Journal of Cell Science ieNcl use al .DsodadRyTruant* Ray and Desmond R. Carly 1 Munsie, Nicole stress Lise during formation rod cofilin–actin affects shuttling nuclear–cytoplasmic Cofilin Article Research h ulu pncl tes oii skont eactively be to known is karyopherin or Cofilin importin classic stress. a through nucleus cell the into imported give upon nucleus will the stresses related aging neurodegeneration. for mechanisms and disease into actin insights dendrites important disease understanding and under Thus, synapses 2010). dynamics healthy Hoogenraad, is of and cytoskeleton maintenance (Hotulainen actin for the critical dynamics actin of are as diseases, neurodegenerative regulation in focus Improper a becoming 2006). (Bernstein uses al., in cellular and immediate tread-milling et more actin other (Minamide for stop ATP to stress up means free to potential turn a response as in 2000) al., cytoplasm et or in nucleus form can the rods stress either Cofilin–actin 2000). al., Bamburg, et Minamide and 2007; (Maloney the (AD) (Munsie in disease involved Alzheimer’s lymphocytes are a of rods progression patient cofilin–actin cytoplasmic in and HD cofilin 2011) al., rods in the et in profile cofilin–actin changes expression and nuclear model previouslyprotein cell have of (HD) We disease involvement Huntington’s neurodegeneration. of the field particular reported of the is to isoform, specific interest non-muscle Bamburg, the and Cofilin1, (Bernstein 2010). mechanisms disease Percipalle, in have implicated and members of family been cofilin regulation (Obrdlik the of the regulation structure improper The and 2011). chromatin 2009) and al., et transcription in (Klamt mitochondrial- involvement apoptosis 2007), al., as dependent et (Han diverse metabolism lipid as and membrane are actin functions to respect These with of tread-milling. function facets multiple their in of independent involved biology be cellular to shown been These now proteins. have modulating proteins and binding actin as proteins characterized expressed best ubiquitously are members family its and Cofilin Introduction and nucleus cofilin the that between propose communicating We dynamically imaging. words: further cofilin live-cell Key We with temporal stress. response by during stress stress dynamics cofilin cell rod stress. affect of cofilin–actin cell and system the vivo during a for in cytoplasm in critical actin and formation bind is nuclear rod to (FLIM) shuttling bipartite cofilin cofilin–actin microscopy active nuclear allow a of imaging that requirement an as lifetime NLS cofilin the cofilin fluorescence cofilin through of define the of Using stress in export NLS localization. nucleus mutations cell nuclear nuclear the subtle the during active for redefine defined the as required also enter characterize epitope We well we can basic (NES). as Fo Here, additional signal Cofilin conditions, stress. an export during state with rods. nuclear rods NLS, steady CRM1-dependent, cofilin–actin actin leptomycin-B-sensitive, typical nuclear forming a during in through accumulating cytoskeleton F-actin, (NLS), actin saturates signal the localization cofilin regulating where in involved conditions is protein Cofilin Summary 10.1242/jcs.097667 doi: 3977–3988 125, ß Science Cell of Journal 2012 April 24 Accepted ( correspondence for *Author eateto iceityadBoeia cecs catrUiest,10 anSre et aitn nai,Cnd,LN3Z5 L8N Canada, Ontario, Hamilton, West, Street Main 1200 University, McMaster Sciences, Biomedical and Biochemistry of Department rtrrsnn nrytase FE)btencflnmite n ci,a ela uoae mg nlssi iecls ehave we cells, live in analysis image automated as well as actin, and moieties cofilin between (FRET) transfer energy resonant ¨rster 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. uigorpeiu tde,w bevdta oii ol enter could cofilin that observed we studies, previous our During LMFE,Atn oii,Ctseeo,Ncerepr,Stress export, Nuclear Cytoskeleton, Cofilin, Actin, FLIM–FRET, [email protected] ) lh/ea( alpha/beta h ulu hog h ula oecmlx(P)with (NPC) complex pore importin receptor nuclear transport nuclear the the of assistance through the nucleus importin the the by recognized protein are that acids amino adapter Importin- basic 1992). of stretch al., a by et defined Iida 1993; al., et n ecple 01 n ta. 93.Drn u live-cell our During cofilin of 1993). exit rapid the al., noted we time, et real in shock Ono heat of imaging Obrdlik 2011; 1987; with al., Percipalle, functions et from (Nishida and state activity aside steady polymerase function RNA or to stress-dependent nucleus respect the a into have actin indicating may rods, transporting nuclear NLS at and the nucleus, the in that in cofilin found of be nucleus majority situations, can the the cell shock, stress the heat in and certain treatment F-actin DMSO During of as 2011). such function Percipalle, the and for (Obrdlik required the likely in is role a elongation and transcription is have II polymerase to RNA it Iida shown to respect 2012; been with how has nucleus al., recently although et in Cofilin (Dopie 1992). actin, unknown al., higher et is of likely entry transport nuclear but this nuclear mediating 2001), the be to for shown al., been has required Cofilin et 2007). al., (Fahrenkrog et (Seibel diffuse cells mammalian yeast freely of to point in able cut-off 2007). diffusion be passive kDa a should al., has that which et protein NPC, the kDa across (Lange 18 complex an the is the Cofilin disassociates in in cytoplasm interaction Ran–GDP the to cargo exchange the nucleotide encourages RanGAP-mediated nuclear binds nucleus. binding proteins the nucleus, export Ran–GTP nuclear in the for where release reversed in is cargo process levels This facilitating compartment. high factors import at maintained nuclear maintained is is transport Ran–GTP, nuclear which Ran. protein, GTP-binding of family Ras small Directionality the through 2007). al., et a / b -eedn L 2-EVKKA-6 (Abe (26-PEEVKKRKKAV-36) NLS )-dependent a n usqetyipre into imported subsequently and a / b dpnetNS are NLSs -dependent b (Lange 3977 , 40 Journal of Cell Science agtn h L a etlrtdb h eladmythus neurodegenerative may some and for cell target the drug by new diseases. a tolerated as be cofilin may by define cofilin NLS not of but the capabilities altering, targeting that rod-forming imply the cofilin also to abolishing of and able completely stress importance was to the rods response highlight to form in data still activity respond These could to stress. that to ability mutant respond cofilin impaired a an but had had stress that formation mutants cofilin– rod cofilin form expressing to abolished cells unable how were Importantly, observed they rods. and when actin in stress stress resulted to during that responded formation NLS cells the rod cofilin model. decreased investigated the cell-based or to a we loss cofilin mutations in RNA, minimal endogenous mutants made (sh) cofilin We with hairpin of coupled activity short actin-binding NLS, by that in and down residues hypothesized knock key NES been mutating cofilin By be has 2010). the could the al., rods it et in persistent (Bamburg down HD, involved therapeutic breaking al., or be and et formation rod may Munsie AD altering 2000; formation of al., rod et progression aberrant (Minamide in Since HD neurotoxic and 2011). be but AD to 2006), thought of al., is models clearance et and (Bernstein formation models improper cellular in a neuroprotective defined NLS. and bipartite redefined a cofilin as as NLS well of cofilin as the sequence sequence we NES acid study CRM1-dependent this conserved amino During 1998). B, the al., leptomycin examined et a by (Kudo at CRM1 residue disrupted mammalian cysteine be inactivates single and (Fornerod can to, cytoplasm cargo pathway links covalently the its This which in transport 1997). cargo to al., the gradient et releasing Ran NPC, the the al., on across et protein, relies export (Bogerd nuclear which acid the CRM1, amino by recognized any is consensus is This x 1996). and acid amino hydrophobic R-/xotndpnetNShsacnevdcnessof consensus conserved that a hypothesized Lx has We classic The NES (NES). stress. signal CRM-1/exportin-dependent export nuclear extended active an have after may cofilin nucleus the from 3978 oii–ci osaetogtt etransiently be to thought are rods Cofilin–actin 2–3 Lx 2–3 ora fCl cec 2 (17) 125 Science Cell of Journal Lx 1–2 ,weeLi yial ecn u a eany be can but leucine a typically is L where L, Results eunei oii htft h uaieCM-eedn NES CRM1-dependent putative single the a identified fits (11- consensus We that cofilin. cofilin in of in presence signal sequence the rapid export hypothesize nuclear to The active us 1). led an export Movie the the nuclear this exit material following of rapidly supplementary nature removed to 1C; observed not (Fig. is is cofilin nucleus stimulus response, stress stress rod the cofilin nuclear If and predicted form. as cells nucleus rods shock of the in heat imaging accumulates undergoing cofilin live-cell stress, and During mCerulean-cofilin our 1B). expressing in (Fig. recapitulate stably could system we which model 1992), cell shock in al., accumulate heat et rapidly (Iida to During nucleus shown 1A). the been (Fig. has cofilin nucleus stresses the other and in levels cell the varying throughout diffuse with is cofilin conditions, state NES steady CRM1-dependent Under conserved a has Cofilin yols,cmae ihte2 D YPwihcnfreely can EYFP which conditions, normal EYFP Under 2B). kDa (Fig. the nucleus 26 to the into EYFP the diffuse of localization with the mediate compared would it cytoplasm, frame if in see to fused EYFP was to VIKVFNDMKV, activity, sequence, NES for NES sequence putative this the test initially (muscle To 2A). cofilin2 and Fig. (non-muscle) enriched; cofilin1 species between different and between cofilin conserved of is sequence This 1997). al., et ndtcal ula oii hnpretncerfluorescent nuclear percent when to cofilin 20 nuclear valine increase detectable NES, significant a the in the caused mutation of full V20A in one The the mutated (V20A). acids alanine of and amino context EYFP hydrophobic to the cofilin this consensus in fused if NES we test an cofilin, To length as 2). works Movie sequence material CRM1-dependent identified supplementary a nucleus, as 2B; the function (Fig. exit can longer NES sequence no but this nucleus, that to the observed indicating into was protein diffuse fusion to the able 1997), be al., et (Kudo predominantly Wolff B 1998; leptomycin al., inhibitor, appeared et CRM1 the VIKVFNDMKV– sequence with expressing treated NES were cells this EYFP an When to 2B). as (Fig. function fused cytoplasmic can sequence revealed EYFP NES Imaging since cofilin 2B). putative (Fig. the cell that the throughout diffuse appears VIK VFND MK mg r iue t42 at each minutes of right are bottom image the in Numbers heat shock. of course the expressing during stably mCerulean–cofilin cells live in imaging ( 10 bars: Scale microscope. at taken Images 60 stress. shock nucleus heat the during enters cofilin of proportion vr 0scns cl a:10 bar: Scale seconds. 60 every hntp ne taysae ( state. steady under arrow) phenotype (dashed cytoplasmic or arrow) STHdh in ( conditions cells. shock heat and state steady under cofilin nucleus. of the Immunofluorescence shuttle of to out ability and the into has Cofilin 1. Fig. C -0 Bgr ta. 96 Fornerod 1996; al., et (Bogerd V-20) 6 eprl100 Temporal ) anfcto nawidefield a on magnification A oii a aeancer(solid nuclear a have can Cofilin ) 6 anfcto widefield magnification ˚ .Clsimaged Cells C. m m. B m large A ) m. Journal of Cell Science ula loecn inlaayi nedgnu oii fe etsokada3hrcvr ntepeec rasneo etmcnB olwdb co by followed B, leptomycin of absence or presence the in recovery h 3 60 at a taken and were shock images heat N blinded after and cofilin random endogenous 10 on immunostaining. analysis signal fluorescent nuclear meitl fe etsokadwr muotie o oii.2 euniliae narno rai h ihwr ae t60 at shown). taken rods were dish with the cells in of area (percentage random counted a were in rods images nuclear sequential with 25 cells cofilin. and for cells immunostained of were number and shock heat after immediately eoe o ro oimnsann.Cflnrmisnceratrha hc nclstetdwt etmcnBadfwrclsfr os ahdl Dashed rods. form cells fewer and B leptomycin with treated cells in shock (white heat mutant after V20A nuclear the ( expressing remains outline. cells Cofilin cell in immunostaining. form whole still to rods prior indicate Endogenous h actin. for 3 immunostained for and recover fixed were Cells h. 1 ( for shocked heat were cells and oiiV0 ne taysaecniin nlv cells. live in conditions state steady under cofilinV20A oii rEF-oiiV0 a rnfce noSHhcls el eeiae ro o n fe etsok 2Amtn cuuae oei h nu the in more accumulates mutant V20A ( shock. stress. heat after during medium and rods the to, nuclear to prior form added imaged was not were B does Cells leptomycin cells. and ng/ml STHdh stress 5 into and before 60 transfected cells at was STHdh taken EYFP-cofilinV20A into images or transfected cofilin All was imaged. NES EYFP–cofilin were cells. cells STHdh Live into transfected were NES EYFP-cofilin h 2Amtn uigsrs,idctn htoeepeso of overexpression that indicating stress, expressing during cells in mutant endogenously, V20A form still the can rods that Table show performed, we material was actin cytoplasm supplementary for immunofluorescence or 2C; when nucleus however, (Fig. S1); the stress either shock in heat rods abilityduring form the inhibited to also EYFP–cofilin mutation This of S1). Table and material S1A,B supplementary Fig. 2C,D; (Fig. performed was analysis signal ( cofilin. of species other of that with sequence. NES NES cofilin2 putative and conserved cofilin1 a human has Cofilin 2. Fig. F 5# Td el eeutetdo rae ih5n/llpoyi o ro oha hc teste muotie o oii,o el eeallowed were cells or cofilin, for immunostained then stress shock heat to prior h 4 for B leptomycin ng/ml 5 with treated or untreated were cells STHdh ) 0clscniin * cells/condition. 30 P , G .05 cl as 10 bars: Scale 0.0035. fet flpoyi ramn ntepretg fclswt ula osimdaeyatrha hc.Clswr fixed were Cells shock. heat after immediately rods nuclear with cells of percentage the on treatment B leptomycin of Effects ) 6 anfcto nawdfedmcocp.( microscope. widefield a on magnification m m. n 5 , ( A 0clsprcntutcniin * construct/condition. per cells 50 D R1dpnetNScnessadmthn eunei oii.Sqec oprsno the of comparison Sequence cofilin. in sequence matching and consensus NES CRM1-dependent ) rp ersnspretncerfursetsga nlssfrEF–oii essEYFP– versus EYFP–cofilin for analysis signal fluorescent nuclear percent represents Graph ) 6 anfcto n el rmteeiae eeqatfe o ecn ula fluorescence. nuclear percent for quantified were images these from cells and magnification B oii E ucin u fcnetadi etmcnBsniie YPaoeor alone EYFP sensitive. 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Journal of Cell Science oii a rvosydfndpttv ula localization al., nuclear et (Iida putative 26-PEEVKKRKKAV-36 acids defined amino at previously (NLS) signal a has NLS bipartite Cofilin conserved a has Cofilin cofilin– into incorporated be or stress. form during V20A to rods mutant ability actin NES the that have and not NES does CRM1-dependent al., a et has Park cofilin 2012; al., 6 et Stu exportin (Dopie and 2011; B-sensitive profilin leptomycin not through since is mediated formation and is rod export to is for nuclear respect CRM1, actin proteins with through rod B, cofilin target B leptomycin affecting many that leptomycin specifically believe vivo has we in export, CRM1 nuclear the Although likely in assay. that affected indicating forming 2010) is al., cofilin–actin rod et for only proteins (Minamide required vitro only the in be formation to shown been have actin and 3980 vne l,20) rmteedt,w ocuethat conclude we data, these From 2003). al., et ¨ven ora fCl cec 2 (17) 125 Science Cell of Journal Dpee l,21;Id ta. 92.Teedt ugs that suggest data These 1992). via al., entry et nuclear import actin Iida for nuclear 2012; critical and al., be to et 1A,C) shown (Dopie (Fig. been also is nucleus cofilin has of cofilin the rods proportion large (supplementary to formed a sequence stress, localized construct During cofilin S1). this wild-type Table material the expressing with cells compared S1), form Table fewer material to supplementary ability 3A,B; although its (Fig. the by stress maintained enter during studies to KR31-32AA rods cofilin previous cofilin of ability and to the done in nucleus Contrary change been no 1992). and observed has we al., 31 what full- others, et the to lysine of (Iida similar context acids previously construct, the amino in EYFP-cofilin KR31-32AA, critical length alanines, to in mutated 32 rods we arginine nuclear nucleus cofilin–actin the system, of enter formation to our the cofilin inhibit of also ability would the inhibiting importin- whether monopartite, test a is which 1992), as 10 bars: N cells. leptomycin-B-treated and control KR31-32AA- RK21-22AA cofilin GFP– and KR31-32AA RK21-22AA EYFP– cofilin EYFP–cofilin, for analysis signal then ( was performed. ng/ml imaging 5 Live with B. treated leptomycin were they and cells KR31- 32AA- RK21-22AA GFP-cofilin or 32AA ( nlssfrec osrc nlv cells. live in N construct each for analysis ( STHdh cells. into transfected were KR31-32AA RK21-22AA EYFP-cofilin entry. or nuclear EYFP-cofilin cofilin affects NLS bipartite ( cells. fixed * construct cells/construct, each for analysis signal nuclear active ( for import. look to cells into STHdh transfected were KR31-32AA 22AA NLS- bipartite GFP-cofilin oiiK3-2Awstasetdinto transfected was cofilinKR31-32AA conserved NLS. highly bi-partite a putative has Cofilin 3. Fig. a,GPcflnbprieNLS- GFP-cofilin-bipartite Gal, 10 bars: Scale microscope. widefield 60 at taken formation. images rod All and shocked signal heat nuclear were showing cells and cells STHdh GFP-SV40NLS- mediates cofilin. of species other ( of that with bi-partite NLS cofilin2 and cofilin1 human the comparison of Sequence cofilin. in sequence ( cells. live in N conditions state steady under KR31-32AA EYFP–cofilin versus EYFP– cofilin for analysis signal fluorescent ( H D C B 5# 5# 5# rp ersnspretnuclear percent represents Graph ) h uaiecflnbprieNLS bipartite cofilin putative The ) matching and consensus NLS Bi-partite ) YPcflnR2-2AKR31- RK21-22AA EYFP-cofilin ) 0clscntut * cells/construct, 30 0clscntut * cells/construct, 30 0prconstruct/condition. per 30 G b Glwr rnfce noSTHdh into transfected were -Gal ecn ula loecn signal fluorescent nuclear Percent ) E m b m. ecn ula fluorescent nuclear Percent ) Glncerety GFP- entry. nuclear -Gal I F ecn ula fluorescent nuclear Percent ) uaigbt at fthe of parts both Mutating ) a b / P b Gl F-oii NLS- GFP-cofilin -Gal, , dpnetNS To NLS. -dependent .5 mgstknof taken Images 0.05. 6 anfcto na on magnification P P ( b A , , GlRK21- -Gal EYFP- ) .5 Scale 0.05. 0.05. b Gland -Gal b N a in Gal 5# b -Gal, m m 30 b - Journal of Cell Science 1991) ai ihrgos eeal eaae y1–2aioacids, amino 10–12 (K/R)(R/K)x by consensus separated the generally with regions, rich active basic optimal for a required are acids or entry. diffusion, amino nuclear simple by additional alone nucleus that the enter may cofilin either RKSSTPEEV D smnmr 2 D sttae) hscntutfrexceeds far construct this tetramer), as kDa 624 monomer, and as kDa GFP of cofilin weight between mutant molecular expressing NLS full-length, fused cells a treated created construct with also we KR31-32AA We total), B. RK21-22AA EYFP leptomycin in EYFP–cofilin of kDa size or (44 cumulative EYFP–cofilin the cofilin to to an due simply fused not nucleus and the NES, from the via part exclusion export in nuclear is active 3F,G), by (Fig. mediated mutant NLS the RK31-32AA, KR21-22AA and S1B Fig. material (supplementary a cofilin S1). not effect length is Table this specific full indicating of type fibroblasts 3T3 context cell NIH the in in functional also mutant material was NLS (supplementary The capable rods S1). longer cytoplasmic no Table or is nuclear mutant forming NLS cofilin of the the with As mutant, S1). NES Table EYFP– cofilin material type supplementary less wild 3F,G; to (Fig. had compared cofilin state signal KR31-32AA fluorescent steady nuclear RK21-22AA percent Under EYFP–cofilin fusion KR31-32AA. the conditions, make to RK21-22AA regions length basic full EYFP–cofilin both the 3T3 type mutated of we context the cell NIH protein, the whether cofilin in not test functions mouse To was sequence NLS S1C). activity in bipartite Fig. NLS material S2. constructs (supplementary the Fig. specific that these material confirmed of supplementary fibroblasts in testing shown fluorescent Additional are nuclear percent analysis for controls signal of and entry nuclear Method mediate 3D,E). longer (Fig. no could sequence the Gal, oii hr r w ai mn cd,R.Tesqec (21- sequence The RK. acids, amino basic two are there cofilin n ewe oii1adcfln Fg C.I re to and were GFP 21–34) order context between of acids out fused acids In amino taken were and amino (cofilin cofilin from 3C). (cofilin NLS sequences (Fig. these bipartite NLS functional, the monopartite cofilin2 or the 26–34) and either if cofilin1 cofilin determine cofilin in of between NLS species different bipartite and between the conserved of evolutionarily parts are basic Both consensus. NLS h ciiyo h oii L euneb t blt to a ability GFP–SV40NLS– As 1999). expressed its Stamminger, we by and control, (Sorg sequence nucleus positive NLS the GFP– into cofilin The tetramer) mediate the NLS. of endogenous heterologously an activity GFP–putative-NLS– lacks the of and fusion tetramerizes, triple kDa, 112 oaaie ntetil uinGFP–( fusion mutated triple were the regions extended in basic nuclear The critical alanines both to the assay. 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FRET from optimal acceptor energy pairs, donor–acceptor of an transfer to resonant measure donor the to by of cells used live efficiency be in scale calculating can nanometer FRET the at measure environment. interactions protein–protein live-cell accurately a in to actin fluorescent (FLIM) employed microscopy we Fo as vivo, imaging strongly measure in lifetime as To interaction S4). bound cofilin–actin Fig. neither the material however (supplementary some cofilin F-actin, have wild-type bind KR31-32AA we 21-22AA to RK assay, and ability vivo. co-sedimentation cellular V20A F-actin critical both in vitro bind other that show hypothesis perform in still to an could our able Using mutants be functions. test these still if therefore to ascertain and actin mutants to that had these and we formation is Therefore, use rod modulate response could to avenue stress we an be import cell may nuclear export cofilin during and regulating that formation hypothesized We rod unknown. requirement absolute the cofilin–actin HD however, for 2010); of approach al., therapeutic et formation novel (Bamburg rod a AD inhibit be or to could rods able down being break that or vivo hypothesized in been actin has bind It can mutant cofilin-NLS-inactive The rtrrsnneeeg rnfr(RT ewe oii and cofilin between (FRET) transfer energy resonance ¨rster natmtt ute eiyteefnig,w etdorcofilin our tested we findings, these verify further to attempt In ` e ta. 07 Lle 2007; al., et res b Glcntutddnt(i.3H,I; (Fig. not did construct -Gal ` e ta. 09.FRET 2009). al., et res , 0,as 30%, Journal of Cell Science oii 2Ahdatnbnln ciiy hra l other all whereas activity, bundling and cofilin actin wild-type only had that K22A found activity and cofilin bundling mutants actin cofilin determine our to of centrifugation, We speed assay S1). co-sedimentation low shock, Table F-actin at vitro material heat in supplementary hour an 5D; 5D; rods performed one (Fig. (Fig. formed additionally cells a which of rods EYFP–cofilin after 37% type cells in cofilin wild mutant of than K22A 2.8% any fewer in the although form rods however formed not S1), still Table did material mutant supplementary depletion, rod- ATP R21A or cofilin/actin shock to heat the of constructs of conditions Under K22A halves cells stress. nucleus subjected both and inducing We mutating R21A the S1). as the enter Table extent expressing material same to these (supplementary the cofilin NLS of to the not of each but ability that 5C) (Fig. the showed affected of signal Quantification mutations fluorescent 5A,B). nuclear in K22A (Fig. actin FLIM–FRET and percent bind to measuring R21A ability these by the the vivo that retained both proteins found expected, fusion mutant As and cofilin cofilin S4A,B). vitro K22A (supplementary Fig. ability and in binding material R21A F-actin F-actin some the bind maintained not of mutants to Table ability did ability material the mutation mutants supplementary measured this 3A; We (Fig. not and S1). did formation KR31- residues localization rod on these nuclear abolish focus mutating EYFP–cofilin found not cofilin had in did affect already resulting We we NLS, as K22A. 32AA first bipartite EYFP–cofilin the on and the in effect R21A mutations of RK21- new minimal same region as two a the formation basic created if have rod We test and KR31-32AA. would to binding 22AA NLS wanted actin cofilin localization, we nuclear vivo, the KR31-32AA, in to RK21-22AA actin mutation mutant, bind still NLS could cofilin stress the during ability Although forming rod affect mutants NLS Cofilin FRET methods. 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BL21 6 5)wr acltdb the by calculated were 256) 6 g 0n) l live-cell All nm). 20 , qa mut of amounts Equal . 05photons/sec, 10e5 t o each for ) m Mand 5 S 3, 9 - Journal of Cell Science ag,A,MLn,L . il,R . eie .E n obt,A H. A. Corbett, and E. S. Devine, E., R. Mills, M., L. McLane, A., Lange, ag,A,Mls .E,Lne .J,Seat . eie .E n obt,A H. A. Corbett, and E. S. Devine, M., Stewart, J., C. Lange, E., E. R. Mills, A. A., Carpenter, Lange, and M. D. Sabatini, R., M. Lamprecht, uo . of,B,Skmt,T,Shenr .P,Ynd,Y,Yngd,M., Yanagida, Y., Yoneda, P., E. Schreiner, T., Sekimoto, R., B., L. Yu, Wolff, B., N., Zhang, Kudo, Y., Zhang, A., Pariser, L., R. Levine, S., Zdanov, F., Klamt, elr .N. J. Keller, arnrg . tflr .adAb,U. Aebi, and D. Stoffler, B., Fahrenkrog, adrn . oet,B . ihrsn .D n mt,A E. A. Smith, and D. W. Richardson, L., B. Roberts, D., Kalderon, I. Yahara, and S. Matsumoto, K., Iida, ri,M . rbr,C . cnie,L,Nesn .B,Lmet .H., F. M. I. Beal, and Lambert, R. R. B., Ratan, P., M. J. 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Piston, and B. Granada, H., G. Springer, A., M. Rizzo, Rajakyla R., Pawłowski, i,J,Le .H,Tyo,J,Vnef,M n rat R. Truant, and M. Vandelft, J., Taylor, H., D. Lee, J., Xia, K. I. Lim, and J. T. Park, H., S. Park, uhr,J . ei,B n uln,R D. R. Mullins, and B. Belin, B., J. Zuchero, Y. Wang, and J. J. Sanglier, B., Wolff, T. Obinata, and R. Nagaoka, H., Abe, S., Ono, ihd,E,Id,K,Ynzw,N,Kys,S,Yhr,I n aa,H. P. Sakai, Percipalle, and and A. I. Obrdlik, Yahara, S., Koyasu, N., R., Yonezawa, J. K., Bamburg, Iida, J., E., E. Nishida, Wild, I., Marsden, S., R. Atwal, N., Caron, L., Munsie, os .C,Dmro .A n a,R N. R. Day, and A. I. Demarco, C., T. Voss, R. Treisman, and B. Larijani, S., Guettler, K., M. Vartiainen, E. Nishida, and F. Toyoshima-Morimoto, E., Taniguchi, or . ry . ice,T,Gu T., Fischer, S., Frey, D., Mohr, Stu iaie .S,Mii . ol,J . ai,R . opne,J . a,Y., Bao, A., J. Coppinger, C., R. Davis, A., R. J. J. Boyle, Bamburg, S., and Maiti, J. S., P. L. Meberg, Minamide, A., J. Boyle, M., A. 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