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

在活细胞的核中，含有DNA的染色质的微小结构波动为蛋白质结合提供了瞬时机会，从而构成了基因表达的初始化学步骤。因此，表征染色质在活细胞核内的小范围运动是很重要的，最终目的是能够实时观察体内的转录活动。为了实现这一点，采用驻波显微镜和双光子图案化光漂白相结合的方法，以获得对活细胞内平移运动的高空间敏感性。利用这种方法，可以测量小于10 0 nm的扩散位移，并观察到用荧光载体Hoechst 33342标记的核内DNA在活细胞中的运动。我们的目标是将介观100 nm运动量与特定纳米级生化过程的存在联系起来，如组蛋白结合和解结合，这对确定DNA的转录可获得性很重要。为了将这两种类型的动力学联系起来，将开发一个双通道扩散实验来平行测量染色质和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其他大分子溶质在核内扩散的影响。这将通过使用惰性探针并测量它们的扩散率作为染色质构象自由的函数来完成。通过研究各种条件下活细胞和分离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和中观长度尺度的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染色质和蛋白质在转录过程中的运动；这种能力应该会大大增加他们对基因转录及其调控的理解。为了让公众能够接触到这项研究，将创建一个便携式演示，允许在课堂环境中可视化单个巨大的DNA分子，目的是向初学理科的学生介绍大学和高中层面的实验生物物理研究。这个外展项目涉及建造一个专门的荧光显微镜，将由一个本科理科专业的团队在首席研究员的监督下进行。这项研究由数学和物理科学局的物理司和多学科活动办公室以及生物科学局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.