We propose to refine and exploit powerful new genetically encoded labeling systems to visualize proteins 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 miniSOG, a small flavoprotein that will do for EM what Green Fluorescent Protein did for LM.
We aim to quantify the sensitivity and spatial resolution of miniSOG and to extend its applicability to low-temperature methods for sample preparation and imaging. Alternative genetically encoded labels will be developed and characterized to allow for two or more proteins of interest to be distinguished in a single EM image by electron energy loss spectroscopy of reaction deposits from oxidation of diaminobenzidine conjugated to different lanthanides. We have also developed reporters, based on the drug- controllable cis-acting protease from hepatitis C viral protease, to distinguish between old and newly synthesized copies of a genetically specified protein of interest. These fusion tags are visible by correlated LM and EM and will be applied to plasticity and disease-related synaptic proteins to reveal their localized appearance and turnover, initially in culture bt eventually in intact mammalian brain. Viral and Cre/lox modular targeting vectors will be created to make cell-type-selective expression of the above EM/LM tags robust and widely applicable to systems as complex as mouse models of disease and learning. We have chosen cell types and proteins important in liver fibrosis and synaptic plasticity as test cases because these biological processes are diverse, engage outstanding local collaborators, and have great biomedical importance. With collaborators we will investigate how hepatocytes, hepatic stellate cells, and endothelial cells change ultrastructural morphology and location of key proteins during fibrogenic injury. Another collaboration will focus on activity-induced changes in morphology and adhesion molecules at synapses in the amygdala during fear conditioning and memory consolidation. Such extension of these new tools into transgenic animals will make it possible to relate ultrastructural location and metabolic turnover of genetically specified proteins to whole- animal behavior and disease.

Public Health Relevance

We propose to refine and exploit powerful new genetically encoded labeling systems to directly visualize and identify proteins by electron microscopy (EM), correlated with light microscopy (LM). These techniques will make it possible to relate ultrastructural location and metabolic turnover of genetically specified proteins to whole-animal behavior and disease.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM086197-07
Application #
8705272
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Deatherage, James F
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Rodriguez, Erik A; Wang, Ye; Crisp, Jessica L et al. (2016) New Dioxaborolane Chemistry Enables [(18)F]-Positron-Emitting, Fluorescent [(18)F]-Multimodality Biomolecule Generation from the Solid Phase. Bioconjug Chem 27:1390-9
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
Martell, Jeffrey D; Yamagata, Masahito; Deerinck, Thomas J et al. (2016) A split horseradish peroxidase for the detection of intercellular protein-protein interactions and sensitive visualization of synapses. Nat Biotechnol 34:774-80
Adams, Stephen R; Mackey, Mason R; Ramachandra, Ranjan et al. (2016) Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species. Cell Chem Biol 23:1417-1427
Rodriguez, Erik A; Campbell, Robert E; Lin, John Y et al. (2016) The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins. Trends Biochem Sci :
Ngo, John T; Adams, Stephen R; Deerinck, Thomas J et al. (2016) Click-EM for imaging metabolically tagged nonprotein biomolecules. Nat Chem Biol 12:459-65
Ou, Horng D; Deerinck, Thomas J; Bushong, Eric et al. (2015) Visualizing viral protein structures in cells using genetic probes for correlated light and electron microscopy. Methods 90:39-48
Lam, Stephanie S; Martell, Jeffrey D; Kamer, Kimberli J et al. (2015) Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods 12:51-4
Palida, Sakina F; Butko, Margaret T; Ngo, John T et al. (2015) PKMζ, but not PKCλ, is rapidly synthesized and degraded at the neuronal synapse. J Neurosci 35:7736-49

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