, and are critical for proper olfactory function. Mislocalization of ciliary proteins and/or the loss of cilia cause impaired olfactory function, which is now recognized as a clinical manifestation of a broad class of human diseases, termed ciliopathies. Future work investigating the mechanisms of olfactory cilia function will provide us important new information regarding the pathogenesis of human sensory perception diseases.

Caveolin regulates kv1.5 trafficking to cholesterol-rich membrane microdomains.

The targeting of ion channels to cholesterol-rich membrane microdomains has emerged as a novel mechanism of ion channel localization. Previously, we reported that Kv1.5, a prominent cardiovascular K(+) channel alpha-subunit, localizes to caveolar microdomains. However, the mechanisms regulating Kv1.5 targeting and the functional significance of this localization are largely unknown. In this study, we demonstrate a role for caveolin in the trafficking of Kv1.5 to lipid raft microdomains where cholesterol modulates channel function. In cells lacking endogenous caveolin-1 or -3, the association of Kv1.5 with low-density, detergent-resistant membrane fractions requires coexpression with exogenous caveolin, which can form channel-caveolin complexes. Caveolin is not required for cell surface expression, however, and caveolin-trafficking mutants sequester Kv1.5, but not Kv2.1, in intracellular compartments, resulting in a loss of functional cell surface channel. Coexpression with wild type caveolin-1 does not alter Kv1.5 current density; rather, it induces depolarizing shifts in steady-state activation and inactivation. These shifts are analogous to those produced by elevation of membrane cholesterol. Together, these results show that caveolin modulates channel function by regulating trafficking to cholesterol-rich membrane microdomains.

Mice lacking sodium channel beta1 subunits display defects in neuronal excitability, sodium channel expression, and nodal architecture.

Sodium channel beta1 subunits modulate alpha subunit gating and cell surface expression and participate in cell adhesive interactions in vitro. beta1-/- mice appear ataxic and display spontaneous generalized seizures. In the optic nerve, the fastest components of the compound action potential are slowed and the number of mature nodes of Ranvier is reduced, but Na(v)1.6, contactin, caspr 1, and K(v)1 channels are all localized normally at nodes. At the ultrastructural level, the paranodal septate-like junctions immediately adjacent to the node are missing in a subset of axons, suggesting that beta1 may participate in axo-glial communication at the periphery of the nodal gap. Sodium currents in dissociated hippocampal neurons are normal, but Na(v)1.1 expression is reduced and Na(v)1.3 expression is increased in a subset of pyramidal neurons in the CA2/CA3 region, suggesting a basis for the epileptic phenotype. Our results show that beta1 subunits play important roles in the regulation of sodium channel density and localization, are involved in axo-glial communication at nodes of Ranvier, and are required for normal action potential conduction and control of excitability in vivo.

Pharmacologic characterization of a kinetic in vitro human co-culture angiogenesis model using clinically relevant compounds.

Angiogenesis, the formation of new vessels from preexisting vessels, involves multiple cell types acting in concert to cause endothelial cell proliferation, migration, and differentiation into microvascular arrays. Under pathologic conditions, microenvironment changes result in altered blood vessel production. Historically, in vitro angiogenesis assays study individual aspects of the process and tend to be variable, difficult to quantify, and limited in clinical relevance. Here, we describe a kinetic, quantitative, co-culture angiogenesis model and demonstrate its relevance to in vivo pharmacology. Similar to in vivo angiogenesis, a co-culture of human umbilical vein endothelial cells with normal human dermal fibroblasts remains sensitive to multiple cytokines, resulting in a concentration-dependent stimulation of tube formation over time. Treatment with axitinib, a selective vascular endothelial growth factor (VEGF) antagonist, inhibited VEGF-mediated tube length and branch point formation and was selective for inhibiting VEGF over basic fibroblast growth factor (bFGF), similar to previous studies. Conversely, an FGFR-1 selective compound, PD-161570, was more potent at inhibiting bFGF-mediated angiogenesis. These results demonstrate the cytokine dynamics, selective pharmacology, and translational application of this model system. Finally, combining quantitative angiogenic biology with kinetic, live-content imaging highlights the importance of using validated in vitro models in drug discovery research.

Development and characterization of a high-throughput in vitro cord formation model insensitive to VEGF inhibition.

BACKGROUND: Anti-VEGF therapy reduces tumor blood vessels, however, some vessels always remain. These VEGF insensitive vessels may help support continued tumor growth and metastases. Many in vitro assays examining multiple steps of the angiogenic process have been described, but the majority of these assays are sensitive to VEGF inhibition. There has been little focus on the development of high-throughput, in vitro assays to model the vessels that are insensitive to VEGF inhibition. METHODS: Here, we describe a fixed end-point and kinetic, high-throughput stem cell co-culture model of cord formation. RESULTS: In this system, cords develop within 24 hours, at which point they begin to lose sensitivity to VEGF inhibitors, bevacizumab, and ramucirumab. Consistent with the hypothesis that other angiogenic factors maintain VEGF-independent vessels, pharmacologic intervention with a broad spectrum anti-angiogenic antagonist (suramin), a vascular disrupting agent (combretastatin), or a combination of VEGF and Notch pathway inhibitors reduced the established networks. In addition, we used our in vitro approach to develop an in vivo co-implant vasculogenesis model that connects with the endogenous vasculature to form functional blood vessels. Similar to the in vitro system, over time these vessels become insensitive to VEGF inhibition. CONCLUSION: Together, these models may be used to identify novel drugs targeting tumor vessels that are not sensitive to VEGF inhibition.

Gene therapy rescues cilia defects and restores olfactory function in a mammalian ciliopathy model.

Cilia are evolutionarily conserved microtubule-based organelles that are crucial for diverse biological functions, including motility, cell signaling and sensory perception. In humans, alterations in the formation and function of cilia manifest clinically as ciliopathies, a growing class of pleiotropic genetic disorders. Despite the substantial progress that has been made in identifying genes that cause ciliopathies, therapies for these disorders are not yet available to patients. Although mice with a hypomorphic mutation in the intraflagellar transport protein IFT88 (Ift88Tg737Rpw mice, also known as ORPK mice)5 have been well studied, the relevance of IFT88 mutations to human pathology is unknown. We show that a mutation in IFT88 causes a hitherto unknown human ciliopathy. In vivo complementation assays in zebrafish and mIMCD3 cells show the pathogenicity of this newly discovered allele. We further show that ORPK mice are functionally anosmic as a result of the loss of cilia on their olfactory sensory neurons (OSNs). Notably, adenoviral-mediated expression of IFT88 in mature, fully differentiated OSNs of ORPK mice is sufficient to restore ciliary structures and rescue olfactory function. These studies are the first to use in vivo therapeutic treatment to reestablish cilia in a mammalian ciliopathy. More broadly, our studies indicate that gene therapy is a viable option for cellular and functional rescue of the complex ciliary organelle in established differentiated cells.

Antifibrillatory agents and potassium channels in the atria: pore block versus channel trafficking.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. The preferred therapy for AF is sustained sinus rhythm control; however, the efficacy of currently used antiarrythmic drugs is limited by adverse side effects resulting from both a lack of ion channel selectivity and nonspecific ventricular activity. The role of the voltage-gated potassium channels in atrial myocyte repolarization and the subsequent control of action potential duration renders them attractive targets for antiarrhythmic drugs in the treatment of AF. Conventional antiarrhythmic drugs generally target the ion permeability of potassium channels. This review discusses the limitations of this traditional approach and introduces, as a novel paradigm for antiarrhythmic pharmacology, the decrease of ion channel cell surface density through the modulation of ion channel trafficking pathways.

The voltage-gated Na+ channel beta3 subunit does not mediate trans homophilic cell adhesion or associate with the cell adhesion molecule contactin.

Voltage-gated Na(+) channel (VGSC) beta1 and beta2 subunits are multifunctional, serving as both channel modulators and cell adhesion molecules (CAMs). The purpose of this study was to determine whether VGSC beta3 subunits function as CAMs. The beta3 extracellular domain is highly homologous to beta1, suggesting that beta3 may also be a functional CAM. We investigated the trans homophilic cell adhesive properties of beta3, its association with the beta1-interacting CAM contactin, as well as its ability to interact with the cytoskeletal protein ankyrin. Our results demonstrate that, unlike beta1, beta3 does not participate in trans homophilic cell-cell adhesion or associate with contactin. Further, beta3 does not associate with ankyrin(G) in a heterologous system. Previous studies have shown that beta3 interacts with the CAM neurofascin-186 but not with VGSC beta1. Taken together, these findings suggest that, although beta1 and beta3 exhibit similar channel modulatory properties in heterologous systems, these subunits differ with regard to their homophilic and heterophilic CAM binding profiles.

Olfactory cilia: our direct neuronal connection to the external world.

An organism's awareness of its surroundings is dependent on sensory function. As antennas to our external environment, cilia are involved in fundamental biological processes such as olfaction, photoreception, and touch. The olfactory system has adapted this organelle for its unique sensory function and optimized it for detection of external stimuli. The elongated and tapering structure of olfactory cilia and their organization into an overlapping meshwork bathed by the nasal mucosa is optimized to enhance odor absorption and detection. As many as 15-30 nonmotile, sensory cilia on dendritic endings of single olfactory sensory neurons (OSNs) compartmentalize signaling molecules necessary for odor detection allowing for efficient and spatially confined responses to sensory stimuli. Although the loss of olfactory cilia or deletion of selected components of the olfactory signaling cascade leads to anosmia, the mechanisms of ciliogenesis and the selected enrichment of signaling molecules remain poorly understood. Much of our current knowledge is the result of elegant electron microscopy studies describing the structure and organization of the olfactory epithelium and cilia. New genetic and cell biological approaches, which compliment these early studies, show promise in elucidating the mechanisms of olfactory cilia assembly, maintenance, and compartmentalization. Importantly, emerging evidence suggests that olfactory dysfunction represents a previously unrecognized clinical manifestation of multiple ciliary disorders. Future work investigating the mechanisms of olfactory dysfunction combining both clinical studies with basic science research will provide us important new information regarding the pathogenesis of human sensory perception diseases.