Bohnert Stephanie, Schiavo Giampietro
Molecular NeuroPathobiology Laboratory, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
J Biol Chem. 2005 Dec 23;280(51):42336-44. doi: 10.1074/jbc.M506750200. Epub 2005 Oct 18.
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.
破伤风毒素特异性结合神经肌肉接头处的运动神经元。在那里，它被内化到经历快速逆行运输至脊髓的囊泡载体中。尽管这种轴突运输途径在健康和疾病中都很重要，但目前缺乏对其分子和生物物理特性的描述。我们试图通过使用破伤风毒素结合片段（TeNT HC）与绿色荧光蛋白的pH敏感变体（比率型pHluorin）的嵌合体来确定活体运动神经元中该区室的pH调节，以填补这一空白。我们已经证明，逆行移动的载体显示出狭窄范围的中性pH值，在运输过程中保持恒定。而静止的TeNT HC阳性细胞器则表现出广泛的pH值范围，从酸性到中性。这种独特的pH调节是由于液泡（H+）ATP酶的靶向差异，在移动的TeNT HC区室中不存在这种酶。因此，在完全消除嗜酸性染料细胞内积累的条件下抑制液泡（H+）ATP酶，并不影响TeNT HC的轴突逆行运输。然而，内吞作用后TeNT HC运输的早期步骤需要功能性的液泡（H+）ATP酶，并且它定位于含有TeNT HC的轴突囊泡中。总之，这些发现表明，液泡（H+）ATP酶在将TeNT HC导向轴突载体而非其随后沿逆行运输途径的进展中发挥特定作用，逆行运输途径可避免酸化并靶向降解性细胞器。
