title: A genetically encoded reporter for real-time imaging of cofilin-actin rods in living neurons.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, and Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Jianjie
authors.initials: J
authors.lastname: Mi
authors.affiliation: Department of Biochemistry and Molecular Biology, and Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Alisa E
authors.initials: AE
authors.lastname: Shaw
authors.affiliation: Department of Biochemistry and Molecular Biology, and Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Chi W
authors.initials: CW
authors.lastname: Pak
authors.affiliation: Department of Biochemistry and Molecular Biology, and Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Keifer P
authors.initials: KP
authors.lastname: Walsh
authors.affiliation: Department of Biochemistry and Molecular Biology, and Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Laurie S
authors.initials: LS
authors.lastname: Minamide
authors.affiliation: Department of Biochemistry and Molecular Biology, and Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Barbara W
authors.initials: BW
authors.lastname: Bernstein
authors.affiliation: Department of Biochemistry and Molecular Biology, and Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado, United States of America ; Department of Chemistry and Biochemistry, University of Alaska, Fairbanks, Alaska, United States of America.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Kuhn
authors.affiliation: Department of Biochemistry and Molecular Biology, and Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: James R
authors.initials: JR
authors.lastname: Bamburg
mesh_terms: D051339:Actin Depolymerizing Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D045744:Cell Line, Tumor; D003199:Computer Systems; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D018374:LLC-PK1 Cells; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis; D013552:Swine; D013569:Synapses / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D051339:Actin Depolymerizing Factors; D000199:Actins; D008164:Luminescent Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein