Intraspecific Variation within the Utricularia amethystina Species Morphotypes Based on Chloroplast Genomes.

Utricularia amethystina Salzm. ex A.St.-Hil. & Girard (Lentibulariaceae) is a highly polymorphic carnivorous plant taxonomically rearranged many times throughout history. Herein, the complete chloroplast genomes (cpDNA) of three U. amethystina morphotypes: purple-, white-, and yellow-flowered, were sequenced, compared, and putative markers for systematic, populations, and evolutionary studies were uncovered. In addition, RNA-Seq and RNA-editing analysis were employed for functional cpDNA evaluation. The cpDNA of three U. amethystina morphotypes exhibits typical quadripartite structure. Fine-grained sequence comparison revealed a high degree of intraspecific genetic variability in all morphotypes, including an exclusive inversion in the psbM and petN genes in U. amethystina yellow. Phylogenetic analyses indicate that U. amethystina morphotypes are monophyletic. Furthermore, in contrast to the terrestrial Utricularia reniformis cpDNA, the U. amethystina morphotypes retain all the plastid NAD(P)H-dehydrogenase (ndh) complex genes. This observation supports the hypothesis that the ndhs in terrestrial Utricularia were independently lost and regained, also suggesting that different habitats (aquatic and terrestrial) are not related to the absence of Utricularia ndhs gene repertoire as previously assumed. Moreover, RNA-Seq analyses recovered similar patterns, including nonsynonymous RNA-editing sites (e.g., rps14 and petB). Collectively, our results bring new insights into the chloroplast genome architecture and evolution of the photosynthesis machinery in the Lentibulariaceae.

The macula densa prorenin receptor is essential in renin release and blood pressure control.

The prorenin receptor (PRR) was originally proposed to be a member of the renin-angiotensin system (RAS); however, recent work questioned their association. The present paper describes a functional link between the PRR and RAS in the renal juxtaglomerular apparatus (JGA), a classic anatomical site of the RAS. PRR expression was found in the sensory cells of the JGA, the macula densa (MD), and immunohistochemistry-localized PRR to the MD basolateral cell membrane in mouse, rat, and human kidneys. MD cell PRR activation led to MAP kinase ERK1/2 signaling and stimulation of PGE2 release, the classic pathway of MD-mediated renin release. Exogenous renin or prorenin added to the in vitro microperfused JGA-induced acute renin release, which was inhibited by removing the MD or by the administration of a PRR decoy peptide. To test the function of MD PRR in vivo, we established a new mouse model with inducible conditional knockout (cKO) of the PRR in MD cells based on neural nitric oxide synthase-driven Cre-lox recombination. Deletion of the MD PRR significantly reduced blood pressure and plasma renin. Challenging the RAS by low-salt diet + captopril treatment caused further significant reductions in blood pressure, renal renin, cyclooxygenase-2, and microsomal PGE synthase expression in cKO vs. wild-type mice. These results suggest that the MD PRR is essential in a novel JGA short-loop feedback mechanism, which is integrated within the classic MD mechanism to control renin synthesis and release and to maintain blood pressure.

Identification of a cis-acting element that localizes mRNA to synapses.

Messenger RNA (mRNA) localization and regulated translation can spatially restrict gene expression to each of the thousands of synaptic compartments formed by a single neuron. Although cis-acting RNA elements have been shown to direct localization of mRNAs from the soma into neuronal processes, less is known about signals that target transcripts specifically to synapses. In Aplysia sensory-motor neuronal cultures, synapse formation rapidly redistributes the mRNA encoding the peptide neurotransmitter sensorin from neuritic shafts into synapses. We find that the export of sensorin mRNA from soma to neurite and the localization to synapse are controlled by distinct signals. The 3' UTR is sufficient for export into distal neurites, whereas the 5' UTR is required for concentration of reporter mRNA at synapses. We have identified a 66-nt element in the 5' UTR of sensorin that is necessary and sufficient for synaptic mRNA localization. Mutational and chemical probing analyses are consistent with a role for secondary structure in this process.

Succinate receptor GPR91 provides a direct link between high glucose levels and renin release in murine and rabbit kidney.

Diabetes mellitus is the most common and rapidly growing cause of end-stage renal disease in developed countries. A classic hallmark of early diabetes mellitus includes activation of the renin-angiotensin system (RAS), which may lead to hypertension and renal tissue injury, but the mechanism of RAS activation is elusive. Here we identified a paracrine signaling pathway in the kidney in which high levels of glucose directly triggered the release of the prohypertensive hormone renin. The signaling cascade involved the local accumulation of succinate and activation of the kidney-specific G protein-coupled metabolic receptor, GPR91, in the glomerular endothelium as observed in rat, mouse, and rabbit kidney sections. Elements of signal transduction included endothelial Ca2+, the production of NO and prostaglandin (PGE2), and their paracrine actions on adjacent renin-producing cells. This GPR91 signaling cascade may serve to modulate kidney function and help remove metabolic waste products through renal hyperfiltration, and it could also link metabolic diseases, such as diabetes, or metabolic syndrome with RAS overactivation, systemic hypertension, and organ injury.

Activation of the succinate receptor GPR91 in macula densa cells causes renin release.

Macula densa (MD) cells of the juxtaglomerular apparatus (JGA) are salt sensors and generate paracrine signals that control renal blood flow, glomerular filtration, and release of the prohypertensive hormone renin. We hypothesized that the recently identified succinate receptor GPR91 is present in MD cells and regulates renin release. Using immunohistochemistry, we identified GPR91 in the apical plasma membrane of MD cells. Treatment of MD cells with succinate activated mitogen-activated protein kinases (MAPKs; p38 and extracellular signal-regulated kinases 1/2) and cyclooxygenase 2 (COX-2) and induced the synthesis and release of prostaglandin E(2), a potent vasodilator and classic paracrine mediator of renin release. Using microperfused JGA and real-time confocal fluorescence imaging of quinacrine-labeled renin granules, we detected significant renin release in response to tubular succinate (EC(50) 350 microM). Genetic deletion of GPR91 (GPR91(-/-) mice) or pharmacologic inhibition of MAPK or COX-2 blocked succinate-induced renin release. Streptozotocin-induced diabetes caused GPR91-dependent upregulation of renal cortical phospho-p38, extracellular signal-regulated kinases 1/2, COX-2, and renin content. Salt depletion for 1 wk increased plasma renin activity seven-fold in wild-type mice but only 3.4-fold in GPR91(-/-) mice. In summary, MD cells can sense alterations in local tissue metabolism via accumulation of tubular succinate and GPR91 signaling, which involves the activation of MAPKs, COX-2, and the release of prostaglandin E(2). This mechanism may be integral in the regulation of renin release and activation of the renin-angiotensin system in health and disease.

Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing.

Single-cell CRISPR screens enable the exploration of mammalian gene function and genetic regulatory networks. However, use of this technology has been limited by reliance on indirect indexing of single-guide RNAs (sgRNAs). Here we present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct-capture Perturb-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries that contain dual-guide expression vectors. We demonstrate the utility of this approach for high-throughput investigations of genetic interactions and, leveraging this ability, dissect epistatic interactions between cholesterol biogenesis and DNA repair. Using direct capture Perturb-seq, we also show that targeting individual genes with multiple sgRNAs per cell improves efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Last, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments.

Comparative genomic analysis of Genlisea (corkscrew plants-Lentibulariaceae) chloroplast genomes reveals an increasing loss of the ndh genes.

In the carnivorous plant family Lentibulariaceae, all three genome compartments (nuclear, chloroplast, and mitochondria) have some of the highest rates of nucleotide substitutions across angiosperms. While the genera Genlisea and Utricularia have the smallest known flowering plant nuclear genomes, the chloroplast genomes (cpDNA) are mostly structurally conserved except for deletion and/or pseudogenization of the NAD(P)H-dehydrogenase complex (ndh) genes known to be involved in stress conditions of low light or CO2 concentrations. In order to determine how the cpDNA are changing, and to better understand the evolutionary history within the Genlisea genus, we sequenced, assembled and analyzed complete cpDNA from six species (G. aurea, G. filiformis, G. pygmaea, G. repens, G. tuberosa and G. violacea) together with the publicly available G. margaretae cpDNA. In general, the cpDNA structure among the analyzed Genlisea species is highly similar. However, we found that the plastidial ndh genes underwent a progressive process of degradation similar to the other terrestrial Lentibulariaceae cpDNA analyzed to date, but in contrast to the aquatic species. Contrary to current thinking that the terrestrial environment is a more stressful environment and thus requiring the ndh genes, we provide evidence that in the Lentibulariaceae the terrestrial forms have progressive loss while the aquatic forms have the eleven plastidial ndh genes intact. Therefore, the Lentibulariaceae system provides an important opportunity to understand the evolutionary forces that govern the transition to an aquatic environment and may provide insight into how plants manage water stress at a genome scale.

Connexin 40 and ATP-dependent intercellular calcium wave in renal glomerular endothelial cells.

Endothelial intracellular calcium ([Ca(2+)](i)) plays an important role in the function of the juxtaglomerular vasculature. The present studies aimed to identify the existence and molecular elements of an endothelial calcium wave in cultured glomerular endothelial cells (GENC). GENCs on glass coverslips were loaded with Fluo-4/Fura red, and ratiometric [Ca(2+)](i) imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single GENC caused a nine-fold increase in [Ca(2+)](i), which propagated from cell to cell throughout the monolayer (7.9 +/- 0.3 microm/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >400 microm. Inhibition of voltage-dependent calcium channels with nifedipine had no effect on the above parameters, but the removal of extracellular calcium reduced Delta[Ca(2+)](i) by 50%. Importantly, the gap junction uncoupler alpha-glycyrrhetinic acid or knockdown of connexin 40 (Cx40) by transfecting GENCs with Cx40 short interfering RNA (siRNA) almost completely eliminated Delta[Ca(2+)](i) and the calcium wave. Breakdown of extracellular ATP using a scavenger cocktail (apyrase and hexokinase) or nonselective inhibition of purinergic P2 receptors with suramin, had similar blocking effects. Scraping cells off along a line eliminated physical contact between cells but did not effect calcium wave propagation. Using an ATP biosensor technique, we detected a significant elevation in extracellular ATP (Delta = 76 +/- 2 microM) during calcium wave propagation, which was abolished by Cx40 siRNA treatment (Delta = 6 +/- 1 microM). These studies suggest that connexin 40 hemichannels and extracellular ATP are key molecular elements of the glomerular endothelial calcium wave, which may serve important juxtaglomerular functions.

The collecting duct is the major source of prorenin in diabetes.

In addition to the juxtaglomerular apparatus, renin is also synthesized in renal tubular epithelium, including the collecting duct (CD). Angiotensin (Ang) II differentially regulates the synthesis of juxtaglomerular (inhibition) and CD (stimulation) renin. Because diabetes mellitus, a disease with high intrarenal renin-Ang system and Ang II activity, is characterized by high prorenin levels, we hypothesized that the CD is the major source of prorenin in diabetes. Renin granular content was visualized using in vivo multiphoton microscopy of the kidney in diabetic Munich-Wistar rats. Diabetes caused a 3.5-fold increase in CD renin, in contrast to less pronounced juxtaglomerular changes. Ang II type 1 receptor blockade with Olmesartan reduced CD renin to control levels but significantly increased juxtaglomerular renin. Using a fluorogenic renin assay, the prorenin component of CD renin content was measured by assessing the difference in enzymatic activity of medullary homogenates before and after trypsin activation of prorenin. Trypsinization caused no change in control renin activity but a 5-fold increase in diabetes. Studies on a CD cell line (M1) showed a 22-fold increase in renin activity after trypsinization and a further 35-fold increase with Ang II treatment. Therefore, prorenin significantly contributes to baseline CD renin. Diabetes, possibly via Ang II, greatly stimulates CD prorenin and causes hyperplasia of renin-producing connecting segments. These novel findings suggest that, in a rat model of diabetes, prorenin content and release from the CD may be more important than the juxtaglomerular apparatus in contrast to the existing paradigm.

Increased renal renin content in mice lacking the Na+/H+ exchanger NHE2.

Macula densa (MD) cells express the Na(+)/H(+) exchanger (NHE) isoform NHE2 at the apical membrane, which may play an important role in tubular salt sensing through the regulation of cell volume and intracellular pH. These studies aimed to determine whether NHE2 participates in the MD control of renin synthesis. Renal renin content and activity and elements of the MD signaling pathway were analyzed using wild-type (NHE2(+/+)) and NHE2 knockout (NHE2(-/-)) mice. Immunofluorescence studies indicated that NHE2(-/-) mice lack NHE3 at the MD apical membrane, so the other apical NHE isoform has not compensated for the lack of NHE2. Importantly, the number of renin-expressing cells in the afferent arteriole in NHE2(-/-) mice was increased approximately 2.5-fold using renin immunohistochemistry. Western blotting confirmed approximately 20% higher renal cortical renin content in NHE2(-/-) mice compared with wild type. No-salt diet for 1 wk significantly increased renin content and activity in NHE2(+/+) mice, but the response was blunted in NHE2(-/-) mice. Renal tissue renin activity and plasma renin concentration were elevated three- and twofold, respectively, in NHE2(-/-) mice compared with wild type. NHE2(-/-) mice also exhibited a significantly increased renal cortical cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase (mPGES) expression, indicating MD-specific mechanisms responsible for the increased renin content. Significant and chronic activation of ERK1/2 was observed in MD cells of NHE2(-/-) kidneys. Removal of salt or addition of NHE inhibitors to cultured mouse MD-derived (MMDD1) cells caused a time-dependent activation of ERK1/2. In conclusion, the NHE2 isoform appears to be important in the MD feedback control of renin secretion, and the signaling pathway likely involves MD cell shrinkage and activation of ERK1/2, COX-2, and mPGES, all well-established elements of the MD-PGE(2)-renin release pathway.