Supplemental Figure 1. Neuronal expression of UAS-n-Syb-pH does not affect
function or morphology at the NMJ. (A) Excitatory postsynaptic potentials (EPSP)
recorded at muscle 6 in segment A3 in wild type (wt) and animals expressing
UAS-n-Syb-pH (n-Syb-pH) under the control of a neuronal GAL4 driver (elaV3E1GAL4). There is no difference in the average EPSP amplitude or the
spontaneous miniature EPSP (mEPSP) amplitudes between these genotypes.
(B) A representative train of EPSPs recorded from an n-Syb-pH expressing
synapse (10 Hz for 3 min, 2.0 mM Ca2+). EPSP amplitudes are constant
throughout the stimulus train, consistent with normal vesicle recycling (n = 5).
Inset: representative EPSPs at two different times during the stimulus train. (C)
Endocytosis is normal in n-Syb-pH animals. Synapses at muscle 6/7 in wild type
(n = 5) and n-Syb-pH (n = 5) animals were bathed in 10 M FM4-64 and
stimulated for 1 min at 30 Hz in HL3 + 0.5 mM Ca2+, then washed in HL3 + 0 mM
Ca2+ for 5 minutes, and imaged (load). Synapses were stimulated again (30 Hz,
1 min in HL3 + 0.5 mM Ca2+) and re-imaged (unload). Mean FM4-64
fluorescence was measured for each condition. There is no significant difference
in FM4-64 fluorescence intensity between genotypes. (D) Morphological analysis
at n-Syb-pH expressing synapses. Synaptic bouton number was counted at
muscles 6 and 7 in segments A2–A5 in wild type (n = 14) and n-Syb-pH (n = 19)
animals. There is no significant difference in bouton numbers.
Supplemental Figure 2. Synapto-pHluorins exhibit pH-sensitivity at the
Drosophila NMJ. (A) Acidic saline (black bar) quenches the fluorescence of
surface n-Syb-pH expression. (B) Membrane-permeable NH4Cl (black bar)
increases fluorescence intensity of n-Syb-pH in the presynaptic terminal,
revealing previously quenched n-Syb-pH at synaptic vesicles. Representative
trials of individual synapses are shown. (C) Syt I4C-bound FlAsH bleaches
more rapidly than n-Syb-pH. Values are calculated as the change in
fluorescence relative to background (∆F/F) and normalized to the first data point.
Syt I4C expressing synapses (closed circles) were labeled with FlAsH and
illuminated for 120 seconds. FlAsH fluorescence bleaches to near background
levels during this time. N-Syb-pH expressing synapses (open circles) were
imaged identically for 120 seconds in the absence of stimulation. This
fluorescence represents the basal surface expression of n-Syb-pH that has been
observed in other systems17-19. During the 120 seconds of illumination, the
surface n-Syb-pH fluorescence bleaches by less than 1%. (D) All genetic
backgrounds used throughout this study show the indistinguishable n-Syb-pH
recycling. Genotypes shown: genotype 1 = UAS-n-Syb-pH,elaV3E1 , genotype 2
= UAS-Syt4C,sytAD4/sytD27;UAS-n-Syb-pH/elaV3E1 , genotype 3 = w-,shits2;UASSyt4C,sytAD4/sytD27;UAS-n-Syb-pH/elaV3E1.
Supplemental Figure 3. Images demonstrating n-Syb-pH intensity changes at
the NMJ following the shift from the restrictive to the permissive temperature.
Synapses are from the same gentoype in the top panels (control, without FlAsH
ligand) and bottom panels (FlAsH, with FlAsH ligand). Scale represents arbitrary
fluorescence units.
Supplemental Movie 1. Time lapse movie showing changes in n-Syb-pH fluorescence
intensity for the synapse shown in Figure 1a, b.