Exploring gene transcription mechanisms using single-molecule fluorescence imaging in vitro and in vivo by Professor Achillefs Kapanidis, senior lecturer and a Professor of Biological Physics at the University of Oxford, UK
As part of the Imaging ONEWORLD series, the focus of these lectures is on microscopy and image analysis methods and how to apply these to your research. Almost all aspects of imaging such as sample preparation, labelling strategies, experimental workflows, ‘how-to’ image and analyse, as well as facilitating collaborations and inspiring new scientific ideas will be covered. Speakers will be available for questions and answers. The organisers, core facility staff from the University of Cambridge, Gurdon Institute, MRC-LMB and the ICR/Royal Marsden Trust are also able to continue the discussion and provide advice on your imaging projects.
Alex heads the Imaging Facility at the Gurdon Institute, which includes a variety of microscopy techniques including confocal, high throughput and deconvolution. He is keen to raise the level of microscopy understanding and application, and runs and takes part in various microscopy courses.
Stefanie Reichelt, PhD has been head of the light microscopy facility at the CRUK Cambridge Institute. The core provides state-of-the-art imaging resources, training courses for scientists and students and develop new imaging systems as well as user-friendly analysis and acquisition tools for specific research applications. Stefanie is now Public Engagement Manager for the Biomedical Schools and teaches academically at Cambridge University, in scientific workshops and out-reach events. (http://cargocollective.com/StefanieReichelt)
Dr Alessandro Esposito obtained a PhD in Biophysics in 2006 working at the University of Utrecht and the European Neuroscience Institute in Goettingen for which he was awarded the ‘Sergio Ciani’ award by the Italian Society of Pure and Applied Biophysics. At the University of Cambridge, he then developed novel analytical tools contributing to redefining models of red blood cells homeostasis infected by P. falciparum (malaria). In recognition of his early work, in 2009 Alessandro was awarded a Life Science Interface fellowship by the EPSRC to establish foster the development of heavily multiplexed biochemical imaging. Soon after he moved to the MRC Cancer Unit where he lead the ‘Systems Microscopy initiative’ and retrained in cancer biology. During these years, Alessandro’s work developed into two research streams: i) the study of cellular responses to DNA damage and mutations in signalling pathways and ii) the innovation of biochemical imaging technologies. His team contributed to revealing the vast cell-to-cell variability in stress responses of genetically identical cells, a feature of biological systems that hinder the efficacy of disease management and therapeutic efficacy. Since 2019, Alessandro leads a transdisciplinary research programme at the MRC Cancer Unit in Cambridge devoted to understanding how DNA damage and mutations in KRAS derange homeostatic programmes leading to cancer. His group combines multi-omics data with single-cell biochemical imaging techniques aiming to achieve a deeper understanding of cancer phenotypes during the earliest stages of carcinogenesis, with particular attention to cell-to-cell variability of non-genetic origin and cell-to-cell communication.
Kirti Prakash is a computer scientist by training (Bachelors and Masters degree) but a biologist at heart (PhD degree). Kirti aspires to be an inventor and develop new imaging tools for cell biology and neuroscience. Kirti did his Masters in Computer Science from Aalto University (Finland) and PhD in Biology from Heidelberg University (Germany). During his PhD, he developed a new method to image DNA which led to the first high-resolution images of the epigenetic landscape of meiotic chromosomes and mechanisms behind chromosome condensation. The doctoral research earned him several awards including Springer Best PhD Thesis Prize. After his PhD, he did a couple of postdocs at Carnegie Institution for Science (USA) and University of Cambridge (UK). The primary highlights of his research here were laser-free superresolution microscopy and development of a high-content imaging pipeline to quantify single-cell gene expression. Formerly at the National Physical Laboratory (NPL), and currently working at the Institute for Cancer Research (ICR) and Royal Marsden Trust, he is working on microscope development and image analysis.
Single-molecule studies offer unprecedented and direct access to biologically important heterogeneity and dynamics, and provide dynamic views of biological machines at work; this holds especially true for reactions inside the complex biological milieu of living cells. During the past few years, we have developed and used a wide variety of in vitro and in vivo single-molecule fluorescence methods (single-molecule FRET, super-resolution imaging, single-particle tracking) to answer long-standing questions in gene transcription and DNA repair. Here, I will discuss examples of applications of such methods to unravel the mechanisms of bacterial gene transcription. I will first discuss our studies using single-molecule FRET and unwinding-induced fluorescence enhancement studies to elucidate the intricate sequence of conformational changes that allow RNA polymerase to open the double-stranded promoter DNA for transcription initiation. I will then discuss examples of our in vivo single-molecule work inside living bacteria, focusing on the exploration of the interplay between chromosome organization, gene expression, and bacterial growth.
Senior lecturer and Professor of Biological , Oxford UniversityAchillefs Kapanidis studied Chemistry at the Aristotelian University of Thessaloniki (Greece) and completed his MSc in Food Chemistry and his PhD in Biological Chemistry at Rutgers University (USA). After holding research scientist positions in single-molecule biophysics at the Lawrence Berkeley National Laboratory (Berkeley, California) and at the University of California at Los Angeles (UCLA), he became a senior lecturer at Oxford University in 2005, and a Professor of Biological Physics in 2013; Prof Kapanidis has also been a grant holder of prestigious ERC grants and is currently a Wellcome Trust Investigator.