Abstract
Most single-molecule fluorescence experiments are performed in vitro, using tightly controlled conditions and well-defined concentrations of few interacting components. However, to understand biological processes as they occur in vivo while maintaining the extra information provided by single-molecule detection, we need to perform single-molecule experiments in cellular contexts, and in particular, inside living cells.
Towards this goal, we have developed general localization-based super-resolution imaging methods (based on photo-activated localization microscopy, PALM) to study the subcellular localization, diffusion properties, abundance and clustering of DNA-binding proteins in bacterial cells. We have also developed simple electroporation methods for delivering fluorescent biomolecules (such as DNA, RNA, and proteins) in living bacteria and performing single-molecule tracking and FRET studies in the bacterial cytoplasm; these novel methods allow us, for the first time, to see conformations and conformational changes within single proteins and DNA molecules inside living bacteria.
I will discuss examples of using single-molecule tracking to understanding the subcellular localization and intracellular mobility of proteins interacting with DNA and RNA substrates, focusing mainly on the mechanisms of transcription by the Escherichia coli RNA polymerase. I will also present examples of using single-molecule FRET to study the conformations of promoter-DNA fragments as they are being processed in vivo, and to provide strong evidence for the presence of extensive pausing during transcription initiation.
Biography
After completing a degree in Chemistry at the Aristotelian University of Thessaloniki (Greece), Achilles Kapanidis obtained a Master’s in Food Science and a Doctorate in Biological Chemistry, both at Rutgers University (New Jersey, USA). After holding research scientist positions in single-molecule biophysics at Berkeley and UCLA, he started a research group as a senior lecturer at Oxford University in 2005, and became of Professor of Biological Physics in 2013; Prof Kapanidis has also been a fellow of the European Research Council since 2011.
Prof Kapanidis is currently leading a group of physical and biological scientists, known as the “Gene Machines” group. The group studies biological machinery involved in gene expression, maintenance, and regulation, focusing mainly on gene transcription, DNA replication, and DNA recombination. The main tool of the group is single-molecule fluorescence microscopy, a technique that measures nanometre distances and molecular interactions in real time; we also use the same method to perform super-resolution imaging and single-molecule tracking inside living cells. The work of the group is multidisciplinary, combining optics/photonics, imaging, biochemistry, molecular biology, modelling, and signal processing. Prof Kapanidis has also been pursuing compact single-molecule imaging since 2006, a project that culminated with the development of the NanoImager platform