Nitric oxide delivery and heme-assisted S-nitrosation by the bedbug nitrophorin.

Nitrophorins are heme proteins used by blood feeding insects to deliver nitric oxide (NO) to a victim, leading to vasodilation and antiplatelet activity. Cimex lectularius (bedbug) nitrophorin (cNP) accomplishes this with a cysteine ligated ferric (Fe(III)) heme. In the acidic environment of the insect's salivary glands, NO binds tightly to cNP. During a blood meal, cNP-NO is delivered to the feeding site where dilution and increased pH lead to NO release. In a previous study, cNP was shown to not only bind heme, but to also nitrosate the proximal cysteine, leading to Cys-NO (SNO) formation. SNO formation requires oxidation of the proximal cysteine, which was proposed to be metal-assisted through accompanying reduction of ferric heme and formation of Fe(II)-NO. Here, we report the 1.6 Å crystal structure of cNP first chemically reduced and then exposed to NO, and show that Fe(II)-NO is formed but SNO is not, supporting a metal-assisted SNO formation mechanism. Crystallographic and spectroscopic studies of mutated cNP show that steric crowding of the proximal site inhibits SNO formation while a sterically relaxed proximal site enhances SNO formation, providing insight into specificity for this poorly understood modification. Experiments examining the pH dependence for NO implicate direct protonation of the proximal cysteine as the underlying mechanism. At lower pH, thiol heme ligation predominates, leading to a smaller trans effect and 60-fold enhanced NO affinity (Kd = 70 nM). Unexpectedly, we find that thiol formation interferes with SNO formation, suggesting cNP-SNO is unlikely to form in the insect salivary glands.

Ankmy2 Prevents Smoothened-Independent Hyperactivation of the Hedgehog Pathway via Cilia-Regulated Adenylyl Cyclase Signaling.

The mechanisms underlying subcellular targeting of cAMP-generating adenylyl cyclases and processes regulated by their compartmentalization are poorly understood. Here, we identify Ankmy2 as a repressor of the Hedgehog pathway via adenylyl cyclase targeting. Ankmy2 binds to multiple adenylyl cyclases, determining their maturation and trafficking to primary cilia. Mice lacking Ankmy2 are mid-embryonic lethal. Knockout embryos have increased Hedgehog signaling and completely open neural tubes showing co-expansion of all ventral neuroprogenitor markers, comparable to the loss of the Hedgehog receptor Patched1. Ventralization in Ankmy2 knockout is completely independent of the Hedgehog pathway transducer Smoothened. Instead, ventralization results from the reduced formation of Gli2 and Gli3 repressors and early depletion of adenylyl cyclase III in neuroepithelial cilia, implicating deficient pathway repression. Ventralization in Ankmy2 knockout requires both cilia and Gli2 activation. These findings indicate that cilia-dependent adenylyl cyclase signaling represses the Hedgehog pathway and promotes morphogenetic patterning.

Tulp3 Regulates Renal Cystogenesis by Trafficking of Cystoproteins to Cilia.

Polycystic kidney disease proteins, polycystin-1 and polycystin-2, localize to primary cilia. Polycystin knockouts have severe cystogenesis compared to ciliary disruption, whereas simultaneous ciliary loss suppresses excessive cyst growth. These data suggest the presence of a cystogenic activator that is inhibited by polycystins and an independent but relatively minor cystogenic inhibitor, either of which are cilia dependent. However, current genetic models targeting cilia completely ablate the compartment, making it difficult to uncouple cystoprotein function from ciliary localization. Thus, the role of cilium-generated signaling in cystogenesis is unclear. We recently demonstrated that the tubby family protein Tulp3 determines ciliary trafficking of polycystins in kidney collecting duct cells without affecting protein levels or cilia. Here, we demonstrate that embryonic-stage, nephron-specific Tulp3 knockout mice developed cystic kidneys, while retaining intact cilia. Cystic kidneys showed increased mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), mTOR, and persistently high cyclic AMP (cAMP) signaling, suggesting contribution of multiple factors to cystogenesis. Based on kidney-to-body-weight ratio, cystic index, and epithelial proliferation in developing tubules or cysts, the severity of cystogenesis upon Tulp3 deletion was intermediate between that caused by loss of polycystin-1 or cilia. However, concomitant Tulp3 loss did not inhibit cystogenesis in polycystin-1 knockouts, unlike ciliary disruption. Interestingly, ciliary trafficking of the small guanosine triphosphatase (GTPase) Arl13b, loss of which causes cystogenic severity similar to ciliary loss, was reduced prior to cyst initiation. Thus, we propose that cystogenesis in Tulp3 mutants results from a reduction of ciliary levels of polycystins, Arl13b, and Arl13b-dependent lipidated cargoes. Arl13b might be the ciliary factor that represses cystogenesis distinct from polycystins.

Using Primary Neurosphere Cultures to Study Primary Cilia.

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins.

The primary cilium is a paradigmatic organelle for studying compartmentalized signaling; however, unlike soluble protein trafficking, processes targeting integral membrane proteins to cilia are poorly understood. In this study, we determine that the tubby family protein TULP3 functions as a general adapter for ciliary trafficking of structurally diverse integral membrane cargo, including multiple reported and novel rhodopsin family G protein-coupled receptors (GPCRs) and the polycystic kidney disease-causing polycystin 1/2 complex. The founding tubby family member TUB also localizes to cilia similar to TULP3 and determines trafficking of a subset of these GPCRs to neuronal cilia. Using minimal ciliary localization sequences from GPCRs and fibrocystin (also implicated in polycystic kidney disease), we demonstrate these motifs to be sufficient and TULP3 dependent for ciliary trafficking. We propose a three-step model for TULP3/TUB-mediated ciliary trafficking, including the capture of diverse membrane cargo by the tubby domain in a phosphoinositide 4,5-bisphosphate (PI(4,5)P2)-dependent manner, ciliary delivery by intraflagellar transport complex A binding to the TULP3/TUB N terminus, and subsequent release into PI(4,5)P2-deficient ciliary membrane.

Trafficking to the primary cilium membrane.

The primary cilium has been found to be associated with a number of cellular signaling pathways, such as vertebrate hedgehog signaling, and implicated in the pathogenesis of diseases affecting multiple organs, including the neural tube, kidney, and brain. The primary cilium is the site where a subset of the cell's membrane proteins is enriched. However, pathways that target and concentrate membrane proteins in cilia are not well understood. Processes determining the level of proteins in the ciliary membrane include entry into the compartment, removal, and retention by diffusion barriers such as the transition zone. Proteins that are concentrated in the ciliary membrane are also localized to other cellular sites. Thus it is critical to determine the particular role for ciliary compartmentalization in sensory reception and signaling pathways. Here we provide a brief overview of our current understanding of compartmentalization of proteins in the ciliary membrane and the dynamics of trafficking into and out of the cilium. We also discuss major unanswered questions regarding the role that defects in ciliary compartmentalization might play in disease pathogenesis. Understanding the trafficking mechanisms that underlie the role of ciliary compartmentalization in signaling might provide unique approaches for intervention in progressive ciliopathies.

Smoothened determines ß-arrestin-mediated removal of the G protein-coupled receptor Gpr161 from the primary cilium.

Dynamic changes in membrane protein composition of the primary cilium are central to development and homeostasis, but we know little about mechanisms regulating membrane protein flux. Stimulation of the sonic hedgehog (Shh) pathway in vertebrates results in accumulation and activation of the effector Smoothened within cilia and concomitant disappearance of a negative regulator, the orphan G protein-coupled receptor (GPCR), Gpr161. Here, we describe a two-step process determining removal of Gpr161 from cilia. The first step involves ß-arrestin recruitment by the signaling competent receptor, which is facilitated by the GPCR kinase Grk2. An essential factor here is the ciliary trafficking and activation of Smoothened, which by increasing Gpr161-ß-arrestin binding promotes Gpr161 removal, both during resting conditions and upon Shh pathway activation. The second step involves clathrin-mediated endocytosis, which functions outside of the ciliary compartment in coordinating Gpr161 removal. Mechanisms determining dynamic compartmentalization of Gpr161 in cilia define a new paradigm for down-regulation of GPCRs during developmental signaling from a specialized subcellular compartment.

A Mechanical Switch Couples T Cell Receptor Triggering to the Cytoplasmic Juxtamembrane Regions of CD3ζζ.

The eight-subunit T cell receptor (TCR)-CD3 complex is the primary determinant for T cell fate decisions. Yet how it relays ligand-specific information across the cell membrane for conversion to chemical signals remains unresolved. We hypothesized that TCR engagement triggers a change in the spatial relationship between the associated CD3ζζ subunits at the junction where they emerge from the membrane into the cytoplasm. Using three in situ proximity assays based on ID-PRIME, FRET, and EPOR activity, we determined that the cytosolic juxtamembrane regions of the CD3ζζ subunits are spread apart upon assembly into the TCR-CD3 complex. TCR engagement then triggered their apposition. This mechanical switch resides upstream of the CD3ζζ intracellular motifs that initiate chemical signaling, as well as the polybasic stretches that regulate signal potentiation. These findings provide a framework from which to examine triggering events for activating immune receptors and other complex molecular machines.

Studying G protein-coupled receptors: immunoblotting, immunoprecipitation, phosphorylation, surface labeling, and cross-linking protocols.

Primary cilia are signaling organelles that have been shown to coordinate cellular responses to extracellular cues during physiological processes ranging from organ patterning to cell cycle regulation. A variety of receptors, including G protein-coupled receptors (GPCRs), downstream effectors (adenylyl cyclases), and second messengers, such as calcium, accumulate in the ciliary compartment. Isolation of GPCRs is essential for studying posttranslational modifications, intracellular trafficking, and protein-protein interactions that are important in downstream signaling. However, the presence of multiple hydrophobic transmembrane domains, and the inherent conformational flexibility of GPCRs make their extraction from membranes and solubilization particularly challenging. Here, we describe detailed methods for immunoblotting and immunoprecipitation of GPCRs from whole cell extracts. These methods are applicable for studying other multipass transmembrane proteins (such as adenylyl cyclases). We also describe methods for determining GPCR phosphorylation, surface labeling by biotinylation, and cross-linking to detect transient interactions with other proteins. These methods are amenable for studying both ciliary and nonciliary GPCRs in the context of cellular signaling pathways.

Increased Akt signaling in the mosquito fat body increases adult survivorship.

Akt signaling regulates diverse physiologies in a wide range of organisms. We examine the impact of increased Akt signaling in the fat body of 2 mosquito species, the Asian malaria mosquito Anopheles stephensi and the yellow fever mosquito Aedes aegypti. Overexpression of a myristoylated and active form of A. stephensi and Ae. aegypti Akt in the fat body of transgenic mosquitoes led to activation of the downstream signaling molecules forkhead box O (FOXO) and p70 S6 kinase in a tissue and blood meal-specific manner. In both species, increased Akt signaling in the fat body after blood feeding significantly increased adult survivorship relative to nontransgenic sibling controls. In A. stephensi, survivorship was increased by 15% to 45%, while in Ae. aegypti, it increased 14% to 47%. Transgenic mosquitoes fed only sugar, and thus not expressing active Akt, had no significant difference in survivorship relative to nontransgenic siblings. Expression of active Akt also increased expression of fat body vitellogenin, but the number of viable eggs did not differ significantly between transgenic and nontransgenic controls. This work demonstrates a novel mechanism of enhanced survivorship through increased Akt signaling in the fat bodies of multiple mosquito genera and provides new tools to unlock the molecular underpinnings of aging in eukaryotic organisms.

Piecing together the family portrait of TCR-CD3 complexes.

The pre-T-cell receptor (TCR)-, αßTCR-, and γδTCR-CD3 complexes are members of a family of modular biosensors that are responsible for driving T-cell development, activation, and effector functions. They inform essential checkpoint decisions by relaying key information from their ligand-binding modules (TCRs) to their signaling modules (CD3γε + CD3δε and CD3ζζ) and on to the intracellular signaling apparatus. Their actions shape the T-cell repertoire, as well as T-cell-mediated immunity; yet, the mechanisms that underlie their activity remain an enigma. As with any molecular machine, understanding how they function depends upon understanding how their parts fit and work together. In the 30 years since the initial biochemical and genetic characterizations of the αßTCR, the structure and function of the individual components of these family members have been extensively characterized. Cumulatively, this information has allowed us to piece together a portrait of the αßTCR-CD3 complex and outline the form of the remaining family members. Here we review the known structural and functional characteristics of the components of these TCR-CD3 complex family members. We then discuss how these data have informed our understanding of the architecture of the αßTCR-CD3 complex as well as their implications for the other family members. The intent is to provide a framework for considering: (i) how these thematically similar complexes diverge to execute their specific functions and (ii) how our knowledge of the form and function of these distinct family members can cross-inform our understanding of the other family members.

Defects in coatomer protein I (COPI) transport cause blood feeding-induced mortality in Yellow Fever mosquitoes.

Blood feeding by vector mosquitoes provides the entry point for disease pathogens and presents an acute metabolic challenge that must be overcome to complete the gonotrophic cycle. Based on recent data showing that coatomer protein I (COPI) vesicle transport is involved in cellular processes beyond Golgi-endoplasmic reticulum retrograde protein trafficking, we disrupted COPI functions in the Yellow Fever mosquito Aedes aegypti to interfere with blood meal digestion. Surprisingly, we found that decreased expression of the γCOPI coatomer protein led to 89% mortality in blood-fed mosquitoes by 72 h postfeeding compared with 0% mortality in control dsRNA-injected blood-fed mosquitoes and 3% mortality in γCOPI dsRNA-injected sugar-fed mosquitoes. Similar results were obtained using dsRNA directed against five other COPI coatomer subunits (α, ß, ß', δ, and ζ). We also examined midgut tissues by EM, quantitated heme in fecal samples, and characterized feeding-induced protein expression in midgut, fat body, and ovary tissues of COPI-deficient mosquitoes. We found that COPI defects disrupt epithelial cell membrane integrity, stimulate premature blood meal excretion, and block induced expression of several midgut protease genes. To study the role of COPI transport in ovarian development, we injected γCOPI dsRNA after blood feeding and found that, although blood digestion was normal, follicles in these mosquitoes were significantly smaller by 48 h postinjection and lacked eggshell proteins. Together, these data show that COPI functions are critical to mosquito blood digestion and egg maturation, a finding that could also apply to other blood-feeding arthropod vectors.