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Super-resolution microscopy is often associated with observing tiny details in cells that are fixed, or in other words, frozen in time and in space. These methods are extremely useful as they provide insights that would not be available otherwise. With that said, the main limitation of these approaches is that they rely on fixed cells, and as such, cannot capture dynamic processes.
One field that can be addressed with dynamic single-molecule approaches is protein-protein binding, for example, in the context of drug discovery. Among the most common targets for drug discovery is the family of G-Protein Coupled Receptors, GPCR, which are cell surface receptors that are involved in a multitude of communication processes, from endocrine activity through immune signaling to sensing the environment around us using virtually all of our senses. It is no wonder that they are attractive drug targets. Briefly, the binding of the ligand to the extracellular domain of the receptor initiates a series of events involving changes to protein conformations and to protein-protein association and dissociation, eventually leading to a cellular signal transduction. Probing these events is a key step in the development of assays to manipulate GPCR activity. One possibility for probing is to fluorescently label two nearby GPCR subunits to generate a FRET signal, which will be lost should a drug successfully disrupt the subunit association. Single-molecule FRET (smFRET) allows scientists to accurately perform such assays at a single cell resolution. This can be particularly useful, for example, when trying to discern heterogeneity of different cells and even different receptors on the cell surface1,2. Additionally, planning a single-cell drug discovery experiment enables scientists to use a very small format with very low quantities of the drug at question. This is particularly helpful in early stages of the development, when it is unclear what compounds are effective.
Single-molecule techniques can also be used to study the kinetics of biomolecule movement around the cell and between organelles. Single-particle tracking (SPT) is the most famous among these, allowing scientists to monitor the movement of labeled molecules in live cells and gain extensive information on the biophysics behind the movement. For instance, it can be used to describe molecular mechanisms of membrane channels and transporters. Two recent, yet, very different examples include the mechanism behind the extended translocon in the bacterial Type 9 Secretion Secretion System3 and the mechanism behind the regulation of Aquaporin-4 (AQP4) circulation of the cerebrospinal fluid4.
This image shows PALM-SPT imaging of the mRNA binding protein Hfq in E.coli bacteria taken with the Nanoimager6.
Fluorophore Localization Imaging with Photobleaching (FLImP) builds upon the concepts of SPT and uses sequential bleaching of individual fluorophores to determine protein conformation and structural changes in protein complexes. The group that developed FLImP has been using it to study the growth factor receptor EGFR and explain how mutations to the gene are affecting its activity and its sensitivity to drugs5. This approach can also be used to study other dynamic processes such as response to different environmental cues or stimuli.
In essence, cells are ever-changing and they constantly adapt their behavior according to the environmental changes around them. Probing these dynamic processes at a single-molecule level can shed light on the molecular mechanisms behind what we know as cell biology, as well as behind what we are yet to discover.