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• W3025709774 abstract "The hallmarks of neurodegenerative diseases, including neural fibrils, reactive oxygen species, and cofilin–actin rods, present numerous challenges in the development of in vivo diagnostic tools. Biomarkers such as β-amyloid (Aβ) fibrils and Tau tangles in Alzheimer's disease are accessible only via invasive cerebrospinal fluid assays, and reactive oxygen species can be fleeting and challenging to monitor in vivo. Although remaining a challenge for in vivo detection, the protein–protein interactions underlying these disease-specific biomarkers present opportunities for the engineering of in vitro pathology-sensitive biosensors. These tools can be useful for investigating early stage events in neurodegenerative diseases in both cellular and animal models and may lead to clinically useful reagents. Here, we report a light- and cellular stress–gated protein switch based on cofilin–actin rod formation, occurring in stressed neurons in the Alzheimer's disease brain and following ischemia. By coupling the stress-sensitive cofilin–actin interaction with the light-responsive Cry2-CIB blue-light switch, referred to hereafter as the CofActor, we accomplished both light- and energetic/oxidative stress–gated control of this interaction. Site-directed mutagenesis of both cofilin and actin revealed residues critical for sustaining or abrogating the light- and stress-gated response. Of note, the switch response varied depending on whether cellular stress was generated via glycolytic inhibition or by both glycolytic inhibition and azide-induced ATP depletion. We also demonstrate light- and cellular stress–gated switch function in cultured hippocampal neurons. CofActor holds promise for the tracking of early stage events in neurodegeneration and for investigating actin's interactions with other proteins during cellular stress. The hallmarks of neurodegenerative diseases, including neural fibrils, reactive oxygen species, and cofilin–actin rods, present numerous challenges in the development of in vivo diagnostic tools. Biomarkers such as β-amyloid (Aβ) fibrils and Tau tangles in Alzheimer's disease are accessible only via invasive cerebrospinal fluid assays, and reactive oxygen species can be fleeting and challenging to monitor in vivo. Although remaining a challenge for in vivo detection, the protein–protein interactions underlying these disease-specific biomarkers present opportunities for the engineering of in vitro pathology-sensitive biosensors. These tools can be useful for investigating early stage events in neurodegenerative diseases in both cellular and animal models and may lead to clinically useful reagents. Here, we report a light- and cellular stress–gated protein switch based on cofilin–actin rod formation, occurring in stressed neurons in the Alzheimer's disease brain and following ischemia. By coupling the stress-sensitive cofilin–actin interaction with the light-responsive Cry2-CIB blue-light switch, referred to hereafter as the CofActor, we accomplished both light- and energetic/oxidative stress–gated control of this interaction. Site-directed mutagenesis of both cofilin and actin revealed residues critical for sustaining or abrogating the light- and stress-gated response. Of note, the switch response varied depending on whether cellular stress was generated via glycolytic inhibition or by both glycolytic inhibition and azide-induced ATP depletion. We also demonstrate light- and cellular stress–gated switch function in cultured hippocampal neurons. CofActor holds promise for the tracking of early stage events in neurodegeneration and for investigating actin's interactions with other proteins during cellular stress. Actin–cofilin bundles or rods, formed in neurons as part of a cellular stress response, impact synaptic function and may play a significant early role in the progression of neurodegenerative disorders (1Bamburg J.R. Bernstein B.W. Davis R.C. Flynn K.C. Goldsbury C. Jensen J.R. Maloney M.T. Marsden I.T. Minamide L.S. Pak C.W. Shaw A.E. Whiteman I. Wiggan O. ADF/Cofilin-actin rods in neurodegenerative diseases.Curr. Alzheimer Res. 2010; 7 (20088812): 241-25010.2174/156720510791050902Crossref PubMed Scopus (121) Google Scholar). Prior studies have shown that actin and cofilin combine in a 1:1 ratio in rods, with actin assuming a highly twisted conformation (2Minamide L.S. Maiti S. Boyle J.A. Davis R.C. Coppinger J.A. Bao Y. Huang T.Y. Yates J. Bokoch G.M. Bamburg J.R. Isolation and characterization of cytoplasmic cofilin-actin rods.J. Biol. Chem. 2010; 285 (20022956): 5450-546010.1074/jbc.M109.063768Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). This unusual arrangement excludes most other potential binding partners, with particular isoforms of 14-3-3 protein being a notable exception (2Minamide L.S. Maiti S. Boyle J.A. Davis R.C. Coppinger J.A. Bao Y. Huang T.Y. Yates J. Bokoch G.M. Bamburg J.R. Isolation and characterization of cytoplasmic cofilin-actin rods.J. Biol. Chem. 2010; 285 (20022956): 5450-546010.1074/jbc.M109.063768Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 3Kim J.-S. Huang T.Y. Bokoch G.M. Reactive oxygen species regulate a slingshot-cofilin activation pathway.Mol. Biol. Cell. 2009; 20 (19339277): 2650-266010.1091/mbc.e09-02-0131Crossref PubMed Scopus (144) Google Scholar). Although cofilin–actin rods are frequently observed in postmortem brain slices from Alzheimer's disease patients, their transient nature, along with the multifaceted activities of both cofilin and actin, makes them particularly challenging to study in vivo (1Bamburg J.R. Bernstein B.W. Davis R.C. Flynn K.C. Goldsbury C. Jensen J.R. Maloney M.T. Marsden I.T. Minamide L.S. Pak C.W. Shaw A.E. Whiteman I. Wiggan O. ADF/Cofilin-actin rods in neurodegenerative diseases.Curr. Alzheimer Res. 2010; 7 (20088812): 241-25010.2174/156720510791050902Crossref PubMed Scopus (121) Google Scholar). Thus, methods for the induction of cofilin–actin rods in cell culture have been developed, including in primary neurons exposed to Aβ fibrils (4Davis R.C. Marsden I.T. Maloney M.T. Minamide L.S. Podlisny M. Selkoe D.J. Bamburg J.R. Amyloid beta dimers/trimers potently induce cofilin-actin rods that are inhibited by maintaining cofilin-phosphorylation.Mol. Neurodegener. 2011; 6 (21261978): 1010.1186/1750-1326-6-10Crossref PubMed Scopus (68) Google Scholar) and HeLa cells subjected to ATP-depleting conditions (2Minamide L.S. Maiti S. Boyle J.A. Davis R.C. Coppinger J.A. Bao Y. Huang T.Y. Yates J. Bokoch G.M. Bamburg J.R. Isolation and characterization of cytoplasmic cofilin-actin rods.J. Biol. Chem. 2010; 285 (20022956): 5450-546010.1074/jbc.M109.063768Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Both oxidative and energetic stress, separately, have been shown to induce cofilin–actin rods. Exposure of cells to H2O2 induces cofilin–actin rod formation through an ROS-dependent mechanism via activation of the cofilin slingshot phosphatase, whereas exposure to sodium azide/Deoxy-d-glucose induces cofilin–actin rod formation through mitochondrial inhibition, subsequent ATP depletion, and activation of the cofilin phosphatase chronophin (5Whiteman I.T. Gervasio O.L. Cullen K.M. Guillemin G.J. Jeong E.V. Witting P.K. Antao S.T. Minamide L.S. Bamburg J.R. Goldsbury C. Activated actin-depolymerizing factor/cofilin sequesters phosphorylated microtubule-associated protein during the assembly of Alzheimer-like neuritic cytoskeletal striations.J. Neurosci. 2009; 29 (19828813): 12994-1300510.1523/JNEUROSCI.3531-09.2009Crossref PubMed Scopus (90) Google Scholar). Prior work shows that mutagenesis of only one or two amino acids in cofilin (e.g. S3E, S3A/S120A) can significantly reduce the affinity of cofilin for native actin structures, both in vivo and in vitro (6Hughes R.M. Lawrence D.S. Optogenetic engineering: Light-directed cell motility.Angew. Chem. Int. Ed. Engl. 2014; 53 (25156888): 10904-1090710.1002/anie.201404198Crossref PubMed Scopus (17) Google Scholar, 7Moriyama K. Yahara I. The actin-severing activity of cofilin is exerted by the interplay of three distinct sites on cofilin and essential for cell viability.Biochem. J. 2002; 365 (12113256): 147-15510.1042/bj20020231Crossref PubMed Scopus (45) Google Scholar, 8Moriyama K. Yahara I. Two activities of cofilin, severing and accelerating directional depolymerization of actin filaments, are affected differentially by mutations around the actin-binding helix.EMBO J. 1999; 18 (10581248): 6752-676110.1093/emboj/18.23.6752Crossref PubMed Scopus (91) Google Scholar, 9Moriyama K. Iida K. Yahara I. Phosphorylation of Ser-3 of cofilin regulates its essential function on actin.Genes Cells. 1996; 1 (9078368): 73-8610.1046/j.1365-2443.1996.05005.xCrossref PubMed Scopus (314) Google Scholar). These mutant cofilins are subsequently able to function as biosensors and bioactuators of cofilin-dependent cell functions (10Ghosh M. Song X. Mouneimne G. Sidani M. Lawrence D.S. Condeelis J.S. Cofilin promotes actin polymerization and defines the direction of cell motility.Science. 2004; 304 (15118165): 743-74610.1126/science.1094561Crossref PubMed Scopus (554) Google Scholar, 11Ghosh M. Ichetovkin I. Song X. Condeelis J.S. Lawrence D.S. A new strategy for caging proteins regulated by kinases.J. Am. Chem. Soc. 2002; 124 (11890784): 2440-244110.1021/ja017592lCrossref PubMed Scopus (44) Google Scholar, 12Zebda N. Bernard O. Bailly M. Welti S. Lawrence D.S. Condeelis J.S. Phosphorylation of ADF/cofilin abolishes EGF-induced actin nucleation at the leading edge and subsequent lamellipod extension.J. Cell Biol. 2000; 151 (11086013): 1119-112810.1083/jcb.151.5.1119Crossref PubMed Scopus (168) Google Scholar). Recently, a cofilin mutant (R21Q) was described that enables real-time tracking of cofilin–actin rod formation and dissipation in cell culture models when expressed as a fusion with the fluorescent protein mCherry (13Mi J. Shaw A.E. Pak C.W. Walsh K.P. Minamide L.S. Bernstein B.W. Kuhn T.B. Bamburg J.R. A genetically encoded reporter for real-time imaging of cofilin-actin rods in living neurons.PLoS One. 2013; 8 (24391794): e8360910.1371/journal.pone.0083609Crossref PubMed Scopus (15) Google Scholar). Unlike overexpressed WT cofilin, the R21Q mutant resists incorporation into cofilin–actin rods in the absence of oxidative and energetic stress; however, it readily incorporates into cofilin–actin bundles under sources of oxidative and energetic stress (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, Aβ, ATP depletion). In light of the R21Q study, we asked whether a cofilin mutant with substantially reduced affinity for F-actin could be the basis for an optogenetic switch that enables the bundling of cofilin and actin in response to both light and cellular stress stimuli. The requirement for such a switch would be that it resists background incorporation into cofilin–actin bundles under both normal and cellular stress conditions, yet readily forms cofilin–actin bundles when exposed to a combination of cellular stress and light. However, in contrast to native cofilin–actin bundle formation, reversal of the light-induced cofilin–actin bundles could be accomplished without removal of the source of cellular stress. With the advantage of rapid and reversible light-mediated induction, the resulting switch would allow for tracking the formation and movement of cofilin–actin bundles in living cells, including neurons. Prior studies demonstrated that point mutants of cofilin-1 (e.g. S3E, S3A/S120A) exhibit diminished actin-binding capabilities, both when expressed as stand-alone cofilins and when incorporated into protein fusions (cofilin–cryptochrome 2–mCherry) (6Hughes R.M. Lawrence D.S. Optogenetic engineering: Light-directed cell motility.Angew. Chem. Int. Ed. Engl. 2014; 53 (25156888): 10904-1090710.1002/anie.201404198Crossref PubMed Scopus (17) Google Scholar). We investigated whether the same cofilin-containing protein fusions exhibited a similar response to energetic stress in the presence of an ATP-depletion medium, as has been previously reported for both endogenous and overexpressed cofilins (2Minamide L.S. Maiti S. Boyle J.A. Davis R.C. Coppinger J.A. Bao Y. Huang T.Y. Yates J. Bokoch G.M. Bamburg J.R. Isolation and characterization of cytoplasmic cofilin-actin rods.J. Biol. Chem. 2010; 285 (20022956): 5450-546010.1074/jbc.M109.063768Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 13Mi J. Shaw A.E. Pak C.W. Walsh K.P. Minamide L.S. Bernstein B.W. Kuhn T.B. Bamburg J.R. A genetically encoded reporter for real-time imaging of cofilin-actin rods in living neurons.PLoS One. 2013; 8 (24391794): e8360910.1371/journal.pone.0083609Crossref PubMed Scopus (15) Google Scholar, 14Bernstein B.W. Shaw A.E. Minamide L.S. Pak C.W. Bamburg J.R. Incorporation of cofilin into rods depends on disulfide intermolecular bonds: Implications for actin regulation and neurodegenerative disease.J. Neurosci. 2012; 32 (22573689): 6670-668110.1523/JNEUROSCI.6020-11.2012Crossref PubMed Scopus (50) Google Scholar). Following literature procedures, cofilin (WT, S3E, S3A, S3A/S120A)-Cry2PHR-mCh protein fusions expressed in HeLa cells were incubated with either PBS or ATP depletion media for 1 h, followed by fixation and imaging. As a result of the applied cellular stress, cofilins with full actin-binding capability (WT and S3A) were readily incorporated into clusters, whereas cofilins with impaired actin-binding ability (S3A/S120A and S3E) failed to incorporate into clusters (Fig. 1A and B). Via immunostaining with an actin antibody, we demonstrated that the observed cofilin clusters were co-localized with endogenous β-actin (Fig. 1B). Further exploration of the ATP-depletion buffer revealed that using the full complement of mitochondrial inhibition (10 mm NaN3/6 mm 2-Deoxy-d-Glucose) resulted in abundant cofilin–actin clusters distributed throughout the cell body, whereas treatment with glycolytic inhibition only (0 mm NaN3/6 mm 2-DG) resulted in the accumulation of cofilin–actin bundles around the cell periphery (Fig. 2 and Figs. S1–S3). We attribute this effect to naturally occurring ROS production, which is higher at the highly dynamic cell edges (15Cameron J.M. Gabrielsen M. Chim Y.H. Munro J. McGhee E.J. Sumpton D. Eaton P. Anderson K.I. Yin H. Olson M.F. Polarized cell motility induces hydrogen peroxide to inhibit cofilin via cysteine oxidation.Curr. Biol. 2015; 25 (25981793): 1520-152510.1016/j.cub.2015.04.020Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Experiments were also conducted to establish the minimum time and concentrations necessary for induction of cofilin–actin clustering, demonstrating that 15 min in full ATP-depletion medium was sufficient for inducing robust cofilin–actin bundling (Fig. S1–S3).Figure 2Response to variation in stress-inducing buffers in HeLa cells expressing Cof. A and B, WT-Cry2-mCh subjected to (A) 6 mm 2-Deoxy-d-glucose alone for 15 min at 37°C or (B) 6 mm 2-Deoxy-d-glucose/10 mm sodium azide for 15 min at 37°C. Bottom images depict 3D surface plots for each image (FIJI). Scale bar = 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Having established that the cofilin-Cry2 fusions responded as expected to cofilin–actin bundle–forming conditions (i.e. ATP depletion), we investigated whether the fusions would respond to both cellular stress and light inputs in the presence of the Cry2-binding partner CIB. To test this, both N- and C-terminal cofilin-Cry2 fusion proteins and a β-actin–CIB-GFP protein fusion were co-expressed in HeLa cells. Cells were incubated in the presence of ATP-depletion medium for 15 min prior to imaging on a widefield fluorescent microscope. Of the various combinations of cofilin-Cry2 and actin-CIB, the N-terminal Cof/S3A/S120A and the C-terminal Cof/S3E exhibited robust light- and stress-dependent cofilin–actin fusion protein clustering that was reversible in the absence of blue light (Table 1). As the response of the Cry2-mCh-CofS3E fusion was somewhat more robust (Fig. 3 and Movie S1), this construct was carried forward in conjunction with the β-actin–CIB-GFP construct for the remaining studies.Table 1Screening of optogenetic constructs in presence of light + ATP depletion: Survey of light-stimulated cofilin–actin cluster formationProtein pairDMEM/10% FBSATP-depletion mediaN-terminal cofilinsCof-WT-Cry2/ β-actin -CibNo clusters++++; ICof-S3A/S120A-Cry2/ β-actin -CibNo clusters+++; RCof-S3E-Cry2/ β-actin -CibNo clusters++; RCof-S3A/S120A-Cry2/Cib-ONLYNo clusters—C-terminal cofilinsCry2-Cof-WT/ β-actin -CibNo clusters++++; ICry2-Cof-S3A/S120A/β-actin-CibNo clusters+++; ICry2-Cof-S3E/β-actin-CibNo clusters++++; RNo cofilin controlsCry2-mCh/β-actin -CibNo clusters+; RCry2-mCh/Cib-ONLYNo clusters— Open table in a new tab As the interaction of the WT cofilin construct with endogenous actin varied in response to changing cellular stress levels, the same treatments were applied to the optogenetic cofilin–actin bundling proteins (Fig. 4 and Movies S2–S5). In response to a full complement of azide, but no glycolysis inhibitor (10 mm azide/0 mm 2-DG), light-activated cofilin–actin bundles were formed throughout cell bodies but were shorter lived than those formed in the presence of 10 mm azide/6 mm 2-DG. In response to full inhibition of glycolysis, but no azide (0 mm azide/6 mm 2-DG), light-activated cofilin–actin bundles formed chiefly around the cell periphery but were also shorter lived than those under full ATP depletion conditions. We note that this is an analogous response to that observed with incorporation of WT cofilin into endogenous actin bundles (Fig. 2). Finally, although control cells (in the absence of ATP depletion) form sparse light + stress–induced clusters (Fig. 4B), weak light-activated localization between cytosolic Cry2-mCh-CofS3E and β-actin–CIB-GFP can still be observed in a subset of transfected cells (14.5% (10.7); n = 5 replicates (Fig. 5 and Movie S5).Figure 5Stress-independent co-localization of Cry2-mCh-Cof.S3E and β-actin–CIB-GFP at the cell periphery. HeLa cells expressing Cry2-mCh-Cof.S3E/actin-CIB-GFP are shown. A–C, before blue-light stimulation (A), after 10 min of blue-light stimulation (B), and after an additional 10 min without blue light stimulation (C). D–G, zoomed region of cell from A shows cell periphery (D) mCherry and (F) GFP before and (E) mCherry and (G) GFP 2 min post light stimulation. H, co-localization of images shown in E and G. Scale bar = 10 μm. I, time course of co-localization of cropped region shows change in mCherry fluorescence intensity versus time at the cell periphery. Error bars (S.E.) are determined from three different peripheral regions. A, H, and I, scale bars = 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The light- and cellular stress–dependent clustering of the cofilin and actin fusions indicated that site-directed mutagenesis of both cofilin and actin might provide insight into the mechanism of interaction between the two proteins. One of the primary driving forces of native cofilin–actin bundling is the switch from primarily ATP-actin to ADP-actin that occurs as a result of mitochondrial inhibition-induced depletion of ATP. Thus, we investigated whether alteration of the ATP-binding site of actin via site-directed mutagenesis might alter its ability to bind cofilin in the absence of ATP depletion by mimicking actin in its ADP-bound state. Inspection of a crystal structure of actin with a nonhydrolysable ATP mimic (PDB ID 1NWK (16Graceffa P. Dominguez R. Crystal structure of monomeric actin in the ATP state. Structural basis of nucleotide-dependent actin dynamics.J. Biol. Chem. 2003; 278 (12813032): 34172-3418010.1074/jbc.M303689200Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar)) revealed two potential contacts that might be critical for binding the tertiary phosphate group of ATP: Ser-14 (a predicted hydrogen bond contact) and Val-159 (potentially critical for maintaining binding cavity shape and hydrophobic packing) (Fig. 6). We proposed that removal of the Ser-14–phosphate hydrogen bond–forming ability might create an actin mutant with the properties of an ADP-actin, whereas increasing the steric bulk of the Val-159 residue might have a similar effect by occluding the binding site for the tertiary phosphate of ATP. In addition to the proposed actin mutants, we also considered whether further mutagenesis of cofilin could impact the function of the light-activated switch. Cofilin has four cysteine residues, two of which have previously been demonstrated to be critical for cofilin–actin rod stability via the formation of intermolecular disulfide bonds (Cys-39 and Cys-147; Fig. 6C) (14Bernstein B.W. Shaw A.E. Minamide L.S. Pak C.W. Bamburg J.R. Incorporation of cofilin into rods depends on disulfide intermolecular bonds: Implications for actin regulation and neurodegenerative disease.J. Neurosci. 2012; 32 (22573689): 6670-668110.1523/JNEUROSCI.6020-11.2012Crossref PubMed Scopus (50) Google Scholar, 17Pope B.J. Zierler-Gould K.M. Kühne R. Weeds A.G. Ball L.J. Solution structure of human cofilin: Actin binding, pH sensitivity, and relationship to actin-depolymerizing factor.J. Biol. Chem. 2004; 279 (14627701): 4840-484810.1074/jbc.M310148200Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 18Bamburg J.R. Bernstein B.W. Actin dynamics and cofilin-actin rods in Alzheimer disease.Cytoskeleton. 2016; 73 (26873625): 477-49710.1002/cm.21282Crossref Scopus (93) Google Scholar). Simultaneous mutation of cofilin cysteines 39 and 147 to alanine has been shown to interfere with efficient incorporation into cofilin–actin rods (14Bernstein B.W. Shaw A.E. Minamide L.S. Pak C.W. Bamburg J.R. Incorporation of cofilin into rods depends on disulfide intermolecular bonds: Implications for actin regulation and neurodegenerative disease.J. Neurosci. 2012; 32 (22573689): 6670-668110.1523/JNEUROSCI.6020-11.2012Crossref PubMed Scopus (50) Google Scholar). We hypothesized that if cofilin–actin bundling with CofActor was similar to native cofilin–actin rod formation, then mutagenesis of cofilin Cys-39 and Cys-147 in CofActor should also interfere with switch function in the presence of cellular stress. Single and double mutant actins and cofilins were generated and tested for their ability to participate in light- and stress-activated clustering when co-transfected with the corresponding Cry2-mCh-CofS3E or β-actin–CIB-GFP (Table 2). In the case of the V159I single mutant (β-actinV159I–CIB-GFP), light + cellular stress were both required for cofilin–actin cluster formation. However, the S14V mutant (β-actinS14V–CIB-GFP) gave a robust, reversible light-activated clustering response in the absence of ATP-depletion media (Fig. 7 and Movie S6). Notably, the S14V mutant formed rodlike bundles with dimensions similar to those observed with native cofilin–actin rods (avg. 2 rods per cell (± 1); length of 7.42 μm (± 0.68)). These structures are present prior to light-activated recruitment of Cry2, indicating that they are formed independently of optogenetic cofilin. Optogenetic cofilin undergoes light-activated recruitment to these rodlike structures in addition to forming cofilin–actin clusters analogous to those observed in the presence of nonmutant actin (Fig. 7A–F). Double mutants (S14V/V159L and S14V/V159I) exhibited a stress-independent response analogous to that of the S14V mutant, forming numerous light-independent rodlike structures present in cells (Fig. 7D). Interestingly, while the V159I single mutant (β-actinV159I–CIB-GFP) did not efficiently form stress-independent clusters as observed with the S14V mutant, it did exhibit enhanced nuclear localization and formed numerous nuclear-localized light-activated cofilin–actin clusters in the presence of cellular stress (Fig. 8 and Movie S7), indicating that the presence of a hydrophobic side chain at the 159 position may enhance nuclear import of the actin construct. In the case of the cofilin mutants (Fig. 9A–G), light- and stress-activation of cofilin–actin bundles were not significantly reduced in the C39A and C147A single mutants, but were highly diminished in the C39A/C147A double mutant, consistent with a previous report (14Bernstein B.W. Shaw A.E. Minamide L.S. Pak C.W. Bamburg J.R. Incorporation of cofilin into rods depends on disulfide intermolecular bonds: Implications for actin regulation and neurodegenerative disease.J. Neurosci. 2012; 32 (22573689): 6670-668110.1523/JNEUROSCI.6020-11.2012Crossref PubMed Scopus (50) Google Scholar). Conversely, light + stress activation of bundle formation was eliminated in the C39D and C39D/C147D mutants, but not in the C147D mutant (Fig. 9G).Table 2Summary of blue-light activation responses of actin and cofilin mutants under normal and energetic stress conditionsPBSATP depletedActin mutantsV159I—++++S14V+++++++V159L/ S14V++++++++V159I/ S14V++++++++Cofilin mutantsS3E/C39A—+++S3E/C147A—+++S3E/C39A/C147A—+S3E/C39D——S3E/C147D—+++S3E/C39D/C147D—— Open table in a new tab Figure 8Light-inducible nuclear cofilin–actin cluster formation. A and B, HeLa cells expressing Cry2-mCh-Cof-S3E/actin-V159Ile-CIB-GFP in 10 mm azide/6 mm 2-DG/PBS are shown (A) pre- and (B) post 5 min of blue-light stimulation. C, nuclear-localized cofilin–actin clusters. Cry2-mCh-Cof.S3E (mCherry fluorescence). D, Actin-V159I-CIB-GFP (GFP fluorescence). E, overlay of mCherry and GFP channels. Scale bar = 10 μm. See also Movie S7.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9Mutation of cofilin Cys-39 and Cys-147 impairs actin-cofilin bundle formation. Cells are shown before and after 10 min of blue-light stimulation. A, HeLa cells expressing Cof-S3E-C39A/actin-CIB-GFP. B, HeLa cells expressing Cof-S3E-C147A/actin-CIB-GFP. C, HeLa cells expressing Cof-S3E-C39A-C147A/actin-CIB-GFP. D, HeLa cells expressing Cof-S3E-C39D/actin-CIB-GFP. E, HeLa cells expressing Cof-S3E-C147D/actin-CIB-GFP. F, HeLa cells expressing Cof-S3E-C39D-C147D/actin-CIB-GFP. G, quantification of A–F as % cells forming light inducible clusters. Error bars are S.D. from minimum six replicate measurements. *, p < 0.001 versus S3E control; one-way ANOVA, Holm-Sidak method. All scale bars = 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, to demonstrate the potential utility of CofActor in neurons, we tested switch responsivity in primary hippocampal neuron cultures prepared from newborn mice. These cultures are routinely used as in vitro models to study cellular mechanisms of neurodegeneration (19Szatmari E.M. Oliveira A.F. Sumner E.J. Yasuda R. Centaurin-α1-Ras-Elk-1 signaling at mitochondria mediates β-amyloid-induced synaptic dysfunction.J. Neurosci. 2013; 33 (23516302): 5367-537410.1523/JNEUROSCI.2641-12.2013Crossref PubMed Google Scholar). Prior to light stimulation, dissociated neuron cultures transfected with the CofActor system exhibited 8.14 (± 2.49) actin rods and 2.14 (± 0.55) cofilin rods in soma and 54.86 (± 16.17) actin rods and 25.14 (± 4.69) cofilin rods in neuronal processes (Fig. 10A–C). Upon stimulation with light in ATP-depletion medium, the number of rods significantly increased in both subcellular compartments (Fig. 10B). In the soma, the number of actin rods increased by 267% (21.71 ± 5.32; p = 0.018), whereas the number of cofilin rods increased by 327% (7 ± 2.02; p = 0.032). In neuronal processes, the number of actin rods increased by 168% (92.43 ± 15.7; p = 0.004), whereas the number of cofilin rods increased by 223% (56.14 ± 12.73; p = 0.039). Taken together, the data presented here show that the CofActor optogenetic sensor reports on conditions required for the formation of native actin–cofilin rods in cells exposed to ATP-depletion conditions and therefore is a promising tool to study the cellular mechanisms that lead to abnormal cytoskeletal reorganization and are associated with pathological conditions, including neurodegeneration and excitotoxicity. Finally, we investigated whether CofActor would respond to alternate sources of cellular stress (ROS-generating H2O2), and whether that response could be inhibited with a recently reported inhibitor of cofilin–actin rod formation in neurons (20Tam S.-W. Feng R. Lau W.K.-W. Law A.C.-K. Yeung P.K.-K. Chung S.K. Endothelin type B receptor promotes cofilin rod formation and dendritic loss in neurons by inducing oxidative stress and cofilin activation.J. Biol. Chem. 2019; 294 (31248984): 12495-1250610.1074/jbc.RA118.005155Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). In HeLa cells treated with 500 μm H2O2, we observed a light-induced CofActor response analogous to that observed with ATP-depleted cells (Fig. 11 and Movie S8). The CofActor response was somewhat less robust in H2O2 than in azide/2-DG, consistent with previous observations of endogenous cofilin–actin rod induction (21Davis R.C. Maloney M.T. Minamide L.S. Flynn K.C. Stonebraker M.A. Bamburg J.R. Mapping cofilin-actin rods in stressed hippocampal slices and the role" @default.
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• W3025709774 date "2020-08-01" @default.
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• W3025709774 title "CofActor: A light- and stress-gated optogenetic clustering tool to study disease-associated cytoskeletal dynamics in living cells" @default.
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