Experimental Study of the Molecular Basis for Chromatin Motion in Live Cells and Model Systems

在活细胞的细胞核中，含有dna的染色质的微小结构波动为蛋白质结合提供了短暂的机会，从而构成了基因表达的初始化学步骤。因此，描述活细胞细胞核内染色质的小规模运动是很重要的，其最终目的是能够实时观察体内转录活性。为了实现这一点，驻波显微镜和双光子模式光漂白的组合被用来获得高空间灵敏度的平移运动在活细胞内。使用该方法，可以测量小于100 nm的扩散位移，并在活细胞中观察到用荧光团Hoechst 33342标记的核内DNA的运动。目标是将介观100纳米运动的量与特定纳米级生化过程（如组蛋白结合和解结合）的存在联系起来，这对于确定DNA的转录可用性很重要。为了将两种类型的动力学联系起来，将开发双通道扩散实验来并行测量染色质和gfp标记的组蛋白的动力学。此外，还将评估其他环境因素的影响，如乙酰化和其他类型核蛋白的存在。最后，将测量染色质运动对核内其他大分子溶质扩散的影响。这将通过使用惰性探针和测量它们的扩散率作为染色质构象自由的函数来完成。通过在各种条件下研究活细胞和分离细胞核中的染色质动力学，应该有可能弥合分子水平核小体动力学的体外研究与介观长度尺度上染色质扩散的体内研究之间的差距。这个项目的目标是使研究人员能够看到转录过程中染色质和蛋白质的运动；这种能力将大大增加他们对基因转录及其调控的理解。为了使这项研究能够向公众开放，将创建一个便携式演示，使单个巨大的DNA分子在课堂环境中可视化，目的是向大学和高中的初级理科学生介绍实验生物物理研究。该外联项目将在首席研究员的指导下，由一组理科生组成的团队进行，该项目涉及建造一台专用荧光显微镜。这项研究由数学和物理科学理事会的物理司和多学科活动办公室以及生物科学理事会的分子和细胞生物科学司共同资助。

In the nucleus of a live cell, small structural fluctuations in the DNA-containing chromatin provide transient opportunities for protein binding, and thus comprise the initial chemical steps in gene expression. It is therefore important to characterize the small-scale motions of chromatin inside the nuclei of live cells, with the ultimate aim of being able to observe in vivo transcriptional activity in real time. To accomplish this, a combination of standing wave microscopy and two-photon patterned photobleaching is employed to attain high spatial sensitivity to translational motion inside living cells. Using this method, diffusive displacements of less than 100 nm can be measured, and the motion of intranuclear DNA labeled with the fluorophore Hoechst 33342 has been observed in live cells. The goal is to correlate the amount of mesoscopic, 100 nm motion with the presence of specific nanoscale biochemical processes like histone binding and unbinding, which are important for determining the transcriptional availability of DNA. To relate the two types of dynamics, a two channel diffusion experiment will be developed to measure the dynamics of chromatin and GFP-labeled histone proteins in parallel. In addition, the effects of other environmental factors like acetylation and the presence of other types of nuclear proteins will be assessed. Finally, the effects of chromatin motion on the diffusion of other macromolecular solutes in the nucleus will be measured. This will be done by using inert probes and measuring their diffusivity as a function of chromatin conformational freedom. By studying chromatin dynamics in both live cells and isolated nuclei under a variety of conditions, it should be possible to bridge the gap between in vitro studies of molecular-level nucleosome dynamics and in vivo studies of chromatin diffusion on mesoscopic lengthscales. This project has as its goal enabling investigators to see the movements of chromatin and proteins during transcription; this ability should greatly increase their understanding of gene transcription and its regulation. In order to make the research accessible to the general public, a portable demonstration will be created that will allow the visualization of single, giant DNA molecules in a classroom environment, with the aim of introducing beginning science students to experimental biophysical research, both at the college and high school levels. The outreach project, which involves the construction of a specialized fluorescence microscope, will be carried out by a team of undergraduate science majors under the supervision of the principal investigator. This research is being jointly funded by the Physics Division and the Office of Multidiscipinary Activities in the Mathematical and Physical Sciences Directorate, and by the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.