Dimensional Reduction for Single-Molecule Imaging of DNA and Nucleosome Condensation by Polyamines, HP1α and Ki-67.
Dimensional Reduction for Single-Molecule Imaging of DNA and Nucleosome Condensation by Polyamines, HP1α and Ki-67.
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DOI：
10.1021/acs.jpcb.2c07011
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发表时间：
2023-03-09
影响因子：
3.3
通讯作者：
Ha, Taekjip
中科院分区：
文献类型：
--
作者：
Benning, Nils A.;Kaestel-Hansen, Jacob;Rashid, Fahad;Park, Sangwoo;Urteaga, Raquel Merino;Liao, Ting-Wei;Hao, Jingzhou;Berger, James M.;Hatzakis, Nikos S.;Ha, Taekjip
大分子组织成离散的无膜区室。越来越多的证据表明，核小体以及DNA本身可以进行聚类或浓缩，以调节基因组活动。目前的体外缩合研究提供了对缩合物的物理性质的深入了解，例如表面张力和扩散。然而，需要提供多组分缩合的复杂动力学研究所需的分辨率的方法。在这里，我们使用支持的脂质双层平台串联全内反射显微镜观察DNA和核小体的二维运动在单分子分辨率。这种三维研究的降维使我们能够观察到这些早期冷凝物在生理冷凝剂存在下的初始冷凝事件和溶解。使用多胺，我们观察到，最初的缩合发生在几分钟的时间尺度上，而溶解发生在几秒钟内电荷反转后。多胺化合价，DNA长度，和GC含量影响阈值多胺浓度缩合。基于蛋白质的核小体凝聚剂HP 1 α和Ki-67的凝聚阈值浓度比基于电荷的凝聚剂低得多，Ki-67是最有效的，需要低至100 pM的核小体凝聚。此外，即使在检测的最高浓度HP 1 α和Ki-67下，我们也未观察到冷凝物溶解。我们还介绍了一种双色成像方案，其中高密度的核小体标记在一种颜色被用来标定冷凝物的边界和相同的核小体的另一种颜色在低密度可以跟踪相对于Ki-67介导的冷凝后的边界。我们的平台应该能够在规定的物理化学条件下的染色质组分的凝聚动力学研究的单分子的最终决议。
Macromolecules organize themselves into discrete membrane-less compartments. Mounting evidence has suggested that nucleosomes as well as DNA itself can undergo clustering or condensation to regulate genomic activity. Current in vitro condensation studies provide insight into the physical properties of condensates, such as surface tension and diffusion. However, methods that provide the resolution needed for complex kinetic studies of multicomponent condensation are desired. Here, we use a supported lipid bilayer platform in tandem with total internal reflection microscopy to observe the two-dimensional movement of DNA and nucleosomes at the single-molecule resolution. This dimensional reduction from three-dimensional studies allows us to observe the initial condensation events and dissolution of these early condensates in the presence of physiological condensing agents. Using polyamines, we observed that the initial condensation happens on a time scale of minutes while dissolution occurs within seconds upon charge inversion. Polyamine valency, DNA length, and GC content affect the threshold polyamine concentration for condensation. Protein-based nucleosome condensing agents, HP1α and Ki-67, have much lower threshold concentrations for condensation than charge-based condensing agents, with Ki-67 being the most effective, requiring as low as 100 pM for nucleosome condensation. In addition, we did not observe condensate dissolution even at the highest concentrations of HP1α and Ki-67 tested. We also introduce a two-color imaging scheme where nucleosomes of high density labeled in one color are used to demarcate condensate boundaries and identical nucleosomes of another color at low density can be tracked relative to the boundaries after Ki-67-mediated condensation. Our platform should enable the ultimate resolution of single molecules in condensation dynamics studies of chromatin components under defined physicochemical conditions.
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DOI：
10.1083/jcb.201302044
发表时间：
2013-04-29
期刊：
The Journal of cell biology
影响因子：
--
作者：
Li YR;King OD;Shorter J;Gitler AD
通讯作者：
Gitler AD
影响因子：
16.6
作者：
Ambadipudi S;Biernat J;Riedel D;Mandelkow E;Zweckstetter M
通讯作者：
Zweckstetter M
影响因子：
4.6
作者：
Akhunzada, Muhammad Jan;D'Autilia, Francesca;Brancato, Giuseppe
通讯作者：
Brancato, Giuseppe
影响因子：
64.5
作者：
Feric M;Vaidya N;Harmon TS;Mitrea DM;Zhu L;Richardson TM;Kriwacki RW;Pappu RV;Brangwynne CP
通讯作者：
Brangwynne CP
影响因子：
64.8
作者：
Larson AG;Elnatan D;Keenen MM;Trnka MJ;Johnston JB;Burlingame AL;Agard DA;Redding S;Narlikar GJ
通讯作者：
Narlikar GJ