EMBO/SourceData huggingface dataset:
Figure 2Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z197 and Z360 were used, which harbor glmY'-lacZ and glmZ'-lacZ fusions, respectively.Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Expression of glmY'-lacZ in strain Z197 and the ∆rapZ mutant Z225 is compared.Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z190 and Z201 were addressed, which transcribe glmY'-lacZ either from the σ54-promoter or the σ70 promoter, respectively.Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z190 and the ∆qseF mutant Z196 are compared, both of which transcribe the glmY'-lacZ fusion solely from the σ54 promoter.Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Complementation experiment analyzing the requirement of rapZ for glmY expression in cells grown to exponential phase under standard conditions in flask cultures. Strains Z197 and the ∆rapZ mutant Z225 were used. Tested plasmids were pFDX4291 (vector control for pFDX4324 and pYG82 = VC1), pFDX4324 (rapZ), pYG82 (rapZquad), pBGG237 (vector control for pBGG164 = VC2) and pBGG164 (strep-rapZ).
Figure 3A BACTH assay addressing interaction of RapZ with QseF and QseE. The following plasmid combinations were tested using reporter strain RH785 (columns 1-6): pBGG352/pBGG349, pBGG353/pBGG348, pYG199/pBGG349, pYG246/pBGG348, pKT25/pUT18C (negative control), pKT25-zip/pUT18C-zip (positive control). , β-galactosidase activities are presented as mean ± SD.B SPR spectroscopy experiments addressing interaction of RapZ with the cytoplasmic part (aa 196-475) of kinase QseE and response regulator QseF. Strep-RapZ was captured onto a sensor chip and increasing concentrations of QseE'-His10 or QseF-His10 were injected.C To assess the role of RapZ for QseF activity, β-galactosidase activities were determined from strains Z196 (∆qseF) and Z1110 (∆qseF ∆rapZ) at indicated times during growth. Strains harbored the glmY'-lacZ fusion that is solely expressed from the σ54 promoter and the following plasmids: pKESK23 (VC = vector control; black and grey; note that activities are too low for display), pYG89 (qseF, green), pYG90 (qseF-D56E, purple), pYG93 (qseF-D56A, red). , β-galactosidase activities are presented as mean ± SD.D In vitro phosphorylation assays addressing autophosphorylation of 1 μM His10-tagged QseE' (aa 196-475) in presence of various concentrations of RapZ. Samples were removed following [γ-32P]-ATP addition at indicated times and separated on 12.5 % SDS-PAA gels, which were analyzed by phospho imaging.E Analysis of QseE' autophosphorylation (lanes 1-6) and phosphoryl group transfer to QseF (lanes 7-12) in absence or presence of 5 μM Strep-RapZ. To assess phosphoryl group transfer, 1 μM QseF-His10 was added to the assay.F To analyse the role of GlcN6P, 5 μM RapZ or the equivalent volume of buffer was pre-incubated with the indicated GlcN6P concentration for 5 min and subsequently 1 μM QseE'-His10 was added. Following an additional incubation for 5 min, [γ-32P]-ATP was added and the reactions were stopped after 1 min.
Figure 4A β-Galactosidase activities of strains Z197 (wild type) and Z1118 (∆glmY ∆glmZ), which carry the chromosomal glmY'-lacZ fusion, were determined during growth.B Strains Z197 (wild type) and Z225 (∆rapZ) were transformed with the following plasmids expressing the mentioned sRNAs: pBR-plac (vector control = VC), pYG83 (glmY), pYG84 (glmZ) and pSD69 (gcvB). sRNA expression was induced with 1 mM IPTG and β-galactosidase activities were determined in the exponential growth phase.C To assess the impact of GlmY* and GlmZ on stimulation of QseE' autophosphorylation by RapZ, 1 μM QseE'-His10 was incubated with [γ-32P]-ATP in absence or presence of 5 μM Strep-RapZ and/or the sRNAs GlmY*, GlmZ and GcvB. In lanes 3-11 QseE' was co-incubated with RapZ as well as the indicated sRNAs provided at 0.5 μM, 1.25 μM and 2.5 μM. In lanes 14-16, QseE' was incubated with 2.5 μM of each sRNA without RapZ. Samples were removed 1 min after addition of [γ-32P]-ATP and separated on 12.5 % SDS-PAA gels, which were analyzed by phospho imaging.
Figure 6Northern blot experiments (A-C) assessing GlmY* half-life under GlcN6P replete (+GlcN) and depletion conditions (-GlcN). Transcription was stopped by addition of rifampicin and samples were harvested at indicated times for Northern analysis. A Analysis of the ∆glmS strain Z1126. The GlcN6P depleted culture was split 8 min after rifampicin addition and one of the sub-cultures was resupplied with GlcN (indicated by arrow). B The ∆glmS ∆rapZ double mutant Z1127 was tested. C Semi-logarithmic plots of GlmY* decay for half-life determination. D EMSA experiments addressing the role of GlcN6P for GlmY*/RapZ interaction. Radiolabelled GlmY* was incubated with incremental concentrations of RapZ (left panel) or RapZ-CTD (right panel) in absence or presence of 7.5 mM GlcN6P. Binding reactions were separated on native PAA gels and analyzed by phospho-imaging. The RapZ/GlmY* complex is indicated by an arrow.E EMSA following incubation of GlmY* with 1200 nM RapZ in presence of various GlcN6P concentrations ranging from 0 (lane 2) to 8 mM (lane 10). The fraction of GlmY* remaining in the gel pocket is marked with an asterisk. The RapZ/GlmY* complex is indicated by an arrow.F EMSA following incubation of GlmY* with 1200 nM RapZ in absence or presence of 7.5 mM of the indicated metabolite. The RapZ/GlmY* complex is indicated by an arrow.
Figure 2Western blot for phosphorylated and total ERK in HEK293 cells transfected with native and ΔA781_N783 (left) or P824R (right) CSF-1R and treated with CSF-1 (D, E) or IL-34 (F, G) for 10 minutes. Horizontal line indicates untreated cells, with increasing concentrations 10, 50 and 100 ng/ml. Corresponding densitometry displays the ratio of phospho-ERK to total ERK, normalised to the ratio of the untreated control for the native receptor (**** p < 0.0001, *** p < 0.0005, ** p < 0.01, * p < 0.05, one-way ANOVA with Sidak's post-test for multiple comparison, n = 3 biological replicates, , error bars indicate SEM).Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with rapamycin. Horizontal line indicates untreated cells, with concentrations 0.1, 0.5, 1, 5, 10μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM). Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with 3-methyladenosine. Horizontal line indicates untreated cells, with increasing concentrations 0.1, 0.2, 0.5, 1mM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, * p = 0.0114, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM). Western blot for CSF-1R and Actin in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with MG132 or PLX3397. Horizontal line indicates untreated cells, 20μM PLX3397, with increasing concentrations 0.1, 0.5, 0.1, 5μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (*** p = 0.0002, ** p = 0.002, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM).
Figure 3Immunocytochemistry of microglia isolated from Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ P0 mouse pup brains. Microglia were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker(red). Arrowheads (white) indicate bead+ cells. Quantification of mouse microglial phagocytic activity expressed as percentage bead+ cells (n = 4 assays, error bars indicate SEM).L) Immunocytochemistry of macrophages differentiated in vitro from Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mouse bone marrow. Macrophages were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker (red) and DAPI (blue). Arrowheads (white) indicate bead+ cells. M) Quantification of mouse macrophage phagocytic activity expressed as percentage bead+ cells. (* p = 0.024, Student's t-test with Welch's correction, n = 4 assays, error bars indicate SEM).
Figure 4A) Immunohistochemistry of Csf-1rflx/wt;Cx3cr1-Cre- (top) and Csf-1rflx/wt;Cx3cr1-Cre+ (bottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). Scale bars indicate 50 μm. B) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice. (Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, obliques (#) indicate comparison between Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice, ## or ** p < 0.003, #### or **** p < 0.0001, n = 5 mice per group,error bars indicate SEM).C) Immunohistochemistry of Csf-1rflx/wt;Tie2-Cre- (top) and Csf-1rflx/wt;Tie2-Cre+ mice (ottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). D) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Tie2-Cre- and Csf-1rflx/wt;Tie2-Cre+ mice. (n = 5 mice per group. Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, *p < 0.01, *** p < 0.0005, error bars indicate SEM)).
Figure 5qPCR of wild type (blue) or Csf-1r+/- (red) endothelial cells treated with control, Csf-1r+/+ MCM or Csf-1r+/- MCM. Statistical analyses of inter-genotype changes. (**** p < 0.0001, ** p < 0.005, Scatter plots represent technical replicates of n = 2 independent primary cell isolations and microglia conditionings, Two-way ANOVA with multiple comparisons and Sidak post-test, error bars indicate SEM)).
Figure 6IHC of ALSP patient post mortem tissue for amyloid-β (green) and CLD5 (red), (B) human IgG (green) and PDGFRβ (red), (C) GFAP (green) and CLD5 (red). ΔA781_N783 and P824R indicate the CSF-1R variant present.(D) Iba1 (green) and GFAP (red), (E) amyloid-β (green) and CD68 (red), (f) CD163 (green) and CLD5 (red), imaging of an ALSP patient with the ΔA781_N783 CSF-1R variant. ΔA781_N783 and P824R indicate the CSF-1R variant present.(G) Fibrinogen (green) and CLD5 (red) and (H) FLAIR imaging of an ALSP patient with the ΔA781_N783 CSF-1R variant. ΔA781_N783 and P824R indicate the CSF-1R variant present.
Figure 1B: PK vs. PIPLC treatment to determine prion loads in infected cells in 384-well plates. Both treatments discriminate between RML prion infected CAD5 and non-infectious brain homogenate (NBH) treated CAD5 cells. **** = p-value < 0.0001 (Student's t-test). Shown are mean ± SD, n ≤ 30 individually cultured wells.C: The infectivity of PIPLC and PK-treated cell lysates was determined by infecting CAD5 cells with lysates as indicated. The signal intensity of the highest dilution was measured and compared to untreated RML CAD5 cell lysate. PIPLC-treated cells and PK-treated lysates showed similar infectivity titers. Prion-infected and NBH, as well as RML on CAD5ΔPrnp were used for control.D: RML-infected CAD5 and GT-1/7 cells were transfected with non-targeting (NT) or Prnp targeting siRNAs in a 384-well plate and subjected to PK or PIPLC treatment. Z'-factors were calculated for each condition. Shown are mean ± SD, n = 22 individually cultured wells.
Figure 5A: Western Blot of RML prion infected COCS treated with 1 µM PSA or DMSO. PSA treatment was started two weeks after infection and continued until lysis. PSA reduced the amount of PrPSc. Right panel: Quantification of the Western Blot; n = 5 biological replicates. Values: mean ± SD. * p = 0.0211 (Student's t-test).B: Western Blot analysis of NBH-treated COCS. PSA treatment was identical to the samples of A. PSA did not affect PrPC expression. Right panel: Quantification of the Western Blot; n = 5 biological replicates. Values represent mean ± SD. n.s. = not significant (Student's t-test). ⍺: antiC: Negative geotaxis climbing assay in prion infected Drosophila. Flies were treated with DMSO, 0.5 mM PSA, 0.75 mM PSA, or 1 mM PSA at the larval stage and during adulthood for the duration of the assay. Climbing ability was assessed on groups of flies (n=3 x 15) three times a week and expressed as a performance index. Statistical analysis on the difference between PG127 prion infected versus control prion-free treatment group data in each graph was performed by an unpaired t test: DMSO: p = 0.0002; PSA: 0.5 mM: p = 0.0186; 0.75 mM: p = n.s.; 1 mM: p = n.s. n.s. = not significant. Shown are mean ± SDD: RT-QuIC analysis of whole-head homogenates of prion infected Drosophila. For each sample, 10 male and 10 female heads from the same treatment group were homogenized, 1:20 diluted and applied to the RT-QuIC. Shown are the RT-QuIC reactions of three independent homogenates per treatment group, each assessed in quadruplicates. For quantification, the lag-time of each reaction was calculated and plotted in a graph. Shown are mean ± SD. The assays were performed for 100 hours, samples not yielding a positive reaction are considered negative. *** = p ≤ 0.0006, n.s. = not significant, flies fed with NBH and treated with DMSO were used as a negative control, a standard prion-free and a prion-containing sample were used as assay controls. To control for potential interference of DMSO or PSA with the RT-QuIC reaction, prion positive sample was spiked with 1 µM PSA.
Figure 1SHAPE analysis of the complete sfRNAs of ZIKV (D) Nucleotides with SHAPE reactivity greater than 0.85 are labeled in red, and moderately reactive site (0.4-0.85) are labeled in cyan. Unlabeled sites have low or no SHAPE reactivity (<0.4). The SHAPE reactivity were also annotated onto the corresponding secondary structures in (B) Nucleotides which SHAPE reactivity was not determined are labeled in gray in (B)SHAPE analysis of the complete sfRNAs of WNV (E). Nucleotides with SHAPE reactivity greater than 0.85 are labeled in red, and moderately reactive site (0.4-0.85) are labeled in cyan. Unlabeled sites have low or no SHAPE reactivity (<0.4). The SHAPE reactivity were also annotated onto the corresponding secondary structures in (C). Nucleotides which SHAPE reactivity was not determined are labeled in gray in (C).
Figure 2(A)-(D) The scattering profiles (A), pair distance distribution functions (PDDFs) (B), dimensionless Kratky plots (C) and Porod-Debye plots (D) of the complete sfRNAs of DENV2 (cyan line), ZIKV (red line) and WNV (blue line). The fine features of P1 in the scattering profiles in (A) arise from helical interstrand pair-distance correlation. The PDDF profiles in (B) were calculated using GNOM (qmax=0.3). The dimensionless Kratky plot and Porod-Debye plot for ZIKV xrRNA1 (red dashed line) were included in (C) and (D) for comparison, indicating that the structures of sfRNAs are more extended and open than that of ZIKV xrRNA1.
Figure 3(C, D) The scattering profiles (left), the PDDFs (middle) and the dimensionless Kratky plots (right) of individual xrRNA1s (C) and xrRNA2s (D) of DENV2 (cyan), ZIKV (red), WNV (blue) and MVEV (green). The insets in (C) and (D) show the Guinier regions of the respective scattering profiles with a linear fit line. The back-calculated scattering profile of ZIKV- xrRNA1 crystal structure (black, open circle) can be nicely fitted onto its experimental SAXS data (red line) in (C), while the back-calculated scattering profile of MVEV- xrRNA2 crystal structure (black, open circle) fits poorly to its experimental SAXS data (green line) in (D). The asymmetric PDDFs of xrRNA1 (C) and xrRNA2 (D), indicate slightly elongated molecules with asymmetric shapes. The dimensionless Kratky plots of xrRNA1 (C) and xrRNA2 (D) of DENV2, ZIKV, WNV and MVEV suggest all individual xrRNA1s and xrRNA2s are well-folded and of reduced flexibility in solution.
Figure 4(A)-(C) The scattering profiles (A), the PDDFs (B) and the dimensionless Kratky plots (C) of xrRNA1-2s from DENV2 (cyan), ZIKV (red) and WNV (blue). The inset in (A) shows the guinier regions of the respective scattering profiles with linear fit lines. The dimensionless Kratky plot of ZIKV xrRNA1 (red dashed line) was included in (C) for comparison, indicating that the structures of xrRNA1-2s are all well folded.
Figure 5(A)-(C) The scattering profiles (A), the PDDFs (B) and the dimensionless Kratky plots (C) of DB12s from DENV2 (cyan), ZIKV (red) and WNV (blue). The inset in (A) shows the Guinier regions of the respective scattering profiles with linear fit lines. The dimensionless Kratky plot of ZIKV xrRNA1 (red dashed line) was included in (C) for comparison, indicating that the structures of DB12s of ZIKV and WNV are well folded, but that of DB12 of DENV2 is partially folded. The Kratky plots for the two PK3 mutants of DB12s, the ZIKV- DB12-PK3∆ (green) and WNV- DB12-PK3∆ (black), respectively, are also included for comparison, suggesting reduced compactness upon PK3 mutations.
Figure 6(C) E protein expression of ZIKV and the mutants showed by IFA.(D) The progeny virus RNA in cultured supernatants of ZIKV and the mutants were detected by qRT-PCR in three technical replicates. Two-way ANOVA and Dunnett's multiple comparison test was used for statistical analysis. The error bars represent the standard deviation. *** p<0.001, **** p<0.0001.(E) The luciferase activity of the ZIKV-Rep and the mutants were measured at different time points after transfection in three technical repelicates. The error bars represent the standard deviation.
Figure 7(A)-(C) The scattering profiles (A), the PDDFs (B) and the dimensionless Kratky plots (C) of 3'SLs from DENV2 (cyan), ZIKV (red), WNV (blue) and WNV-3'SLM (438UAG441A→438GUC441U) mutant (green). The inset in (A) shows the guinier regions of the respective scattering profiles with linear fit lines.
Figure 8(A)-(C) The fitting chi2 are plotted against the ensemble size for complete sfRNAs of DENV2 (A), ZIKV (B) and WNV (C), suggesting optimized minimal structural ensembles with 3, 3 ,3 conformers, respectively. The respective insets show the distribution of the minimal ensembles and the initial pools of 10000 random conformers as a function of Rg.
figf1(a) The Hiw-associated complex was purified by TAP (see Online Methods) and analyzed by one-dimensional SDS-PAGE gel followed by Sypro Ruby staining. Mass spectrometry identified the full-length Hiw, β-tubulin, Rae1 and Fsn, indicated by arrows.(b) The Rae1-associated complex purified from fly brains by TAP was analyzed by one-dimensional SDS-PAGE gel followed by Coomassie blue G-250 staining. Mass spectrometry identified Hiw, Nup98 and Rae1, indicated by arrows.(c) Larval brain lysate of wild-type (WT), hiw null mutant (hiwΔN) and Fsn mutant (Fsnf06595/Df(2R)7872) flies was subject to co-immunoprecipitation with antibody to Hiw (Hiw2b). Both the input and the immunoprecipitated complexes were analyzed by western blots with antibodies to Hiw or Rae1. The membrane was also blotted with antibody to β-tubulin to ensure equal amount of total proteins in the input samples. Full-length blots are presented in Supplementary Figure 8.
figf2(a) The gene structure of Rae1. The black-arrowed boxes indicate exons. A transposable element, P{GawB}NP3499, located ∼400 bp upstream of the first exon of Rae1 was used to generate imprecise excision mutants of Rae1. PCR analysis indicates that ∼900 bp and a ∼1.5-k bp DNA fragment in the Rae1 promoter region are missing in Rae1EX28 and Rae1EXB12, respectively. (b) Western blot analysis on Rae1 protein in the brain lysate of wild-type, Df(2R)5764/+, Rae1EX28/+, or late second instar Rae1EX28, Rae1EX28/Df(2R)5764(/Df) and Rae1EXB12 /Df(2R)5764 larvae. β-tubulin was used as the loading control. Full-length blots are presented in Supplementary Figure 9. (c) Quantification of Rae1 protein levels in the Rae1 heterozygous and homozygous mutant brains. Rae1 protein levels were first normalized to β-tubulin protein levels and are presented as percentage of wild-type level. *P 0.05, **P 0.001. Error bars denote s.e.m.
figf3(a) Representative confocal images of segment A3 muscle 6/7 synapses stained for both DVGLUT (green) and FasII (red), in late 2nd/early 3rd instar wild-type, Rae1EX28, Rae1EX28 presynaptic rescue (Rae1EX28; elav-Gal4/UAS-NTAP-Rae1) and Rae1EX28 wallenda suppression (Rae1EX28; wnd3) larvae. Scale bar represents 10 μm. (b,c) Quantification of bouton number (b) and size (c) in wild-type, Rae1EX28/Df, Rae1EX28, Rae1EX28 rescue, Rae1EX28; wnd3 suppression and hiwΔN larval NMJs (segment A3 muscle 6/7; n = 20, 21, 18, 20, 20 and 11, respectively, for bouton number; n = 271, 465, 548, 726, 486 and 826, respectively, for bouton size). There was no significant difference in either bouton number or bouton size between Rae1EX28 and Rae1EX28/Df (P > 0.1 for both comparisons). *P 0.001. Error bars denote s.e.m.
figf4(a) Representative confocal images of muscle 4 synapses stained for both DVGLUT (green) and FasII (red) in wild-type, Rae1EX28/+, hiwND51, hiwND51; Rae1EX28/+, hiwΔN and hiwΔN; Rae1EX28/+ larvae. Scale bar represents 10 μm. (b) Quantification of bouton number at muscle 4 NMJs in wild-type, Rae1EX28/+, hiwND51, hiwND51; Rae1EX28/+, hiwΔN and hiwΔN; Rae1EX28/+ larvae (n = 27, 23, 42, 46, 23 and 25 cells, respectively). There was a significant increase in the number of boutons formed in hiwND51; Rae1EX28/+ compared with hiwND51 3rd instar larval NMJs (*P 0.001). The number of boutons in the hiwΔN; Rae1EX28/+ double mutant was not significantly different from that in hiwΔN (**P > 0.5), demonstrating a lack of enhancement of the hiwΔN phenotype by Rae1EX28/+. Error bars denote s.e.m.