Neumann Sylvia, Campbell George E, Szpankowski Lukasz, Goldstein Lawrence S B, Encalada Sandra E
Department of Molecular and Experimental Medicine, Dorris Neuroscience Center, The Scripps Research Institute.
Department of Cellular and Molecular Medicine, University of California San Diego; Department of Bioengineering, University of California San Diego.
J Vis Exp. 2014 Oct 30(92):e52029. doi: 10.3791/52029.
Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate ("map") the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. "Cargo mapping" consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to "map" them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.
要理解分子马达如何协调其活动以在神经元内运输囊泡货物，需要在单个囊泡水平上对马达/货物关联进行定量分析。本实验方案的目标是使用定量荧光显微镜将活货物的移动位置和方向与同一货物相关联的马达的组成和相对数量进行关联（“映射”）。“货物映射”包括对在微流控装置上培养的轴突中移动的荧光标记货物进行实时成像，然后在记录实时移动过程中进行化学固定，随后用抗马达抗体对同一轴突区域进行免疫荧光（IF）染色。通过将亚像素位置坐标分配给马达和货物通道，通过将高斯函数拟合到表示单个荧光点源的衍射极限点扩散函数，来评估货物与其相关马达之间的共定位。随后将固定的货物和马达图像叠加到货物移动图上，以将它们“映射”到其跟踪轨迹。该实验方案的优势在于结合了实时和IF数据，既能记录活细胞中囊泡货物的运输，又能确定与这些完全相同的囊泡相关的马达。这项技术克服了以前使用生化方法确定纯化的异质大量囊泡群体的平均马达组成的挑战，因为这些方法无法揭示单个移动货物上的组成。此外，该实验方案可适用于分析其他细胞类型中的其他运输和/或 trafficking 途径，以将单个细胞内结构的移动与其蛋白质组成相关联。该实验方案的局限性在于，由于培养的原代神经元转染效率低，通量相对较低，且高分辨率成像的视野有限。未来的应用可能包括增加表达荧光标记货物的神经元数量的方法。
