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

<div><p>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.</p></div

Visualization of rod formation and disappearance in neurons.

<p>Panels from time lapse imaging of neurons expressing cofilin-wt-mRFP (A) or cofilinR21Q-mRFP (B, C). In (A), neurons expressing cofilin-wt-mRFP had spontaneous rods present before imaging and acquired more rods due to photostress during the 2 h of imaging at 30 s intervals. Under identical imaging conditions, neurons expressing cofilinR21Q-mRFP had few spontaneous rods before imaging and did not form new rods during imaging (B). When cofilinR21Q-mRFP expressing neurons were treated with Aβd/t, formation of induced rods could be followed (C) that disappeared following removal of the Aβd/t (image of washout (w/o) is at 60 min). Scale bars  = 10 μm. (D) Disappearance of cofilinR21Q-mRFP rods induced by 18 h Aβd/t-treatment in images taken at 4 min intervals after Aβd/t washout (filled triangles). Total area of 18 rods in three separate neurons is plotted vs time after washout as a percent of the area at washout. The half-life of rods following Aβd/t removal is ∼ 47 min. One rod, in a separate neurite of a neuron that contained reversible rods in other neurites, was found to increase in size (open squares) and is presumed to be a spontaneous rod.</p

CofilinR21Q-mRFP incorporates into virtually all AMPA-induced rods, but only into half of rods in Aβd/t-treated neuronal cultures.

<p>CofilinR21Q-mRFP fluorescence image, immunolabeled (Alexa 488) image, and overlay in neurons treated 30 min with 150 μM AMPA showing virtually all rods (immunolabel) have incorporated cofilinR21Q-mRFP. Similar results (not shown) were obtained for ATP-depleted neurons. CofilinR21Q-mRFP fluorescence image, immunolabeled image (Alexa 647 but colorized green), and overlay in neurons treated 24 h with Aβd/t. Immunolabeled rods that incorporated cofilinR21Q-mRFP were quantified from many different cultures and co-labeled rods accounted for 48±4% (std. deviation) of the total rods. Immunolabeled rods that do not contain mRFP are shown by arrowheads. Scale bars  = 10 μm.</p

Cofilin structure and position of residues for which site directed mutants were made.

<p>Side view of cofilin model created in PyMOL showing approximate F-actin binding interface for both upper and lower subunit contacts, and a rotated view showing the position of the residues for which mutants were made, as well as the C-terminal residue through which the mRFP is connected.</p

Live-imaging of AMPA-induced cofilinR21Q-mRFP labeled rods and their effect on vesicular transport before and after reversal.

<p>CofilinR21Q-mRFP and amyloid precursor protein (APP)-YFP dual infected neuronal cultures were treated with 25 μM AMPA for 45 min before imaging. Neurons expressing both fluorescent protein chimeras, which formed or did not form rods, were imaged once in the red channel to locate rods and then continuously for 12 sec (50 frames) in the YFP channel. In the AMPA-treated neuron that did not form rods (A), APP-YFP-containing vesicles (B) remain dynamic, as indicated by the retrograde (leftward) and anterograde (rightward) movements of the labeled vesicles in the kymographs (C, D) obtained from the regions of the neurite with red and purple line-scans (colored arrows demarcate the ends of the line scans) corresponding to the colored box on the kymographs. In the AMPA-treated neuron that formed rods (E, yellow arrows), APP-YFP-containing vesicles (F) stopped moving, as indicated by the lack of retrograde (leftward) and anterograde (rightward) movements of the labeled vesicles in the kymographs (G, H) obtained from the regions of the neurite with green and red line-scans (F) corresponding to the colored box on the kymographs. (Scale bars on images A, B, E, F are 15 μm and on kymographs C, D, G, H are 8 μm). In another set of experiments cofilinR21Q-mRFP and APP-YFP expressing neurons were treated 30 min with 25 μM AMPA to induce rods and then washed once with fresh medium and treated with 10 μM DNQX to prevent further AMPA-receptor activity. Neurons with rods were identified in the red channel. One hour after washout rods were still present in many processes (I) and APP-YFP containing vesicles were generally not dynamic, although a few exceptions were observed in kymographs taken along two different line scans (J, K). By three hours after washout (L), most rods have disappeared and APP-YFP vesicles are more dynamic (M, N). Line scans in I and L are not identical due to slight morphological changes that occur during recovery. (Scale bars on I, L = 10 μm and on kymographs J, K, M, N = 5 μm).</p

Quantification of rods in cultured neurons expressing either WT or R21Q cofilin-mRFP from promoters of different strengths.

<p>(A) Cofilin-immunolabled hippocampal neurons (5 days in vitro) untreated (control) or treated with Aβd/t for 24 h, which induced rods in some neurites. Inset: higher magnification of the rod boxed in the figure (B) The percentage of neurons (percent rod index) that form spontaneous rods is significantly above control levels and proportional to the level of expression of wild type cofilin-mRFP. Controls for this and all other experiments for which proteins are expressed via adenovirus infection are neurons infected with adenovirus expressing mRFP alone, which in all cases gave percent and number rod indexes identical to untreated cultures (not shown). The percent rod index remains at control levels and is independent of the level of expression of cofilinR21Q-mRFP. (C) The number of rods (rod number index) forming per neuron (or field for equal density cell cultures) is significantly above background and proportional to the level of expression of wild type cofilin-mRFP. The number rod index is not increased above control level for neurons expressing cofilinR21Q-mRFP. (**Significant at p<0.005, compared to the CMV-RFP control group).</p

CofilinR21Q-mRFP reports on rod formation in hippocampal neurons in response to Aβd/t treatment.

<p>(A) The fraction of cofilinR21Q-mRFP expressing neurons that formed rods after treatment with Aβd/t is 2 to 3 fold higher than for untreated neurons, regardless of which promoter drives expression, although more rods are detected when cofilin-R21Q-mRFP expression is greatest. (B) The number of rods per field in Aβd/t treated neurons expressing cofilinR21Q-mRFP is about 2 fold higher than the corresponding non-Aβd/t treated controls regardless of which promoter drives expression. (*Significant at p<0.05, **Significant at p<0.005, compared to their appropriate non-Aβd/t-treated control group).</p

Rod formation in cells overexpressing either cofilinR21Q-mRFP or cofilinK22Q-mRFP is dramatically reduced compared to expression of cofilin-wt-mRFP.

<p>Pig kidney LLC-PK_A4.8 cells and HeLa cells were infected with adenovirus for expression of a cofilin shRNA (grey bars) or a control adenovirus (black bars), then 3 days later the cells were transfected or infected with plasmids or adenovirus for expressing cofilin-mRFP chimeras. Two days after this, the cells were ATP-depleted. The percentage of mRFP-positive cells forming rods is shown. Uninfected LLC-PK_A4.8 or HeLa cells that are ATP-depleted, fixed and immunolabeled for cofilin contain no rods <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083609#pone.0083609-Ashworth1" target="_blank">[22]</a>. Based upon quantification of cofilin on Western blots of cell extracts (data not shown), the silencing in both cell types is >95%.</p