Tetanus toxin is transported in a novel neuronal compartment characterized by a specialized pH regulation
Tetanus toxin is transported in a novel neuronal compartment characterized by a specialized pH regulation
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DOI：
10.1074/jbc.m506750200
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发表时间：
2005-12-23
影响因子：
4.8
通讯作者：
Schiavo, G
中科院分区：
文献类型：
--
作者：
Bohnert, S;Schiavo, G
破伤风毒素与神经肌肉交界处的运动神经元特异性结合。在那里，它被内化成囊泡载体，快速逆行运输到脊髓。尽管这种轴突运输途径在健康和疾病中很重要，但它的分子和生物物理特征目前尚不清楚。我们试图通过使用破伤风毒素结合片段(TeNTHC)的嵌合体和绿色荧光蛋白的pH敏感变体(比率PHluorin)来确定活的运动神经元中这个隔室的pH调节来填补这一空白。我们已经证明，逆行移动的载体显示的中性pH值范围很小，在运输过程中保持不变。固定的TeNTHC阳性细胞器表现出从酸性到中性的广泛的pH值范围。这种不同的pH调节是由于对液泡(H+)ATPase的不同靶向，这种靶向在移动的帐篷H-C隔间中不存在。因此，在完全消除嗜酸性染料在细胞内积累的条件下抑制液泡(H+)ATPase并不影响帐篷H-C的轴突逆行运输。然而，功能性空泡(H+)ATPase是内吞作用后帐篷H-C运输的早期步骤所必需的，它定位于含有帐篷H-C的轴突小泡。综上所述，这些发现表明，液泡(H+)ATPase在早期的分选活动中发挥着特定的作用，将TeNTHC导向轴突载体，但不是在它们随后沿着逆行运输路线进行的过程中，后者逃避酸化并靶向降解的细胞器。
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 (TeNTHC) 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 TeNTHC-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 H-C 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 H-C. However, a functional vacuolar (H+) ATPase is required for early steps of TeNT H-C trafficking following endocytosis, and it is localized to axonal vesicles containing TeNT H-C. Altogether, these findings indicate that the vacuolar (H+) ATPase plays a specific role in early sorting events directing TeNTHC to axonal carriers but not in their subsequent progression along the retrograde transport route, which escapes acidification and targeting to degradative organelles.