Tetanus toxin binds specifically to motor neurons at the neuromuscular junction. There, it is internalized into vesicular carriers undergoing fast retrograde transport to the spinal cord. Despite the importance of this axonal transport pathway in health and disease, its molecular and biophysical characterization is presently lacking. We sought to fill this gap by determining the pH regulation of this compartment in living motor neurons using a chimera of the tetanus toxin binding fragment (TeNT HC) and a pH-sensitive variant of the green fluorescent protein (ratiometric pHluorin). We have demonstrated that moving retrograde carriers display a narrow range of neutral pH values, which is kept constant during transport. Stationary TeNT HC-positive organelles instead exhibit a wide spectrum of pH values, ranging from acidic to neutral. This distinct pH regulation is due to a differential targeting of the vacuolar (H+) ATPase, which is not present on moving TeNT HC compartments. Accordingly, inhibition of the vacuolar (H+) ATPase under conditions that completely abolish the intracellular accumulation of acidotrophic dyes does not affect axonal retrograde transport of TeNT HC. However, a functional vacuolar (H+) ATPase is required for early steps of TeNT HC trafficking following endocytosis, and it is localized to axonal vesicles containing TeNT HC. Altogether, these findings indicate that the vacuolar (H+ ATPase plays a specific role in early sorting events directing TeNT HC to axonal carriers but not in their subsequent progression along the retrograde transport route, which escapes acidification and targeting to degradative organelles.
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