The most prominent feature in elp3 mutant

The most prominent feature in elp3 mutant HTS assay boutons is the occurrence of sizable T bars with large protrusions that extend into the cytoplasm ( Figures 4D–4G, arrows). Quantification of T bar top lengths (platforms) in controls indicates that they never exceed 300 nm, while in elp3 mutants we observe more than 20% of the T bars with a platform that is larger than 300 nm and up to 400 nm in length ( Figures 4D–4G, arrowheads; Figure 4H).

Thus, TEM indicates an increase in T bar size in elp3 mutants, and these data are consistent with the extensive “tentacles” extending into the cytoplasm in elp3 mutants that we observe in electron tomograms of elp3 mutant boutons ( Figures 4I–4O, arrows). In line with these data, we measure a concomitant increase in the number of synaptic vesicles that are in direct contact with the BKM120 datasheet T bar ( Figure 4P). Hence, the elaboration of the dense projections of the T bar in elp3 mutants results in an increased number of T bar-tethered vesicles. To determine functional consequences associated with the loss of elp3 at the NMJ, we measured synaptic transmission using two electrode voltage clamp. The average excitatory junctional current (EJC) amplitude

in 0.45 mM calcium is significantly increased in elp3 mutants ( Figures 5A and 5B), and also current clamp recordings indicate increased excitatory junctional potential amplitudes

in elp3 mutants Urease compared to controls ( Figure S4). To determine quantal content, we measured spontaneous vesicle fusion (mEJC) and quantified the quantal amplitude. As shown in Figures 5C–5F, mEJC amplitudes are significantly increased in elp3 mutants compared to controls, while the mEJC frequency trends toward an increase, but this is not statistically significant. The quantal content (in 0.45 mM calcium) also trends toward an increase but is not significantly different in controls and mutants (EJC/mEJC; controls, 45.3 ± 3.5 quanta; elp3Δ3/Δ4, 54.6 ± 6.7 quanta). Increased mEJC amplitude can be caused by larger synaptic vesicles that harbor more neurotransmitter or by a more elaborate postsynaptic glutamate receptor field. Given that synaptic vesicle size distribution in elp3 mutants is not different from controls, we labeled elp3 mutant NMJs with anti-GluRIIA8B4D2 antibodies and with anti-GluRIII/IIC antibodies that each recognize different glutamate receptor subunits ( DiAntonio et al., 1999 and Marrus et al., 2004). While we did not observe a difference in GluRIII/IIC labeling between elp3 mutants and controls ( Figures 5G, 5H, and 5M), the GluRIIA labeling in elp3 mutants is increased compared to controls, and this defect is rescued by a genomic fragment that harbors wild-type elp3 ( Figures 5I–5M).

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