| The nucleus is an organelle of central importance to the mammalian cell. However, our understanding of the organizations and dynamics of many nuclear structures and processes remains inadequate, largely due to the difficulty in probing them in situ, with single-molecule sensitivity as well as ultra-high resolutions in space and time. In this dissertation, we develop approaches to interrogate, through imaging and modeling, the spatio-temporal organizations and dynamics of two key nuclear processes: gene expression and DNA replication. We first describe a novel fluorescence imaging technique, named reflected light-sheet (RLS) microscopy, that is capable of detecting single molecules with superior signal-to-background ratio inside the mammalian nucleus. By selectively illuminating only a thin section of the nucleus using a light-sheet reflected off a miniature mirror, RLS microscopy combines the capabilities of 3D optical sectioning, fast imaging speed, and applicability to single, normal-sized adherent cells. As demonstration, we apply RLS microscopy to directly monitor the DNA binding dynamics and spatio-temporal colocalization of single mammalian transcription factor molecules in live cells. By measuring their diffusion constants, DNA-bound fraction, as well as in vivo residence times, we resolve three distinct modes of their interaction with genomic DNA.;Furthermore, we take advantage of the prowess of RLS illumination for super-resolution microscopy (SRM), attaining resolution improvements critical for resolving nuclear structures with high molecular density. Using RLS-SRM, we map the distribution of RNA polymerase II (RNAP II), the main workhorse of mammalian transcription, which has been proposed to heterogeneously cluster into spatially discrete foci termed "transcription factories". Leveraging on the photophysics of rhodamine-based dyes, we also develop an image analysis algorithm capable of accurately counting the copy number of RNAP II molecules in these foci. We found that majority of the foci originate from single RNAP II molecules, which exhibit no significant clustering within the length scale of the reported diameters of "transcription factories", arguing against the prevalent existence of such "factories" as previously believed. We also super-resolve in the mammalian nucleus individual DNA replication domains (RDs), and quantitatively characterize their physical morphology and propagation on a global scale. Our results support a spatio-temporal model for RD dynamics across different stages of S-phase, in which the progression of replicons along chromosomes as well as the nuclear lamina constrains the distribution of DNA synthesis sites and drives the spreading of RDs in specific spatial patterns.;Lastly, to better understand the catalytic mechanism of DNA replication at the molecular level, we simulate the dynamics of DNA polymerase, whose catalytic action is accompanied by a large nucleotide-induced movement of its finger domain, using a Langevin-type Gaussian Network Model. Our model captures the induced conformational dynamics of the polymerase upon substrate binding, and reveals its close coupling to the advancement towards transition-state along the reaction coordinate. These results demonstrate the precise role of conformational dynamics in achieving catalysis of the polymerization reaction, and indicate that the mechanism for lowering the reaction barrier through conformational motion is encoded in the structural topology of DNA polymerase. Overall, the strategies developed in this dissertation pave the way for quantitative mapping and characterization of nuclear processes at unprecedented levels of detail, both in space and in time. |
