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.
Highlights
In all eukaryotic cells, proteins of the actin-depolymerizing factor (ADF)/cofilin family are key regulators of actin dynamics and actomyosin contractility [1,2,3]
To identify a genetically encoded marker for visualizing rods in live cells, we first examined the possibility of expressing monomeric Red Fluorescent Protein chimeras of wild type cofilin
We determined if limiting the expression levels of cofilin-wtmRFP would reduce numbers of neurons in which spontaneous rods form and/or reduce the numbers of rods in these neurons
Summary
Proteins of the actin-depolymerizing factor (ADF)/cofilin family are key regulators of actin dynamics and actomyosin contractility [1,2,3]. Cofilin is the prominent isoform expressed in mammalian neurons [4]. Under conditions of cellular stress, cofilin forms complexes with actin that can alter cell function [7]. Hippocampal neurons, subjected to energy stress (ATP depletion, excitotoxic glutamate, hypoxia/ischemia) [8], oxidative stress (peroxide, NO) [8,9], extracellular ATP [10], and soluble forms of the Alzheimer’s disease b-amyloid peptides (Ab) [11,12], form within their neurites cofilin-actin (1:1) filament bundles called rods [13]. Cofilin-actin rods can grow to occlude completely the neurite in which they form, causing microtubule loss [8] and synaptic dysfunction [15,16]. Rods are observed in brains from human Alzheimer disease subjects and may even represent a common mechanism compromising synapse function in other neurodegenerative diseases
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
