Sensing molecular dynamics in living cells

The process of secretion is central to all normal physiology, as it is one of the main ways that cells can communicate with each other over both short and long distances in the body. For example, neurotransmission, the process where nerve cells communicate a signal, is mediated by the secretion of a neurotransmitter (the chemical carrier of the signal) into the tiny space between nerves: this chemical carrier then transmits the impulse to the next nerve cell in the chain. Over long distances, cells communicate using chemical carriers called hormones. Hormonal communication underlies reproduction, appetite, respiration, fight-or-flight and sleep, amongst a variety of other essential physiological functions. The protein molecules that mediate both hormonal and neurotransmitter secretion are the same and over the last 40 years or so, biochemists have defined a complete catalogue of proteins involved in this process. The final trigger for secretion is almost always an increase of the calcium concentration near the secretory machinery in cells, mediated by ion channels. If any part of this process becomes mis-regulated, this leads variously to a number of diseases and conditions, including asthma, diabetes, epilepsy, growth and sleep disorders and hypertension. What is missing at this stage in most of cell biology is information describing the "wheres and whens": where are these molecules - not just for example on the surface of a cell, but how are the single protein molecules arranged, when do they interact, function, and how are molecules regulated dynamically?

Recently, tremendous advances have been made in microscopy and imaging spectroscopy. It has become possible to examine the precise locations of many 100000s of single molecules inside living cells. It is also possible to track the movements of these molecules, and measure where and when these molecules interact with one another. These approaches are still to be widely adopted, perhaps because to fully exploit them, substantial input from physicists and mathematicians is required. Combining these very advanced imaging approaches with electrophysiology (the study of the tiny electrical currents passed by single ion channels) presents another technical hurdle and requires the correct team of experts to be present in one place. This work, accelerating at Heriot Watt, employs advanced imaging techniques (eg fluorescence lifetime imaging, fluorescence correlation spectroscopy, single molecule localisation microscopies, total internal reflectance fluorescence microscopy) to visualise and quantify the localisations, dynamics, interactions and underlying spatio-temporal patterns of large cohorts of single molecules in the membranes of living cells. Specifically, the molecules thought to drive membrane fusion are studied here – the SNAREs and SM proteins – and in particular in neuroendocrine cells responsible for hormonal secretion. The techniques required for such studies are technically demanding and cutting-edge; my group were early adopters and developers. The massive datasets delivered by such approaches are extremely content rich; the input of physical and mathematical scientists as well as of engineers is essential to maximise the impact of such approaches as well as to extract the full amount of information from such complex datasets.

This talk will describe the pros and cons of some of these approaches and their use in studying the molecular dynamics that underpin secretion.