Tracking vesicular transport in neurons

描述（由申请人提供）：理解突触传递对于理解健康和疾病的神经元基础至关重要。突触连接的发育和功能在很大程度上取决于囊泡靶向和转运的机制。这包括突触囊泡的运输以及突触受体的运输。水疱转运具有高度特异性，但其特异性的分子机制尚不清楚。在这个项目中，将开发一种方法，可以在很长一段时间内跟踪单个分子。过去已经通过过表达gfp标记的蛋白来研究水泡运输和靶向。该方法的一个缺点是运输的物种必须高度过表达。另一个缺点是荧光团的漂白。更重要的是，要想更好地了解回收过程，就需要对单个分子进行长时间的高精度跟踪。在这里，一种方法将使用具有长寿命的荧光纳米颗粒。功能化的荧光颗粒结合特定的蛋白质将被微注射到完整视网膜中的视网膜无突细胞中，并将使用多光子显微镜进行长时间的跟踪观察。

DESCRIPTION (provided by applicant): Understanding synaptic tranmission is essential in understanding the neuronal basis of health and disease. Development and function of synaptic connections profoundly depend on the mechanisms of vesicular targeting and transport. This includes transport of synaptic vesicles as well as trafficking of synaptic receptors. Vesicular transport is highly specific, yet the molecular mechanisms of its specificity are not well understood. In this project a method will be developed that allows tracking of individual molecules over extended periods of time. Vesicular transport and targeting has in the past been studied by overexpressing GFP-labeled proteins. One disadvantage of the method is that the transported species must be highly overexpressed. Another disadvantage is the bleaching of the fluorophores. More importantly a better understanding of the recycling would require tracking of single molecules over extended periods of time with high accuracy. Here an approach will be used using fluorescent nanoparticles with long lifetime. Fluorescent particles functionalized to bind specific proteins will be microinjected into retinal amacrine cells in the intact retina and will be followed over extended periods of time using multiphoton microscopy.