Department of Chemistry, Stanford University, Stanford, California.
Department of Chemistry, Stanford University, Stanford, California.
Biophys J. 2018 Jul 17;115(2):230-241. doi: 10.1016/j.bpj.2018.05.026. Epub 2018 Jun 19.
One of the fundamental features that govern the cooperativity of multiple dyneins during cargo trafficking in cells is the spatial distribution of these dyneins on the cargo. Geometric considerations and recent experiments indicate that clustered distributions of dyneins are required for effective cooperation on micron-sized cargos. However, very little is known about the spatial distribution of dyneins and their cooperativity on smaller cargos, such as vesicles or endosomes <200 nm in size, which are not amenable to conventional immunostaining and optical trapping methods. In this work, we present evidence that dyneins can dynamically be clustered on endosomes in response to load. Using a darkfield imaging assay, we measured the repeated stalls and detachments of retrograde axonal endosomes under load with <10 nm localization accuracy at imaging rates up to 1 kHz for over a timescale of minutes. A three-dimensional stochastic model was used to simulate the endosome motility under load to gain insights on the mechanochemical properties and spatial distribution of dyneins on axonal endosomes. Our results indicate that 1) the distribution of dyneins on endosomes is fluid enough to support dynamic clustering under load and 2) the detachment kinetics of dynein on endosomes differs significantly from the in vitro measurements possibly due to an increase in the unitary stall force of dynein on endosomes.
在细胞内货物运输过程中，控制多个动力蛋白协同作用的基本特征之一是这些动力蛋白在货物上的空间分布。几何考虑因素和最近的实验表明，对于微米大小的货物（如囊泡或内体），需要动力蛋白的簇状分布才能实现有效的协同作用。然而，对于尺寸小于 200nm 的较小货物（如囊泡或内体），动力蛋白的空间分布及其协同作用知之甚少，因为这些货物不适合传统的免疫染色和光阱方法。在这项工作中，我们提供了证据表明，动力蛋白可以在响应负载时动态地在内体上聚集。我们使用暗场成像测定法，以<10nm 的定位精度，在高达 1kHz 的成像速率下，对负载下逆行轴突内体的反复停顿和脱离进行了测量，测量时间超过几分钟。使用三维随机模型对内体在负载下的运动进行了模拟，以深入了解轴突内体上动力蛋白的机械化学特性和空间分布。我们的结果表明：1）内体上动力蛋白的分布具有足够的流动性，可以在负载下支持动态聚集；2）动力蛋白在内体上的脱离动力学与体外测量有很大的不同，这可能是由于内体上动力蛋白的单位停顿力增加所致。
