A Genetically Encoded Reporter for Real-Time Imaging of Cofilin-Actin Rods in Living Neurons: e83609

• Published in: PloS one Vol. 8; no. 12
• Main Authors: Mi, Jianjie, Shaw, Alisa E, Pak, Chi W, Walsh, Keifer P, Minamide, Laurie S, Bernstein, Barbara W, Kuhn, Thomas B, Bamburg, James R
• Format: Journal Article
• Language: English
• Published: 01.12.2013
• ISSN: 1932-6203
• DOI: 10.1371/journal.pone.0083609

Filament bundles (rods) of cofilin and actin (1:1) form in neurites of stressed neurons where they inhibit synaptic function. Live-cell imaging of rod formation is hampered by the fact that overexpression of a chimera of wild type cofilin with a fluorescent protein causes formation of spontaneous and persistent rods, which is exacerbated by the photostress of imaging. The study of rod induction in living cells calls for a rod reporter that does not cause spontaneous rods. From a study in which single cofilin surface residues were mutated, we identified a mutant, cofilinR21Q, which when fused with monomeric Red Fluorescent Protein (mRFP) and expressed several fold above endogenous cofilin, does not induce spontaneous rods even during the photostress of imaging. CofilinR21Q-mRFP only incorporates into rods when they form from endogenous proteins in stressed cells. In neurons, cofilinR21Q-mRFP reports on rods formed from endogenous cofilin and induced by all modes tested thus far. Rods have a half-life of 30-60 min upon removal of the inducer. Vesicle transport in neurites is arrested upon treatments that form rods and recovers as rods disappear. CofilinR21Q-mRFP is a genetically encoded rod reporter that is useful in live cell imaging studies of induced rod formation, including rod dynamics, and kinetics of rod elimination.