Fast and parallel nanoscale three-dimensional tracking of heterogeneous mammalian chromatin dynamics.
Fast and parallel nanoscale three-dimensional tracking of heterogeneous mammalian chromatin dynamics.
复制标题
异质哺乳动物染色质动力学的快速并行纳米三维追踪。
DOI：
10.1091/mbc.e21-10-0514
复制
发表时间：
2022-05-15
影响因子：
3.3
通讯作者：
Moerner, W. E.
中科院分区：
文献类型：
--
作者：
Gustavsson, Anna-Karin;Ghosh, Rajarshi P.;Petrov, Petar N.;Liphardt, Jan T.;Moerner, W. E.
染色质组织和动力学对于基因调控至关重要。在这项工作中，我们提出了一种方法，快速和并行的三维（3D）跟踪多个染色体位点的选择超过数千帧的各种时间尺度。我们通过开发和结合荧光和可降解纳米抗体阵列，工程点扩散函数和光片照明实现了这一点。结果是温和的活细胞3D跟踪，在整个哺乳动物细胞核中具有出色的时空分辨率。对样品漂移和核翻译的校正促进了对染色质动力学的准确长期跟踪。我们演示了快速动态（50 Hz）和时间尺度延伸到几个小时的跟踪，我们发现细胞之间的大异质性和轴向方向的动态明显的各向异性。我们进一步量化了抑制肌动蛋白聚合对动力学的影响，并发现在这种处理后，表观扩散系数D* 和异常扩散指数α总体增加，并在3D中过渡到更各向同性的动力学。我们认为，在未来，我们的方法将使研究人员能够更好地了解染色质动力学以及它在疾病进展期间和细胞功能扰动后如何改变。
Chromatin organization and dynamics are critical for gene regulation. In this work we present a methodology for fast and parallel three-dimensional (3D) tracking of multiple chromosomal loci of choice over many thousands of frames on various timescales. We achieved this by developing and combining fluorogenic and replenishable nanobody arrays, engineered point spread functions, and light sheet illumination. The result is gentle live-cell 3D tracking with excellent spatiotemporal resolution throughout the mammalian cell nucleus. Correction for both sample drift and nuclear translation facilitated accurate long-term tracking of the chromatin dynamics. We demonstrate tracking both of fast dynamics (50 Hz) and over timescales extending to several hours, and we find both large heterogeneity between cells and apparent anisotropy in the dynamics in the axial direction. We further quantify the effect of inhibiting actin polymerization on the dynamics and find an overall increase in both the apparent diffusion coefficient D* and anomalous diffusion exponent α and a transition to more-isotropic dynamics in 3D after such treatment. We think that in the future our methodology will allow researchers to obtain a better fundamental understanding of chromatin dynamics and how it is altered during disease progression and after perturbations of cellular function.