New Molecular Tools for Protein Detection and Dynamics in Electron Microscopy Daniela Boassa 1,2 , John T. Ngo 3 , Mason R. Mackey 1,2 , Ranjan Ramachandra 1,2 , Thomas J. Deerinck 1,2 , Mark H. Ellisman 1,2 and Stephen R. Adams 4* 1. Department of Neurosciences, University of California San Diego, La Jolla, CA, USA. 2. National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, CA, USA. 3. Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, MA USA. 4. Department of Pharmacology, University of California San Diego, La Jolla, CA, USA. * Corresponding author: sadams@ucsd.edu Genetically-encoded tags such as miniSOG and APEX have become valuable tools for marking protein localization by deposition of osmiophilic DAB for electron microscopy (EM) [1,2]. To visualize protein- protein interactions, we have now developed split miniSOG, analogous to the well-known fluorescent complementation assay with a split fluorescent protein but generating an EM-visible mark by photooxidation of DAB. We identified two fragments, N-terminal segment termed “mSOG1-94” (10.96kDa) and a 46 amino acid C-terminal polypeptide designated “mSOG-Jα95-140” (5.15 kDa) that when fused to interacting proteins enable binding of FMN, the endogenous fluorescent photosensitizer. We have demonstrated its reconstitution with leucine zipper domains of bFos and bJun, subunits of the AP-1 transcriptional complex that interact constitutively as nuclear heterodimers, and visualized neurotoxic assemblies of α-synuclein, a neuronal protein involved in Parkinson’s disease. We recently introduced a method for highlighting multiple specific proteins and distinguishing them by electron energy-loss spectroscopy (EELS) to give a version of color EM [3]. Briefly, proteins are labeled with antibodies, genetically-encoded tags (miniSOG or APEX) and localized precipitates of lanthanide (Ln) chelates of DAB are sequentially formed by orthogonal activation of the photosensitizers or peroxidases. Energy-filtered transmission EM (EFTEM) gives lanthanide elemental maps that can be overlaid on a conventional TEM to reveal the desired proteins localization. To increase the methods sensitivity, we have developed 2 nd generation DAB-chelates, that precipitate >4 times more lanthanide than their predecessors, with histone H2B nuclear and with mitochondrial matrix using Ce2-DAB. Additionally, we have devised a new strategy that involves precipitation of a DAB-alkyne followed by a reaction to a lanthanide-chelate azide. This bypasses the difficulty in precipitating polar DAB Ln chelates and expands our choice of known metal chelates, including potentially short polymers of them that would prevent DAB precipitation if attached before (photo)oxidation. These methods give greater metal deposition than our first-generation Ln-DAB2 enabling the collection of multi-tilt EELS tomograms and multi-spectral EELS imaging of the more intense ‘low loss’ spectral peaks that permit shorter acquisitions (less beam damage), greater signal (e.g. for tomograms) and potentially new multi-color modes for SEM. The use of peroxidases in LM and EM to generate localized deposition includes exogenous horse radish peroxidase (HRP) and more recently genetically-encoded ascorbate peroxidases, APEX. We have now adapted a small molecule peroxidase based on a ferric-macrocycle (Fe-TAML) to EM that is very small (M.W. ~500); about 100 times smaller than HRP and the size of a fluorophore molecule, but capable of generating an EM visible reaction product, and this is an advantage in terms of penetration into fixed cells and tissues for 3D labeling. To demonstrate its use, we used a synthesized azide derivative that can be 1042 doi:10.1017/S1431927619005944 Microsc. Microanal. 25 (Suppl 2), 2019 © Microscopy Society of America 2019 https://doi.org/10.1017/S1431927619005944 Downloaded from https://www.cambridge.org/core. IP address: 107.173.179.9, on 07 Aug 2019 at 10:13:53, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms.