It is possible to observe gene expression within single cells using a tetracycline inducible promoter for activation. Transcription can be observed by using a fluorescent fusion protein to bind nascent RNA. Ultimately, it is desirable to activate a reporter gene within a single cell with only photons. This is achieved by preparing a chemically altered transcription factor that is functionally unable to activate a reporter gene until it is exposed to photon excitation. We apply two-photon imaging to visualize tumor cells expressing a transgene and ultimately this approach will provide the means to activate a specific gene within a single cell within any tissue to ultimately observe its functional significance in situ.
"Structural analysis, single-molecule approaches, and biochemical characterization of RNAPII activity in reconstituted transcription systems have shed light on processes such as transcriptional translocation, pausing, arrest, and RNAPII backtracking at the level of individual polymerases (Arndt and Kane, 2003; Herbert et al., 2008; Kornberg, 2007). On the other hand, techniques such as chromatin immunoprecipitation (ChIP) and live cell imaging have provided insight into the average behavior of large populations of polymerases on active genes in vivo (Saunders et al., 2006; Singer et al., 2005; Struhl, 2007). However, the gap of knowledge between these extremes, including how RNAPII elongation complexes dynamically interact, has not yet been filled. "
[Show abstract][Hide abstract] ABSTRACT: Multiple RNA polymerase II (RNAPII) molecules can transcribe a gene simultaneously, but what happens when such polymerases collide--for example due to polymerase pausing or DNA damage? Here, RNAPII collision was characterized using a reconstituted system for simultaneous transcription by two polymerases. When progression of leading polymerase is obstructed, rear-end collision entails a transient state in which the elongation complexes interact, followed by substantial backtracking of trailing polymerase. Elongation complexes remain stable on DNA, with their activity and the integrity of transcription bubbles remaining intact. Subsequent TFIIS-stimulated transcript cleavage allows resumed forward translocation, resulting in trailing polymerase oscillating at the obstruction. Conversely, if leading polymerase is merely stalled at a pause site, collision and TFIIS cooperate to drive it through. We propose that dynamic interactions between RNAPII elongation complexes help regulate polymerase traffic and that their conformational flexibility buffers the effect of collisions with objects on DNA, thereby maintaining stability in the face of obstacles to transcription.
"The detailed view of gene expression dynamics arises from experiments typically performed in single living cells grown in tissue culture plates. Obviously, an important step in understanding the flow of gene expression is to study the dynamics within the context of the whole organism (Singer et al, 2005). This is a vital question since we do not yet know whether the dynamic behaviors we observe in tissue culture cells are of the same characteristics within tissues and living organisms, in which different types of cells are adjacent to each other and might be exchanging cell-to-cell signals. "
[Show abstract][Hide abstract] ABSTRACT: Molecular imaging in living cells or organisms now allows us to observe macromolecular assemblies with a time resolution sufficient to address cause-and-effect relationships on specific molecules. These emerging technologies have gained much interest from the scientific community since they have been able to reveal novel concepts in cell biology, thereby changing our vision of the cell. One main paradigm is that cells stochastically vary, thus implying that population analysis may be misleading. In fact, cells should be analyzed within time-resolved single-cell experiments rather than being compared to other cells within a population. Technological imaging developments as well as the stochastic events present in gene expression have been reviewed. Here, we discuss how the structural organization of the nucleus is revealed using noninvasive single-cell approaches, which ultimately lead to the resolution required for the analysis of highly controlled molecular processes taking place within live cells. We also describe the efforts being made towards physiological approaches within the context of living organisms.
The EMBO Journal 09/2006; 25(15):3469-79. DOI:10.1038/sj.emboj.7601226 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Modern imaging technology, now utilized in most biomedical research areas (bioimaging), enables the detection and visualization of biological processes at various levels of the molecule, organelle, cell, tissue, organ and/or whole body. In toxicologic pathology, the impact of modern imaging technology is becoming apparent from digital histopathology to novel molecular imaging for in vivo studies. This overview summarizes recent progresses in digital microscopy imaging and newly developed digital slide techniques. Applications of virtual microscopy imaging are discussed and compared to traditional optical microscopy reading. New generation digital pathology approaches, including automatic slide inspection, digital slide databases and image management are briefly introduced. Commonly used in vivo preclinical imaging technologies are also summarized. While most of these new imaging techniques are still undergoing rapid development, it is important that toxicologic pathologists embrace and utilize these technologies as advances occur.
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