Professor Achillefs Kapanidis

Abstract:

Diseases caused by viruses represent a serious global health concern. In this thesis, I present novel methods for labelling, imaging and analysing virus particles. I also describe mathematical and computational models that can be used to describe biological structures, such as viruses. In studies of viral structure, pleomorphism, and articularly filamentous morphologies, are frequently overlooked.To characterise and better understand viral filaments, I developed and optimised an imaging protocol for individual virus particles that captured the heterogeneity of viral shape. I also produced analysis pipelines that took this image data to study the structure of viruses at the individual virion level in order to find an overall picture of the populations of virions that exist. These pipelines investigated virus size, patterning of surface proteins and the location of viral genome. I also demonstrated a novel virus labelling strategy that specifically labels virus that will aid in the future study of viral assembly and infection. I also used mathematical modelling to further understand the role of viral morphologies. I modelled virus particles as a pressure vessel to study engineering stress in relation to virus, bacteria, and cellular morphology. Together these advancements will further our ability to study viruses and our knowledge of virus structure

Abstract:

Bacterial transcription by RNA polymerase (RNAP) is spatially organized. RNAPs transcribing highly expressed genes locate in the nucleoid periphery, and form clusters in rich medium, with several studies linking RNAP clustering and transcription of rRNA (rrn). However, the nature of RNAP clusters and their association with rrn transcription remains unclear. Here we address these questions by using single-molecule tracking to monitor the subcellular distribution of mobile and immobile RNAP in strains with a heavily reduced number of chromosomal rrn operons (Δrrn strains). Strikingly, we find that the fraction of chromosome-associated RNAP (which is mainly engaged in transcription) is robust to deleting five or six of the seven chromosomal rrn operons. Spatial analysis in Δrrn strains showed substantial RNAP redistribution during moderate growth, with clustering increasing at cell endcaps, where the remaining rrn operons reside. These results support a model where RNAPs in Δrrn strains relocate to copies of the remaining rrn operons. In rich medium, Δrrn strains redistribute RNAP to minimize growth defects due to rrn deletions, with very high RNAP densities on rrn genes leading to genomic instability. Our study links RNAP clusters and rrn transcription, and offers insight into how bacteria maintain growth in the presence of only 1–2 rrn operons.

Abstract:

Although viral protein structure and replication mechanisms have been explored extensively with X-ray crystallography, cryo-electron microscopy, and population imaging studies, these methods are often not able to distinguish dynamic conformational changes in real time. Single-molecule fluorescence resonance energy transfer (smFRET) offers unique insights into interactions and states that may be missed in ensemble studies, such as nucleic acid or protein structure, and conformational transitions during folding, receptor–ligand interactions, and fusion. We discuss the application of smFRET to the study of viral protein conformational dynamics, with a particular focus on viral glycoprotein dynamics, viral helicases, proteins involved in HIV reverse transcription, and the influenza RNA polymerase. smFRET experiments have played a crucial role in deciphering conformational changes in these processes, emphasising the importance of smFRET as a tool to help elucidate the life cycle of viral pathogens and identify key anti-viral targets