Tracking vesicular transport in neurons

描述(由申请人提供)：了解突触传递对于了解健康和疾病的神经基础是必不可少的。突触连接的发育和功能在很大程度上依赖于囊泡的靶向和转运机制。这包括突触小泡的运输以及突触受体的运输。囊泡转运具有高度的特异性，但其特异性的分子机制还不是很清楚。在这个项目中，将开发一种方法，允许在更长的时间段内跟踪单个分子。过去，人们通过过度表达GFP标记的蛋白来研究囊泡的运输和靶向。这种方法的一个缺点是，运输的物种必须高度过度表达。另一个缺点是荧光团的漂白。更重要的是，要更好地理解回收利用，就需要在更长的时间内以高精度跟踪单分子。这里将使用一种使用寿命长的荧光纳米颗粒的方法。结合特定蛋白质的荧光颗粒将被显微注射到完整视网膜的视网膜无长突细胞中，并将使用多光子显微镜进行长时间的跟踪。

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.