Observation of spatial propagation of amyloid assembly from single nuclei
Observation of spatial propagation of amyloid assembly from single nuclei
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DOI：
10.1073/pnas.1105555108
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发表时间：
2011-09-06
影响因子：
11.1
通讯作者：
Weitz, David A.
中科院分区：
文献类型：
--
作者：
Knowles, Tuomas P. J.;White, Duncan A.;Weitz, David A.
淀粉样蛋白生长的关键早期阶段，其中通常可溶的蛋白质转化为纤维状纳米结构，是具有挑战性的研究使用常规技术，但在许多常见的病理中涉及的蛋白质聚集现象是至关重要的。与所有的成核和生长现象一样，在传统的宏观实验中难以跟踪单个核，这些实验探测样品的整体时间演化，但由于它们将独立的随机事件混合到系综测量中，因此不会产生关于初级成核步骤的详细信息。为了克服这一限制，我们已经开发了微滴测定，使我们能够检测单个初级成核事件，并监测其随后的空间和时间演变，我们发现这两者都是由二次成核现象决定的。通过变形液滴高纵横比，我们可视化的实时传播波的蛋白质组装从离散的初级成核位点发出。我们表明，与经典的凝胶化现象相反，初级成核步骤的特征是对系统尺寸的显着依赖，并且丝状蛋白质自组装过程涉及聚集体的高度不均匀的空间分布。这些发现明显偏离了淀粉样蛋白生长的现状，并揭示了一种普遍的驱动力，源于限制，与生物质量控制机制一起，帮助蛋白质保持可溶性，因此在自然界中具有功能。
The crucial early stages of amyloid growth, in which normally soluble proteins are converted into fibrillar nanostructures, are challenging to study using conventional techniques yet are critical to the protein aggregation phenomena implicated in many common pathologies. As with all nucleation and growth phenomena, it is difficult to track individual nuclei in traditional macroscopic experiments, which probe the overall temporal evolution of the sample, but do not yield detailed information on the primary nucleation step as they mix independent stochastic events into an ensemble measurement. To overcome this limitation, we have developed microdroplet assays enabling us to detect single primary nucleation events and to monitor their subsequent spatial as well as temporal evolution, both of which we find to be determined by secondary nucleation phenomena. By deforming the droplets to high aspect ratio, we visualize in real-time propagating waves of protein assembly emanating from discrete primary nucleation sites. We show that, in contrast to classical gelation phenomena, the primary nucleation step is characterized by a striking dependence on system size, and the filamentous protein self-assembly process involves a highly nonuniform spatial distribution of aggregates. These findings deviate markedly from the current picture of amyloid growth and uncover a general driving force, originating from confinement, which, together with biological quality control mechanisms, helps proteins remain soluble and therefore functional in nature.