Article Figures & Data
Figures
- Figure 1.
Generation of pallidin null mutations and synaptic localization of Pldn protein. A, Schematic of the workflow utilized to generate the excision of the pallidin locus. Black arrows indicate primers used for PCR confirmation of the excision. B, PCR confirmation of the deletion of the pldn locus. C, Immunoblot analysis of adult heads lysates from wild type (w1118), pldnΔ1 mutants (w1118;pldnΔ1), and neuronal pldn overexpression (pldn-OE; c155-Gal4/Y;UAS-pldn-3xflag/+), which reveals a band running at 19 kDa, the predicted molecular weight of Drosophila Pldn. This band (indicated by arrowhead) is absent in pldnΔ1 and increased in pldn-OE. Anti-α-tubulin immunoblot was used as loading control. D, Representative images of third-instar larval NMJs from wild-type and pldnΔ1 mutants immunostained for Pldn (green) and the neuronal microtubule marker Futsch (magenta). The neuronal membrane is immunolabeled with anti-HRP (blue). E, Magnified images of area 1 and area 2 (F) marked in D, exhibiting a high degree of colocalization between Pldn and Futsch. G, Representative images of third-instar larval muscle immunostained for Pldn (green) and F-actin (phalloidin; magenta), showing Pldn localization to the muscle Z band.
- Figure 2.
Synaptic growth and structure is unperturbed in pallidin mutants. Representative images of muscle 6/7 NMJs from wild type (w1118) (A) and pldnΔ1 mutants (B) immunostained with anti-vGlut (synaptic vesicle marker; green), anti-HRP (white). Below, wild-type and pldnΔ1 NMJs on muscle 4 immunostained with anti-BRP (active zone marker; green) and GluRIII (postsynaptic glutamate receptor marker; magenta). No significant differences are observed in bouton number (C), BRP density (D), BRP number/NMJ (E), or HRP area (F) in wild-type (n = 12), pldnΔ1 (n = 12), and pldnΔ1/Df (w1118;pldnΔ1/Df(3L)BSC675; n = 10). p > 0.05; one-way ANOVA for all parameters.
- Figure 3.
Pallidin stability is dependent on dysbindin and blos1. A, Representative images of Pldn immunostaining (green) in NMJs of wild type, pldnΔ1, dysb1 (w1118;dysb1), dysb+pldn-OE (w1118;OK6/UAS-pldn-3xflag;dysb1), and blos1ex2 (w1118;blos1ex2) mutants. Inset, neuronal membrane (HRP; white). B, Quantification of Pldn signal intensity, normalized to HRP intensity, for the indicated genotypes (n = 13-20). C, Immunoblot analysis of adult head lysates probed for Pldn in wild type, pldnΔ1, dysb1, blos1ex2. D, Quantification of Pldn immunoblot intensity normalized to α-tubulin for the genotypes indicated (n = 3). E, Representative images of Venus-Dysb immunostaining (green) in NMJs of wt+venus-dysb-OE (w1118;OK6/+;UAS-venus-dysb/+) and pldn+venus-dysb-OE (w1118;OK6/+;UAS-venus-dysb, pldnΔ1/pldnΔ1) . The intensity of Venus-Dysb in pldn+venus-dysb-OE (1.74 ± 0.11 a.u., n = 15) is significantly increased compared with that of wt+venus-dysb-OE (0.91 ± 0.09 a.u., n = 9); p < 0.001; Student’s t test.
- Figure 4.
Baseline synaptic transmission is normal in pallidin mutants. A, Representative electrophysiological traces (EPSP and mEPSP traces) from wild-type and pldnΔ1 mutant synapses. B, Quantal content was determined across a range of extracellular calcium concentrations for wild-type and pldnΔ1 synapses. No significant difference in the slope of the line, indicating the apparent calcium cooperativity of synaptic transmission, was observed. No significant differences were observed in the EPSP amplitude (C), mEPSP frequency (D), mEPSP amplitude (E), asynchronous release (assayed by determining the mEPSP frequency within the 2 s immediately after EPSP stimulation) (F), or probability of release measured by failure analysis (assayed by % EPSP failure in 0.1mM Ca2+) (G) in wild-type (n = 10), pldnΔ1 (n = 10), and pldnΔ1/Df (n = 10) mutant synapses. H, Representative EPSC traces (top) and cumulative EPSC amplitudes (bottom) using two electrode voltage clamp evoked by 60-Hz stimulation (60 stimuli) in wild-type and pldnΔ1 mutant synapses. No significant differences were observed in the estimated readily releasable synaptic vesicle pool (RRP) between wild type (n = 7) and pldnΔ1 (n = 9) (I). p > 0.05; one-way ANOVA for all parameters.
- Figure 5.
pallidin mutants retain the capacity to express presynaptic homeostatic potentiation. A, Representative EPSP and mEPSP traces from wild-type and pldnΔ1 mutant synapses after PhTx application (20 µM). B, Normalized mEPSP amplitude and quantal content values of wild-type (n = 10) and pldnΔ1 (n = 12) mutants following PhTx application. Data are normalized to values of each genotype in the absence of PhTx. No deficit in acute presynaptic homeostatic potentiation was observed in pldnΔ1 mutants. C, Representative EPSP and mEPSP traces from GluRIIA (w1118;GluRIIAsp16) and GluRIIA;pldnΔ1 (w1118;GluRIIAsp16;pldnΔ1) mutant synapses. D, Normalized mEPSP amplitude and quantal content values of GluRIIA (n = 18) and GluRIIA;pldnΔ1 (n = 8). Data are normalized to wild-type values. No deficit in chronic presynaptic homeostatic potentiation was observed in pldnΔ1 mutants. E, Absolute values of mEPSP amplitude, EPSP amplitude (F), and quantal content (G) from the normalized data in B and D for the indicated genotypes.
- Figure 6.
BLOC-1 mutants fail to sustain neurotransmitter release during high-frequency stimulation. A, Increased rate of depletion and slowed recovery of the synaptic vesicle pool is observed under high-frequency stimulation in pldnΔ1 mutants (n = 15; p < 0.01; Student’s t test) compared with wild type (n = 11). Presynaptic overexpression of pallidin in pldnΔ1 mutants (pldn+pldn-OE: w1118;OK6-Gal4/UAS-pldn;pldnΔ1; n = 12) significantly slows the rate of depletion and increases the rate of recovery. Synapses were stimulated at 10 Hz in 2 mM extracellular calcium for 10 min, then allowed to recover, taking a test pulse at 0.2 Hz for the following 10 min. EPSP amplitudes for each time point were binned for 2 s, normalized to prestimulus amplitudes, and plotted as a function of time. B, A similar increase in depletion and slowing of recovery is observed in dysb1 (n = 9; p < 0.05; Student’s t test) and blos1ex2 mutants (n = 13; p < 0.05; Student’s t test). C, Both overexpression of dysb in pldnΔ1 mutants (pldn+dysb-OE: w1118;OK6-Gal4/UAS-3xflag-dysb;pldnΔ1, n = 12) and overexpression of pldn in dysb1 mutants (dysb+pldn-OE: w1118;OK6-Gal4/UAS-pldn-3xflag;dysb1, n = 12) fail to rescue the increased rundown during high-frequency stimulation. D, Determination of the total releasable synaptic vesicle pool. Control (shits1, n = 6), pldnΔ1 (shits1;pldnΔ1, n = 5), and dysb1 (shits1;dysb1, n = 5) mutants were stimulated at 10 Hz in 2 mM calcium at 32°C to deplete the total releasable synaptic vesicle pool. EPSP amplitudes at each time point were plotted as a percentage of the starting EPSP amplitude. E, No significant difference was observed in the total quanta released between the three genotypes. p > 0.05; one-way ANOVA.
- Figure 7.
Activity-dependent loss of FYVE-positive synaptic endosomes in pallidin mutants. A, GFP-2xFYVE puncta are observed in synapses from control (w1118;OK6-Gal4/UAS-GFP-myc-2xFYVE) and pldnΔ1 mutants (w1118;OK6-Gal4/UAS-GFP-myc-2xFYVE; pldnΔ1) at rest and following a 5-min incubation in 90 mM KCl (high K+). Similar GFP-2xFYVE density and intensity are observed in controls before and after stimulation, whereas pldnΔ1 NMJs have reduced GFP-2xFYVE density and intensity following stimulation. Quantification of GFP-2xFYVE density (B), size (C), and intensity (D) in wild-type (n = 13), pldnΔ1 (n = 8), and dysb1 mutants (w1118;OK6-Gal4/UAS-GFP-myc-2xFYVE; dysb1; n = 9) at rest and following high K+ stimulation. *p < 0.05; ** p < 0.01; ***p < 0.001; paired Student’s t test.
- Figure 8.
Activity-dependent accumulation of tubular endosomal structures in pallidin mutants. A, Representative electron micrographs of NMJs at rest in wild-type, pldnΔ1, and dysb1 mutants. B, Increased tubular endosomal structures are observed in BLOC-1 mutants following incubation in high K+ (90 mM K+, 5 min), while rarely observed in controls. Tubular endosomes (white arrows) and cisternal endosomes (black arrows) are noted. Quantification of the density of synaptic vesicles (C), cisternal endosomes (D), and tubular endosomes (E) at rest and following high K+ stimulation in wild type (n = 16 rest and high K+), pldnΔ1 (n = 13 rest and n = 28 high K+), and dysb1 (n = 7 rest and n = 28 high K+). Note that synaptic vesicle and cisternal endosome densities are reduced, while the tubular endosome density is increased in pldnΔ1 and dysb1 mutants following stimulation. F, Three-dimensional serial EM reconstruction of tubular endosomal structures (red) near synaptic vesicles (yellow), demonstrating they are not continuous with the plasma membrane (green). *p < 0.05; **p < 0.01; ***p < 0.001; Student’s t test.
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