Labeling cellular proteins for multi-scale microscopy and other applications

Abstract The Beatty group's research program is investigating how cellular proteins organize and interact in space and time. We use the principles of chemical biology to create new technologies for labeling and imaging proteins with an innovative set of reporters. Recent advances in imaging instrumentation and computational analysis have created new opportunities for examining the molecular basis of disease with exceptional detail. Researchers can now interrogate features ranging in size from single molecules (e.g., drugs or proteins) to whole organisms, uncovering how proteins organize to create living systems. The central obstacle that has limited progress in such studies is the shortage of methods for labeling proteins for fluorescence microscopy (FM), electron microscopy (EM), or correlative light and EM (CLEM). As a result, multi-color imaging studies most often rely on immunolabeling— a method with many drawbacks. Imaging across size dimensions, termed “multiscale microscopy”, would benefit from new genetic tags for labeling proteins with bright organic fluorophores (for FM), electron-dense nanoparticles (for EM), or other chemical reporters. Ideal tags would be small, specific, and biocompatible. The Beatty group has successfully tackled this challenge. In the last 5 years, our team created new genetic tags named versatile interacting peptide (VIP) tags. VIP tags have the high affinity and specificity of an antibody, but are an order of magnitude smaller in size. A small tag (4.3-6.2 kDa) reduces the impact on protein structure or function, particularly when compared to 27 kDa fluorescent proteins (e.g., GFP). VIP tags enable the effortless switching from conventional FM to high-resolution imaging, including EM, without changing the genetic tag. We now propose to build upon this concept and further develop this technology. With the support of NIGMS, we will expand the set of VIP tags to enable the imaging of multiple proteins at the same time. We will add a new class of genetic tags that use novel dimerization motifs to facilitate multi-color, multiscale microscopy. We will use new tags in optimized, user-friendly workflows for imaging receptors across size scales and platforms, including FM, EM, and CLEM (i.e., multiscale microscopy). Throughout our work, new tags and methods will be validated through in-depth studies of two receptors that control iron uptake: transferrin receptor 1 (TfR1) and transferrin receptor 2 (TfR2). We will make new discoveries on TfR2's function, binding interactions, and trafficking by labeling receptors with environmental sensors, catalysts (e.g., for proximity-based labeling), nanoparticles, and other small molecule reporters.