Research ArticleNew Research, Development

Rab3-GEF Controls Active Zone Development at the Drosophila Neuromuscular Junction

Haneui Bae, Shirui Chen, John P. Roche, Minrong Ai, Chunlai Wu, Aaron Diantonio and Ethan R. Graf
eNeuro 7 March 2016, 3 (2) ENEURO.0031-16.2016; https://doi.org/10.1523/ENEURO.0031-16.2016

Article Figures & Data

Figures

  • Figure 1.
    Figure 1.

    Bruchpilot distribution is disrupted in rab3-GEF mutant NMJs. A, Schematic representations of the cloned WT Drosophila rab3-GEF gene, the rab3-GEFMA18 and rab3-GEFMA20 mutant genes generated by EMS mutagenesis, and the rab3-GEFSC225 mutant gene generated by P-element excision. Magenta exonic regions correspond to the tripartite DENN domain composed of the uDENN, DENN, and dDENN modules. The green exonic region corresponds to the DD. Arrowheads identify the Q1862STOP and G460E point mutations associated with the rab3-GEFMA18 and rab3-GEFMA20 mutant alleles, respectively. Boxed region of the rab3-GEFSC225 mutant allele denotes the part of the gene that is excised. B, Confocal images of muscle 4 NMJs costained with antibodies against the presynaptic AZ protein Brp (magenta) and the postsynaptic receptor DGluRIII (green) from WT (CS), rab3-GEFMA18 mutant (rab3-GEFMA18 /Df(1)ED7289) and rab3-GEFMA20 mutant (rab3-GEFMA20 /Df(1)ED7289) third-instar larvae. Scale bar, 2 µm.

  • Figure 2.
    Figure 2.

    The altered distribution of Brp in rab3-GEFSC225 mutant NMJs is identical to the rab3 mutant and results in the clustering of Ca2+ channels at a small number of release sites. A, Images of NMJs costained with α-Brp (magenta) and α-DGluRIII (green) from CS, homozygous rab3-GEFSC225 mutant larvae, rab3-GEFSC225 /Df(1)ED7289 mutant larvae, and rab3rup /Df(2R)ED2076 mutant larvae. Scale bar, 2 µm. B, C, Histograms show (B) the average percentage of DGluRIII clusters apposed to Brp puncta per NMJ and (C) the average area of individual Brp puncta for the genotypes listed in A. n = 10 NMJs for all genotypes; (B) *p≪0.000001a versus CS; (C) *p≪0.000001b versus CS. D, Images of NMJs expressing cacophony-GFP (green) driven by dvglut NMJX-Gal4 in from WT (dvglut NMJX-Gal4/+; UAS-Cacophony-GFP/+) and rab3-GEFSC225 mutant (dvglutNMJX-Gal4, rab3-GEFSC225 /Df(1)ED7289; UAS-Cacophony-GFP/+) larvae. NMJs are costained with α-Brp (magenta) and α-DGluRIII (blue) antibodies. Scale bar, 2 µm. E, Histogram shows the average area of cacophony-GFP clusters for the genotypes listed in D. n = 10 NMJs for both genotypes; *p≪0.000001c versus WT.

  • Figure 3.
    Figure 3.

    α-Rab3-GEF immunostaining reveals Rab3-GEF protein at the NMJ and in ventral nerve cord cell bodies and neuropil. A, Images of NMJs immunostained with α-Rab3-GEF (green) and α-Brp (magenta) from CS and rab3-GEFSC225 /Df(1)ED7289 mutant larvae. Inset, Magnified view of boxed region, indicating partial overlap of α-Rab3-GEF and α-Brp signal but little colocalization. Scale bars: lower-magnification images, 2 µm; higher-magnification inset, 1 µm. B, Image of a single bouton of a WT NMJ expressing UAS-rab3-YFP (magenta) driven by the dvglutNMJX-Gal4 driver and costained with α-Rab3-GEF (green) and α-Brp (blue). Scale bar, 1 µm. C, Single optical sections of ventral nerve cords from CS and rab3-GEFSC225 /Df(1)ED7289 mutant larvae immunostained with α-Rab3-GEF (green) and α-Brp (magenta). In WT brains, Rab3-GEF immunostaining is prominent in the neuropil, as well as in the surrounding neuronal cell bodies visualized by cytosolic Rab3-GEF surrounding immuno-negative nuceli. Scale bar, 20 µm.

  • Figure 4.
    Figure 4.

    Neuronal expression of transgenic rab3-GEF rescues Brp distribution in rab3-GEFSC225 mutant NMJs. A, Images of NMJs costained with α-Brp (magenta) and α-DGluRIII (green) from WT (dvglutNMJX-Gal4/+), the rab3-GEFSC225 mutant (dvglutNMJX-Gal4, rab3-GEFSC225 /Df(1)ED7289), and rescue larvae corresponding to the rab3-GEFSC225 mutant with neuronal expression of UAS-rab3-GEF (dvglutNMJX-Gal4, rab3-GEFSC225 /Df(1)ED7289; UAS-rab3-GEF/+). Scale bar, 2 µm. B, C, Histograms show (B) the average percentage of DGluRIII clusters apposed to Brp puncta per NMJ and (C) the average area of individual Brp puncta for the genotypes listed in A. n = 11 NMJs for all genotypes; (B) *p≪0.000001i versus both WT and rescue larvae; (C) *p≪0.000001j versus both WT and rescue larvae.

  • Figure 5.
    Figure 5.

    Short-term facilitation is defective in the rab3-GEFSC225 mutant. A Representative EJC traces of a train of five stimuli given at a frequency of 20 Hz in 0.40 mm Ca2+ from WT (dvglutNMJX-Gal4/+), the rab3-GEFSC225 mutant (dvglutNMJX-Gal4, rab3-GEFSC225 /Df(1)ED7289), rescue larvae corresponding to the rab3-GEFSC225 mutant with neuronal expression of UAS-rab3-GEF (dvglutNMJX-Gal4, rab3-GEFSC225 /Df(1)ED7289;;UAS-rab3-GEF/+), and rab3 mutant larvae (dvglutNMJX-Gal4/+; rab3rup /Df(2R)ED2076). The stimulation artifact has been removed for clarity. B, Histogram showing quantification of the average FI for each of the four genotypes listed in A, calculated by dividing the amplitude of the fifth EJC by the amplitude of the first EJC in a 20 Hz stimulus train. WT, n = 13; rab3-GEFSC225 mutant, n = 12; rab3-GEFSC225 with rescue, n = 14; rab3 mutant, n = 9; *p≪0.000001 m versus both WT and rescue larvae; #p=1.83 × 10−6 m versus WT.

  • Figure 6.
    Figure 6.

    Simultaneous disruption of both rab3 and rab3-GEF does not enhance the Brp distribution phenotype. A, Images of NMJs costained with α-Brp (magenta) and α-DGluRIII (green) from rab3-GEFSC225 mutant, rab3rup /Df(2R)ED2076 mutant, and rab3-GEFSC225 ; rab3rup /Df(2R)ED2076 double-mutant larvae. Scale bar, 2 µm. B, C, Histograms show (B) the average percentage of DGluRIII clusters apposed to Brp puncta per NMJ and (C) the average area of individual Brp puncta for the genotypes listed in A. n = 10 NMJs for all genotypes.

  • Figure 7.
    Figure 7.

    Rab3-GEF is required for proper trafficking of Rab3. A, Images of NMJs costained with α-Brp (magenta) and α-Rab3 (green) from CS and rab3-GEFSC225 /Df(1)ED7289 mutant larvae. Scale bar, 2 µm. B, Images of motor nerve segments immunostained with α-Rab3 (green) and α-HRP (magenta) from from CS and rab3-GEFSC225 /Df(1)ED7289 mutant larvae. Scale bar, 2 µm. C, Histogram shows the average intensity of α-Rab3 signal throughout the entire NMJ for the genotypes listed in A. n = 10 NMJs for both genotypes; *p≪0.000001 p versus CS. D, Histogram shows the average intensity of α-Rab3 signal in Brp-positive and Brp-negative regions of NMJs for the genotypes listed in A. n = 10 NMJs for both genotypes; *p≪0.000001 q versus α-Rab3 intensity at the Brp-positive regions in WT NMJs; #p≪0.000001 r versus α-Rab3 intensity at the Brp-positive regions in rab3-GEF mutant NMJs. E, Histogram shows the average intensity of α-Rab3 signal per nerve segment as defined by α-HRP staining for CS (n = 24 nerve segments) and rab3-GEFSC225 /Df(1)ED7289 mutant larvae (n = 12 nerve segments); *p≪0.000001 s versus CS.

  • Figure 8.
    Figure 8.

    Transgenic expression of WT Rab3 or a GTP-locked variant of Rab3 increases Rab3 accumulation at the NMJ but fails to rescue Brp distribution in the rab3-GEFSC225 mutant. A, Images of NMJs immunostained with α-Rab3 (green) from WT (dvglutNMJX-Gal4/+), the rab3-GEFSC225 mutant (dvglutNMJX-Gal4, rab3-GEFSC225 /Df(1)ED7289), the rab3-GEFSC225 mutant expressing UAS-rab3 (dvglutNMJX-Gal4, rab3-GEFSC225 /Df(1)ED7289;; UAS-rab3/+), and the rab3-GEFSC225 mutant expressing UAS-rab3Q80L (dvglutNMJX-Gal4, rab3-GEFSC225 /Df(1)ED7289;; UAS-rab3Q80L/+). Scale bar, 2 µm. B, Histogram shows the average intensity of α-Rab3 signal throughout the entire NMJ for the genotypes listed in A. n = 10 NMJs for all genotypes; *p= 6.7 × 10−7 t versus WT, 1.35 × 10−6 t versus the rab3-GEF mutant expressing UAS-rab3, and 5.12 × 10−7 t versus the rab3-GEF mutant expressing UAS-rab3Q80L. C, Images of NMJs costained with α-Brp (magenta) and α-DGluRIII (green) from the genotypes listed in A. Scale bar, 2 µm. D, E, Histograms show (D) the average percentage of DGluRIII clusters apposed to Brp puncta per NMJ and (E) the average area of individual Brp puncta for the genotypes listed in A. n = 10 NMJs for all genotypes; (D) *p≪0.000001 u versus WT; (E) *p≪0.000001 v versus WT.

Tables

  • Table 1.

    Statistical Table

    Data structureType of testPower (α=0.05)
    a (Fig. 2B)Normally distributedANOVA followed by Bonferroni’s test1
    b (Fig. 2C)Normally distributedANOVA followed by Bonferroni’s test1
    c (Fig. 2E)Normally distributedStudent’s t test1
    dNormally distributedANOVA followed by Bonferroni’s test1
    eNormally distributedANOVA followed by Bonferroni’s test1
    fNormally distributedStudent’s t test0.05241
    gNormally distributedANOVA followed by Bonferroni’s test1
    hNormally distributedANOVA followed by Bonferroni’s test1
    i (Fig. 4B)Normally distributedANOVA followed by Bonferroni’s test1
    j (Fig. 4C)Normally distributedANOVA followed by Bonferroni’s test1
    kNormally distributedANOVA followed by Bonferroni’s test0.07662
    lNormally distributedANOVA followed by Bonferroni’s test0.15701
    m (Fig. 5B)Normally distributedANOVA followed by Bonferroni’s test1
    nNormally distributedANOVA followed by Bonferroni’s test0.15085
    oNormally distributedANOVA followed by Bonferroni’s test0.13093
    p (Fig. 7C)Normally distributedStudent’s t test1
    q (Fig. 7D)Normally distributedStudent’s t test1
    r (Fig. 7D)Normally distributedStudent’s t test1
    s (Fig. 7E)Normally distributedStudent’s t test1
    t (Fig. 8B)Normally distributedANOVA followed by Bonferroni’s test1
    u (Fig. 8D)Normally distributedANOVA followed by Bonferroni’s test1
    v (Fig. 8E)Normally distributedANOVA followed by Bonferroni’s test1
    wNormally distributedStudent’s t test0.05575
    xNormally distributedStudent’s t test0.0937
    yNormally distributedStudent’s t test1
    zNormally distributedStudent’s t test0.99976
    aaNormally distributedStudent’s t test0.0588
    bbNormally distributedStudent’s t test0.13625
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Rab3-GEF Controls Active Zone Development at the Drosophila Neuromuscular Junction
Haneui Bae, Shirui Chen, John P. Roche, Minrong Ai, Chunlai Wu, Aaron Diantonio, Ethan R. Graf
eNeuro 7 March 2016, 3 (2) ENEURO.0031-16.2016; DOI: 10.1523/ENEURO.0031-16.2016
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