Development and Characterisation of Novel Microscopy Reagents to Study Hormone Action

The ubiquitous small molecule, cyclic nucleotide, cyclic 3’,5’-adenosine monophosphate (cAMP), mediates the intracellular actions of a wide range of cell surface receptors for hormones, neurotransmitters and growth factors, as well as sensory stimuli. cAMP signalling is now known to occur in discrete sub-cellular compartments, or cAMP nanodomains, and is now widely viewed as the paradigm for “compartmentalised” cellular signalling in biological systems. Exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular enzyme activated by cAMP that is involved in a wide variety of cellular and physiological processes, however reagents are lacking to study its association with nanodomains. For example, there is only one available EPAC1-specific antibody (5D3) that has been used for immunofluorescent staining to localise active EPAC1 in cells engineered to express EPAC1, but its uses in microscopy of non-engineered cells remain limited. To overcome the limits of antibodies, a number of alternatives have recently become available, including non-antibody scaffold proteins known as Affimers, which, unlike antibodies, do not require an animal host. Affimers are high affinity reagents that can be developed quickly with a number of advantages, including their small size (12-15 kDa), which makes super-resolution imaging a possibility, and structural stability even under the harshest conditions. I will describe the characterisation of five promising EPAC1-Affimer binder candidates with the aim of further developing the best Affimers as a tools for cell culture and microscopy.