An ELF4 hypomorphic variant results in NK cell deficiency.

NK cell deficiencies (NKD) are a type of primary immune deficiency in which the major immunologic abnormality affects NK cell number, maturity, or function. Since NK cells contribute to immune defense against virally infected cells, patients with NKD experience higher susceptibility to chronic, recurrent, and fatal viral infections. An individual with recurrent viral infections and mild hypogammaglobulinemia was identified to have an X-linked damaging variant in the transcription factor gene ELF4. The variant does not decrease expression but disrupts ELF4 protein interactions and DNA binding, reducing transcriptional activation of target genes and selectively impairing ELF4 function. Corroborating previous murine models of ELF4 deficiency (Elf4-/-) and using a knockdown human NK cell line, we determined that ELF4 is necessary for normal NK cell development, terminal maturation, and function. Through characterization of the NK cells of the proband, expression of the proband's variant in Elf4-/- mouse hematopoietic precursor cells, and a human in vitro NK cell maturation model, we established this ELF4 variant as a potentially novel cause of NKD.

Recurrent high-impact mutations at cognate structural positions in class A G protein-coupled receptors expressed in tumors.

G protein-coupled receptors (GPCRs) are the largest family of human proteins. They have a common structure and, signaling through a much smaller set of G proteins, arrestins, and effectors, activate downstream pathways that often modulate hallmark mechanisms of cancer. Because there are many more GPCRs than effectors, mutations in different receptors could perturb signaling similarly so as to favor a tumor. We hypothesized that somatic mutations in tumor samples may not be enriched within a single gene but rather that cognate mutations with similar effects on GPCR function are distributed across many receptors. To test this possibility, we systematically aggregated somatic cancer mutations across class A GPCRs and found a nonrandom distribution of positions with variant amino acid residues. Individual cancer types were enriched for highly impactful, recurrent mutations at selected cognate positions of known functional motifs. We also discovered that no single receptor drives this pattern, but rather multiple receptors contain amino acid substitutions at a few cognate positions. Phenotypic characterization suggests these mutations induce perturbation of G protein activation and/or ß-arrestin recruitment. These data suggest that recurrent impactful oncogenic mutations perturb different GPCRs to subvert signaling and promote tumor growth or survival. The possibility that multiple different GPCRs could moonlight as drivers or enablers of a given cancer through mutations located at cognate positions across GPCR paralogs opens a window into cancer mechanisms and potential approaches to therapeutics.

Super-resolution microscopy reveals photoreceptor-specific subciliary location and function of ciliopathy-associated protein CEP290.

Mutations in the cilium-associated protein CEP290 cause retinal degeneration as part of multiorgan ciliopathies or as retina-specific diseases. The precise location and the functional roles of CEP290 within cilia and, specifically, the connecting cilia (CC) of photoreceptors, remain unclear. We used super-resolution fluorescence microscopy and electron microscopy to localize CEP290 in the CC and in the primary cilia of cultured cells with subdiffraction resolution and to determine effects of CEP290 deficiency in 3 mutant models. Radially, CEP290 localizes in close proximity to the microtubule doublets in the region between the doublets and the ciliary membrane. Longitudinally, it is distributed throughout the length of the CC whereas it is confined to the very base of primary cilia in human retinal pigment epithelium-1 cells. We found Y-shaped links, ciliary substructures between microtubules and membrane, throughout the length of the CC. Severe CEP290 deficiencies in mouse models did not prevent assembly of cilia or cause obvious mislocalization of ciliary components in early stages of degeneration. There were fewer cilia and no normal outer segments in the mutants, but the Y-shaped links were clearly present. These results point to photoreceptor-specific functions of CEP290 essential for CC maturation and stability following the earliest stages of ciliogenesis.

Defining the layers of a sensory cilium with STORM and cryoelectron nanoscopy.

Primary cilia carry out numerous signaling and sensory functions, and defects in them, "ciliopathies," cause a range of symptoms, including blindness. Understanding of their nanometer-scale ciliary substructures and their disruptions in ciliopathies has been hindered by limitations of conventional microscopic techniques. We have combined cryoelectron tomography, enhanced by subtomogram averaging, with superresolution stochastic optical reconstruction microscopy (STORM) to define subdomains within the light-sensing rod sensory cilium of mouse retinas and reveal previously unknown substructures formed by resident proteins. Domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of subcompartments, including the lumen and walls of the axoneme, the membrane glycocalyx, and the intervening cytoplasm. Within this framework, we report spatial distributions of key proteins in wild-type (WT) mice and the effects on them of genetic deficiencies in 3 models of Bardet-Biedl syndrome.

Critical Role for Phosphatidylinositol-3 Kinase Vps34/PIK3C3 in ON-Bipolar Cells.

Purpose: Phosphatidylinositol-3-phosphate (PI(3)P), and Vps34, the type III phosphatidylinositol 3-kinase primarily responsible for its production, are important for function and survival of sensory neurons, where they have key roles in membrane processing events, such as autophagy, endosome processing, and fusion of membranes bearing ubiquitinated cargos with lysosomes. We examined their roles in the most abundant class of secondary neurons in the vertebrate retina, the ON-bipolar cells (ON-BCs). Methods: A conditional Vps34 knockout mouse line was generated by crossing Vps34 floxed mice with transgenic mice expressing Cre recombinase in ON-BCs. Structural changes in the retina were determined by immunofluorescence and electron microscopy, and bipolar cell function was determined by electroretinography. Results: Vps34 deletion led to selective death of ON-BCs, a thinning of the inner nuclear layer, and a progressive decline of electroretinogram b-wave amplitudes. There was no evidence for loss of other retinal neurons, or disruption of rod-horizontal cell contacts in the outer plexiform layer. Loss of Vps34 led to aberrant accumulation of membranes positive for autophagy markers LC3, p62, and ubiquitin, accumulation of endosomal membranes positive for Rab7, and accumulation of lysosomes. Similar effects were observed in Purkinje cells of the cerebellum, leading to severe and progressive ataxia. Conclusions: These results support an essential role for PI(3)P in fusion of autophagosomes with lysosomes and in late endosome maturation. The cell death resulting from Vps34 knockout suggests that these processes are essential for the health of ON-BCs.

SPATA7 maintains a novel photoreceptor-specific zone in the distal connecting cilium.

Photoreceptor-specific ciliopathies often affect a structure that is considered functionally homologous to the ciliary transition zone (TZ) called the connecting cilium (CC). However, it is unclear how mutations in certain ciliary genes disrupt the photoreceptor CC without impacting the primary cilia systemically. By applying stochastic optical reconstruction microscopy technology in different genetic models, we show that the CC can be partitioned into two regions: the proximal CC (PCC), which is homologous to the TZ of primary cilia, and the distal CC (DCC), a photoreceptor-specific extension of the ciliary TZ. This specialized distal zone of the CC in photoreceptors is maintained by SPATA7, which interacts with other photoreceptor-specific ciliary proteins such as RPGR and RPGRIP1. The absence of Spata7 results in the mislocalization of DCC proteins without affecting the PCC protein complexes. This collapse results in destabilization of the axonemal microtubules, which consequently results in photoreceptor degeneration. These data provide a novel mechanism to explain how genetic disruption of ubiquitously present ciliary proteins exerts tissue-specific ciliopathy phenotypes.

ß2-Adrenergic receptor activation mobilizes intracellular calcium via a non-canonical cAMP-independent signaling pathway.

Beta adrenergic receptors (ßARs) are G-protein-coupled receptors essential for physiological responses to the hormones/neurotransmitters epinephrine and norepinephrine which are found in the nervous system and throughout the body. They are the targets of numerous widely used drugs, especially in the case of the most extensively studied ßAR, ß2AR, whose ligands are used for asthma and cardiovascular disease. ßARs signal through Gαs G-proteins and via activation of adenylyl cyclase and cAMP-dependent protein kinase, but some alternative downstream pathways have also been proposed that could be important for understanding normal physiological functioning of ßAR signaling and its disruption in disease. Using fluorescence-based Ca2+ flux assays combined with pharmacology and gene knock-out methods, we discovered a previously unrecognized endogenous pathway in HEK-293 cells whereby ß2AR activation leads to robust Ca2+ mobilization from intracellular stores via activation of phospholipase C and opening of inositol trisphosphate (InsP3) receptors. This pathway did not involve cAMP, Gαs, or Gαi or the participation of the other members of the canonical ß2AR signaling cascade and, therefore, constitutes a novel signaling mechanism for this receptor. This newly uncovered mechanism for Ca2+ mobilization by ß2AR has broad implications for adrenergic signaling, cross-talk with other signaling pathways, and the effects of ßAR-directed drugs.

Phosphatidylinositol-3-phosphate is light-regulated and essential for survival in retinal rods.

Phosphoinositides play important roles in numerous intracellular membrane pathways. Little is known about the regulation or function of these lipids in rod photoreceptor cells, which have highly active membrane dynamics. Using new assays with femtomole sensitivity, we determined that whereas levels of phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate were below detection limits, phosphatidylinositol-3-phosphate (PI(3)P) levels in rod inner/outer segments increased more than 30-fold after light exposure. This increase was blocked in a rod-specific knockout of the PI-3 kinase Vps34, resulting in failure of endosomal and autophagy-related membranes to fuse with lysosomes, and accumulation of abnormal membrane structures. At early ages, rods displayed normal morphology, rhodopsin trafficking, and light responses, but underwent progressive neurodegeneration with eventual loss of both rods and cones by twelve weeks. The degeneration is considerably faster than in rod knockouts of autophagy genes, indicating defects in endosome recycling or other PI(3)P-dependent membrane trafficking pathways are also essential for rod survival.

Efficient mutagenesis of the rhodopsin gene in rod photoreceptor neurons in mice.

Dominant mutations in the rhodopsin gene, which is expressed in rod photoreceptor cells, are a major cause of the hereditary-blinding disease, autosomal dominant retinitis pigmentosa. Therapeutic strategies designed to edit such mutations will likely depend on the introduction of double-strand breaks and their subsequent repair by homologous recombination or non-homologous end joining. At present, the break repair capabilities of mature neurons, in general, and rod cells, in particular, are undefined. To detect break repair, we generated mice that carry a modified human rhodopsin-GFP fusion gene at the normal mouse rhodopsin locus. The rhodopsin-GFP gene carries tandem copies of exon 2, with an ISceI recognition site situated between them. An ISceI-induced break can be repaired either by non-homologous end joining or by recombination between the duplicated segments, generating a functional rhodopsin-GFP gene. We introduced breaks using recombinant adeno-associated virus to transduce the gene encoding ISceI nuclease. We found that virtually 100% of transduced rod cells were mutated at the ISceI site, with ∼85% of the genomes altered by end joining and ∼15% by the single-strand annealing pathway of homologous recombination. These studies establish that the genomes of terminally differentiated rod cells can be efficiently edited in living organisms.

Distribution of RGS9-2 in neurons of the mouse striatum.

Regulators of G protein signaling (RGS) proteins negatively modulate G protein-coupled receptor (GPCR) signaling activity by accelerating G protein hydrolysis of GTP, hastening pathway shutoff. A wealth of data from cell culture experiments using exogenously expressed proteins indicates that RGS9 and other RGS proteins have the potential to down-regulate a significant number of pathways. We have used an array of biochemical and tissue staining techniques to examine the subcellular localization and membrane binding characteristics of endogenous RGS9-2 and known binding partners in rodent striatum and tissue homogenates. A small fraction of RGS9-2 is present in the soluble cytoplasmic fraction, whereas the majority is present primarily associated with the plasma membrane and structures insoluble in non-ionic detergents that efficiently extract the vast majority of its binding partners, R7BP and G(beta5). It is specifically excluded from the cell nucleus in mouse striatal tissue. In cultured striatal neurons, RGS9-2 is found at extrasynaptic sites primarily along the dendritic shaft near the spine neck. Heterogeneity in RGS9-2 detergent solubility along with its unique subcellular localization suggests that its mechanism of membrane anchoring and localization is complex and likely involves additional proteins beside R7BP. An important nuclear function for RGS9-2 seems unlikely.

Signal transducing membrane complexes of photoreceptor outer segments.

Signal transduction in outer segments of vertebrate photoreceptors is mediated by a series of reactions among multiple polypeptides that form protein-protein complexes within or on the surface of the disk and plasma membranes. The individual components in the activation reactions include the photon receptor rhodopsin and the products of its absorption of light, the three subunits of the G protein, transducin, the four subunits of the cGMP phosphodiesterase, PDE6 and the four subunits of the cGMP-gated cation channel. Recovery involves membrane complexes with additional polypeptides including the Na(+)/Ca(2+), K(+) exchanger, NCKX2, rhodopsin kinases RK1 and RK7, arrestin, guanylate cyclases, guanylate cyclase activating proteins, GCAP1 and GCAP2, and the GTPase accelerating complex of RGS9-1, G(beta5L), and membrane anchor R9AP. Modes of membrane binding by these polypeptides include transmembrane helices, fatty acyl or isoprenyl modifications, polar interactions with lipid head groups, non-polar interactions of hydrophobic side chains with lipid hydrocarbon phase, and both polar and non-polar protein-protein interactions. In the course of signal transduction, complexes among these polypeptides form and dissociate, and undergo structural rearrangements that are coupled to their interactions with and catalysis of reactions by small molecules and ions, including guanine nucleotides, ATP, Ca(2+), Mg(2+), and lipids. The substantial progress that has been made in understanding the composition and function of these complexes is reviewed, along with the more preliminary state of our understanding of the structures of these complexes and the challenges and opportunities that present themselves for deepening our understanding of these complexes, and how they work together to convert a light signal into an electrical signal.

Rhodopsin-EGFP knock-ins for imaging quantal gene alterations.

We have developed an imaging approach to monitor changes in gene structure in photoreceptors. We review here, the strategy and recent progress. Knock-in mice bearing a human rhodopsin-EGFP fusion gene potentially allow detection of a single molecular event: correction of a single copy of a gene within an entire retina. These mice can also be used for imaging rhodopsin distribution, membrane structure, and trafficking in normal mice or in disease states, using confocal or multiphoton fluorescence imaging techniques. They represent tools for studying molecular triggers of photoreceptor development, for following stem cell populations, and for evaluating retinal transplantation experiments.

ABCA4 mutations causing mislocalization are found frequently in patients with severe retinal dystrophies.

ABCA4, also called ABCR, is a retinal-specific member of the ATP-binding cassette (ABC) family that functions in photoreceptor outer segments as a flipase of all-trans retinal. Homozygous and compound heterozygous ABCA4 mutations are associated with various autosomal recessive retinal dystrophies, whereas heterozygous ABCA4 mutations have been associated with dominant susceptibility to age-related macular degeneration in both humans and mice. We analyzed a cohort of 29 arRP families for the mutations in ABCA4 with a commercial microarray, ABCR-400 in addition to direct sequencing and segregation analysis, and identified both mutant alleles in two families (7%): compound heterozygosity for missense (R602W) and nonsense (R408X) alleles and homozygosity for a complex [L541P; A1038V] allele. The missense mutations were analyzed functionally in the photoreceptors of Xenopus laevis tadpoles, which revealed mislocalization of ABCA4 protein. These mutations cause retention of ABCA4 in the photoreceptor inner segment, likely by impairing correct folding, resulting in the total absence of physiologic protein function. Patients with different retinal dystrophies harboring two misfolding alleles exhibit early age-of-onset (AO) (5-12 years) of retinal disease. Our data suggest that a class of ABCA4 mutants may be an important determinant of the AO of disease.

Enhancement of phototransduction g protein-effector interactions by phosphoinositides.

Light responses in photoreceptor cells are mediated by the action of the G protein transducin (G(t)) on the effector enzyme cGMP phosphodiesterase (PDE6) at the surface of disk membranes. The enzymatic components needed for phosphoinositide-based signaling are known to be present in rod cells, but it has remained uncertain what role phosphoinositides play in vertebrate phototransduction. Reconstitution of PDE6 and activated G(alphat), on the surface of large unilamellar vesicles containing d-myo-phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), stimulated PDE activity nearly 4-fold above the level observed with membranes containing no phosphoinositides, whereas G protein-independent activation by trypsin was unaffected by the presence of phosphoinositides. PDE activity was similarly stimulated by d-myo-phosphatidylinositol-3,4-bisphosphate and d-myo-phosphatidylinositol-4-phosphate (PI(4)P), but much less by d-myo-phosphatidylinositol-5-phosphate (PI(5)P) or d-myo-phosphatidylinositol-3,5-bisphosphate. Incubation of rod outer segment membranes with phosphoinositide-specific phospholipase C decreased G protein-stimulated activation of endogenous PDE6, but not trypsin-stimulated PDE activity. Binding experiments using phosphoinositide-containing vesicles revealed patterns of PDE6 binding and PDE6-enhanced G(alphat)-GTPgammaS binding, consistent with the activation profile PI(4,5)P(2) > PI(4)P > PI(5)P approximately control vesicles. These results suggest that enhancement of effector-G protein interactions represents a possible mechanism for modulation of phototransduction gain by changes in phosphoinositide levels, perhaps occurring in response to longterm changes in illumination or other environmental cues.

Acceleration of key reactions as a strategy to elucidate the rate-limiting chemistry underlying phototransduction inactivation.

PURPOSE: A reconstituted system was used to establish a strategy to determine the rate-limiting chemistry responsible for recovery of the dim-flash response in rod photoreceptors. METHODS: A general approach for identifying the rate-limiting step in a series of reactions is to evaluate the consequences of accelerating each step separately, while monitoring the rate of formation of the end product of the series. This strategy was applied to the reactions involved in quenching phototransduction in bovine rod outer segment (bROS) homogenates. The decay of photoactivated rhodopsin (R*) and inactivation of transducin by guanosine triphosphate (GTP) hydrolysis are the leading candidates for limiting the rate of phototransduction turn-off. These reactions were accelerated separately and together by adding hydroxylamine and/or the regulator of G-protein signaling-9 catalytic domain (RGS9d) while monitoring phosphodiesterase (PDE) activity triggered by a pulse of light in bROS homogenates. RESULTS: PDE activity in bROS homogenates triggered by a flash of light returned to its dark value with a rate constant of 0.087 +/- 0.002 seconds in this system. The rate of PDE recovery increased to 0.11 +/- 0.004 seconds when R* decay was accelerated with 10 to 50 mM hydroxylamine, suggesting that R* inactivation limits the rate of phototransduction turn-off under these conditions. Adding both hydroxylamine and RGS9d, a factor that accelerates transducin inactivation, increased the rate of PDE decay even further. RGS9d had no effect on PDE recovery kinetics unless quenching of R* was also accelerated. CONCLUSIONS: Under in vitro conditions in bROS homogenates, the quenching of R* normally limits the rate of phototransduction shut-off. If R* decay is accelerated, inactivation of transducin by GTP hydrolysis becomes rate limiting. This study offers a general approach that could be used to investigate the rate-limiting chemistry of phototransduction turn-off in vivo.