The Microprocessor, containing the RNA binding protein Dgcr8 and RNase III enzyme Drosha, is responsible for processing primary microRNAs to precursor microRNAs. The Microprocessor regulates its own levels by cleaving hairpins in the 5′UTR and coding region of the Dgcr8 mRNA, thereby destabilizing the mature transcript.To determine whether the Microprocessor has a broader role in directly regulating other coding mRNA levels, we integrated results from expression profiling and ultra high-throughput deep sequencing of small RNAs. Expression analysis of mRNAs in wild-type, Dgcr8 knockout, and Dicer knockout mouse embryonic stem (ES) cells uncovered mRNAs that were specifically upregulated in the Dgcr8 null background. A number of these transcripts had evolutionarily conserved predicted hairpin targets for the Microprocessor. However, analysis of deep sequencing data of 18 to 200nt small RNAs in mouse ES, HeLa, and HepG2 indicates that exonic sequence reads that map in a pattern consistent with Microprocessor activity are unique to Dgcr8.We conclude that the Microprocessor's role in directly destabilizing coding mRNAs is likely specifically targeted to Dgcr8 itself, suggesting a specialized cellular mechanism for gene auto-regulation. MicroRNA maturation involves two processing steps Dgcr8 mRNA levels raises the possibility that this mechanism may affect many other mRNAs.The Microprocessor was recently shown to have an additional role in directly destabilizing a mRNA target. Specifically, it can cleave hairpins in the 5′UTR and coding region of the Dgcr8 mRNA, which in turns destabilizes the mature transcript To further test whether there is a broader role of the Microprocessor in the direct regulation of mRNAs, we evaluated the mRNA and small non-coding RNA profiles of wild-type, Dgcr8 KO and Dicer KO cells as well as a recently published data set of small RNAs less than 200 nucleotides from human Hela and HepG2 cell lines mRNAs regulated by a direct Microprocessor cleavage mechanism should be upregulated in cells deficient for the Microprocessor, but not in Dicer deficient cells. Therefore, we evaluated coding mRNA profiling data from wild-type, Dgcr8 KO and Dicer KO mouse ES cells. Normalized mRNA levels in Dgcr8 KO and Dicer KO cells were compared to wild-type ES cells Figure 1If genes specifically upregulated in Dgcr8 KO cells are normally cleaved by the Microprocessor, there should be hairpin substrates for the complex within these mRNAs. Therefore, we searched for evolutionary conserved hairpins within these mRNAs using predictions generated by the EvoFold algorithm . None of the reads mapping to the coding region hairpin were found in either Dgcr8 or Dicer KO libraries confirming their Dgcr8- and Dicer-dependence  had small RNAs mapping across the exon without shared 5′ or 3′ ends consistent with degradation . However, the mRNA levels of the host genes of these three miRNAs were not altered in the Dgcr8 and Dicer KO ES cells. Therefore, production of these miRNAs does not appear to influence the overall levels of the annotated host mRNAs. Together, these detailed analyses of both mRNA expression profiling and small RNA sequencing data from ES cells failed to uncover any genes other than Dgcr8 that are directly destabilized by the Microprocessor.A small number of annotated miRNAs map to exonic regions of coding genes (∼37 in mice) , thereby confirming that these libraries contain hairpin products of the Microprocessor cleavage. These findings show that the analysis of the Hela and HepG2 small RNA data sets should identify other hairpins that are cleaved by the Microprocessor even if they are not further processed.It is possible that 18–32 nucleotide small RNA sequencing missed Microprocessor-cleaved exonic hairpins that are sequestered and/or are not processed by Dicer. Microprocessor miRNAs are typically 60–75 nucleotides in length. Therefore, to directly identify these hairpins, we analyzed ultra high-throughput sequencing data sequence sets produced from small RNAs less than 200 nucleotides in length from Hela and HepG2 cells . Only four of these hairpins had at least 5 sequence reads. Furthermore, none of the small RNA reads in these hairpins mapped in a manner consistent with Microprocessor cleavage. That is, they had heterogeneous 5′ and 3′ends and/or the ends went beyond the extremes of the hairpins . Notably, 15 out of the 31 were genes that encode ribosomal protein subunits, which are highly abundant in cells. Out of the 31, 11 transcripts had small RNA reads distributed over the exon, as would be expected for degradation products. The remaining 20 transcripts had small RNA reads clustering in small window(s) within exons. However, further examination of the regions in these 20 transcripts using RNAfold did not reveal the presence of any good hairpin structures, in contrast to the Dgcr8 small RNA mapping-regions. In summary, analysis of the ultra high-throughput sequence reads of RNAs less than 200 nucleotides, like the ES cell small RNA dataset, showed that a role of the Microprocessor in direct mRNA regulation is likely limited to Dgcr8.Again, limiting the analysis to Evofold predicted hairpins would miss non-conserved hairpins. Therefore, we mapped small RNAs from HeLa and HepG2 libraries to exons of transcripts upregulated over 2-fold with siRNA-mediated knockdown of both Drosha and Dgcr8 relative to siGFP. Expression information was extracted from recently published microarray data in HeLa cells see  [14]. AsOur findings show a focused role for the Microprocessor in destabilizing coding mRNAs by the direct cleavage and destabilization of spliced transcripts. Indeed, we only find evidence for the cleavage and destabilizaton of Dgcr8. Similar to previous reports, our mRNA profiling analysis of wild-type, Dgcr8, and Dicer deficient cells identifies many mRNAs that are specifically upregulated with the loss of Dgcr8 . A number of these annotated exonic miRNAs were not present in our small RNA libraries even though the host gene is clearly expressed. This finding may be the result of mis-annotation of these sequences as miRNAs or that processing of the hairpins is somehow suppressed in ES cells. One example of an annotated exonic miRNA that is present in large numbers in ES cells is miR-21. Its host gene, Tmem49, is not upregulated in Dgcr8 or Dicer knockout ES cells. Possible explanations include: 1) there are alternative transcripts responsible for miR-21 production either from an alternative promoter or an alternative splicing event or 2) only a small subset of the Tmem49 transcripts is processed by the Microprocessor to produce the pre-miR-21 hairpin.The absence of Dgcr8-dependent upregulation of the host transcripts carrying annotated exonic miRNAs is worth noting  http://www-stat.stanford.edu/~tibs/SAM/). Specifically, two sets of genes were determined: 1) Genes upregulated in Dgcr8 KO relative to Dicer KO and 2) Genes upregulated in Dgcr8 KO relative to wild-type. Overlapping transcripts between these two sets of genes were assigned to the group upregulated in Dgcr8 KO relative to both Dicer KO and WT. Genes downregulated in Dgcr8 KO relative to Dicer and WT were determined using the same approach. For the analysis of overlap between small RNAs or predicted hairpins and protein coding mRNAs, we excluded Affy transcripts annotated only as miRNAs, transcripts mapping to the mitochondrial genome, chromosome Y and transcripts missing gene ID annotations.Microarray experiments on the wild-type, Dgcr8 KO, and Dicer KO cells were performed by the Gladstone Genomic Core Facility using the Affymetrix 1.0 mouse gene ST arrays with 3 biological replicates per genotype (wild-type (v6.5), HeLa cell microarray data was previously published Small RNA reads from the Solexa sequencing dataset were first mapped to the genome  using Eland. Uniquely mapping small RNA reads were mapped to exons by examining overlap between genomic coordinates of a small RNA read and each exon. Any small RNA overlapping with beginning and end of an exon as well as lying within in an exon was included as a positive hit. Exon information was determined using annotations from the UCSC Known Genes and Ensemble databases  and all transcripts were collapsed to match to Affy ID annotationsFor analysis of data from HeLa and HepG2 cells, small RNAs from all libraries were first mapped to the genome (hg18) using Eland. Sequence length of HeLa cell libraries ranged from 15 to 26 nt. Sequence length of HepG2 cell libraries ranged from 15 to 36 nt. Genomic coordinates of the small RNAs were then mapped to exons of transcripts upregulated with siDrosha and siDgcr8 relative to siGFP. Exon information was determined using RefSeq annotations, which were matched to Affy IDs. The positive hits were further filtered manually of snoRNAs. The remaining exons were ranked based on the number of small RNA reads and exons containing >10 small RNAs were analyzed further using custom tracks at the UCSC genome browser. For exons with small RNA reads localized to a small window, sequences surrounding the small RNA reads were extracted and fold predictions were generated using RNAfold.Genomic locations of miRNA hairpins were extracted from miRBase and converted to the hg18 assembly using the liftover tool. Genomic coordinates of small RNA sequences (25 to 36 nt) from HeLa and HepG2 cells were mapped to miRNA hairpin locations.http://www.cbse.ucsc.edu/~jsp/EvoFold/) Lists of long CDS and 5′UTR hairpins and their location in the human genome (mapping based on May 2004 release) were downloaded from the EvoFold database Click here for additional data file.Table S2(3.62 MB TIF)Click here for additional data file.Table S3(1.56 MB TIF)Click here for additional data file.Figure S1Distribution of small RNA reads from Dgcr8 KO and WT libraries across the Hn1 exon. Small RNA locations are presented as in (0.66 MB TIF)Click here for additional data file.Figure S2Distribution of small RNA reads from Dgcr8 KO and WT libraries across the Atbf1 exon, presented as in (0.31 MB TIF)Click here for additional data file.Figure S3Distribution of small RNA reads from Dgcr8 KO and WT libraries across the Adam23 exon, presented as in (0.26 MB TIF)Click here for additional data file.Figure S4Distribution of small RNA reads from Dgcr8 KO and WT libraries across the Zfp462 exon, presented as in (0.43 MB TIF)Click here for additional data file.Figure S5Distribution of small RNA reads from Dgcr8 KO and WT libraries across the Arrdc3 exon, presented as in (0.45 MB TIF)Click here for additional data file.Figure S6Distribution of small RNA reads from Dgcr8 KO and WT libraries across the Arrdc3 exon, presented as in (0.55 MB TIF)Click here for additional data file.Figure S7Distribution of 25 to 36 nt sequence reads from HeLa and HepG2 cell <200nt libraries across pre-miRNA hairpins. Locations are presented as in (0.71 MB TIF)Click here for additional data file.


Using FRET, we previously demonstrated that two ARE-BPs, HuR and AUF1, form stable homomeric and heteromeric associations in the nucleus and cytoplasm. In the current study, we use immuno-FRET of endogenous proteins to examine the intracellular localization and interactions of HuR and AUF1 as well as KSRP, TIA-1, and Hedls. These results were compared to those obtained with their exogenously expressed, fluorescently labeled counterparts.A number of RNA binding proteins (BPs) bind to A+U rich elements (AREs), commonly present within 3'UTRs of highly regulated RNAs. Individual RNA-BPs proteins can modulate RNA stability, RNA localization, and/or translational efficiency. Although biochemical studies have demonstrated selectivity of ARE-BPs for individual RNAs, less certain is the in vitro transcribed Cy3-labeled RNA was transfected into cells. Interestingly, Cy3-RNA was found to coalesce in P body like punctate structures and, by FRET, was found to interact with the RNA decapping proteins, Hedls and Dcp1.All ARE-BPs examined were found to colocalize and to form stable associations with selected other RNA-BPs in one or more cellular locations variably including the nucleus, cytoplasm , or in stress granules or P bodies. Interestingly, FRET based interaction of the translational suppressor, TIA-1, and the decapping protein, Hedls, was found to occur at the interface of stress granules and P bodies, dynamic sites of intracellular RNA storage and/or turnover. To explore the physical interactions of RNA-BPs with ARE containing RNAs, Biochemical methodologies, such as co-immunoprecipitation, and cell biological approaches such as standard confocal microscopy are useful in demonstrating the possibility of proteins and/or proteins and RNAs interacting. However, as demonstrated herein, colocalization of proteins and proteins and RNA is not always indicative of interaction. To this point, using FRET and immuno-FRET, we have demonstrated that RNA-BPs can visually colocalize without producing a FRET signal. In contrast, proteins that appear to be delimited to one or another intracellular compartment can be shown to interact when those compartments are juxtaposed. RNA binding proteins (BPs) are involved in numerous aspects of RNA maturation, turnover, translational efficiency, and in movement of transcripts throughout the cell. RNA BPs have roles during normal cellular growth and division, as well as during times of cellular stress, conditions that may alter the "normal" status of protein/protein and protein/mRNA interactions. Our studies focus on a class of RNA binding proteins known as ARE binding proteins. By recognizing and binding to A+U rich elements (AREs) in the 3'UTR of RNA transcripts the ARE-BPs control the cellular half life of numerous gene classes of RNA including, proto oncogenes, cytokines, chemokines, and G protein coupled receptors .Several ARE-BPs have been studied extensively including: AUF1/hnRNP D , BRF1/2 Coimmunoprecipitation experiments have demonstrated that various ARE-BPs can directly interact with one another including: HuR/AUF1 ; HuR/TIAThe regulation of ARE containing RNAs extends well beyond simple interactions between various ARE-BPs. ARE-BPs are themselves subject to regulation by phosphorylation, an effect that has been shown to drive their intracellular localization and/or to alter their ability to bind to target RNAs -30.Here we expand upon previous studies focusing on AUF1 and HuR to include the ARE-BPs KSRP and TIA-1. Beyond simply expanding the number of proteins, we wanted to investigate endogenously expressed proteins and their interactions and compare this to results obtained with fluorescently tagged proteins. To achieve this goal, we used the technique of immuno-FRET. This previously described but minimally utilized technique brings together immunocytochemistry and FRET to visualize endogenous proteins and their interactions in individual cells .Using immunocytochemistry, we demonstrate that endogenous HuR, AUF1, KSRP and TIA-1 all colocalize in the nucleus of DDT1-MF2 cells with variable expression of each in the cytoplasm. We also show via immuno-FRET the physical interactions of all pair-wise combinations of these proteins. To extend these findings, we examined the intracellular localization and interactions of these ARE-BPs under conditions of cellular stress (induction of stress granules (SGs)) and MAP Kinase stimulation (induction of nucleocytoplasmic shuttling). Upon oxidative stress, HuR, AUF1, KSRP and TIA-1 were all found to localize to SGs. MAPK stimulation, known to induce nucleocytoplasmic shuttling of HuR and AUF1 , also cain vitro transcribed, fluorescently labeled, stabilized mRNA transcripts coding for the β adrenergic receptor are present in the cytoplasm of DDT1-MF2 cells as punctate bodies. These transcripts were shown to recruit the mRNA degradation enzymes Hedls and Dcp1a to their cellular location.We also investigated Processing bodies, , cytoplasmic compartments associated with RNA degradation, for the presence of each ARE-BP . CertainIn summary, we conclude that a number of ARE-BPs are capable of interacting with one another; each protein is subject to differential regulation in terms of rate and degree of relocalization from nucleus to cytoplasm. Further distinctions arise at the level of discrete intracellular compartments, i.e., SGs and PBs. In this context, we demonstrate the utility of the immuno-FRET method to visualize the interaction between endogenous ARE-BP in a number of discrete cellular locations. Using this method we have shown that all of the four ARE-BP investigated interact in pairs in the cell within specific intracellular compartments. Moreover, by using immuno-FRET, we were able to differentiate between mere colocalization of the endogenous proteins and specific, FRET-detectable, interactions. Importantly, immuno-FRET results recapitulate substantially those obtained with fluorescently labeled proteins.FRET is a powerful technique capable of identifying interactions between pairs of fluorescently labeled proteins with appropriately overlapping excitation and emission spectra. In this study we used both 'traditional' and immuno-FRET methods to examine the interactions of several ARE-BPs in both live and fixed cells. For traditional FRET, eCFP and eYFP tagged versions of proteins were used. For immuno-FRET, endogenously expressed proteins were detected via 2° Abs labeled with Cy3 and Cy5 . As with traditional FRET, immuno-FRET requires that the fluorescently labeled antibodies be FRET compatible pairs. Traditional and immuno-FRET each have advantages and disadvantages. With traditional FRET, expression levels of transiently transfected tagged proteins can be difficult to control. Results have the potential to be erroneous due to protein overexpression, which can lead to aggregation and mis localization, something that can be caused by the presence of the fluorescent protein tag itself . With imIn a previous report , we inveCytoplasmic SGs have been described as sites of triage for RNA transcripts when cells are undergoing stress -43. It iAs demonstrated by the relocalization of TIA-1, induction of oxidative stress with arsenite induces the rapid formation of cytoplasmic SGs . SimilarFigure Figure Figure Figure Figure Figure In previous experiments, as well as in the current Figure Pertaining to KSRP, the reason(s) underlying the variability of exogenous protein expression in SG is currently unknown. Two potential explanations might be level of KSRP protein expression or epitope tagging of the protein. The non-uniformity of response between exogenous and endogenous protein demonstrates the utility of immuno-FRET over transient transfection in studying cellular protein localization.PBs are generally recognized as sites associated with RNA degradation. They are distributed throughout the cytoplasm and contain numerous proteins involved in RNA decay ,47,48. IFigure To investigate the potential for interaction of proteins compartmentalized to SGs and PBs, SGs were first induced by oxidative stress. Figure TIA-1 was detected in PBs during both unstimulated and MAPK stimulated conditions. In unstimulated cells, a portion of the cytoplasmic TIA-1 appears to be present in PBs leading to the generation of a FRET signal between TIA-1 and Hedls. In stressed cells, TIA-1 is present in both SGs and PBs. In this case, the strongest FRET signal was generated at the interface of TIA-1 containing SGs and Hedls containing PBs. Why TIA-1, along with TTP ,51 and BStimulation of MAP kinase pathways has the biochemical effect of stabilizing ARE containing RNAs -54. MAPKIn the current study, we extended this exploration to examine the effects of MAPK stimulation on KSPR and TIA-1. As TIA-1 possesses a general cytoplasmic distribution in unstimulated cells, we speculated that MAPK stimulation might not have a discernable effect on its distribution and indeed, it does not, Figures Using FRETc, we are able to demonstrate that all of the ARE-BPs studied interact in all the pair-wise combinations tested when localized to the nucleus and in the cytoplasm when relocalization is due to MAPK stimulation. These results are supportive of the notion that ARE-BPs are bound to multiple AREs as part of multiprotein complexes. One implication of this conclusion is that fine-tuning of the half-life of an RNA molecule is almost certainly tightly regulated by a large number of factors and conditions that remain to be elucidated.Figure In Figure in vitro transcription and transfection of fluorescently labeled RNAs with or without 2'F (dUTP) chemical stabilization. For this set of experiments, we chose the latter approach. To visualize target RNA, full length, 2'F-dUTP modified, 5' capped, polyadenylated, hamster β2 adrenergic receptor (β-AR) RNA was in vitro transcribed, utilizing an appropriate stoichiometry of Cy3-labeled UTP. Importantly, by nondenaturing gel shift assay, the fully modified RNA was able to bind ARE-BPs with normal (low nM) affinity (data not shown). As is evident in Figure Direct investigation of ARE-containing RNA molecules allows us to begin to interconnect data for ARE-BP localization with specific RNAs. Currently, overcoming the obstacle of visualizing RNA molecules with short half-lives can be approached in one of two ways: plasmid based expression of RNAs encoding stem loops recognized by the MS2-coat protein  or in viFigure We speculate that what underlies this result is the ribonuclease resistant RNA is attracting or sequestering Hedls protein. Figure It has long been recognized that certain RNA-BPs undergo nuclear/cytoplasmic shuttling and are capable of localizing in discrete intracellular compartments. Based on the seminal work of Anderson and others ,43,56,58Although immuno-FRET of ARE binding proteins is being described herein for the first time, there is certainly ample precedent demonstrating the usefulness and veracity of this methodology ,59-62. AIn previous investigations , we demoInduction of oxidative stress with arsenite causes proteins to aggregate in SGs to varying degrees with further distinctions between endogenous and exogenous proteins. Specifically, arsenite-induced stress caused rapid formation of cytoplasmic SGs with the presence of endogenous HuR, AUF1, KSRP and TIA-1 being readily detected. However, robust FRET signals in SGs were limited to the interactions between TIA-1/HuR and TIA-1/KSRP and to a lesser extent, TIA-1/AUF1, HuR/AUF1, KSRP/AUF1 and KSRP/HuR. Interestingly, and in contrast to their endogenous counterparts, the exogenous pair of TIA-1-YFP/AUF1-CFP does not demonstrate a readily detectable FRET signal.2-adrenergic receptor ARE [The presence and interaction all four ARE-BPs in SGs may seem anomalous given that they have putatively different roles when bound to an ARE. When preceding experiments failed to detect AUF1 in SGs, we initially reasoned that this result was consistent with the biology of AUF1 generally being considered an mRNA destabilizing protein; hence, it might not be expected to be bound to an RNA being sequestered and stabilized within an SG. This reasoning could be extended to KSRP. However, in biochemical studies, we have shown that AUF1 and HuR are able to bind to each other and to co-reside on the βptor ARE . Thus, tPotentially adding to the uncertainty of ARE-BP localization and interactions is the discrepancy, upon cellular activation, between endogenous and exogenous proteins. As our studies show, this appears to be the case for KSRP. This particular result underscores one of the potential problems associated with transient transfection of exogenous proteins and the advantage, when possible, of visualizing endogenous proteins by immuno-FRET.An unanticipated finding was the strong FRET signal between endogenous TIA-1 and Hedls, Figure Studies indicate that mRNAs cycle between PBs and SGs, depending on the availability of translation initiation or degradation machinery. A working model, called the mRNA cycle, was proposed by Balagopal and Parker . In thisAs RNAs are dynamic between PBs and SGs so then too are the proteins associated with them. The complete proteomic composition of PBs and SGs is still being elucidated, however, a number of proteins have been identified and localized to one or both of these cytoplasmic granules . RelevanA somewhat disappointing finding was the more or less general, diffuse effect of MAP kinase stimulation on the relocalization of RNA binding proteins, Figures There is also biochemical evidence in the literature that MAPK activation causes the relocalization of RNA and associated RNA binding proteins in a phosphorylation-dependent manner ,68-73. FIn addition to HuR, phosphorylated KSRP , TTP 7777, AUF1 in vitro transcribed, capped, polyadenylated, chemically stabilized (2'F) fluorescently labeled (Cy3-UTP) RNA was transfected into DDT1-MF2 cells. Two outcomes are noted. First, that the 2'F/Cy3 RNA is remarkably stable demonstrating virtually no change in abundance at 24 hrs post transfection. Secondly, the transfection of RNA forms distinct cytoplasmic aggregates. This finding is entirely consistent with that of Barreau et al [Drosophila, the localization and trafficking of capped, fluorescently labeled run RNA.In a related experimental direction, we attempted to address a long-standing interest, that of colocalization of ARE-containing mRNAs and RNA binding proteins. To perform these experiments, au et al , where aau et al , who desin vitro transcribed, chemically stabilized RNA is probably trapped in PB due to its inability to undergo appropriate degradation. In contrast, FRET was not observed between labeled RNA and the ARE binding protein, HuR. This is perhaps not surprising given the absence of HuR in PBs.Going beyond this observation, we used immuno-detection of the decapping proteins, Dcp1 and Hedls, to demonstrate that the aggregated RNA and the decapping enzymes were colocalized. In fact, we were able to observe robust FRET between Cy3-labeled β-AR mRNA and Dcp1. These results lead to a couple of conclusions. First, that interaction of labeled RNA and protein can be observed, a potentially highly useful proof of principal. Secondly, that transfection of At least two other papers have been published describing FRET between RNA and RNA binding proteins. Lorenz  infers FIn summary, we have demonstrated herein that the interaction between a number of RNA binding proteins, both endogenous and exogenous, can be detected by FRET. Further, there is a reasonable degree of concordance between the shuttling and intracellular localization of endogenous and exogenous ARE-BPs, with exceptions duly noted. There is also substantial concordance of the intracellular localization of ARE-BPs by imaging and biochemical techniques. Future tests of usefulness and validity of these methods will invariably involve knockdown experiments and site directed mutagenesis of ARE-BPs to examine factors such as phosphorylation state as a modulator of localization and protein/protein interaction. Our current work cannot determine whether the interactions we detect are the primary or the most functionally relevant interactions of any given RNA-BP. Additional methods, such as 3-FRET  may alloConstruction of the HuR-eYFP, HuR-eCFP, p37AUF1-eCFP and p37AUF1-eYFP vectors was as described previously . An Xho12 in Dubelcco's Modified Eagle Medium (Invitrogen) supplemented with 5% Fetal Bovine Serum (Hyclone) and 0.1% penicillin/streptomycin solution (Invitrogen). For transient transfection DDT1-MF2 cells were grown on 25 mm glass cover slips to approximately 80% confluence. For immunocytochemistry DDT1-MF2 cells were grown on 18 mm glass cover slips to approximately 60% confluence. Where indicated, cells were treated with anisomycin (75 nM) or sodium arsenite (0.5 mM).DDT1-MF2 Hamster smooth muscle cells were grown in 100 mm dishes at 37°C, 5% COVectors were transfected into DDT1-MF2 using Fugene 6 (Roche) following the manufacturers protocol. The amount of plasmid DNA transfected was adjusted empirically to yield the desired 1:1 molar ratio of the CFP to YFP protein.2, pH 6.9), treated with 0.1% sodium borohydride (Sigma) and blocked with 5% horse serum. Cells were incubated at 4°C, overnight with 1°Ab and in 2°Ab for 1 hour at room temperature in the dark. Cover slips were mounted on glass slides using a 12.5% solution of Mowiol  in 50% glycerol. Detection of HuR was via a 1:400 dilution of monoclonal 1°Ab (Santa Cruz Biotechnology) or a 1:200 dilution of a rabbit polyclonal 1°Ab; detection of AUF1 was via a 1: 500 dilution of a polyclonal 1°Ab (Upstate) which recognizes all 4 isoforms of AUF1; detection of TIA-1 was via a 1:500 dilution of goat polyclonal 1°Ab (Santa Cruz Biotechnology); detection of KSRP was via a 1:1000 dilution of a mouse monoclonal 1°Ab (courtesy of Dr. D.L. Black) and detection of Hedls was via a 1:8000 dilution of a rabbit polyclonal 1°Ab (courtesy of Dr. J. Lykke-Anderson). Secondary antibodies used were either a 1:200 dilution of goat anti-rabbit 2°Ab labeled with Cy5; a 1:400 dilution of a goat anti-rabbit 2°Ab labeled with Cy3; a 1:800 dilution of goat anti-mouse 2°Ab labeled with Cy5; a 1:1000 dilution of goat anti-mouse 2°Ab labeled with Cy3; a 1:2000 dilution of goat anti-mouse 2° Ab labeled with Alexa Fluor 488; a 1:200 dilution of a donkey anti-goat Cy3 or a 1:400 dilution of a donkey anti-goat 2°Ab labeled with Alexa Fluor 647. With the exception of TIA-1, all 1° and 2° Abs were applied together. To prevent cross-reaction between 2°Ab, the TIA-1 donkey anti-goat 2°Ab was added first and separately. All Cy3 and Cy5 labeled 2° antibodies were F(ab')2 IgG fragments from Jackson Immuno Research; Alexa Fluor dye labeled antibodies were F(ab')2 IgG fragments from Molecular Probes.DDT1-MF2 cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences), permeabilized with 0.5% Triton X-100 in PHEM buffer  conjugated to 2° Ab were used. In this instance Cy3 is the donor fluorophor and Cy5 or Alexa Fluor 647 is the acceptor fluorophor. In DDT1-MF2 cells, the signal intensity ratio of Cy3 to Cy5 required for energy transfer to occur and generate a FRET signal was determined to be 1:1 or greater (data not shown). The requirements for FRET; a FRET fluorophor pair, specific distance between fluorophors and fluorophor alignment, are not changed due to placing the fluorophor pair on 2° Ab.Images of live and fixed cells were captured at 100× magnification under oil immersion, , using 2 × 2 or 4 × 4 binning mode on an inverted Nikon Eclipse TE3000 fluorescence microscope using a Cooke SensiCam QE CCD camera running Slidebook software . All images were collected at room temperature using an integration time greater than 25 ms. Additionally, all images were background corrected by subtracting the intensity of a cell "devoid" area from each individual image. For live cell images, imaging medium was DMEM and integration time was between 25 and 250 ms. For fixed cell and immunostained images, integration time was between 25 and 1000 ms. Live cells containing CFP and YFP labeled proteins were viewed via the CFP channel to prevent photobleaching of YFP. All images presented had a signal intensity greater than 100 after background correction. FRETc images were produced using Slidebook software  using spectral bleed through numbers that had been experimentally determined as detailed below. Images were edited using Adobe Illustrator.All experimental data was collected from a minimum of two separate experiments. For each immunocytochemical experiment, a minimum of 400 cells was examined. For each transfection experiment, a minimum of 200 cells was examined. In general, images were captured at a level that would provide best resolution of the nucleus.The CFP and YFP spectral bleed through numbers were calculated as indicated previously . AdditioTo calculate FRETc, the following equation was used:FRETc is the corrected FRET value, raw FRET is the uncorrected FRET intensity, D is the donor fluorophor intensity, X is the donor fluorophor spectral bleed through number, A is the acceptor fluorophor intensity, and Y is the acceptor fluorophor spectral bleed through number.7G cap analogue (Promega) and 3' polyadenylation to the stabilized, fluorescent RNA molecule. A DNA template (1 μg) with an incorporated T7 promoter sequence was transcribed using Durascribe T7 enzyme (2 μl) in a 20 μl reaction containing 5 mM ATP, 1 mM GTP, 5 mM 2'F-dCTP, 5 mM 2'F-dUTP, 0.75 mM Cy3-UTP, 10 mM DTT and 4 mM m7G cap analogue for 2 hours at 37°C. Polyadenylation was carried out subsequent to the in vitro transcription. After removal of a small aliquot (1 μl) for gel electrophoresis, 3.125 mM MnCl2, 1.25 mM ATP, 2 ul E-PAP enzyme, 10 ul 5× E-PAP buffer and 1 μl SUPERase, (Ambion) were added directly to the in vitro transcription reaction tube, total volume 40 μl, incubate 2 hours at 37°C. This reaction creates a poly A tail of ~100-200 nucleotides. Based on a calculation of the base:dye ratio , the capped and polyadenylated Cy3-RNA products contain ~2-4 Cy3 molecules per transcript. The optimal concentration for observation of diffuse Cy3 signal in cells has been determined empirically to be ~200 pM of RNA when DDT1-MF2 cells were transfected using Lipofectamine 2000, as per the manufacturer's guidelines (Invitrogen).To create ribonuclease resistant RNA labeled with a fluorescent Cy3 moiety, we incorporated Cy3-UTP (Amersham) and 2'F substituted CTP and UTP (Epicenter Biotechnologies) into β1 Adrenergic Receptor mRNA molecules via T7 RNA polymerase (Epicenter Technologies). We altered previously published methods , to inco2 prior to the media being changed back to DMEM (37°C). Cells were left to recover for 4 hours prior to any subsequent treatment.Cy3-labeled RNAs were transfected into DDT1-MF2 cells using Lipofectamine 2000 (Invitrogen). Briefly, DDT1-MF2 cells were resuspended and plated at 80% confluence onto 100 mm plates containing 25 mm cover slips and allowed to adhere, about 2 hours. The RNA was melted at 90°C for 1 min and allowed to cool slowly to room temperature prior to transfection. The Lipofectamine 2000 and Cy3-labeled RNA (2 μg) were diluted separately into Optimem 1 media (4 fold and 140 fold respectively) (Invitrogen) and incubated at room temperature for 30 minutes. DDT1-MF2 cells were washed with Optimem 1 and changed into this media. The Lipofectamine 2000 and RNA solutions were mixed together and added to the media bathing the cells. The cells were incubated overnight at 37°C, 5% COPDG performed all FRET and immuno-FRET experiments between RNA binding proteins, performed all the image analysis, created all the figures, and wrote various drafts of the manuscript. MAT performed all FRET experiments between fluorescently labeled RNA and RNA binding proteins, created preliminary figures, and assisted in writing the methods section relevant to these experiments. JDP is the laboratory director, and oversaw all aspects of the project. JDP worked with PDG to design and analyze the data, and wrote major portions of the manuscript as well as performing all final editing. All authors approve of the final manuscript.PDG is a post-doctoral fellow in the JDP lab and has been working in the area of RNA binding protein imaging for several years. MAT is a graduate student in pharmacology, currently working in the area of cancer biology. JDP has been a Professor of Medicine and Pharmacology at the University of Colorado School of Medicine since 1991. His laboratory has been focused on RNA turnover and signaling pathways that modulate the function of RNA binding proteins for many years. JDP has co-organized and participated in numerous meetings related to RNA turnover and RNA biology.Video 1. Interactions of PBs and SGs in DDT1-MF2 cells subjected to oxidative stress. DDT1-MF2 cells were treated with 0.5 mM sodium arsenite for 30 minutes prior to immunostaining. PBs were detected by Hedls (red) and SGs by KSRP (green). Images were collected along the Z axis of the cell at 100× magnification under oil immersion, , using 2 × 2 binning mode on an inverted Nikon Eclipse TE3000 fluorescence microscope using a Cooke SensiCam QE CCD camera running Slidebook software . Capture time was 500 ms and step size was 0.5 microns. 3D stack was "deconvolved" using the nearest-neighbors method.Click here for file


Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein.  Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo.  RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport.  To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes.  After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE .  We have extended this technique through co-localization with additional RNAs and proteins , and in combination with disruption of motor proteins and the cytoskeleton  to probe mechanisms underlying mRNA localization. The DNA template must have upstream promoter sites for transcription by T7, SP6, or T3 RNA polymerase.Linearize plasmid DNA containing the RNA localization element or other relevant sequence and resuspend at 1 μg/μl with DEPC-treated HAdd the following reagents to a sterile 1.5mL tube: Mix reagents gently and centrifuge briefly (10 sec. in a microcentrifuge).Incubate for 2-4 hours at 37° C, covering the tube with aluminum foil to prevent photobleaching of fluorophore.Add 1 μl 1 mg/ml RNase-free DNase (Promega) to degrade template DNA.Incubate 15 minutes at 37° C.Add 79 μl 20 mM EDTA (pH 8.0) to stop the reaction.Remove 1 μl to a separate tube, labeled as "input" and save.Spin reaction through a 1 ml G50 column , which will include unincorporated nucleotides while excluding full length mRNA.3COONH4, 1 μl carrier RNA  or 1 μl glycogen (20 mg/ml).Add 250 μl 100% ethanol, 10 μl 7M CHMix well and freeze until solid at -80° C for >30 minutes or on dry ice for 15 minutes. Spin at maximum speed in microcentrifuge for 15 minutes, decant supernatant.Wash with 150 μl of 75% ethanol.Spin for 3 minutes at maximum speed in microcentrifuge, decant supernatant.Dry the pellet at 37° C for 3 minutes, ensuring that all ethanol has evaporated.Concentrate the RNA by ethanol precipitation: 2O and remove 1 μl to a separate tube, labeled as "incorporated".Resuspend in 11 μl DEPC-HDetermine the percent incorporation and dilute the RNA to 50 nM . The RNA should be frozen in 5 μl aliquots, for single use to avoid freeze / thaw cycles and can be stored for several weeks at -80° C.RNA Preparation:Thaw an aliquot of RNA (50 nM), and denature the RNA at 70° C for 3-5 minutes. Spin for 10 minutes in room temperature microcentrifuge at maximum speed to remove any particulates, and keep on ice.Xenopus laevis females (Nasco). Surgically remove ovary from Xenopus laevis ovary into a 50 ml conical tube containing 25 ml of Collagenase solution . Trim pieces of Shake gently at 18° C for 15 minutes, or until oocytes are visibly separated from ovary. Due to batch to batch variation, collagenase treatment time may vary and should be monitored carefully through visual inspection to ensure defoliculation. Allow oocytes to settle in tube, remove solution and wash the oocytes with MBSH buffer . Repeat wash twice more for a total of three washes. This oocyte isolation protocol is similar to that detailed in Cohen et al., 2009. Manually sort stage III/IV oocytes  in MBSH buffer under a standard dissecting microscope. Stage III oocytes are completely opaque but white, while stage IV oocytes are slightly larger and speckled with pigment. Oocytes that are slightly transparent are too young and oocytes that fully pigmented or exhibit polarized pigment distribution are too old.Oocyte Preparation: Needle Preparation:Pull and bevel needles to an outer diameter of ~ 0.05 mm. (We use Drummond Scientific 3.5 inch capillaries (order # 3-000-203-G/X), a Sutter Instrument Co. micropipette puller and a WPI Inc. beveller.)2O to deliver 2 nl per injection. We front load our needle using a gas-driven microinjector , and calibrate drop size using a micrometer. Calibrate needle with DEPC-HLoad RNA into needle.Place sorted oocytes into an injection dish containing MBSH buffer. We microinject on a dish with a layer of black foam rubber. The pale oocytes stand out well on a white background.Carefully inject each oocyte with 2 nl of RNA at 50 nM.2O, and load next RNA for injection.Expel RNA, rinse needle with DEPC-HPlace oocytes in a well of a sterile 24 well plate (Sigma Aldrich). We culture up to a thousand oocytes per well. Remove buffer and replace with 400 μl Oocyte Culture Medium  per well. Place culture plate inside an air-tight plastic container with wet paper towel to maintain moist environment during culture.Incubate oocytes at 18° C for time points ranging between 8 and 48 hours.Sort out any dead oocytes and place surviving oocytes in glass vials. We routinely observe >90% survival. Place oocytes in MEMFA fixative  and rock for 20 minutes. Protect the oocytes from light by covering with aluminum foil.Wash oocytes by removing fixative and replacing with equal volume of MBSH buffer. Repeat this wash once more for a total of two washes.Remove half of the volume, replace with methanol.Repeat step "a" twice.Remove all of the solution, replace with methanol.Repeat methanol wash.Wash oocytes into anhydrous methanol: Oocytes can be stored at -20° C until ready to image.Before imaging, add Murray s Clearing Medium  to a glass bottom FluoroDish (WPI Inc.) to cover the imaging surface. Carefully transfer the oocytes from methanol to FluoroDish, minimizing the volume of methanol transferred.Image on an inverted confocal microscope (We have successfully used a Zeiss LSM510 and a Leica TCS SP2).          Figure 1: Visualization of Subcellular RNA Localization by Microinjection of Fluorescently Labelled Transcripts.  A.  Following microinjection, RNA labeled with Alexa Fluor 546 is initially restricted to the nucleus. B. After eight hours of culture, a fluorescently labeled control RNA can be seen uniformly distributed in the cytoplasm of the oocyte. C. However, fluorescently labeled RNA containing sequences that recruit the transport machinery can be seen in the process of subcellular localization to the vegetal pole of the oocyte (towards bottom). Scale bar = 50 μm.          Xenopus oocytes. This method, using fluorescently labeled RNA transcripts has higher signal to noise ratio than previously obtained with digoxigenin-labeled transcripts and is simpler and faster than in-situ based approaches . Using this method, we can engineer RNA sequence mutations and rapidly test for in vivo function. Further, by using additional Alexa-UTP fluorophores, multiple RNA species can be visualized in the same oocyte. We have found that in Xenopus oocytes Alexa Fluor 546-14-UTP gives superior results as compared with Alexa Fluor 488-5-UTP because of autofluorescence in the 488 nm wavelength range; however, dual imaging of two RNAs is nonetheless possible . Also, this technique works well in conjunction with immunostaining protocols to detect protein colocalization, as a wide variety of compatible fluorescent secondary antibodies are available . Finally, disruption of the RNA localization process through over-expression of dominant negative factors or small molecule interference can be combined with this assay to visualize subtle defects in the mRNA localization pathway . This technique has proven to be a relatively simple and robust assay for detecting mRNA distribution in vivo and can be integrated easily into existing biochemical, molecular and cell biological techniques for probing mechanisms of mRNA transport.Here we have presented a protocol for visualizing mRNA localization in


Caenorhabditis elegans NALCN homologues NCA-1 and NCA-2 in the propagation of neuronal activity from cell bodies to synapses. Loss of NCA activities leads to reduced synaptic transmission at neuromuscular junctions and frequent halting in locomotion. In vivo calcium imaging experiments further indicate that while calcium influx in the cell bodies of egg-laying motorneurons is unaffected by altered NCA activity, synaptic calcium transients are significantly reduced in nca loss-of-function mutants and increased in nca gain-of-function mutants. NCA-1 localizes along axons and is enriched at nonsynaptic regions. Its localization and function depend on UNC-79, and UNC-80, a novel conserved protein that is also enriched at nonsynaptic regions. We propose that NCA-1 and UNC-80 regulate neuronal activity at least in part by transmitting depolarization signals to synapses in C. elegans neurons.Voltage-gated cation channels regulate neuronal excitability through selective ion flux. NALCN, a member of a protein family that is structurally related to the α1 subunits of voltage-gated sodium/calcium channels, was recently shown to regulate the resting membrane potentials by mediating sodium leak and the firing of mouse neurons. We identified a role for the  C. elegans nervous system, we reveal that a putative channel complex, consisting of ion-conducting, pore-forming proteins called NCAs and two auxiliary components called UNC-79 and UNC-80, regulates neuronal function. We first show that an increase or decrease of the activity of this channel causes physiological changes that indicate corresponding alterations in neuronal activity. We then demonstrate by in vivo calcium imaging that the NCA channel, localizing along axons, specifically regulates excitation of synapses. We speculate that this channel participates in the propagation of electric signals that activate synapses.Neurons communicate to their targets through synapses that are activated by the electrical signals conveyed along neuronal processes. The tightly regulated ion flux across the cell membrane drives the generation of these electrical signals; it is therefore important to identify ion channels that regulate the excitability of neurons. In the  C. elegans neurons. Components conserved throughout evolution, these channel complexes may play similar roles in other nervous systems.A putative cation channel NCA/UNC-79/UNC-80 modulates the propagation of excitation signals along axons in  By allowing selective ion flux across the plasma membrane, cation channels regulate the excitation and function of neurons. In most nervous systems, action potentials, the traveling and rapidly reversing membrane potentials, are induced by the opening of voltage-gated sodium channels and are modulated by voltage-gated sodium, potassium, and occasionally calcium , one L-type α1 subunit (EGL-19), and one T-type α1 subunit (CCA-1) [2+ transients that define sarcomere excitability [2+ transients in cultured mechanosensory neuron cell bodies [ (CCA-1) –19. UNC- (CCA-1) . The los (CCA-1) . In phar (CCA-1) ,17. EGL-tability ,15,18. Il bodies .C. elegans [Drosophila [A rat cDNA clone encoding a protein with homology to the α1 subunit of voltage-gated calcium and sodium channels was first isolated by a degenerative oligo-based PCR screening . Homolog elegans , Dmα1U/Cosophila , and Vgcosophila ,26 in moosophila  and EEEEosophila ,29, respDrosophila Dmα1U mutants are viable but display altered sensitivity to anesthetics and abnormal circadian rhythm [C. elegans nca-1;nca-2 double knockout mutants also display abnormal halothane sensitivity and more frequent pauses during locomotion, a phenotype termed “fainter” [NALCN knockout mice are neonatal lethal due to a disrupted respiratory rhythm [+ leak currents and decreased firing, suggesting that NALCN functions as a Na+ leak channel and regulates neuronal excitability by affecting membrane potentials [n rhythm . C. elegfainter” . The phyy rhythm . Mutant tentials .C. elegans. Our genetic and phenotypic analyses of nca loss- and gain-of-function mutants show that NCA proteins affect synaptic function by modulating the transmission of depolarization signals. This function depends on two novel auxiliary proteins: UNC-79 and UNC-80. Thus, a putative NCA channel regulates neuronal activity in C. elegans neurons, at least in part by facilitating axonal conductance of depolarizing signals from the cell body to the synapse.In this study, we describe the physiological and cell biological characterization of the NCA proteins in C. elegans, we identified and analyzed the phenotypes of animals carrying dominant and recessive mutations in nca-1 and its homologue, nca-2. Both dominant and recessive mutations in NCAs have clear effects on C. elegans behavior. gk9 and gk5, the two deletion alleles for nca-1 and nca-2, respectively, were generated by the C. elegans gene knockout consortium. Removing part of the essential pore-forming domain of NCA-1 and NCA-2, both mutations are predicted to cause severe losses of protein functions (nca-1(gk9);nca-2(gk5) double mutants are fainters that fail to sustain sinusoidal locomotion and succumb to long periods of halting .To investigate the function of NCA channels in unctions A. While halting   .We identified two gain-of-function alleles of ca-1 see A. One of neurons   , both hpceforth) . Moreove mutants . In summnca(lf) and nca(gf) mutants suggest that NCA activity regulates synapse function. To address this possibility, we recorded spontaneous and evoked postsynaptic currents in body wall muscles as an indirect measure for presynaptic activities of GABAergic and cholinergic neurons NMJs [2+, nca(lf) mutants displayed a significant decrease in the frequency of spontaneous release   as compared to wild-type animals (55.6 ± 5.3 Hz at 5mM Ca2+ and 39.7 ± 6.5 Hz at 1mM Ca2+)  mutants, the amplitude of ePSC was reduced by 60% at 5mM Ca2+ , and by 75% at 1 mM Ca2+  (nca(lf) mutants.The locomotion defects of both ons NMJs ,34. In tmM Ca2+) A and 2C.1mM Ca2+ B and 2D. = 0.01) B and 2D.nca(gf) mutations affect synaptic transmission. At 5 mM extracellular Ca2+, some nca(gf) animals , others (n = 6). No ePSC could be evoked in any of the two groups (nca(gf) animals also show aberrant synaptic activity and further establish the link between NCA channels and synaptic function.We also examined how o groups F and 2H.2+ sensor, in live C. elegans [nca(gf) mutants  mutants, HSN cell bodies generated trains of Ca2+ spikes undistinguishable from those in wild-type soma for spike frequency , interval  and amplitude   mutants showed no Ca2+ transients at all (nca(lf), synapse, top trace). The rest of the nca(lf) mutants retained Ca2+ transient trains (nca(lf), synapse, bottom trace). This resulted in an overall significant decrease of synaptic spike frequency (1.2 ± 0.7 spikes/min in nca(lf)) compared to wild-type synapses , and to the spike frequency of nca(lf) cell bodies . Remarkably, the remaining trains of Ca2+ transients in the nca(lf) mutants maintained temporally correlated in spike interval (5.2 ± 0.4 s) with the cell body of nca(lf) mutants . They also display comparable amplitude (6.8 ± 0.7%) to those in wild-type synapses   C–3H. At  > 0.05) C–3H. Thunca(gf) mutants, HSN cell bodies also displayed trains of calcium spikes similar to those in wild-type animals in their frequency , interval  and amplitude  , and interval  with those in nca(gf) cell bodies. However, the amplitude of Ca2+ transients was significantly increased at synapses (9.5 ± 1.3% for nca(gf) versus 5.2 ± 0.7% for wild-type, p = 0.029). Although the mean amplitude appears only moderately bigger than in wild-type animals, nca(gf) mutants exhibited a fraction of unusually large Ca2+ transients at synapses that were well above the range seen in wild-type animals (In  > 0.05) B–3E. At nca(lf) and (gf) mutants specifically altered Ca2+ transients at the presynaptic regions, indicating that under our assay conditions, NCA activity does not alter the excitation at HSN soma but affects presynaptic activity. The decrease of Ca2+ transients in nca(lf) suggests that NCA is required to initiate presynaptic activation in response to depolarization signals. The elevated Ca2+ transients in nca(gf) mutants further suggests that the gain-of-function mutations enhance NCA's activity in presynaptic activation.In summary, both nca(gf) mutants (see nca(gf) coilers to fainters and fully suppressed their synaptic morphology defects , as well as two previously identified unc-80 alleles, e1272 and e1069, exhibit recessive and fully penetrant fainter phenotypes identical to that of the nca(lf) double mutant (nca(lf);unc-80 triple mutants are indistinguishable from either nca(lf) double mutants or unc-80 single mutants in behavior (nca(gf);unc-80 double mutants display the same fainter phenotype as unc-80 single mutants (unc-80 mutations epistatic to nca(lf) alleles, suggesting that NCA activity depends on UNC-80.To identify proteins that modulate NCA activities, we performed a genetic suppressor screen for mutations that reverted locomotion defects of ants see . We iden defects . One suphenotype  and latehenotype . unc-80 e mutant . We founbehavior . Further mutants . Therefounc-80 was recently cloned based on the observation that RNAi knockdown of an open reading frame F25C8.3 in wild-type animals resulted in a fainter phenotype and the identification of missense mutations in F25C8.3 from unc-80 alleles [F25C8.3 rescued the fainter phenotype of unc-80 mutants  mutants from fainters to nca(gf) locomotion patterns . The HSN cell bodies displayed trains of Ca2+ transients with normal frequency , interval  and amplitude  , with an overall reduction in frequency (0.9 ± 0.7 spikes/min) when compared to unc-80 cell bodies , and to wild-type synapses . The remaining trains of Ca2+ transients at synapses maintained temporally correlated with cell body transients in spike interval . They were also comparable in amplitude with wild-type synaptic transients   mutants, unc-80 mutants also displayed identical changes in presynaptic activation. This indicates that UNC-80 either mediates or functions together with the putative NCA channel to regulate presynaptic activation.We confirmed that  > 0.05) B–3E. Likunc-80 and nca-1 genes  and unc-80 mutants, respectively (Punc-25 [nca(gf) mutants  promoter reporter constructs, which contain their predicted upstream genomic sequences, revealed similar expression patterns in the nervous system, including many sensory neurons and all motoneurons, for both the -1 genes A. Exprespromoter  was ableectively  and S12. deletion mutants . hpIs98 (Punc-80-UNC-80::RFP) restored NCA-1 expression at the nerve ring and along the nerve cords in unc-80 mutants (nca(gf) mutants, the staining was also eliminated or greatly reduced in nca(gf);unc-80 mutants (nca-1 transcripts were present at wild-type level in unc-80 mutants (nca(lf) mutants, UNC-80::RFP staining was also significantly reduced (nca(lf);hpIs98 panel), suggesting that UNC-80 localization along the axon is also dependent on the presence of NCA protein. This NCA-1–dependent localization of UNC-80::RFP, together with the fact that no transmembrane motifs are present in UNC-80, is consistent with the possibility that UNC-80 functions as an auxiliary subunit that regulates the transport, stability, or clustering of NCA at the membrane.To further determine how UNC-80 regulates NCA-1 activity, we examined the distribution of NCA-1 in  mutants A, indica mutants A, sugges mutants B. Togethunc-79 loss-of-function mutants also have a fainter phenotype, and have been reported to contain lower than normal levels of NCA-1 protein by Western blot analyses [unc-80 mutants, we observed reduced or completely diminished NCA-1 staining in the unc-79 mutants (unc-79 mutants (nca(lf), unc-80, and unc-80;nca(lf) mutants, no UNC-79 staining was detectable in neuronal processes . This effect was not induced by the expression of other channels , and was abolished when transfected cells were incubated with 100 μM verapamil or 10 μM gadolinium . In contrast, co-transfection of NCA-1 and UNC-80 constructs induced significant cell death . This effect was abolished when the UNC-80 expression vector was co-transfected with a NCA-1 clone carrying a deletion in the coding region . Moreover, the increased cell death in NCA-1 and UNC-80 co-transfected cells was also blocked in the presence of 100 mM verapamil  or 10 mM gadolinium . Therefore the co-expression of UNC-80 and NCA-1 induced the same effect, with similar blocker responses as NALCN in HEK293T cells, suggesting that the putative NCA/UNC-80 channel complex likely shares similar ion leak properties.Using this same assay, we examined whether 3T cells . cDNAs e3T cells . Transfe2+ transients at serotonergic NMJs in nca(lf) mutants suggest a decreased presynaptic activity in the absence of the putative NCA channels. The calcium imaging analyses further suggest that this synaptic defect is related to a failure to initiate presynaptic activity. In wild-type animals, the calcium spikes at HSN cell bodies and synapses are temporally correlated. In both nca(lf) and unc-80 mutants, at least under our assay conditions, despite the normal calcium dynamics in cell bodies, the number of Ca2+ transients was reduced at synapses. The NCA channel is unlikely to conduct Ca2+ transients at synapses, since the remaining transients in nca(lf) mutants were normal in amplitude and maintained temporal correlation with the depolarization signals in cell bodies. Together with their nonsynaptic localization along nerve processes, these results strongly indicate that NCA channel activity is required to transmit depolarization signals to synapses  and unc-80 mutants is incomplete; however, the molecular lesions in these mutants predict severe loss of protein functions. All nca(lf) and unc-80 alleles are behaviorally indistinguishable from each other and fully penetrant for the fainter phenotype, which strongly argues against an allelic effect on phenotype penetrance. The partial loss of Ca2+ transients and the variable degree of the decrease of mPSC frequency in nca(lf) mutants therefore more likely suggest that while some depolarization signals depend on NCA channels to induce presynaptic activation, other signals reach synapses independently of NCA activity  and unc-80 mutants in our physiological analyses, there is little behavioral variability among individual animals. Because every animal alternates between a state of normal sinusoidal movement and quiescence, we speculate that C. elegans neurons alternately fire NCA/UNC-80–dependent and -independent depolarization signals. Perhaps due to the necessary experimental manipulation  and the short assay time, we measured neuronal activity fixed in one “mode,” resulting in the appearance of two distinct populations.It is worth noting that although we detected two distinct, active versus quiescent populations in nca gain-of-function mutants. Specifically, we found that these mutant animals showed a coiler uncoordinated phenotype, and exhibited larger calcium transients at synaptic sites. Gain-of-function mutations in NCA-1 do not affect the temporal correlation of calcium transients between HSN cell bodies and synapses. Whole-mount staining with antibodies against NCA-1 showed no obvious changes in the subcellular distribution or intensity of the staining signals in nca(gf) mutants, indicating that these mutations likely alter the activity rather than the abundance of the NCA-1 protein. The calcium imaging phenotype is consistent with the NCA(gf) channel further increasing the membrane excitability, which leads to enhanced activation of calcium channels at HSN synapses ) was able to induce similar locomotion defects as NCA-1(gf) proteins in C. elegans  mutants, they do not enhance nca(lf) mutants, and they suppress defects exhibited by nca(gf) mutant. By contrast, unc-80 mutants do not phenocopy VGCC loss-of-function mutants or display obvious genetic epistasis with VGCC gain-of-function mutants  mutants, but also a complete suppression of nca(gf) locomotion defects and the disappearance of NCA-1 and UNC-80 along nerve processes. Furthermore, UNC-79 is dependent on the presence of both NCA and UNC-80 for its localization along neurites. Therefore, both UNC-80 and UNC-79 are likely conserved auxiliary components of the NCA family channels.We observed identical genetic interaction between n motifs . SimilarNCA-1 and NCA-2 have close sequence homologues in other invertebrate and vertebrate, including human. The mammalian member of this family, NALCN, has recently been characterized physiologically in HEK293 cells . In spitC. elegans NCA and its mammalian homologues share common functional properties. First, C. elegans NCA proteins show at least similar properties to NALCN proteins when heterologously expressed in mammalian cell culture. In HEK293T cells, transfecting NALCN, or co-transfecting NCA-1 and UNC-80 induced cell death that was blocked by the NALCN blockers verapamil and gadolinium. Conversely, expressing mammalian NALCN proteins in C. elegans could substitute functionally for the NCA proteins. Specifically, wild-type C. elegans expressing a mouse cDNA that carries the hp102 equivalent mutation (NALCN(R329Q)) in neurons exhibited a locomotion pattern with exaggerated body bends, reminiscent of the nca(gf) mutants  mutants may result from a combination of deficits in the propagation of depolarization signals, neuronal firing, and even vesicle release. The mouse NALCN affects the resting potential and controls the excitability/firing rate of hippocampal neurons [Given the conservation in the functional properties of NCA family members, it is reasonable to speculate that these channels may also carry out similar functions in neurons. Our current studies suggest a specific function for the NCA channel in transmitting and regulating excitability along  neurons ; whetherhp102 was originally identified in a genetic screen for hpIs3 defective mutants [unc-80(hp369) and unc-79(hp424) were identified in a hp102 suppressor screen and outcrossed three times against N2. e625, e1069, and e1272 were identified through abnormal locomotion in previous C. elegans screens [gk9 and gk5 were generated by the Gene Knockout Consortium and were outcrossed three times against N2. tm1591 was generated by National Bioresource Project for the Nematode and was outcrossed once against N2.All strains were cultured at 22 °C unless specified otherwise.  mutants  and was  screens . gk9 andhp102 mutants were identified from an active zone marker hpIs3 screen [hp102 was rough mapped to Chromosome IV between E03H12 (1.40 cM) and D2096 (3.74 cM) by SNP mapping against CB4856. During the mapping, we noticed that uncoordinated)-77(e625)unc. We determined that e625 and hp102 were alleles of the same gene due to the genetic interactions displayed by these two mutants: while hp102/e625 heterozygous animals showed fully penetrant coiling locomotion as either homozygous mutants, hp102/+ or e625/+ heterozygous mutants showed only slightly more exaggerated body bends compared with wild-type animals. This conclusion was confirmed when we mapped both mutants to the same genetic locus, rescued both mutants with the same genetic fragments, and identified mutations in the same open reading frame (see below).3 screen . Based oe mutant  that washp102 and e625 mutations were then further fine-mapped between B0273 (1.74 cM) and C49A9 (3.08 cM) based on the following data. From dpy-13unc-77/CB4865, three out of 20 Unc non Dpy animals had their recombination breakpoints between B0273 (1.74 cM) and F38A5 (3.21 cM), placing unc-77 to the right of B0273 (1.74 cM). From unc-77bli-6/CB4856 animals, three out of three Unc non Bli recombinants and two out of two Bli non Unc recombinants had their recombination breakpoints between C49A9 (3.08 cM) and F38A5 (3.21 cM), placing unc-77 to the left of C49A9 (3.08 cM). From unc-5unc-77/CB4856 heterozygous animals, two out of two unc-77 non unc-5 recombinants had their recombination breakpoints between C31H1 (2.56 cM) and C49A9 (3.08 cM), placing unc-77 between C31H1 (2.56 cM) and C49A9 (3.08 cM). Cosmids and PCR fragments amplified from the genomic sequence covering this region  were injected into hp102;hpIs3 and e625;hpIs3 animals. Only DNA fragments covering the C11D2.6 (nca-1) genomic region rescued the locomotion and hpIs3 marker defects. We further confirmed that unc-77 corresponds to nca-1 by sequencing the entire predicted genomic regions  of hp102 and e625 mutants and identifying a single missense mutation in the coding region of each mutant  and e625;nca-2(gk5) mutants displayed the same behavior as hp102 and e625 homozygous mutants. Furthermore, PCR fragments amplified from the nca-1 genomic region from hp102 when expressed in wild-type animals induced the same coiling locomotory defects as hp102 mutants  background. If they showed fainter phenotype, the suppressor was confirmed as intragenic revertants. From the progenies of 13,000 mutagenized F1 hermaphrodites , we identified four intragenic suppressors reverting hp102 mutants to wild-type locomotion. We also identified multiple alleles of two different extragenic suppressors that reverted hp102 animals to fainters, and failed to complement unc-80 and unc-79 mutants, respectively.unc-80 was first rough mapped between F21D9 (21.82 cM) and F38A6 (27.08 cM) on Chromosome V through two-factor SNP mapping against CB4856. Two out of four unc-80 recombinants from unc-51unc-80/CB4856 broke between Y113G7A (24.71 cM) and F38A6 (27.08 cM), placing unc-80 to the right of Y113G7A (24.71 cM). 0/9 Rol non Unc recombinants from unc-51rol-9/unc-80 animals segregated unc-80, placing it to the right of rol-9 . In two-factor mapping against pha-4 and CB4856, we could not find breakage between unc-80 and pha-4 (25.60 cM), placing unc-80 between 25.12 cM and 27.08 cM, tentatively near 25.60 cM. Clones that cover this region were generated by PCR to be tested for rescuing of unc-80 mutants. Kim Schuske (University of Utah) determined that RNAi knockdown of F25C8.3 (which lies within this region) in wild-type animals was able to induce a fainter phenotype . We generated and shared with the Schuske group DNA fragments spanning F25C8.3 . Whole-mount immunofluorescent staining was carried out as previously described [Clontech), SAD-1, SNB-1, and UNC-10  were used in 1:10, 1:10, 1:200, 1:200, 1:100 and 1:2000 dilutions, respectively.Antibodies against aa1731–1914 of the predicted NCA-1d isoform, and a combination of aa506–608 and aa1205–1851 of UNC-79 were generated in rat , a condition that stimulates constitutive egg-laying thus spontaneous activation of HSN neurons. Recording was carried out as previously described [escribed . All recescribed  and Text2, 95% air. Lipofectamine 2000 was used to transfect the HEK293T cells following the standard procedure (Invitrogen). 0.4 μg total DNA was used for each transfection experiment. Medium was replaced 4 h after transfection, during which the culture was split into three sets with equal density, two sets were exposed to 100 μM verapamil or 10 μM Gd3+, respectively.HEK293T cells were grown in α-MEM (GIBCO) medium supplemented with 10% FBS (GIBCO) at 37 °C in a humidified atmosphere of 5% COCell death assays were performed 48 h after transfection. Culture medium was replaced by extracellular solution containing 50 μg/ml of propidium iodide (PI) (Invitrogen). After 30 min incubation at 37 °C, fluorescence intensity in each well was measured with a plate reader  as described previously ,46. The Figure S1nca(gf), nca(lf), unc-80, and nca(gf);unc-80 mutants. The coiling position of nca(gf) was fully restored in nca(gf);unc-80 mutants.(A) Images of the body morphology of hp102, unc-80, and nca-1(hp102); unc-80 animals, visualized by SYD-2::GFP (hpIs3). Inserts are magnified views of regions marked by the dotted line. nca(gf) mutants showed abnormal clustering (arrowheads) and gaps between active zone marker puncta. This defect was fully suppressed in nca(gf);unc-80 mutants.(B) Active zone marker morphology of DD GABAergic synapses in L2 larvae of wild-type, n = 10 for each strain) for hpIs3 , where nca(gf);unc-80 mutants showed wild-type level puncta.(C) Quantification of the average number  hp102 mutants (bottom panels) compared to wild-type animals (top panels). This phenotype was rescued in nca(gf);unc-80 mutants.(D) Morphology of HSN synapses visualized by an active zone marker nca(gf) hp102 mutants display constitutive egg-laying, resulting in fewer eggs  and younger eggs  retained in uterus of hp102 animals compared to wild-type animals. n = 15, the number of eggs was counted from animals 24-h post L4 larval stage. Error bar: SEM. Statistic comparisons were performed against the wild-type dataset using the Tukey-Kramer multiple comparison test. *p < 0.01, **p < 0.001, Scale bar: 5μm.(E and F) (805 KB PDF)Click here for additional data file.Figure S2unc-13(e51) mutants, where synaptic transmission is severely abolished, HSN cell bodies were capable of generating trains of calcium spikes. (A) Representative cameleon trace displayed by HSN cell bodies of unc-13(e51) mutants as in unc-13(e51) mutants showed no statistically difference by the Kolmogorov-Smirnov rank test (p > 0.1). The number at the bottom of each bar represented the number of animals examined.A manuscript describing this work has been submitted. We present here only one piece of supporting evidence. In (52 KB PDF)Click here for additional data file.Figure S3x-axis represents the recording time in seconds, and the y-axis represents fluorescent intensity in arbitrary units of pixel intensity. The ratio between YFP and CFP fluorescent signals at each time point was plotted against the same x-axis, resulting in a third trace that represents the YFP/CFP ratio change. Calcium spikes display a characteristic asymmetric shape, with a fast, linear rising phase followed by a slower, exponential decaying phase (arrows); whereas peaks due to noise, or random fluorescent ratio fluctuations, typically show a symmetric shape, with linear rising and decaying phases (arrowheads). Reciprocal changes in YFP and CFP intensity were observed for many ratio peaks (examples were shown by dashed lines), though reciprocity was sometimes obscured by motion artifacts caused by movement of the neurons during egg-laying. Despite the fluctuations in the absolute YFP and CFP intensity levels over the recording period, the YFP/CFP ratio-metric trace revealed similar ratio changes for most calcium spikes in wild-type animals, suggesting that the calcium spike size is insensitive to fluorescent baseline changes and accurately reflects changes in calcium concentration. The right panel shows raw YFP and CFP traces for HSN synaptic trace shown in The left panel, top two lines, show raw YFP and CFP traces for HSN cell body recordings shown in (49 KB PDF)Click here for additional data file.Figure S4(A) A schematic representation of the gene structure of unc-80 (F25C8.3) adapted from the Wormbase. The exons are shown as pink boxes. The genetic lesions of unc-80 alleles  are shown. (B) Protein structure and similarity of UNC-80 family members. C.e.: C. elegans; D.m.: Drosophila melanogaster; R.n.: Rattus norvagicus; M.m.: Mus musculus; H.s.: Homo sapiens.(57 KB PDF)Click here for additional data file.Figure S5hpIs3 in wild-type and nca(gf) respectively. In wild-type animals, SYD-2::GFP puncta are round and regularly spaced, whereas nca(gf) animals show regions lacking puncta, as well as clustering of puncta. Genomic DNAs containing the nca-1 gene, or expression of NCA-1 from a GABAergic neuron-specific promoter, restored the hpIs3 phenotype of nca(gf) to wild-type morphology. Inserts show a higher magnification of the region underlined by the dotted line. Scale bar: 5 μm.Active zone morphology was examined using the SYD-2::GFP marker (246 KB PDF)Click here for additional data file.Figure S6(A) Wild-type animals were co-stained with anti-NCA-1 antibody (red) and either anti-SNB-1 or anti-SAD-1 antibodies (green). (B) unc-80; hpIs98 (UNC-80::RFP) animals were co-stained with anti-RFP antibody (red) and either anti-SNB-1 or anti-SAD-1 antibodies (green). Poor colocalization was observed in all cases. Scale bar = 5 μm.(366 KB PDF)Click here for additional data file.Figure S7unc-80 animals were stained with anti-NCA-1 (red) and anti-UNC-10 (green) antibodies. Punctate NCA-1 staining pattern along the ventral and dorsal nerve cords was observed in wild-type but diminished in unc-80 animals. UNC-10 staining pattern was similar between wild-type and unc-80 mutant animals. The expression of UNC-80::RFP from its own promoter (hpIs98) restored the punctate expression pattern of NCA-1 along the nerve cords.(A) UNC-80 regulates NCA-1 protein localization. Wild-type and unc-80 mutations do not affect the transcript level of nca-1. Total RNA was isolated from mixed staged wild-type, unc-80(e1069) and unc-80(e1272) animals, and the transcript levels of nca-1 were analyzed by RT-PCR (with a control reaction containing no template). nca-1 transcript was present at similar levels in all strains. Scale bar: 5 μm.(B) (480 KB PDF)Click here for additional data file.Figure S8unc-79 animals (bottom panel). Specific staining was observed in the nerve ring of wild-type but not in unc-79 mutant animals. *ns: non-specific staining. (B) The dorsal (top panel) and ventral (bottom panel) nerve cords of wild-type animals stained with anti-UNC-79 antibody showed punctate staining pattern.(A) An antibody against UNC-79 was used to stain wild-type (top panel) and (201 KB PDF)Click here for additional data file.Figure S9nca(gf), nca(lf), unc-80, nca(lf);unc-80, and nca(gf);unc-80 animals were simultaneously stained with anti-UNC-79 antibody. UNC-79 signals were detected in the nerve ring (NR) of wild-type and nca(gf) animals but disappeared in other mutants.Wild-type, (399 KB PDF)Click here for additional data file.Table S1nca(lf) or unc-80 mutants in combination with cca-1, egl-19 (lf and gf) and unc-2 mutants display enhanced or additive locomotion defects than either single mutants. Similarly, nca(gf) mutants in unc-2 and egl-19(lf) backgrounds lead to more severe or additive locomotion phenotypes. In particular, egl-19(gf) mutations do not suppress or improve the locomotion defects of unc-80 and nca(lf) mutants. nca(gf) mutations do not improve the locomotion defects of egl-19(lf) or unc-2(lf) mutants. This suggests that NCA/UNC-80 and VGCCs do not function in a linear pathway. Their genetic interactions are most consistent with these two types of channels regulating different aspects of neuronal excitation.(37 KB DOC)Click here for additional data file.Text S1(53 KB DOC)Click here for additional data file.Video S1A wild-type animal displays sinusoidal forward and backward locomotion patterns, also called body bends(797 KB MOV)Click here for additional data file.Video S2nca-2(gk5);nca-1(gk9) mutant animal displays a locomotion deficit termed as “Fainter”. The animal was capable of moving in a sinusoidal fashion upon stimulation , but stopped after only a couple of body bends.An (980 KB MOV)Click here for additional data file.Video S3unc-77(hp102), nca(gf) animals display constitutive and exaggerated body bends that make them appear “coiling” during both forward and backward movements.(835 KB MOV)Click here for additional data file.Video S4nca-1 genomic fragments harboring the hp102 mutation displayed the exaggerated body bends similar to those by unc-77(hp102) mutants Click here for additional data file.Video S54× real time. The cameleon signal was pseudo-colored based on the YFP/CFP ratio value, with red representing the highest value. Each pink flash reflected the peak of one calcium spike.(410 KB MOV)Click here for additional data file.Video S6unc-80(e1272) mutant animal displayed the same fainter locomotion deficit as nca-2(gk5);nca-1(gk9) mutants (An  mutants .(981 KB MOV)Click here for additional data file.Video S7nca-2(gk5);nca-1(gk9);unc-80(e1272) mutant animal displayed the same fainter locomotion deficit as in nca(gk5);nca-1(gk9) (unc-80(e1272) (An a-1(gk9)  and in u0(e1272)  mutants.(985 KB MOV)Click here for additional data file.Video S8unc-77(hp102); unc-80(e1272) mutant animal displayed the same fainter phenotype as in unc-80(e1272) mutants (An  mutants .(1 MB MOV)Click here for additional data file.Video S9unc-80(e1272) mutant animal carrying an extrachromosomal array that consisted of genomic fragments covering the unc-80 locus displayed continuous locomotion, and did not ‘faint'.An (807 KB MOV)Click here for additional data file.Video S10unc-77(hp102); unc-80(e1272) mutant animal carrying an extrachromosomal array that consisted of genomic fragments covering the unc-80 locus displayed continuous and exaggerated sinusoidal (coiling) locomotion pattern, similar to that of unc-77(hp102) mutants (A  mutants .(479 KB MOV)Click here for additional data file.Video S11nca-2(gk5); nca-1(gk9) mutant animal carrying an extrachromosomal array expressing NCA-1 Driven by a pan-neuronal promoter displayed continuous and sinusoidal locomotion, and did not ‘faint.'An (794 KB MOV)Click here for additional data file.Video S12unc-80(e1272) mutant animal carrying an extrachromosomal array expressing UNC-80 driven by a pan-neuronal promoter displayed continuous and sinusoidal locomotion, and did not ‘faint.'An (972 KB MOV)Click here for additional data file.Video S13hp102 and e625 animals.A wild-type animal carrying an extrachromosomal array that expresses mouse NALCN(R329Q) driven by a pan-neuronal promoter displayed exaggerated body bends, which is characteristic of (159 KB MOV)Click here for additional data file.Video S14unc-77(hp102) mutants Click here for additional data file.


Coronavirus sequence analyses demonstrate distinctive circulating strains in natural populations. , causing FIP. To resolve these alternative hypotheses, we isolated viral sequences from FCoV-infected clinically healthy and sick cats ; 735 sequences from 4 gene segments were generated and subjected to phylogenetic analyses. Viral sequences from healthy cats were distinct from sick cats on the basis of genetic distances observed in the membrane and nonstructural protein 7b genes. These data demonstrate distinctive circulating virulent and avirulent strains in natural populations. In addition, 5 membrane protein amino acid residues with functional potential differentiated healthy cats from cats with FIP. These findings may have potential as diagnostic markers for virulent FIP-associated FCoV.Feline coronavirus (FCoV) is endemic in feral cat populations and cat colonies, frequently preceding outbreaks of fatal feline infectious peritonitis (FIP). FCoV exhibits 2 biotypes: the pathogenic disease and a benign infection with feline enteric coronavirus (FECV). Uncertainty remains regarding whether genetically distinctive avirulent and virulent forms coexist or whether an avirulent form mutates in vivo Feline infectious peritonitis (FIP) is an uncommon, fatal, progressive, and immune-augmented disease of cats caused by feline coronavirus (FCoV) infection. Although FCoV is common in most domestic, feral, and nondomestic cat populations worldwide , FIP will develop in <10% of FCoV seropositive cats  and FECV-positive but asymptomatic cats (controls). Cases were infected with feline coronavirus (FCoV) and had the clinical disease of feline infectious peritonitis (FIP). Controls were also infected with FCoV, but were clinically asymptomatic (FECV-asymptomatic). The prediction was that phylogenetic analysis of viral gene sequences would demonstrate paraphyly for FIP case-cats and FECV-asymptomatic cats if the in vivo mutation hypothesis was supported, and monophyly of the 2 if the circulating virulent/avirulent hypothesis was supported . AdditioA total of 56 live, euthanized, or recently deceased domestic cats were clinically examined and sampled in Maryland veterinary hospitals during 2004–2006 . Blood  were stained with hematoxylin and eosin (HE). Tissues were also flash frozen in liquid nitrogen (–220°C) for RNA extraction and stored at either –220°C or –70C°.For complete blood counts, fresh (<12 hours) whole-blood samples were assessed by Antech veterinary diagnostic laboratory by using an automated cell counter . Biochemical analysis  and ELISA for feline immunodeficiency virus , and coronavirus  antibodies were also performed.HE-stained slides of spleen, liver, lymph node, intestine, and kidney sections were evaluated for evidence of granulomatous and pyogranulomatous lesions . Formalin-fixed sections (3 μm thick) were cut from paraffin blocks and placed on glass slides for immunohistochemical (IHC) testing, as previously described, with CoV p56, a cross-reacting antibody for the demonstration of feline coronavirus (FECV and FIPV biotypes) .RNA from 160 μL ascites fluid or frozen feces suspended 10% in phosphate-buffered saline was extracted by using the QIAamp virus RNA mini kit  following the manufacturer’s instructions. RNA from tissue was extracted from ≈60 mg of frozen liver, lung, spleen, colon, jejunum, or lymph node by using RNAeasy (QIAGEN) following the manufacturer’s instructions. Extracted RNA was eluted in 35 μL of RNase-free water and stored at –70ºC. cDNA was reverse transcribed using 9 μL of eluted RNA (10 pg–5 μg) in an initial 12-μL reaction mixture containing 50 ng of random hexamers and 0.5 mmol/L of dNTPs. After incubation at 65ºC for 5 min to denature the RNA, 10 mmol/L of dithiothreitol, 5× Synthesis Buffer, 40 U of RNaseOUT, and 15 units of Thermoscript RT were added on ice . Reaction mixtures were incubated in thermocycler at 25ºC for 10 min, followed by 50ºC for 30 min. cDNA was stored at –20ºC.2, 0.25 mmol/L concentrations of dNTPs , 2 mmol/L concentrations of each primer, and 2.5 units of Platinum Taq DNA polymerase (Invitrogen). PCR was conducted on a geneAmp PCR system 9700 thermocycler  with the following touchdown conditions: 2 min at 94ºC then a touchdown, always starting with 20 sec at 94ºC, then 30 sec at 60ºC (3 cycles), 58ºC (5 cycles), 56ºC (5 cycles), 54ºC (5 cycles), 52ºC (22 cycles), and then 1 min at 72ºC for extension, and with a final extension at 72ºC for 7 min and a hold at 4ºC. PCR products were visualized by electrophoresis on a 1% agarose gel and primers and unincorporated dNTPs were removed by using Microcon YM .Primers amplifying 7b (736 bp), membrane protein (575 bp), polymerase (386 bp), and spike-NSP3   were desEcoRI), and sequences were obtained from clones with the correct insert by using standard ABI BigDye terminator reactions (Applied Biosystems). Anticontamination measures were taken at all steps of reverse transcription–PCR (RT-PCR) amplification and post-PCR processing.Representative PCR products were cloned and sequenced , panel B INSERT SHAPE  INSERT SHAPE  INSERT SHAPE  INSERT SHAPE  INSERT SHAPE  INSERT SHAPE  INSERT SHAPE pol 1a, spike-NSP3, membrane, and NSP7b were analyzed separately. Nucleotide sequences were compiled and aligned for subsequent phylogenetic analysis by using ClustalX .Sequences from pol 1a, membrane, NSP 7b, and spike-NSP3 were deposited in GenBank under accession nos. EU663755–EU664317.Variable sites and parsimoniously informative sites were computed in MEGA version 3.0 , and was indistinguishable from FCoV isolated from other cats with FIP. This finding suggested that the pathogenic FIP-case type of FCoV infected this cat subsequent to its infection with an avirulent FECV and apparently replaced it.Samples from 1 cat, Fca-4590, were particularly informative. The virus was isolated from the cat predisease, and then again 7 months later postdisease. Fca-4590 was asymptomatic but infected with FECV in May 2004. FCoV sampling from that month showed strong (high bootstrap) affiliation with the FECV-asymptomatic clades for the membrane and the Tissue-specific differentiation within each cat was minimal . By contmembrane and NSP 7b generated in this study were translated to amino acid sequences  or YIIAL (II); infected cats without clinical FIP had the HIIVI (III), HIIVL (IV), HVIAL (V), YVVAL (VI), or YIVAL (I) haplotype. No FIP cases had haplotype III, IV, V, or VI, whereas 3 FECV-asymptomatic cats carried the YIVAL signature found predominately in FIP cases  genes from feline infectious peritonitis virus (FIPV) cases (gray shaded), from feline enteric coronavirus (FECV) asymptomatic cats, from feline coronavirus (FCoV)-Aju, and reference sequences for severe acute respiratory syndrome coronavirus (CoV), MHV-1, IBV-Beu, BVC-K, HcoV-229E, TGEV-Purdue, and FCoV 79-1146 . FCoV reference sequences for FECVUCD, FIPVUCD1, FIPV791146, and FIPVUCD3 are also included. Diagnostic sites are highlighted in the membrane. For membrane, cat ID and 2-digit year of sampling is listed and the number of original clones is in parenthesis; the frequency of unique amino acid sequences is reported in column 2. No diagnostic sites were found correlating with FIPV and FECV biotype in NSP 7b.Midpoint rooted maximum likelihood (ML) tree of unique pol1a 386-bp sequences . Cloned sequences from feline infectious peritonitis (FIP) cases are shown in red, feline enteric coronavirus (FECV) asymptomatic cats are shown in blue, and feline coronavirus (FCoV) virulent strain from Aju-92 (cheetah) is in green. Each sequence is labeled as follows: source farm , 4-digit cat identification number, tissue source , 2-digit year , and number of clones for each sequence. Scale bar indicates number of substitutions per site.Feline coronavirus (FCoV) sequence from cats from the Weller farm. Maximum likelihood phylogenetic tree of unique membrane and nonstructural protein 7b (NSP 7b) FCoV gene sequences showing monophyly correlating to disease status. Cloned sequences from feline infectious peritonitis virus (FIPV) biotypes are shown in red; feline enteric coronavirus (FECV) biotypes are shown in green. A) membrane (maximum likelihood [ML] -ln L = 2646.84352 best tree found by maximum parsimony (MP): length = 270, CI = 0.789, retention index [RI] = 0.971; mid-point rooted); B) NSP 7b . The number of cats is indicated in parenthesis in the key. Each sequence is labeled as follows: 4-digit cat identification number, tissue source , 2-digit year , and the unique 3-4-digit sequence number. The number of clones for each sequence is indicated after the sequence label in parenthesis. Where ML tree was congruent with MP tree, branch lengths are indicated below branches; the number of homoplasies is in parenthesis after the branch length. Bootstrap values are shown (MP/minimum evolution/ML) above branches. Virus sequence obtained from cat 4590 in May 2004 and at the time of death due to FIPV in December 2004 is indicated by box. The 2 distinct virus genotypes isolated from this case pre- and postdisease in both the membrane and NSP 7b genes are consistent with the dual circulating virulent and avirulent strains in FCoV pathogenesis. Scale bars indicate substitutions/site.


Here, we show that coral larvae respond to acoustic cues that may facilitate detection of habitat from large distances and from upcurrent of preferred settlement locations. Using in situ choice chambers, we found that settling coral larvae were attracted to reef sounds, produced mainly by fish and crustaceans, which we broadcast underwater using loudspeakers. Our discovery that coral larvae can detect and respond to sound is the first description of an auditory response in the invertebrate phylum Cnidaria, which includes jellyfish, anemones, and hydroids as well as corals. If, like settlement-stage reef fish and crustaceans, coral larvae use reef noise as a cue for orientation, the alleviation of noise pollution in the marine environment may gain further urgency.Free-swimming larvae of tropical corals go through a critical life-phase when they return from the open ocean to select a suitable settlement substrate. During the planktonic phase of their life cycle, the behaviours of small coral larvae (<1 mm) that influence settlement success are difficult to observe  Most nearshore site-attached marine organisms complete an early larval stage in the open ocean before settling to benthic habitats. Chemical compounds produced by reef organisms provide important settlement cues for coral larvae 2 = 30.50, df = 4, p<0.0001). When the chambers were placed 0.5 m below the speakers, larvae moved towards the upper surface of the chambers  . When the speakers were silent, larvae distributed themselves randomly throughout the chambers independent of the loudspeakers' position . In sum, coral larvae displayed directional movement both horizontally and vertically towards underwater speakers broadcasting reef noise.In each trial, using six chambers directed towards underwater speakers playing a compilation of day and night reef sounds , free-swThe possibility that the directional movement of larvae was caused by moonlight, tides, or chemical cues with onshore-offshore gradients was eliminated by the radial arrangement of the speakers and chambers . RegardlThis study reports the first known behavioural response to a water-bourne acoustic cue in a marine larva of the invertebrate phylum Cnidaria, which in addition to jellyfish, anemones and hydroids, includes the corals responsible for the formation of the largest biological structures on earth: coral reefs. Other major sensory modalities which enable detection of light (photoreception), substrates (mechanoreception) and chemicals (chemoreception) have all previously been demonstrated in coral larvae e.g. . The extAll animal manipulations were approved by the Department of Environment & Nature (MINA) of the government of the Netherlands Antilles.Montastraea faveolata during the 2008 mass spawning in Curaçao, Netherlands Antilles. Larvae were raised from gamete bundles collected at Playa Kalki  on 20 September 2008 and maintained in 0.45 µm-filtered seawater in 2 L polystyrene containers. To accurately time the field experiments to the onset of larval settlement , a subset of larvae were reared in polystyrene Petri dishes  were arranged in a triangular pattern, and two transparent PLEXIGLAS® chambers  were placed in front of each speaker with the near end of the chamber at a distance of 1 m . We intrref). Using p = ρcv , the gradient in particle velocity in the choice chamber was from 9.64×10−8 m s−1 at the near end to 9.35×10−8 m s−1 at the far end.To determine whether coral larvae exhibited a response to general reef noise (rather than to a specific source of reef noise), we broadcast a compilation of recordings of coral reef sounds, which incorporated variation in reef noise due to time-of-day, season, habitat, and depth. Reef sounds were recorded using an omnidirectional hydrophone  and an Edirol R-1 24-Bit recorder . Recorded sounds were played back using Electrovoice UW-30 underwater speakers , and were broadcast from the 3 speakers in synchrony. The broadcast sound consisted of 15 different 3-minute recordings to avoid potential pseudoreplication introduced by using a single recording in playback experiments