Cellular machinery in situ by correlative microscopy

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Molecular machines in cells are essential for life. Malfunctions of these machines cause diseases such as cancer and dementia. Conversely, machinery blockage in pathogens can be used to tackle global health threats, including antibiotic resistance and parasitic diseases. While these machines operate at the atomic level, the consequences of their function and dysfunction manifest in cells and tissues that are many orders of magnitude larger and more complex than the machines themselves. Imaging capabilities at different biological scales including light and electron microscopies are therefore required to fully understand molecular machinery operation and function. Cryo-electron microscopy (cryo-EM) is a Nobel Prize-winning transformative technology that is essential for studying molecular machines. Cryo-EM samples are rapidly frozen using cryogenic liquids and examined at very low temperature. With this type of preparation, samples can be imaged in their natural, hydrated state. However, no information is available in cryo-EM data alone about the molecular identity of the imaged sample. Within complex cellular and tissue samples, the use of fluorescent labels observed using light microscopy allows individual components of interest to be localised. This means that specific regions of cell or tissue samples can be efficiently targeted for cryo-EM data collection. The ISMB is internationally recognised as a centre of excellence for cryo-EM. We wish to apply for funds for a new cryo-fluorescence microscope that enables high-precision fluorescence localisation in frozen samples to identify regions of interest to target with cryo-EM. The cryo-fluorescence microscope would allow seamless transfer of fragile samples between different imaging systems and would integrate localisation of regions of interest in both fluorescence and cryo-EM imaging modalities. Together with its technical capabilities, our planned purchase of a next-generation microscope will enhance throughput and facilitate non-expert access. Specific projects that will benefit from this new equipment include: 1) Studies of the molecular mechanisms of bacterial conjugative transfer, the main means by which antibiotic resistance genes spread among bacteria; 2) Studies of amyloid fibre assembly and disassembly that will reveal how the cell's quality control machinery handles protein misfolding associated with neurodegenerative disease; 3) Studies of the molecular machinery that controls the trafficking of cargo and metabolites through cells, which when disrupted can have detrimental physiological consequences including compromised cardiac function; 4) Studies of the structure and regulation of the neuronal microtubule cytoskeleton that will reveal how they adapt their dynamics during brain development and how this is disrupted in human diseases; 5) Studies of regulatory proteins which when disrupted by naturally occurring mutations, cause protein polymerisation that disrupts liver and lung cell function, resulting in cirrhosis and lung disease respectively.