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.


Type-1 T cells are critical for effective anti-tumor immune responses. The recently discovered microRNAs (miRs) are a large family of small regulatory RNAs that control diverse aspects of cell function, including immune regulation. We identified miRs differentially regulated between type-1 and type-2 T cells, and determined how the expression of such miRs is regulated.in vitro differentiated murine T helper type-1 (Th1) and T helper type-2 (Th2) cells to identify differentially expressed miRs. We used quantitative RT-PCR to confirm the differential expression levels. We also used WST-1, ELISA, and flow cytometry to evaluate the survival, function and phenotype of cells, respectively. We employed mice transgenic for the identified miRs to determine the biological impact of miR-17-92 expression in T cells.We performed miR microarray analyses on + T cells derived from miR-17-92 transgenic mice demonstrated superior type-1 phenotype with increased IFN-γ production and very late antigen (VLA)-4 expression when compared with counterparts derived from wild type mice. Human Jurkat T cells ectopically expressing increased levels of miR-17-92 cluster members demonstrated increased IL-2 production and resistance to activation-induced cell death (AICD).Our initial miR microarray analyses revealed that the miR-17-92 cluster is one of the most significantly over-expressed miR in murine Th1 cells when compared with Th2 cells. RT-PCR confirmed that the miR-17-92 cluster expression was consistently higher in Th1 cells than Th2 cells. Disruption of the IL-4 signaling through either IL-4 neutralizing antibody or knockout of signal transducer and activator of transcription (STAT)6 reversed the miR-17-92 cluster suppression in Th2 cells. Furthermore, T cells from tumor bearing mice and glioma patients had decreased levels of miR-17-92 when compared with cells from non-tumor bearing counterparts. CD4The type-2-skewing tumor microenvironment induces the down-regulation of miR-17-92 expression in T cells, thereby diminishing the persistence of tumor-specific T cells and tumor control. Genetic engineering of T cells to express miR-17-92 may represent a promising approach for cancer immunotherapy. We have focused on the development of effective immunotherapeutic strategies for central nervous system (CNS) tumors, such as glioblastoma multiforme (GBM). Preclinical studies have demonstrated that tumor-specific T helper type-1 (Th1) and T cytotoxic type-1 (Tc1) cells, but not type-2 counterparts, can efficiently traffic into CNS tumor sites and mediate effective therapeutic efficacy, recruited via the type-1 chemokine CXCL10  and the MicroRNAs (miRs) are a novel class of endogenous small single-stranded RNA molecules which are 18-24 nucleotides in length . Mature In the current study, in an effort to understand the potential roles of miRs in anti-tumor immunity, we examined miRs differentially expressed in Th1 and Th2 cells. Our miR microarray and RT-PCR analyses revealed that of all analyzed miRs, members of the miR-17-92 cluster (miR-17-92) are of the most significantly over-expressed miRs in murine Th1 cells when compared with Th2 cells. The miR-17-92 transcript encoded by mouse chromosome14 (and human chromosome 13) is the precursor for 7 mature miRs  ,25. ThismiRs in the miR-17-92 cluster are amplified in various tumor types, including B cell lymphoma and lung cancer, and promote proliferation and confer anti-apoptotic function in tumors, thereby promoting tumor-progression -31. KnocWe show here that miR-17-92 is up-regulated in Th1 cells when compared with Th2 cells. IL-4 and STAT6 signaling mediate the down-regulation of miR-17-92. Tumor-bearing host conditions also suppress the miR-17-92 cluster expression in T cells, which is associated with a loss in ability to produce IFN-γ. This led us to hypothesize that miR-17-92 cluster overexpression might enhance type-1 responses. Indeed, type-1 T cells derived from miR-17-92 transgenic mice demonstrated a more pronounced type 1 phenotype including enhanced IFN-γ production and increased VLA-4 expression when compared with control type-1 T cells. These findings suggest that miR-17-92 plays a critical role in type-1 adaptive immunity.γ (R4-6A2), IL-4 (11B11), CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7) and CD49d (R1-2) were all purchased from BD Pharmingen. Purified mAbs against CD3 (UCHT1) and CD28 (CD28.2) and IL-4 (MP4-25D2) were purchased from Biolegend. RT-PCR reagents and primers were purchased from Applied Biosystems and analyzed on a BioRad IQ5. WST-1 reagent was purchased from Roche. For isolation of T cells, immunomagenic isolation kits from Miltenyi Biotec were used. All reagents and vectors for lentiviral production were purchased from System Biosciences with the exception of Lipofectamine 2000, which was from Invitrogen.RPMI 1640, FBS, L-glutamine, sodium pyruvate, 2-mercaptoethanol, nonessential amino acids, and penicillin/streptomycin were obtained from Invitrogen Life Technologies. Recombinant murine (rm) IL-12 was purchased from Cell Sciences Technologies. RmIL-4, recombinant human (rh) IL-4 and rhIL-2 were purchased from PeproTech. Purified monoclonal antibodies (mAbs) against IL-12 (C15.6), IFN-Stat6tm1Gru/J; The Jackson lab) (both 5-9 wk of age) were purchased from The Jackson Laboratory. C57BL/6-background miR-17-92 transgenic (TG) mice Rsky/J; The Jackson Lab) were maintained in the Hillman Cancer Center Animal Facility at University of Pittsburgh as breeding colonies and bred to C57BL/6-background mice transgenic for Cre recombinase gene under the control of the Lck promoter  to obtain mice, in which T cells expressed miR-17-92 at high levels (miR-17-92 TG/TG). For mouse tumor experiments, C57BL/6 mice and C57BL/6 background STAT6-/- mice received subcutaneous injection of 1 × 106 B16 tumor cells resuspended in PBS into the right flank. On day 15 following tumor inoculation, mice were sacrificed and splenic T cells were isolated. Animals were handled in the Hillman Cancer Center Animal Facility at University of Pittsburgh per an Institutional Animal Care and Use Committee-approved protocol.C57BL/6 mice and C57BL/6 background STAT6 deficient mice  in the presence or absence of rhIL-4(10 ng/ml). RT-PCR analyses were performed with both healthy donor- and patient-derived T cells to determine the expression of miR-17-92 as described in the relevant section.This study was approved by the local ethical review board of University of Pittsburgh. All healthy donors and patients with GBM signed informed consent before blood samples were obtained. To determine the impact of IL-4, healthy donor-derived CD4+ splenic T cells. Magnetic activated cell separation (MACS) was carried out using positive selection. Briefly, spleens were minced in complete media, resuspended in red blood cell lysis buffer and stained with immunomagnetically labeled anti-CD4 antibody. Cells were then washed and placed through the magnetic column in 500 μl of MACS buffer. The column was then washed 3 times with buffer and then removed from the magnet and labeled cells were extracted in 3 ml of MACS buffer.Th1 and Th2 cells were differentiated from immunomagnetically-separated CD4+ cells were stimulated in 48 well plates with anti-CD3 mAb (5 μg/ml) in the presence of irradiated C57BL/6 spleen cells (3000 Rad) as feeder cells. RmIL-12 (4 ng/ml), rmIFN-γ (4 ng/ml), anti-IL-4 (10 μg/ml) mAb and rhIL-2 (100 IU/ml) were added for Th1 development. Th2 cells were generated from the same CD4+ cell precursors stimulated with anti-CD3 mAb and feeder cells in the presence of rmIL-4 (50 ng/ml), two anti-IFN-γ mAbs (10 μg/ml), anti-IL-12 mAb (10 μg/ml) and rhIL-2 (100 IU/mL). After 10 days cells were stained for IL-4 and IFN-γ to confirm differentiation. Neutral cell culture included anti-CD3, feeder cells and rhIL-2. For studies involving IL-4 blockade, 12.5 ng/ml anti-human IL-4 mAb (Biolegend) was used in human experiments and 2.5 μg/ml anti-mouse IL-4 mAb (11B11) in murine studies. IFN-γ and IL-4 in the culture supernatants were measured using specific ELISA kits (R&D Systems). For FACs analysis, cells were incubated with mAb at 4°C for 30 min, washed twice in staining buffer, and fixed in 500 μl of 2% paraformaldehyde in PBS. Cells were stored in the dark at 4°C until analysis. Flow cytometry was carried out on the Coulter XL four-color flow cytometer at the flow cytometry core facility of the University of Pittsburgh Cancer Institute.For differentiation of T cells, purified CD4http://microrna.sanger.ac.uk/, version 9.1) plus 2 internal controls with 7 serial dilutions printed in house  [Total RNA was isolated from Th1 and Th2 cells using the Trizol reagent and quality was confirmed with an A260/A280 ratio greater than 1.85. Two μg of total RNA was labeled with either Hy5  or Hy3  fluorescent dyes using miRCURY LNA microRNA labeling kit  according to manufacturer's protocol. Labeled miR samples in duplicate were cohybridized on to miR array slides, a custom spotted miR array V4P4 containing duplicated 713 human, mammalian and viral mature antisense microRNA species  . After wCT method [Total RNA was extracted using the Qiagen RNeasy kit and quality was confirmed with a A260/A280 ration greater than 1.85. RNA was subjected to RT-PCR analysis using the TaqMan microRNA Reverse Transcription Kit, microRNA Assays (Applied Biosystems), and the Real-Time thermocycler iQ5 (Bio-Rad). The small nucleolar SNO202 was used as the housekeeping small RNA reference gene for all murine samples and RNU43 for human samples. All reactions were done in triplicate and relative expression of RNAs was calculated using the ΔΔT method .4 cells were cultured in a 96 well plate for 24-48 hours in 100 μl of complete media. Then, 10 μl of WST-1 reagent was added to each well. Cells were incubated at 37°C, 5% CO2 for 4 hours, and placed on a shaker for 1 min. The plates were then read on a micro plate reader with a wavelength of 420 nm and a reference at 620 nm.For WST-1 proliferation assays, 1 × 102 and placed at 4°C with PEG-it Virus Concentration Solution (SBI) for 24 hrs. Supernatants/PEG solutions were then centrifuged and the pellet was resuspended in a reduced volume of media as viral stock. Jurkat cells were further resuspended in the viral stock together with polybrene (8 μg/ml) for 24 hrs. Fresh media was then added to the cells and transduction efficiency was evaluated by GFP expressing cells. For IL-2 production, transduced Jurkat cells were stimulated with Phorbol 12-myristate 13-acetate (PMA) (10 ng/ml) and ionomycin (500 nM) for overnight and supernatant was assayed for IL-2 by a human IL-2 ELIZA kit. For activation induced cell death (AICD), cells were treated with 10 μg/ml purified anti-CD3 mAb (UCHT1) from Biolegend for 24 hours and then cell viability was measured using WST-1 reagent.Jurkat human T cell leukemia cells (American Type Culture Collection) were transduced by either one of the following pseudotype lentiviral vectors: 1) control vector encoding GFP; 2) the 17-92-1 expression vector encoding miR-17 18 and 19a, or 3) the 17-92-2 expression vector encoding miR 20, 19b-1, and 92a-1. All vectors were purchased from SBI. Lentiviral particles were produced by co-transfecting confluent 293TN cells (SBI) with pPACK-H1 Lentivirus Packaging Kit (SBI) and the miR containing expression vectors (SBI) noted above using Lipofectamine 2000 reagent (Invitrogen). Supernatant was collected after 48 hour incubation at 37°C with 5% COp < 0.05. A post test for linear trend test was used to determine linear trend and we considered p < 0.05 to be significant.All statistical analyses were carried out on Graphpad Prism software. The statistical significance of differences between groups was determined using student t- test. We considered differences significant when + T cells, Th1 and Th2 cells were generated as described in Materials and Methods. These T cells exhibited expected cytokine profiles with Th1 cells dominantly producing IFN-γ but not IL-4, while Th2 cells produce mostly IL-4  derived from wild type C57BL/6 mice bearing B16 subcutaneous tumors expressed lower levels of miR-17-5p when compared with those derived from non-tumor bearing mice . These findings suggest that miR-17-92 promotes the type-1 phenotype in differentiating T cells.The data discussed above strongly suggest GBM-associated factors and a type-2 promoting cytokine IL-4) down-regulate miR-17-92 in T cells. miR-17-92 is expected to play pivotal roles in T cell functions. We therefore sought to determine whether ectopic expression of miR-17-92 would promote the type-1 phenotype of T cells. As detailed in Materials and Methods, we produced mice that overexpress miR-17-92 specifically in T cells (miR-17-92 TG/TG). We isolated CD4-5p Fig. . CD4+ ceA-4 Fig. . Althoug down-region Fig. . SimilarmiR-17-92 is expected to play pivotal roles in T cell survival as well as functions. To evaluate these aspects, we transduced Jurkat cells with lentiviral vectors encoding green fluorescence protein (GFP) and either the miR-17-92-1 expression vector encoding miR-17 18 and 19a, or the 17-92-2 expression vector encoding miR 20, 19b, and 92. The control vector encodes GFP, but not miRs. Transduced Jurkat cells were stimulated with PMA and ionomycin for overnight before the supernatants were assayed for IL-2 production by ELISA Fig. . TransduAICD and chemotherapy-induced suppression of T cells represent major obstacles for efficient T cell-based cancer immunotherapy ,37. We nAttaining effective tumor immunity is a major goal of modern biologic therapy, limited by the tumor microenvironment and profound regulatory mechanisms limiting T cell and NK cell effectors. Here we show that the type-2-skewing tumor microenvironment induces down-regulation of miR-17-92 expression in T cells, thereby hampering anti-tumor T cell responses. It also suggests that development of immunotherapy using miR-17-92-transduced T cells is warranted based on our findings demonstrating that ectopic expression of miR-17-92 in T cells leads to improved type-1 functions, including increased VLA-4 expression and IFN-γ production.Blockade of endogenous IL-4 by inhibitory mAb or disruption of STAT6 signaling was sufficient to up-regulate miR-17-92 in T cells Fig . These fSTAT6-/- T cells resistant to tumor-induced inhibition of miR-17-5p, but CD8+ T cells in tumor bearing STAT6-/- mice exhibited higher levels of miR-17-5p when compared with CD8+ T cells obtained from non-tumor bearing STAT6-/- mice. In addition to IL-4, other tumor-derived factors are likely to be involved in these events. Further studies are warranted to elucidate the molecular mechanisms underlying the regulation of miR-17-92 in T cells, especially in the tumor microenvironment.Our findings indicate that the tumor-bearing host down-regulates miR-17-92 in T cells Fig.  and 4. I+ and CD8+ cells, human GBM patients exhibited a statistically significant decrease of miR-17-92 in CD4+ cells but not in CD8+ cells  and Th17 cells, and Th2 cells consistently exhibit the lowest levels of miR-17-92 among the populations tested (data not shown). More studies are warranted on the specific role of miR-17-92 during differentiation.During lymphocyte development, miR-17-92 miRs are highly expressed in progenitor cells, with the expression level decreasing 2- to 3-fold following maturation . In addiex vivo that are resistant to tumor-mediated immune suppression and mediate robust and long-lived anti-tumor responses. miR-17-92 cluster has the potential to confer resistance to tumor-derived immunosuppressive factors and to improve type-1 reactivity. Further characterization of the role of miR-17-92 cluster in tumor antigen (TA)-specific CTLs is clearly warranted and may provide us with ability to develop novel immunotherapy strategies with genetically engineered T cells. Additionally, identification of diminished miR-17-92 expression in the peripheral blood may emerge as an important biomarker in patients with malignancy.These studies reviewed above provide us with critical insights as to what has to be expected if we develop therapeutic strategies by modulating miR-17-92 expression. One major barrier for successful T cell-based cancer immunotherapy is the low persistence of tumor antigen (TA)-specific T cells in tumor-bearing hosts ,66. It smiR: microRNA; VLA-4: very late antigen-4; AICD: apoptosis induced cell death; CNS: central nervous system; GBM: glioblastoma multiforme; ICAM-1: intracellular cell adhesion molecule-1; STAT6: signal transducers and activators of transcription-6; PMA: phorbol myristate acetate; SPC: splenocyte.The authors declare that they have no competing interests.in vivo and in vitro assays, and was one of the two primary writers of the paper. KS was involved in the conception of the study, further designing of the experiments, and took a primary role in culturing the differentiated T cells. AH performed studies using Jurkat cells. MF participated in experimental design, troubleshooting, editing the manuscript and statistical analysis. RU assisted in microRNA array and expression studies and analysis. HM helped with the ELISA and technical editing of the manuscript. TAR, JM and MTL participated in the design of experiments and interpretation of data. EW and FMM performed the miR microarray and assisted with analysis. HO conceived the study, mentored primary authors, was one of the two primary writers of the paper, and heavily participated in experimental design and data analysis. All authors read and approved the final manuscript.GK participated in the conception of the study, experimental design, performed


Double-transgenic mice show negative selection of thymocytes at theCD4+8+TCR10 to CD4+8+TCRhi transition stage. A few CD8 T cells, however, escape clonaldeletion, and in the peripheral lymphoid organs of these mice, they exhibit low levels ofthe transgenic receptor and upregulated levels of the CD44 memory marker. Such cells donot proliferate upon exposure to antigen stimulation in vivo or ex vivo, however, they candevelop low but detectable levels of antigen-specific cytotoxic function after stimulationin vitro in the presence of IL-2.Influenza nucleoprotein (NP)-specific T-cell receptor transgenic mice (F5) were crossedwith transgenic mice expressing the cognate antigenic protein under the control of the H-2K


Caenorhabditis elegans differ in their functions and in their expression of chemosensory receptor genes; in each animal, one AWC randomly takes on one identity, designated AWCOFF, and the contralateral AWC becomes AWCON. Signaling between AWC neurons induces left-right asymmetry through a gap junction network and a claudin-related protein, which inhibit a calcium-regulated MAP kinase pathway in the neuron that becomes AWCON.The left and right AWC olfactory neurons in olrn-1 acts downstream of the gap junction and claudin genes to inhibit the calcium-MAP kinase pathway in AWCON. OLRN-1, a protein with potential membrane-association domains, is related to the Drosophila Raw protein, a negative regulator of JNK mitogen-activated protein (MAP) kinase signaling. olrn-1 opposes the action of two voltage-activated calcium channel homologs, unc-2 (CaV2) and egl-19 (CaV1), which act together to stimulate the calcium/calmodulin-dependent kinase CaMKII and the MAP kinase pathway. Calcium channel activity is essential in AWCOFF, and the two AWC neurons coordinate left-right asymmetry using signals from the calcium channels and signals from olrn-1.We show here that the asymmetry gene olrn-1 and voltage-activated calcium channels are mediators and targets of AWC signaling that act at the transition between a multicellular signaling network and cell-autonomous execution of the decision. We suggest that the asymmetry decision in AWC results from the intercellular coupling of voltage-regulated channels, whose cross-regulation generates distinct calcium signals in the left and right AWC neurons. The interpretation of these signals by the kinase cascade initiates the sustained difference between the two cells. Caenorhabditis elegans are the bilateral (left and right) amphids, which house 12 pairs of ciliated sensory neurons. Each sensory neuron expresses many receptor genes, in contrast with vertebrate olfactory neurons that generally express one receptor gene per cell . A . A ky626s Figure . olrn-1(olrn-1 cDNAs under the AWC-selective odr-3 promoter rescued the 2AWCOFF phenotypes of olrn-1(ky626) mutants , kyIs323 II , zdIs5 [mec-4::GFP]. Mutations used in this study included LG (linkage group) I: nsy-5(ky634), LGII: nsy-1(ky542), nsy-1(ag3),nsy-1(ok593), LGIII: tir-1(tm1111), unc-36(e251), LGIV: egl-19 (n582), egl-19(ad695gf), nsy-4(ky616), unc-43(n1186), unc-43(n408), unc-43(n498gf), LGX: olrn-1(ky626),olrn-1(ut305), unc-2 (lj1),unc-2(e55), sek-1(km4).Germline transformation was carried out as previously described . Co-injeTransgenes maintained as extrachromosomal arrays included the following lines used for mosaic analysis: kyEx914 (line 1) & kyEx918 (line 2) , kyEx1072 (line 1) & kyEx1074 (line 2) , kyEx1097 (line 1) & kyEx1098 (line 2) , kyEx1102 (line 1) & kyEx1103 (line 2) . For unc-2 and unc-36 mosaics, the same extrachromosomal arrays were examined in wild-type and mutant backgrounds: kyEx1628 (line 1) kyEx1629 (line 2) and kyEx1630 (line 3) ; kyEx1229 (line 1) kyEx1387 (line 2) and kyEx1388 (line 3) .Additional transgenic arrays were kyEx822 , kyEx996 , kyEx1075 , kyEx1096 , kyEx1182 , kyEx1320 , kyEx1315 , kyEx1310 .odr-3::olrn-1b::Ch = kyEx1318 (line 1), kyEx1319 (line 2), kyEx1317 (line 3); odr-3::olrn-1bΔ rawR1::Ch = kyEx1337 (line 1), kyEx1345 (line 2), kyEx1344 (line 3); odr-3::olrn-1bΔ TM1,2::Ch = kyEx1297 (line 1), kyEx1298 (line 2); odr-3::olrn-1bΔ rawR2::Ch = kyEx1338 (line 1), kyEx1339 (line 2), kyEx1340 (line 3); odr-3::olrn-1b(G466E)::Ch = kyEx1358 (line 1), kyEx1357 (line 2), kyEx1359 (line 3); odr-3::olrn-1b(ΔRRRR)::Ch = kyEx1332 (line 1), kyEx1296 (line 2), kyEx1300 (line 3), kyEx1299 (line 4); odr-3::olrn-1bΔ Cterm::Ch = kyEx1382 (line 1), kyEx1352 (line 2), kyEx1351 (line 3). These transgenes were injected at 15 ng/μl with 10–15 ng/μl of ofm-1::GFP co-injection marker.The following arrays were used in the deletion analysis: kyIs140 I animals were subjected to ethyl methane sulfonate (EMS) mutagenesis according to standard procedures  in a kyIs140 I, olrn-1(ky626) X mutant. Gain-of-function mosaic analysis was performed on four independent lines in a kyIs140 I wild-type strain. Each data point in Figure odr-1::dsRed, which is expressed in AWC and AWB neurons. The str-2::GFP expression phenotype in mosaic cells was examined under a compound scope at 100–400×. Previous experiments have used a similar strategy  in a kyIs140 [str-2::GFP] I; unc-2(lj1) X mutant. Gain-of-function mosaic analysis for unc-2 was performed on three independent lines in the kyIs140 I strain. Loss-of-function mosaic analysis for unc-36 was performed on three independent lines with unstable extrachromosomal transgenic arrays  in a kyIs140 I; unc-36(e251) III mutant. Gain-of-function mosaic analysis for unc-36 was performed on three independent lines in the kyIs140 strain. Loss of the transgene was inferred by loss of the co-injection marker odr-1::dsRed in AWC neurons. Results from all lines were combined for statistical analysis, which was performed as described above for olrn-1. In all cases the null hypothesis that the two AWCs behaved as independent units could be excluded at P < 0.001. Both the rescued AWC and the non-rescued AWC in unc-2; odr-3::unc-2 mosaics were strongly affected by the contralateral cell – the rescued AWC became AWCOFF more often than predicted by the null hypothesis, and the non-rescued AWC became AWCON more often than predicted (P < 0.001 in both cases). Thus, a single unc-2(+) AWC can bias both AWC neurons. In wild-type mosaics overexpressing unc-2, the wild-type AWC became AWCOFF more often than predicted by the null hypothesis (P = 0.003), but the overexpressing AWC was not affected by the wild-type AWC (P = 0.1943). In unc-36; odr-3::unc-36 mosaics, the wild-type AWC became AWCOFF more often than predicted by the null hypothesis (P < 0.001), but the mutant AWC was not significantly affected (P = 0.075). There was no significant effect of unc-36 overexpression in a wild-type background.Loss-of-function mosaic analysis for To visualize early L1s, 50–100 gravid adults were picked and allowed to lay eggs overnight. Adults and hatched larvae were washed off the plate, and eggs were allowed to hatch for three hours. L1 larvae were examined on a compound microscope at 400–630× magnification or used for confocal microscopy. To obtain late L1/early L2 animals, approximately 50–60 gravid adults were picked to a plate and allowed to lay eggs for 3 hours, after which the adults were removed from the plate. Progeny were examined 30 hours later under the confocal microscope.The author(s) declare that they have no competing interests.olrn-1(ky626) mutant, and IT, TI, and IK cloned olrn-1. AvdL and PS characterized the srsx-3 reporter. SLBH and CIB wrote the paper.SLBH, YS, and CIB designed the experiments, and SLBH and YS performed the experiments. MKV isolated the olrn-1b::Cherry proteins. Confocal images of odr-3::olrn-1b::Cherry proteins in olrn-1(ky626) animals at (a-g) 0–3 hours after hatching and (h-n) L1/L2 stage, 20 hours after hatching. Notched arrowheads indicate AWC axons, flat arrowheads indicate AWC cell bodies. The diagrams show the approximate size and disposition of AWC neurons in the images (anterior is at left). (a-g) Two AWCs are visible in most images; (h-n) only one AWC is visible in most images, but both AWCs have similar OLRN-1b:Cherry levels. Scale bars are 10 μm.Subcellular localization of wild-type and mutant Click here for file


Identification of broad neutralization epitopes in HIV-1 envelope spikes is paramount for HIV-1 vaccine development. A few broad neutralization epitopes identified so far are present on the surface of native HIV-1 envelope spikes whose recognition by antibodies does not depend on conformational changes of the envelope spikes. However, HIV-1 envelope spikes also contain transiently-exposed neutralization epitopes, which are more difficult to identify.+ T cells confers long-term resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion, and the infection of HIV-1 captured and transferred by human DCs.In this study, we constructed single chain Fvs (scFvs) derived from seven human monoclonal antibodies and genetically linked them with or without a glycosyl-phosphatidylinositol (GPI) attachment signal. We show that with a GPI attachment signal the scFvs are targeted to lipid rafts of plasma membranes. In addition, we demonstrate that four of the GPI-anchored scFvs, but not their secreted counterparts, neutralize HIV-1 with various degrees of breadth and potency. Among them, GPI-anchored scFv (X5) exhibits extremely potent and broad neutralization activity against multiple clades of HIV-1 strains tested. Moreover, we show that GPI-anchored scFv (4E10) also exhibited more potent neutralization activity than its secretory counterpart. Finally, we demonstrate that expression of GPI-anchored scFv (X5) in the lipid raft of plasma membrane of human CD4Thus GPI-anchored scFv could be used as a general and effective way to identify antibodies that react with transiently-exposed neutralization epitopes in envelope proteins of HIV-1 and other enveloped viruses. The GPI-anchored scFv (X5), because of its breadth and potency, should have a great potential to be developed into anti-viral agent for HIV-1 prevention and therapy. Human Immunodeficiency Virus type 1 (HIV-1) envelope spike is a trimeric complex consisting of three non-covalently linked heterodimers of gp120 and gp41. Gp120, an exterior glycoprotein, mediates cell attachment, receptor and co-receptor binding. Gp41, a transmembrane glycoprotein, mediates viral and cell membrane fusion, which is critical for viral core to enter target cells. Both gp120 and gp41 are derived by cleavage of a common precursor gp160.HIV-1 envelope spike also elicits antibody responses. Neutralizing antibodies block viral entry by recognizing epitopes on the envelope spike critical for its attachment, receptor and co-receptor interaction, or fusion and appear to be an important component of a protective immune response . DespiteUpon interaction with CD4 receptor, a lipid raft-associated protein -18, on tPreviously, we unexpectedly found that by genetically linking the scFv of an anti-HIV-1 human antibody (TG15) to the transmembrane domain of subunit one of the type 1 interferon receptor, the cell-surface expressed scFv, but not its secretory form, we markedly inhibited HIV-1 entry and HIV-1 envelope-mediated cell-cell fusion ,24. The In nature, over 200 cell surface proteins with various functions are anchored to the plasma membrane by a covalently attached glycosyl-phosphatidylinositol (GPI) anchor . Many GPWe therefore hypothesized that if one can express antibodies that react with transiently-exposed neutralization epitopes in a GPI anchored form and a GPI anchor can target these antibodies into the lipid rafts of plasma membranes of HIV-1-susceptible cells, these antibodies should neutralize infection. If correct, we predict that when the HIV-1 native spike interacts with the CD4 receptor, triggering a series of conformational changes, the transiently-exposed neutralization epitopes will be captured by GPI-anchored antibodies residing in the same lipid raft of the plasma membrane.et al. data not shown). AB31 and AB32 are high affinity antibodies. AB31 recognizes cluster III determinant of gp41 and AB32 interacts with gp120, but its epitope is not well characterized  with VSV-G pseudotyped HIV-1 vector expressing enhanced green fluorescent protein (eGFP) as described before . Because5 and X5 + T cells confers long-term resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion and the infection of HIV-1 captured and transferred by human DCs  does not depend on additional sCD4  to identify transiently-exposed neutralization epitopes in the HIV-1 envelope spike. Second, it is also plausible that GPI-scFvs be used to identify antibodies that recognize transiently-exposed epitopes and then to identify transiently-exposed neutralization epitopes of other viruses, particularly of those enveloped viruses whose entry into target cells depend on lipid raft and the interaction of those envelope glycoprotein with receptor results in conformational changes. It has been shown that glycoproteins of influenza virus, murine leukemia virus, measles virus and Ebola virus are associated with the lipid raft of plasma membrane -52. The GPI anchored scFvs that recognize different transiently-exposed epitopes on HIV-1 envelope spike exhibit various degrees of breadth and potency. Among them, GPI-scFv (X5) that recognizes the epitope near the CD4 binding site and coreceptor (CCR5) binding site, exhibited greatest potency and breadth against multiclades of HIV-1 strains. Our data therefore demonstrate that GPI-scFvs may be developed as a useful tool to identify antibodies that recognize transiently-exposed epitopes in glycoproteins spike of HIV and other enveloped viruses. GPI-scFvs derived from such antibodies could be an effective way to inhibit cell entry of HIV and other enveloped viruses.Proviral plasmids pBru-3, pBru-Yu2, pAD8, pNL4-3 and pMj4 were obtained from the NIH AIDS Research and Reference Reagent Program . Infectious virus stocks of HIV-1 Bru-3 and NL4-3 , Bru-Yu2, and AD8  and Mj4  were produced by transfecting into 293T cells using a calcium phosphate precipitation or a Fugene-6 reagent method . Virus stocks were then propagated by infecting phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMCs) with these infectious clones as described before . Primary+ T cell line CEMss was obtained from Dr. Jon Allan at the Department of Virology and Immunology, the Southwest Foundation for Biomedical Research, San Antonio, TX. TZM.bl and 69Tirev/env cells were obtained from NIH ARRRP, as contributed by J. Kappes and X. Wu  plus G418 (500 μg/ml) (Invitrogen Life Technologies). Human CD4nd X. Wu ,59 and Hnd X. Wu , respectDrosophila Schneider 2 (S2) cells were purchased from Invitrogen Life Technologies and maintained in 75 cm flask (Corning) at the cell density between 0.2 to 2 × 106 cells per ml in the Express FIVE® SFM medium (Invitrogen Life Technologies) supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin and 100 mg/L L-glutamine at 28°C without additional CO2.Sal I site and additional 6 bp were linked by a recursive PCR  were grown in the DMEM plus 1% FBS on the 12-well plate for 24 hours. Cells and supernatant were harvested respectively. Cells were lysed by lysis buffer  in the presence of protease inhibitor cocktail . Proteins in supernatant were precipitated by TCA and dissolved in the equal volume of lysis buffer that to lysed cell pellet. Samples were detected by mouse anti-His tag antibody (Sigma) and mouse anti human beta-tubulin antibody as a reference control.1×106 293T packaging cells were co-transfected with 10 μg of HIV-1-luciferase transfer vector [To generate pseudotypes with the HIV-1 vector, 4 × 10r vector  and 10 μr vector . HIV-1 er vector -67. HIV-r vector . HIV-1 er vector . HIV-1 er vector . HIV-1 eIn a single-cycle assay to measure the infectivity of pseudotypes, 10,000 Mock-, scFv/hinge/his-tag-, scFv/hinge/his-tag/DAF-transduced TZM.bl cells were transduced with HIV-1 pseudotype-containing supernatants equivalent to relative luciferase activity 300,000 to 1,000,000 overnight. Cells were then washed twice with PBS and cultured in complete DMEM medium for 2 days. Cells were then washed once with PBS and lysed in 100 μl of lysis buffer. Luciferase activity in 50 μl of cell suspensions was measured by a BrightGlo Luciferase assay according to the manufacturer's instruction (Promega).50) in a final volume of 0.5 ml overnight. Cells were then washed three times with HBSS and resuspended in 2 ml of the complete DMEM medium and incubated at 37°C for 2 days. Infectivity of HIV-1 was determined by a BrightGlo Luciferase assay (see above).Mock-, scFv/hinge/his-tag-, scFv/hinge/his-tag/DAF-transduced TMZ.bl cells at 5,000 cells per well were seeded onto 96-well plate. After the overnight culture, cells were infected with HIV-1 strains Bru-3, Bru-YU2, AD8, Mj4, RJ407 and JS-JCD  and TZM.bl-GPI-scFv (X5) cells. After 2 days, infectivity of HIV-1 was determined by BrightGlo Luciferase assay (see above).To test the effect of sCD4 on anti-HIV potency of secretory and GPI-anchored scFvs (X5), parental TZM.bl cells, TZM.bl-scFv (X5) and TZM.bl-GPI-scFv (X5)  were seeded onto 96-well plate and incubated overnight. 400 TCID6 CEMss-CCR5 cells transduced with GPI-scFvs (X5 or AB65) and secretory scFvs (X5 or AB65) were infected with HIV-1 strains Bru-3 and Bru-Yu2 (200 TCID50) in a final volume of 0.5 ml overnight. Cells were then extensively washed with HBSS, resuspended in 6 ml of complete DMEM and cultured for 3 to 15 weeks. Every 3 days, 4.5 ml of cell suspensions were harvested and replaced with the fresh medium. The supernatants were then collected. HIV-1 p24 in the supernatants were measured by ELISA (Beckman Coulter) according to the manufacturer's instruction.1×105 per well) with or without 2 μg/ml tetracycline treatment were grown on 12-well plate at 37°C overnight. CEMss-GPI-scFvs (AB65 or X5) (2×105) were then added onto 69TiRev/Env cells. Cell-cell fusion was monitored under the light microscopy at various time intervals. When fusion process reached peak at 20 hours, the results were recorded by CCD digital camera (Leica DM IRB).69TiRev/Env cells . Briefly, 108 PBMCs were resuspended in 800 μl of binding buffer (PBS containing 0.5% BSA and 2 mM EDTA). 200 μl of MACS CD14 microbeads were added, mixed and incubated at 4°C for 15 minutes. The cell-bead mixture was then washed once with binding buffer and resuspended in 1 ml of fresh binding buffer. CD14+ cells were positively selected using an MS+/RS+ column tip. The isolated cells were washed once with complete RPMI and cultured in the complete RPMI supplemented with GM-CSF (1000 U/ml) and IL-4 (1000 U/ml)  for 7 days to generate immature DCs as described before [Peripheral blood was obtained from healthy donors at the Gulf Coast Regional Blood Center, Houston, Texas. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation as described before . To isold before . The expd before .5 per well) were seeded in triplicates in a 96 well U-bottom plate and incubated with 100 TCID50 of NL4-3 in a total volume of 200 μl at 37°C and 5% CO2 for 4 hours. Cells were then washed three times with PBS to remove cell-free virus and resuspended in 2 ml of complete RPMI into wells of 24-well plate with or without adding 1.5 × 105 CEMss-GPI-scFvs (AB65 and X5) cells. Cells were continued to be cultured for 14 days. Every 3 or 4 days, medium was removed from each well and replaced with fresh complete medium. HIV-1 p24 in the supernatants was measured by ELISA as described above.DCs  for 15 min. Cells were washed twice with 500 μl PBS and blocked with blockage buffer (5% goat serum in PBS plus 1%BSA) for 1 hour. Cells were stained with Alexa 555 conjugated CtxB (Invitrogen Life Technologies) at 4°C for 45 min. After washed 3 times with PBS, cells stained with mouse anti-his-tag antibody (Sigma) at 4°C for 45 min, then stained with Alexa 488-conjugated goat anti-mouse IgG antibody (Invitrogen) at 4°C. After cells were washed 3 times with PBS, cells were stained with 4',6-diamidino-2-phenylindole (DAPI) in permeabilization buffer (blockage buffer plus 0.5% saponin) for 5 min. The slides were mounted before being analyzed under confocal fluorescent microscope (Zeiss Model LSM 510).Parental and TZM.bl-GPI-scFvs (AB65 and X5) cells were seeded  onto the Lab-Tek chamber slide  and incubated at 37°C 5% COThe authors declare that they have no competing interests.MW designed and performed the experiments and participated in writing the manuscript. RA contributed to the experiments in human DC transferred and captured HIV part. QHW contributed to GPI anchored scFv (TG15) gene construction. LHL contributed to protein expression and data analysis. JTK participated in the design of the study and helped to draft the manuscript. PZ designed the experiments, wrote and completed the manuscript. All authors read and approved the final manuscript.Supplementary Figure 1. Expression of GPI-scFvs (AB65 and X5) in transduced TZM.bl cells. a. Schematic diagram of the lentiviral vectors pRRL-scFv(4E10)/hinge/his-tag/DAF and pRRL-scFv(4E10)/hinge/his-tag. b. FACS analysis of cell surface expression of scFv/hinge/histag/DAF in mock-, scFvs /hinge/histag/DAF.Click here for fileSupplementary Figure 2. Effect of transgenes on cell surface of expression of HIV-1 receptor and co-receptors in TZM.bl cells. a. Cell surface expression of CD4, CCR5 and CXCR4 of parental TZM.bl cells (mock) and TZM.bl cells transduced with lentiviral vectors expressing GPI-scFvs . b. Cell surface expression of CD4, CCR5 and CXCR4 of parental TZM.bl cells (mock) and TZM.bl cells transduced with lentiviral vectors expressing secretory scFvs . c. Side by side comparison of cell surface expression of CD4, CCR5 and CXCR4 of parental TZM.bl cells (mock) and TZM.bl cells transduced with lentiviral vectors expressing GPI-scFvs  or secretory scFvs .Click here for fileSupplementary Figure 3. Expression of soluble CD4. Coomassie blue staining of purified soluble CD4 by transfected drosophila S2 cells and separated by 12% SDS/PAGE.Click here for fileSupplementary Figure 4. Expression of secretory and GPI-scFvs (AB65 and X5) in transduced CEMss-CCR5 cells. a. Western blot analysis of expression of scFvs (X5 and AB65) in CEMss-CCR5 cells transduced with lentiviral vectors pRRL-scFv/hinge/his-tag/DAF (AB65 and X5) and pRRL-scFv/hinge/his-tag (AB65 and X5). GPI-scFv: GPI-anchored scFv; Sec scFv: secretory scFv; anti-his: anti-his-tag antibody. b. FACS analysis of cell surface expression of scFv/hinge/histag/DAF in mock-, scFvs (X5 and AB65)/hinge/histag/DAF- or m-scFv(TG15)-transduced CEMss-CCR5 cells with or without PI-PLC treatment. c. Cell surface expression of CD4, CCR5 and CXCR4 of parental CEMss-CCR5 cells (mock) and CEMss-CCR5 cells transduced with lentiviral vectors expressing GPI-scFvs (X5 and AB65) and secretory scFvs (X5 and AB65).Click here for file


Arabidopsis thaliana. Mutant alleles of SQUINT (SQN) slightly reduce AGO1 activity and have weak effects on shoot morphology. A screen for mutations that suppress the sqn phenotype produced loss-of-function mutations in the F-box gene FBW2. Mutations in FBW2 not only suppress sqn but also suppress many of the developmental phenotypes of weak, but not null, alleles of AGO1 by increasing AGO1 protein levels. Conversely, over-expression of FBW2 decreases the abundance of the AGO1 protein but not AGO1 messenger RNA, further indicating that FBW2 regulates AGO1 protein levels. fbw2 mutants have no obvious morphological phenotype, but display a reduced sensitivity to abscisic acid (ABA) that can be attributed to increased AGO1 activity. Our results indicate that FBW2 is a novel negative regulator of AGO1 and suggest that it plays a role in ABA signalling and/or response.ARGONAUTE1 (AGO1) mediates microRNA- and small interfering RNA-directed posttranscriptional gene silencing in  Argonaute proteins are core components of the RNA-induced silencing complex (RISC) -3. TheseArabidopsis thaliana . Equal amounts of soluble protein were separated on an 8.5% SDS-PAGE gel, transferred to a nitrocellulose membrane and membranes were blocked [tris buffered saline with tween (TBS-T) with 5% milk]. Anti-AGO1 , anti-FLAG monoclonal (1:2000) (Sigma - F1804), anti-FLAG HRP conjugated , anti-GFP (Invitrogen - A-6455), anti-cMyc rabbit , were incubated overnight in TBS-T + 5% milk at 4°. Anti-actin  (Sigma - A0480) was incubated at room temperature for 2 h.Fourteen-day-old, 20-day-old seedlings, or floral buds were ground using liquid nitrogen and resuspended in 1:3 w/v extraction buffer [20 mM Tris pH 7.5, 300 mM NaCl, 5 mM MgClFBW2 coding or FBW2 genomic region was amplified by PCR using the FBW2 primers  and pEG302 (FLAG), respectively [AGO1::FLAG-AGO1 constructs, genomic AGO1::FLAG-AGO was amplified from the pGreen0229 FLAG-AGO1 construct using AGO1 F and R primers . Plants were scored for greening of the cotyledons. Experiments were repeated at least three times. In order to determine the effect of ABA on root growth, sterilized seeds were plated vertically on 1% sucrose LS plates, imbibed for 3 days, and grown for 5 days under long day conditions . Seedlings of equal size were then transplanted to vertical plates containing either 0 μM or 2.5 μM ABA, and grown for 5 days or more under long day conditions . Primary root length was measured for each plant and root inhibition was determined as the average root length of plants grown at 2.5 μM ABA relative to the average root length of plants gown at 0 μM ABA.ABA: abscisic acid; AGO1: ARGONAUTE1; EMS: ethyl methanesulfonate; mRNA: messenger RNA; miRNA: microRNA; RISC: RNA-induced silencing complex; RT-PCR: real time polymerase chain reaction; siRNA: small interfering RNA; SQN: SQUINT; TBS-T: tris buffered saline tween.The authors declare that they have no competing interests.sqn suppressor screen. MRS and KWE mapped FBW2; RW performed the ABA experiments under the guidance of BDG.KWE performed most of the work described in this paper and co-wrote the manuscript with RSP. MRS performed the Figure S1. (A) Gene structure of FBW2 showing the location of primers used in real time polymerase chain reaction and the abundance of FBW2 messenger RNA in sqn-1 and mutants doubly mutant for sqn-1 and different alleles of fbw2. All target genes were normalized to EIF4. (B) Alignments of the Arabidopsis F-box genes most closely related to At4g08980 (FBW2): At4g05497 (FBW9), At4g05460 (FBL20), At5g57900 (SKIP1). (C) Alignments of the amino acid sequences of FBW2 and its predicted orthologs in other flowering plants. All alignments were made using the following assembled contigs from PlantGDB unless otherwise noted: Citrus sinensis (PUT-157a-Citrus_sinensis-6728477), Gossypium raimondii (PUT-157a-Gossypium_raimondii-11427), Glycine max (PUT-161a-Glycine_max-76896), Oryza sativa Japonica (Genbank AK070008), Asparagus officinalis (Genbank CV288647), Vitis vinifera (PUT-157a-Vitis_vinifera-16230), Nicotiana tabacum (PUT-163a-Nicotiana_tabacum-55992858), Lactuca sativa (PUT-157a-Lactuca_sativa-30499), Pinus taeda (PUT-157a-Pinus_taeda-79282550).Click here for fileFigure S2. (A) Western blot comparing levels of ARGONAUTE1 (AGO1) and yellow fluorescent protein-AGO10 in plants with and without FBW2ox constructs. Anti-AGO1 and anti-GFP antibodies were used to probe each blot. Ponceau staining was used as a loading control. (B) Levels of AGO1-FLAG do not decrease in plants treated with the protease inhibitor MG132. Blots were probed with anti-FLAG monoclonal antibody or anti-ubiquitin antibody. The anti-ubiquitin antibody demonstrates an overall increase in ubiquitination in MG132 treated plants, as expected from a decrease in proteasome activity.Click here for fileTable S1. Polymerase chain reaction primers used in this study.Click here for file