Developing genetically encoded ultra-correlative tags for functional studies employing light and electron microscopy

A key biomedical goal is to understand how organelles and proteins cooperate to make organs work, in both 'normal' and diseased conditions, with the ultimate aim to improve our health. However, current methods to explore these structure-function relationships have a number of drawbacks. Not least amongst these limitations are the resolution limit set by standard electron microscopy approaches and the difficulty associated with correlating functional and structural data across tissue and molecular scale length. We propose to develop a genetically encoded ultra-correlative tag for multiplexing structural and functional analysis of labelled genes.
We will develop novel genetic tags based on heavy metal binding peptides detectable with electron microscopy. This method will give protein localisation precision an order of magnitude greater than established methods and mitigate many of the drawbacks associated with classic electron microscopy approaches.
Further, we will combine it with established light microscopy methods to create a 'dual tag' that enables both correlative light and electron microscopy (CLEM) structural assessments as well as live cell functional imaging on the same genes.
Finally, by integrating this new functional CLEM genetic tagging strategy using gene editing techniques we will achieve truly correlative molecular level resolution and provide quantitative structure-function information.
Importantly, this method is not limited to study the structure-function relationship of a single target protein, in fact using a wide array of compatible covalent labelling tags and specific metal binding peptides, multiple proteins can be simultaneously assessed with precision. The proposed approaches are readily applicable across different biological and disease relevant systems spanning single cell systems through to whole organism physiology.

We aim to develop an ultra-correlative tag detectable by super-resolution and electron microscopy (EM), achieving correlative information at the protein level, by combining a metal binding peptide for EM with a SNAP tag for STORM and verify it in cells.
Objective 1: Characterisation of silver binding peptide (AgBP) as a suitable tag for EM in mammalian cells
We have already identified AgBP as a suitable genetic tag when expressed in hek293T cells fused to laminB1, and will further characterise this by tagging differently distributed proteins in hek293T cells. Vectors will be transiently transfected into hek293T cells, GFP sorted and subjected to EM to confirm predicted localisation patterns. We will use CRISPR-Cas9 to directly tag the validated endogenous genes of interest.
Objective 2: Multiplexing capability
We will explore alternative heavy metal (HM) binding peptides for EM and use in combination with one another to achieve dual EM labeling. We have identified and synthesised a panel of published HM-BP for different metals. In principle we should be able to selectively tag one gene with one HM-BP and another with a second HM-BP (e.g. gold and silver). Elemental analysis combined with 3D EM would achieve tagged protein identification and distribution on selected organelles.
Objective 3: Functional physiological imaging
Correlative light and EM combine cellular fluorescent labeling with the ultrastructural information achieved by EM. Such techniques rely on the use of fluorescent indicators which typically have significant spectral overlap with common fluorescent proteins and labels used in IHC. We propose to obviate this difficulty by using the HM-BP in conjunction with covalent tags such as SNAP and CLIP. A covalent tagging strategy allows the use of fluorescent dyes which are proven to be compatible with super-resolution imaging and can be selected so as not to spectrally overlap with the e.g., calcium indicator, used to monitor cell signalling/function

The development of an ultra-correlative tag represents a much awaited opportunity in the structural and functional biology research community. All researchers interested in studying the relationship between two or more proteins will benefit from our new tagging method. It is important to point out that such a tag will be detectable with STORM microscopy allowing for functional studies as well as electron microscopy allowing for high resolution localisation of the protein of interest.
The first beneficiaries could be for example cardiac physiologists. An interesting problem they could address would be the relationship between the distribution of LTTC channels on the sarcolemma with respect to the distribution of the RyR on the sarcoplasmic reticulum and how this affect the calcium concentration in a cardiac cell. These data would generate a cascade of benefits: for example calcium flux modellers would be able to have calcium flux measurements with corresponding high precision localization of proteins involved in calcium handling, and will be able to produce more realistic models. A better knowledge of calcium handling could ultimately lead to improve drugs design and treatment.
Therefore, anyone with a functional biological question would benefit from our new technological approach: physiologist, modellers and pharmacologists to mention but a few.