Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic range of ∼10,000-fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by two orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a “waterfall” mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially restricted subnuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.

中文翻译：

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通过随机蛋白质标记可视化体内的长期单分子动力学

我们在活细胞中清晰地成像和跟踪单个分子的能力受到在分辨率极限范围内堆积多个拷贝的标记分子的限制。在这里，我们设计了一种通用的遗传策略来精确控制细胞中荧光标记分子的拷贝数。该系统的动态范围约为10,000倍，可以稀疏标记以不同丰度水平表达的蛋白质。结合光稳定的标签，该系统将自动单分子跟踪的持续时间延长了两个数量级。我们证明了在培养的神经元以及完整的斑马鱼中的突触囊泡动力学的长期成像。我们发现轴突初始节段利用“瀑布”机制通过促进顺行转运的持续性来控制突触小泡的转运极性。长期观察还发现，转录因子在空间受限的亚核区域的簇状结合位点之间跳跃，这表明核中的拓扑结构通过螯合机制塑造了局部基因的活性。因此，该策略极大地扩展了活细胞单分子测量的时空长度尺度，从而使新的实验能够定量地了解体内分子动力学的复杂控制。