Split-miniSOG for Spatially Detecting Intracellular Protein-Protein Interactions by Correlated Light and Electron Microscopy

Daniela Boassa; Sakina P Lemieux; Varda Lev-Ram; Junru Hu; Qing Xiong; Sebastien Phan; Mason Mackey; Ranjan Ramachandra; Ryan Emily Peace; Stephen R Adams; Mark H Ellisman; John T Ngo

doi:10.1016/j.chembiol.2019.07.007

Split-miniSOG for Spatially Detecting Intracellular Protein-Protein Interactions by Correlated Light and Electron Microscopy

Cell Chem Biol. 2019 Oct 17;26(10):1407-1416.e5. doi: 10.1016/j.chembiol.2019.07.007. Epub 2019 Aug 1.

Authors

Affiliations

¹ Department of Neurosciences, National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address: dboassa@ucsd.edu.
² Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA.
³ Department of Neurosciences, National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093, USA.
⁴ Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, MA 02215, USA.
⁵ Department of Neurosciences, National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093, USA; Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA.
⁶ Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, MA 02215, USA. Electronic address: jtngo@bu.edu.

Abstract

A protein-fragment complementation assay (PCA) for detecting and localizing intracellular protein-protein interactions (PPIs) was built by bisection of miniSOG, a fluorescent flavoprotein derived from the light, oxygen, voltage (LOV)-2 domain of Arabidopsis phototropin. When brought together by interacting proteins, the fragments reconstitute a functional reporter that permits tagged protein complexes to be visualized by fluorescence light microscopy (LM), and then by standard as well as "multicolor" electron microscopy (EM) via the photooxidation of 3-3'-diaminobenzidine and its derivatives.

Keywords: LOV domain; electron microscopy; protein-protein interactions; split-fluorescent proteins.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

3,3'-Diaminobenzidine / chemistry
Arabidopsis / chemistry
Arabidopsis Proteins / chemistry*
Cells, Cultured
Flavoproteins / chemistry*
HEK293 Cells
HeLa Cells
Humans
Luminescent Proteins / chemistry*
Microscopy, Electron
Microscopy, Fluorescence
Oxidation-Reduction
Photochemical Processes
Protein Binding

Substances

Arabidopsis Proteins
Flavoproteins
Luminescent Proteins
3,3'-Diaminobenzidine

Abstract

Publication types

MeSH terms

Substances

Grants and funding