Roux Sylvie, Colasante Cesare, Saint Cloment Cécile, Barbier Julien, Curie Thomas, Girard Emmanuelle, Molgó Jordi, Brûlet Philippe
Unité d'Embryologie Moléculaire, Institut Pasteur, Unités de Recherche Associées 2578, Centre National de la Recherche Scientifique, 25 rue du Dr Roux, 75724 Paris, France. sroux@nbcm..cnrs-gif.fr
Mol Cell Neurosci. 2005 Sep;30(1):79-89. doi: 10.1016/j.mcn.2005.05.008.
The distribution, dynamics, internalization, and retrograde axonal traffic of a fusion protein composed of green fluorescent protein (GFP) and the atoxic C-terminal fragment of tetanus toxin (TTC) were studied after its in vivo injection. Confocal microscopy and immunogold electron microscopy revealed that the fusion protein (GFP-TTC) rapidly clustered in motor nerve terminals of the neuromuscular junction. Clathrin-coated pits, and axolemma infoldings located between active zones appeared to be involved in the internalization of the fusion protein. Biochemical analysis of detergent-extracted neuromuscular preparations showed that the GFP-TTC fusion protein was associated with lipid microdomains. We suggest that GFP-TTC clustering in these lipid microdomains favors the recruitment of other proteins involved in its endocytosis and internalization in motor nerve terminals. During its retrograde trafficking, GFP-TTC accumulated in different axonal compartments than those used by cholera toxin B-subunit suggesting that these two proteins are transported by different pathways and cargos.
在体内注射由绿色荧光蛋白（GFP）和破伤风毒素无毒C末端片段（TTC）组成的融合蛋白后，研究了其分布、动态变化、内化及逆行轴突运输。共聚焦显微镜和免疫金电子显微镜显示，融合蛋白（GFP-TTC）迅速聚集在神经肌肉接头的运动神经末梢。网格蛋白包被小窝以及位于活性区之间的轴膜内褶似乎参与了融合蛋白的内化过程。对去污剂提取的神经肌肉制剂进行生化分析表明，GFP-TTC融合蛋白与脂质微区相关。我们认为，GFP-TTC在这些脂质微区中的聚集有利于募集其他参与其在运动神经末梢内吞和内化的蛋白质。在逆行运输过程中，GFP-TTC在与霍乱毒素B亚基不同的轴突区室中积累，这表明这两种蛋白质通过不同的途径和货物进行运输。
