We propose to refine and exploit powerful new markers and labeling systems to visualize multiple proteins or other biomolecules by electron microscopy (EM), correlated with light microscopy (LM). EM is one of the most powerful techniques to see cell structures below optical resolution, but has suffered from lack of generally applicable genetically encoded labels until our recent development of new EM-compatible markers such as miniSOG, a small flavoprotein that will do for EM what Green Fluorescent Protein did for LM, and APEX, an engineered ascorbate peroxidase. We have developed split-miniSOG, two fragments that do nothing separately, but when brought back together reversibly regenerate miniSOG and its fluorescence and photooxidative capability. Our newly developed split-miniSOG complementation system allows us to visualize intermolecular interactions at high resolution by EM. We have developed new strategies and techniques to improve the acquisition of element-specific analytical maps in transmission EM to achieve what we refer to as multicolor EM. This technology allows distinct EM- level labeling of multiple species with a different lanthanide element that is separately imaged by electron energy-loss spectrometry (EELS) and displayed in a distinct pseudocolor. Just as multicolor fluorescence has been vital to understanding many cellular functions at optical resolution, we anticipate multicolor EM will be valuable at finer resolution. Our overall goals are to expand and improve EM-compatible reporters, `molecular painting' chemistry, and new instrumentation to improve the resolution, sensitivity, and specificity with which multiple proteins or other biomolecules can be imaged by EM.
We aim to obtain a genetically encoded far-red or near-infrared diaminobenzidine (DAB) photooxidizer analogous to miniSOG but excited at significantly longer wavelengths to facilitate multispecies labeling at high resolution. We will develop controlled living polymerization as an alternative to photooxidative amplification using modern methods such as ATRP, ROMP, or RAFT applied to fixed cells and tissue, to generate lanthanide-containing polymers of defined length and morphology at desired cellular targets. As test cases, these techniques will be applied to fundamental biological problems such as the spatial organization of the genome in the nucleus and aggregation of specified proteins involved in neurodegenerative diseases. We have chosen these biological processes because they are diverse, engage outstanding collaborators, and have great biomedical importance. Ultimately, the combination of photooxidizing, peroxidase-based, and nonphotochemical amplifying systems will give cell and molecular biologists a rich palette for EM, comparable to small-molecule fluorophores plus fluorescent proteins for optical microscopy.

Public Health Relevance

We propose to refine and exploit powerful new markers and labeling systems to directly visualize and identify multiple proteins or other biomolecules by electron microscopy (EM), correlated with light microscopy (LM). These techniques will make it possible to investigate the spatial organization of the genome in the nucleus and aggregation of specified proteins involved in neurodegenerative diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM086197-09A1
Application #
9382800
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Sammak, Paul J
Project Start
2008-09-30
Project End
2021-04-30
Budget Start
2017-08-17
Budget End
2018-04-30
Support Year
9
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of California, San Diego
Department
Pharmacology
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Tsang, Tin Ki; Bushong, Eric A; Boassa, Daniela et al. (2018) High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues. Elife 7:
Han, Shuo; Udeshi, Namrata D; Deerinck, Thomas J et al. (2017) Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells. Cell Chem Biol 24:404-414
Petersen, Mark A; Ryu, Jae Kyu; Chang, Kae-Jiun et al. (2017) Fibrinogen Activates BMP Signaling in Oligodendrocyte Progenitor Cells and Inhibits Remyelination after Vascular Damage. Neuron 96:1003-1012.e7
Hammerling, Babette C; Najor, Rita H; Cortez, Melissa Q et al. (2017) A Rab5 endosomal pathway mediates Parkin-dependent mitochondrial clearance. Nat Commun 8:14050
Rodriguez, Erik A; Campbell, Robert E; Lin, John Y et al. (2017) The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins. Trends Biochem Sci 42:111-129
Goo, Marisa S; Sancho, Laura; Slepak, Natalia et al. (2017) Activity-dependent trafficking of lysosomes in dendrites and dendritic spines. J Cell Biol 216:2499-2513
Sastri, Mira; Darshi, Manjula; Mackey, Mason et al. (2017) Sub-mitochondrial localization of the genetic-tagged mitochondrial intermembrane space-bridging components Mic19, Mic60 and Sam50. J Cell Sci 130:3248-3260
Martell, Jeffrey D; Deerinck, Thomas J; Lam, Stephanie S et al. (2017) Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells. Nat Protoc 12:1792-1816
Rodriguez, Erik A; Tran, Geraldine N; Gross, Larry A et al. (2016) A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat Methods 13:763-9
Loh, Ken H; Stawski, Philipp S; Draycott, Austin S et al. (2016) Proteomic Analysis of Unbounded Cellular Compartments: Synaptic Clefts. Cell 166:1295-1307.e21

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