Simon has a BSc in Biochemistry from Hertiot-Watt University (Edinburgh) and a PhD from the University of East Anglia (Norwich). Simon studied for his PhD at the John Innes Centre in Norwich under the supervision of Prof. J. Allan Downie, investigating the role of calcium signalling during legume symbiosis. It was during this time that Simon first used a confocal microscope, sparking an interest in microscopy and imaging technology.
Following his PhD, Simon went to work as a postdoc in Pete Cullen's lab in the Department of Biochemistry at Bristol University, investigating Ras GTPase-activating proteins. These studies required the use of various microscopy systems and cemented Simon’s passion for biological imaging. Simon moved to the Babraham Institute in 2004 where he established the Institute’s core Imaging Facility.
The Imaging Facility now provides state-of-the-art microscopy services essential for the delivery of Institute science and is an important Babraham Research Campus resource supporting the commercial research community.
We developed a 50-plex imaging protocol using the MACSima platform to characterize the microarchitecture of germinal centers in secondary lymphoid tissues. This workflow combines tissue processing, automated cyclic imaging, image preprocessing, and a new analysis method that detects morphological changes in irregularly shaped stromal cells.
Peptidylarginine deiminase IV (PADI4, PAD4) deregulation promotes the development of autoimmunity, cancer, atherosclerosis and age-related tissue fibrosis. PADI4 additionally mediates immune responses and cellular reprogramming, although the full extent of its physiological roles is unexplored. Despite detailed molecular knowledge of PADI4 activation in vitro, we lack understanding of its regulation within cells, largely due to a lack of appropriate systems and tools. Here, we develop and apply a set of potent and selective PADI4 modulators. Using the mRNA-display-based RaPID system, we screen >10 cyclic peptides for high-affinity, conformation-selective binders. We report PADI4_3, a cell-active inhibitor specific for the active conformation of PADI4; PADI4_7, an inert binder, which we functionalise for the isolation and study of cellular PADI4; and PADI4_11, a cell-active PADI4 activator. Structural studies with PADI4_11 reveal an allosteric binding mode that may reflect the mechanism that promotes cellular PADI4 activation. This work contributes to our understanding of PADI4 regulation and provides a toolkit for the study and modulation of PADI4 across (patho)physiological contexts.
CDS enzymes (CDS1 and 2 in mammals) convert phosphatidic acid (PA) to CDP-DG, an essential intermediate in the de novo synthesis of PI. Genetic deletion of CDS2 in primary mouse macrophages resulted in only modest changes in the steady-state levels of major phospholipid species, including PI, but substantial increases in several species of PA, CDP-DG, DG and TG. Stable isotope labelling experiments employing both 13C6- and 13C6D7-glucose revealed loss of CDS2 resulted in a minimal reduction in the rate of de novo PI synthesis but a substantial increase in the rate of de novo PA synthesis from G3P, derived from DHAP via glycolysis. This increased synthesis of PA provides a potential explanation for normal basal PI synthesis in the face of reduced CDS capacity (via increased provision of substrate to CDS1) and increased synthesis of DG and TG (via increased provision of substrate to LIPINs). However, under conditions of sustained GPCR-stimulation of PLC, CDS2-deficient macrophages were unable to maintain enhanced rates of PI synthesis via the 'PI cycle', leading to a substantial loss of PI. CDS2-deficient macrophages also exhibited significant defects in calcium homeostasis which were unrelated to the activation of PLC and thus probably an indirect effect of increased basal PA. These experiments reveal that an important homeostatic response in mammalian cells to a reduction in CDS capacity is increased de novo synthesis of PA, likely related to maintaining normal levels of PI, and provides a new interpretation of previous work describing pleiotropic effects of CDS2 deletion on lipid metabolism/signalling.