Journal of Cell Science ohaBarry Joshua K clusters voltage-gated in Shaw motors by kinesin conventional of Activation Article Research ta. 91 epl ta. 92 cnte n lc,1997; Block, and Schnitzer 1992; Hirokawa al., 1990; et al., Leopold et the 1991; Block and 1989; increase al., a (Miller al., et to velocity along et the Howard together vesicle not motor 1985; work but Lasek, one one distance, can travel transport Although and motors processivity to dimer. few sufficient a the microtubule, often of KLCs is C-termini two and complex the dimer (KIF5) to chain heavy binding a still of go, consists to kinesin-1 where and ride, exactly to knowing how unclear. ride, transport, remains to selective protein motor during play which 2009; role can al., themselves et important cargos Sobotzik whether 2007; an However, al., 2009). et al., Xu 2010; et al., Song (Maniar et Xu the transport 2012; maintaining selective al., for et kinesin- regulating critical by be of polarity to appear axon-dendrite specificity segment at initial filaments the axon actin and the ankyrin-G and regulate instance, For cytoskeletal transport. also mediated Other 2005). proteins al., Takemura, et associated Burack and 2011; on al., Hirokawa et rely (Nakata 2000; dendrites primarily and axons to to microtubules lead different subcellular that thought between and distinguish is distinct which proteins motors, distances kinesin to of long over transport localized organelles forward are Intracellular receptors compartments. ion including and proteins membrane channels functions, neuronal for Crucial Introduction transport, channel Kv3 of specificity the to function. words: only motor Key regulate not of Kv3 contributing by regulation proteins, T1 motors cargo-mediated channel KIF5 Kv3.1 of the the of Deletion activation to containing tail. Live-cell and puncta. vesicles also they clustering KIF5B tail. anterograde carrier but how Therefore, KIF5B-YFP KIF5B in the in neurons. of the differ cerebellar number number in to VAMP2, mouse motor and binding in and residues the frequency clusters SNAP25 for the KIF5 high- proteins basic (KLC1), increases reduces synaptic 1 the three channels markedly two chain that Kv3.1 and Kv3.1 requires light KLC1 Only reveal kinesin Kv3.1, distribution. KIF5B assays proteins, with KIF5B KIF5-binding biochemical and not K regulate four but Our voltage-gated domain that microtubules, proteins. show (Shaw) T1 and assays KIF5-binding Kv3 domain imaging Kv3.1 the of motor the that the role KIF5 with report potential Endogenous between competes transport. and a binding axonal kinesin, suggesting during function multimeric motors conventional axons, motor KIF5 of affinity regulate along activates chain cargos and clusters clusters How heavy forms protein, neurons. the KIF5-binding often in tetrameric KIF5, locations known on only specific the focus to channel, we cargos Here different many unclear. transports remains motor kinesin conventional The Summary USA 77030, TX Houston, Medicine, of College § Baylor Neurology, of ` Department address: *Present 4 3 2 1 o:10.1242/jcs.122234 doi: 2027–2041 126, Science Cell of Journal 2013 February 19 Accepted hnGu Chen rsn drs:Dprmn fBoeia cecsadPtoilg,Cleeo eeiayMdcn,Vrii ehUiest,Bakbr,V 24 VA Blacksburg, University, Tech Virginia Medicine, Veterinary of College Pathobiology, and Sciences Biomedical of Department address: Present uhrfrcrepnec ( correspondence for Author nertdBoeia rdaePorm h hoSaeUiest,Clmu,O 31,USA 43210, OH Columbus, University, USA State 43210, Ohio OH The USA Columbus, USA Program, 43210, University, 43210, Graduate OH State OH Biomedical Columbus, Ohio Columbus, Integrated University, The University, State Facility, State Ohio Genomics Ohio The Plant-Microbe The Program, Neuroscience, Graduate of Biology Department Developmental and Cellular Molecular, 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. samjratrgaemtroeaigi xn,conventional axons, in operating motor anterograde major a As 1,2,4,§ otg-ae oasu hne,Mlclrmtr xnltasot ag-oo neato,Knsnlgtcan yatcprotein Synaptic chain, light Kinesin interaction, Cargo-motor transport, Axonal motor, Molecular channel, potassium Voltage-gated 1 igunXu Mingxuan , [email protected] ) 2, ,YazegGu Yuanzheng *, 2 nrwW Dangel W. Andrew , + channels lsesaognuie rmtdu oeaiehwKv3 imaging how live-cell of combination biochemistry, examine a protein to Using including function. us KIF5 approaches prompted regulate may neurites channels 2012). along Gu, and clusters Barry al., 2010; al., directly et that et Kaczmarek channel (Xu ion 1999; KIF5 1999; identified to al., first al., binds the et et is (Rudy 2007), Bixby Bean, channel, spiking Kv3.1 1997; 2005; 2005). fast Jan, al., et in and Long 2002; involved Jan al., et 1995; Jahng 2002; al., al., Choe, et responsible et (Li form complex is Xu tetrameric channel which 1992; domains a subfamily, of assembly channel T1 proper the Kv for N-terminal C- a within and Conserved tetramers N- domains. intracellular and terminal segments, membrane-spanning six ar,21) ahK hne ope otisfu voltage- four contains complex pore-forming and channel and along (Gu sensing Kv terminals Each propagation axonal 2011). at Barry, release uni-directional neurotransmitter and and axons, frequency different waveform, axonal sites? distinguish overlapping of KIF5 to binding does transport those How particularly and cargos, organelles. recognition apparent and an to specific proteins leading 2010), for al., KIF5 et conundrum the remains (Xu to domains regulated 2009). bind tail is directly al., C-terminal vesicle proteins et carrier many Laib a Furthermore, 2008; on unknown. al., number et motor the Shubeita How 2007; al., et Vershinin nti td,teosrainta noeosKF often KIF5 endogenous that observation the study, this In xnlK hnesrglt cinptnilinitiation, potential action regulate channels Kv Axonal 3 ee Jukkola Peter , a uuis Each subunits. 4 hnr Shrestha Chandra , 6,USA 061, a uui osssof consists subunit 2, ` 2027 and + Journal of Cell Science rohas n ihu ooaiigFatn(niae ybakarrows). black by 10 (indicated bars: F-actin Scale colocalizing without single-channel and in arrowheads) inverted are Signals ( merged). images. in (blue anti-KIF5B antibody rabbit and tubulin a merged) with phalloidin stained in with was (green KIF5B labeled antibody merged). was in F-actin (red 546 cone. Fluor ( growth Alexa DIV. axonal 10 the at at here (arrowhead) used was hippocampal E18) cultured (at in motors KIF5 endogenous neurons. of Clustering 1. Fig. level lower a at distributed axon KIF5B also distal was the DIV, along KIF5B 10 smoothly 1A). more (Fig. at relatively phalloidin F-actin along culture with with colocalizing also These neuron cones, labeled but growth endings. axonal low-density cones, many in growth axonal a clustered axonal in In at clustered only axons. not and were axons, clusters smoothly rather distal young distributed soma, along both the in from concentrated (days was observed KIF5B DIV be 3 at can neurons clusters neurites. KIF5 along are clusters Endogenous motors form sometimes KIF5 and endogenous smoothly distributed neurons, hippocampal cultured neurons in In motors KIF5 endogenous of pattern Distribution KIF5B- Results 200 anterogradely- to Our up and clusters. recruit dimers. in Kv3.1-containing motor astonishingly activation YFP can a KIF5 punctum that to moving leads indicate tail results KIF5B and Kv3.1 between compelling binding T1 provided multimeric have high-affinity the we that mice, evidence knockout Kv3.1 and assays, 2028 B h o-est utr fhpoaplnuosfo a embryos rat from neurons hippocampal of culture low-density The , C ora fCl cec 2 (9) 126 Science Cell of Journal lseso I5 ln xn ih(niae ywhite by (indicated with axons along KIF5B of Clusters ) m m. nvitro in b tblnwslbldwt os anti- mouse a with labeled was -tubulin A noeosKFBmtr cluster motors KIF5B Endogenous ) n le ern t2 DIV. 21 at neurons older and ) b - a efre ihioae oan fK31adKFBrather KIF5B assay and binding Kv3.1 this of that domains note isolated with to performed important was is tail it KIF5B obtained However, whole was the domain. containing affinity GST-Tail binding and His-31T1 Similar between right). 3B, (Fig. His-31T1 rtis hwn S-aladGTT0 u o GST- not but GST-T70, and GST-Tail T70 showing proteins, His-31T1 to binding the lost 2E). completely (Fig. (Ds), ones acidic three isn 5rsde rmbt nso 7,silple onHis- down pulled still T70, GST-T70 of ends 31T1, both from residues 25 missing GST-Tail oGTT0i ihafnt ( affinity bound His-31T1 high His-31T1 buffer. in binding while a GST-T70 in chip, to surface sensor chip the the over on flowed immobilized were proteins ayKF al n 1ttae a idt,adwa r the are binding. what the and for to, critical how bind tail is, can KIF5 in tetramer binding 2010). elements T1 al., the structural one et strong tails (Xu how KIF5 is tetramers unknown many channel functional remains Kv3 forms it the domain, that tail However, all KIF5 one Among the only binding. to the cargo bind stalk for to domain known a proteins tail microtubules, involved. C-terminal regions, coiled-coil on a through and walking N- dimerization are for an for responsible contains domain domain proteins KIF5C) motor and KIF5B terminal KIF5-binding (KIF5A, KIF5 Mammalian whether wondered arguing a molecules, only contains KIF5 vesicle were carrier motors. of each few that clusters hundreds notion KIF5 current or these many the against contained tens If likely maybe 1B,C). most (Fig. motors, they vesicles, clusters carrier F-actin, with axonal tubulin moving colocalize along not with did sizes they nor and different present with also were clusters trunks However, 1A). (Fig. rmete nseiiae h idn.GST-Tail binding. the eliminated ends either from ( charge positive Tail atascain( association fast idn fiiy( affinity binding h on uatGST-T70 mutant point The (SPR) Resonance proteins, GST-Tail fusion Plasmon GST GST-T70, four Surface GST, and His-31T1 the purified with performed experiment we 31T1, from Blue Colloidal blotting. resulted of western likely sensitivity and different staining most and which affinity binding 3A), lower (Fig. His-31T1 purified I5 al emd eiso S-uinpoen Fg 2A). (Fig. proteins GST-fusion of within series site a T1-binding made Kv3.1 we the tail, in KIF5B residues critical identify To domains T1 tail Kv3.1 KIF5B the between and binding multimeric and High-affinity 11fo atra yae Fg BE.Bt h N-terminal the Both 2B–E). His- (Fig. down lysates (GST-Tail pulled bacterial efficiently from 865–934) 31T1 (a.a. GST-T70 Purified h w hr he ai eius(R residues 2B–D). basic (Fig. three efficiently share less two but The His-31T1, down pulled also T70 opl onHs3T Fg BE.WeesGST-Tail Whereas 2B–E). (Fig. His-31T1 down pull to iascain( disassociation tiig(i.3) upiigy mle rget,GST- fragments, smaller Surprisingly, 3A). Tail (Fig. staining otat GST-Tail contrast, efrhrpromdtepldw sa ihpurified with assay pulldown the performed further We oudrtn h ehns neligKF lseig we clustering, KIF5 underlying mechanism the understand To odtrietebnigafnt ewe S-7 n His- and GST-T70 between affinity binding the determine To RKR 892–934 865–891 rcpttdHs3T eeldb olia blue Colloidal by revealed His-31T1 precipitated , 913–934 865–896 GST-Tail , n GST-Tail and K GST-Tail , RKR n -emnl(GST-Tail C-terminal and ) K K off . 892–934 on d 1.6 : 5.Frhrsotnn ftetoregions two the of shortening Further +5). 3.1 : ihtetrebscrsde uae to mutated residues basic three the with , 2.7 : 865–886 6 6 6 a eryatosn-odrdcinin reduction thousand-fold a nearly had 0.1 892–934 0.4 897–934 865–896 0.8 GST-Tail , RKR 6 6 6 K 10 10 10 d n GST-T70 and , 6.0 : n GST-Tail and , i o lal uldown pull clearly not did , opeeyls h idn to binding the lost completely 2 2 4 3 5 M; M; M; 892 6 n 875–896 1.4 K n n 5 5 893 5 6 )adrltvl slow relatively and 4) )(i.3,middle). 3B, (Fig. 3) )(i.3,lf) In left). 3B, (Fig. 4) 10 R 892–934 894 GST-Tail , 2 RKR 902–934 8 n aeanet a have and ) M; 870–896 S fusion GST . oto of portion ) n l failed all , 5 )with 4) 875–919 892–912 GST- , , , Journal of Cell Science ofre h ietbnigbtenK31T n KIF5B role the and hence and T1 residues basic experiment Kv3.1 three the of between SPR the role critical binding of the context the tail, direct the the in Nonetheless, position domains confirmed rigid T1 complex. more Kv3.1 much the whole-channel a to in due may fixed scenario KIF5B being binding and different Kv3.1 a Full-length have proteins. full-length the than aisfo : o101 npeiiat,teaon fGST-T70 of amount the precipitants, In 100:1. to 0:1 from ratios His-31T1 purified with assay ( pulldown a motor. performed the kinesin we tetramer, of a and value channel first ion the an between provided affinity and binding interaction, electrostatic of , oetmt o ayKFBtisbn ooeK31T1 Kv3.1 one to bind tails KIF5B many how estimate To 0ka n S-7 ( GST-T70 and kDa) 20 v lsesadatvtsKF oos2029 motors KIF5 activates and clusters Kv3 E uaigR His-31T1. Mutating to (E) of binds half 892–934 Neither fragment (D) the His-31T1. of to half binds N-terminal T70 The (C) Kv3.1 site. the of T1-binding region minimal domain. the tail Mapping KIF5B (B) the in site binding vitro eerpae tlattretimes. three least assays at Pulldown repeated kDa. were in left the are on weights indicated Molecular His-31T1. to of T70 binding the negative eliminated to completely positive charges switch to residues acid leadrdbakt,rsetvl.( respectively. with brackets, indicated red are and T1 blue Kv3.1 to crucial binding residues for basic three and motor site The inhibiting lines. the are above fragments indicated tail respectively. of green, numbers or Residue assays black pulldown in in indicated His-31T1 are to or bind bind to that fragments fail tail KIF5B fusion of GST proteins KIF5B. in indicate positions numbers residue The yellow. are in residues highlighted conserved KIF5C, KIF5A, and human KIF5B of In alignment shown. sequence is the domain C-terminal a Kv3.1 and only one dimer simplicity, a For as respectively. shown tetramer, are tail. Kv3.1 KIF5 and in KIF5 (T70) site T1-binding Kv3.1 domains. the tail Kv3.1 KIF5 the and between T1 binding Direct 2. Fig. , 2ka negtdfeetmolar different eight in kDa) 32 idn sast a h v. T1- Kv3.1 the map to assays binding 892 K 893 R 894 otreaspartic three to ( A iga of Diagram ) B – E ) In Journal of Cell Science 6 aa 5–2,cnann h L1bnigst) rGST- or site), binding KLC1 and the T70 tail containing KIF5B 758–820, GST- the GST, (a.a. not in T63 but T1 GST-T70, Kv3.1 and GST-Tail of binding. role microtubule the examined further we aladHsMtrapae ekr(i.4;supplementary 4D; GST- (Fig. between weaker S1C). binding Fig. appeared the material His-Motor although S1A,B), and tail, His-31T1 KIF5B Tail binding with Fig. to compete for did binding KLC1 His-Motor for with contrast, material In compete tail. not KIF5B supplementary His- to does and T1 GST-Tail Kv3.1 His- 4B,C; between suggesting of (Fig. binding His-tagged amounts the different Increasing decrease KLC1 not ratios. two did molar and 31T1 different GST-Tail, in of how proteins determine amount To competitive KIF5B constant to 4A). performed Cai binding (Fig. other’s we 2002; each 2006) affect al., tail, al., T1 et Kv3.1 et and Setou Huang proteins Glater 2000; these 1999; 2005; Stock, Vale, Diefenbach al., and and 1993; Friedman Hackney et 1999; al., 1999; al., et al., et Andrews et Coy and 2010; 1998; Wong al., al., 2010; et et al., Xu et with 2010; (Twelvetrees interactions Rice, for microtubules and and sites auto-inhibition, myosin binding recognition, overlapping tail cargo contain KIF5B for domains to tail binding for C-terminal and KLC1 KIF5 domain with motor not KIF5B but the microtubules with competes T1 dimers). Kv3.1 chain heavy four up (or bind motors can KIF5B four heterotetramers, complex, to tetramer and channel homotetramers Kv3 both one including subfamily, binding channel the Kv3 of the stoichiometry the (His-31T1:GST- is that (AU) complex suggesting 200:320 3C), of and (Fig. ratio increased T70) intensity His-31T1 an of at amount saturated the whereas constant, stayed 2030 yuigmicrotubule using By RKR on omcouue Fg EF supplementary 4E,F; (Fig. microtubules to bound , ora fCl cec 2 (9) 126 Science Cell of Journal , :.SneteT oani ihycnevdwithin conserved highly is domain T1 the Since 1:1. nvitro in nvitro in sebyadpldw assays, pulldown and assembly idn sas iha with assays, binding oiatngtv osrcst irp noeosKIF5 as endogenous used disrupt commonly tail are to KIF5B and cargos, on constructs binding contain effects dominant-negative domains for tail their sites KIF5 binding The examined coexpression. using we distribution neurons, living observed immunostaining was the patterns. Therefore, with clusters different colocalize in rows). proteins in KIF5 KIF5-binding 4th different that colocalization and show results significantly clear 3rd KIF5 5A, clusters, no with (Fig. colocalized in and KIF5, KIF5 KIF5-tail- of less KIF5 with cargo the with colocalized a Whereas VAMP2, colocalized partially row). 2nd extensively SNAP25 Among expressed 5A, binding KLC1 top) (Fig. neurons. some 5A, 2010). clusters (Fig. including al., in DIV, clusters in et 21 proteins KIF5 as (Xu with at colocalized four between motility which neurons Kv3.1b, these colocalization and hippocampal and the cultured distribution examined motor KIF5 we KIF5 in KIF5-binding First, regulating experiments known comparison. four of in of series roles proteins the a determined designed We neurons. we assays, these biochemical evaluate fragments To tail the KIF5 affect of differentially localization KIF5 of proteins Binding oo ciiyb eesn h albnigt ohtemotor the both to KIF5B binding activate tail microtubules. the likely and releasing channels domain by Kv3.1 activity Therefore, motor material supplementary microtubules, S1F). the 4G; in to (Fig. Fig. up microtubules show bind of not did fraction not His-31T1 supplementary pellet concentration does high in 4G; His-31T1 even (Fig. effectively since S1F). in Fig. manner His-31T1 fraction) material P concentration-dependent (the Interestingly, microtubules a from away S1D,E). GST-T70 competed Fig. material et oudrtn o hs rtisbn oKF alin tail KIF5 to bind proteins these how understand to Next, o GST-T70 not moiie S-7 lf) GST-Tail (left), GST-T70 immobilized ( indicated are left. kDa) the (in on weights Molecular staining. Blue Colloidal T70 i.3 idn fiiyadsocimtyo v. 1and T1 Kv3.1 of domains. stoichiometry tail and KIF5B affinity Binding 3. Fig. ifrn ocnrtos(,20n,60n,2 nM, 600 nM, 200 (0, concentrations different 100 nest rp fGTT0(lc ice)adHs3T (red His-31T1 staining and the circles) shows (black panel GST-T70 right of The graph 100:1. intensity 60:1, 30:1, 10:1, 4:1, (3.2 GST-T70 purified panel, left the In SPR. complex. in binding used tail buffer binding the in to ( not proteins but purified solubility, high-concentration the of increase included buffer was stock (0.1%) most X-100 the are Triton in change. His-31T1 buffer of to concentration due highest probably the at traces the h nest fpoenbnswr esrdadsbrce with subtracted and and file value. measured image background were TIFF the bands a protein as of scanned intensity was the gel protein The triangles). mut fprfe i-11 h oa aisbtenHis- between ratios molar ( The 31T1 different His-31T1. with purified incubated of further amounts were (30 which beads assumed), glutathione binding on coated first was C siaino h tihoer fteK31T n KIF5B and T1 Kv3.1 the of stoichiometry the of Estimation ) RKR m )o i-11wr lwdoe h hp h pksin spikes The chip. the over flowed were His-31T1 of M) , rgt,i h P xeiet ouin otiigsix containing Solutions experiment. SPR the in (right), 0ka n S-7 ( GST-T70 and kDa) 20 B RKR idn epnetae fHs3T to His-31T1 of traces response Binding ) nvitro in ulddw uiidHs3T,idctdb the by indicated His-31T1, purified down pulled ( A uiidGTTi n S-7,but GST-T70, and GST-Tail Purified ) eut bandwt protein with obtained results , 2ka ee01 .:,1:1, 0.4:1, 0:1, were kDa) 32 892–934 m oa oue 100% volume; total l mdl)adGST- and (middle) m ,10 M, m and M m g) Journal of Cell Science S-aladGTT0 u o S,GTT3o GST-T70 or GST-T63 GST, not but GST-T70, and GST-Tail n 0 odn oprdt rti eswr sd ( used. were gels protein to compared loading 10% and irtblswr eltd eyfitbnsi h eltln r u ospraatcnaiain vnwe h rti osntbn omicrotubu to bind not assemble does after protein step the washing when no even was contamination, His-31T1. supernatant there arrowheads, to Since red due (bottom). are proteins; antibody lane fusion anti-GST pellet an the using in bands loading) vitro faint (10% very blotting pelleted, western were and microtubules (top) staining Blue Colloidal 100 of presence L1aei : oa ai)mxdwt ifrn mut fHs3T.Mlrrto ewe i-11adHsKC sdwr :,031 :,31ad10 and 3:1 1:1, 0.3:1, 0:1, were used His-KLC1 and His-31T1 between ratios Molar His-31T1. of amounts different with mixed ratio) molar ( 1:1 a in are KLC1 eesandwt olia le(o) S uinpoen eefrhrrvae ihwsenbotn sn nat-S nioy(0 odn)(bo loading) (10% ba antibody Protein anti-GST gel. an SDS-PAGE using a blotting on western loaded with were revealed pellets further dissolved and were supernatants proteins of fusion volume GST equal (top). and Blue ( volume, Colloidal in with supernatant stained the were to 5 (around equal proteins buffer fusion sample GST purified with with incubated then minutes, 20 h L-idn ie(6)i oae ewe eius78ad80 ihihe nbu.TeK3T-idn ie(7)i ewe eius85ad934, and 865 residues between is ( (T70) SNAP25. site and T1-binding microtubules Kv3 domain, The motor blue. KIF5 in for highlighted sites (3.5 820, binding GST-Tail GST-Tail. and with to 758 overlaps binding tail. residues which KIF5B between red, to located binding in for is highlighted KLC1 (T63) not but site microtubules KLC-binding with The competes T1 Kv3.1 4. Fig. C G etr ltigwt nanti-6 an with blotting Western ) uiidGTT0adHs3T eemxdi ordfeetmlrrto,10 :,14ad18 nti xeiet irtblswr sebe nth in assembled were microtubules experiment, this In 1:8. and 1:4 1:1, 1:0, ratios, molar different four in mixed were His-31T1 and GST-T70 Purified ) idn xeiet eerpae tlattretms oeua egt r niae ntelf nka ,spraat ,ple;bakarrowhea black pellet; P, supernatant; S, kDa. in left the on indicated are weights Molecular times. three least at repeated were experiments binding m altxlt ute tblz sebe irtbls h uentnsadpleswr eovdi D-AEadrvae yboth by revealed and SDS-PAGE in resolved were pellets and supernatants The microtubules. assembled stabilize further to paclitaxel M 6 m i nioyi neprmn iia oB xetta oe muto S-al(2.5 GST-Tail of amount lower a that except B, to similar experiment an in antibody His )wsfrtcae nguahoebasadfrhrue opl onprfe i-L1(5.6 His-KLC1 purified down pull to used further and beads glutathione on coated first was g) D nrae muto i-oo eue h muto i-11bnigt S-al ( GST-Tail. to binding His-31T1 of amount the reduced His-Motor of amount Increased ) RKR on omcouue.Mcouue eeassembled were Microtubules microtubules. to bound , m oa)fraohr3 iue n eltdwt ihsedsi.Pleswr resolved were Pellets spin. high-speed a with pelleted and minutes 30 another for total) g v lsesadatvtsKF oos2031 motors KIF5 activates and clusters Kv3 ( A iga fteKFBti oanadfv fisbnigproteins. binding its of five and domain tail KIF5B the of Diagram ) B i-11ddntcmeewt i-L1for His-KLC1 with compete not did His-31T1 ) nvitro in m ) nadtoa odto (30:1) condition additional an g), ihprfe uuisfor tubulins purified with m ,s htGTTi n His- and GST-Tail that so g, E,F e.All les. Purified ) s GST ds, ttom). d nds :1. e in Journal of Cell Science inl eeas eetdi h oao rnfce ern.Saebars: Scale neurons. transfected of ( soma signals. the FRET in 25 strong detected had also YFP-KLC neurons were and 19 signals (blue) of CFP-Tail out between 17 axons (green); along detected were merged) into ( CFP-Tail YFP-KLC1, axons. targeted of distal YFP-VAMP2, expression nor the YFP-SNAP25 YFP-Kv3.1b, Only not proteins. but fusion black YFP axons; various arrows, of Black presence (bottom). axons ( CFP- distal dendrites. brought into arrowheads, merged) merged) in in (green (blue YFP-KLC1 CFP-Tail Tail of expressing Coexpression neurons (top). 8-DIV YFP in and regions somatodendritic colocalizing ( in arrows, puncta. restricted White non-colocalizing proteins red). arrowheads, binding in white its VAMP2; puncta; and and green) SNAP25 in KLC1, antibody (Kv3.1b, H2 (mouse KIF5 endogenous AP,alfie obigCPTi nodsa axons in distal present into were constructs YFP- CFP-Tail YFP axons and bring four distal all YFP-SNAP25 to into although YFP-Kv3.1b, failed 5C), CFP-Tail (Fig. fact, all brought VAMP2, In YFP- YFP, coexpressed, 5B). not mainly When (Fig. dendrites. but was and expressed axons KLC1, YFP-Tail both was when YFP-KLC1 in whereas regions present 2009). neurons, hippocampal somatodendritic cultured Setou, in al., the alone et Setou in and 1999; Scholey, concentrated Konishi and Cross 1997; 2002; al., et (Bi function tail KIF5B regulate differentially localization. proteins KIF5-binding 5. Fig. 2032 m A;80 (A); m ora fCl cec 2 (9) 126 Science Cell of Journal ( D A m togFE inl ivre nsnl hne n e in red and channel single in (inverted signals FRET Strong ) osann fclue ipcma ern t2 I for DIV 20 at neurons hippocampal cultured of Co-staining ) (B). m C F-alitniypoie ln xn nthe in axons along profiles intensity CFP-Tail ) B F-alwas CFP-Tail ) E togFRET Strong ) ifrn rmCPKC n F-v.b ohCFP-SNAP25 motors, both 6D,E). KIF5B CFP-Kv3.1b, (Fig. and them clusters CFP-KLC1 disperse the from to Kv3.1 tends Different of of KLC1 of expression expression bind fraction to whereas Thus, mutant the bottom), small Kv3.1. with 6C, dimerize to (Fig. may very KIF5 axons endogenous a which along in CFP-Kv3.1b Only contained clusters 6C–E). (Fig. clustered ein ihnKFBti,wihcnb sda dominant- as used smaller be can two which identified tail, have KIF5B results within these regions biochemical results Importantly, the with these soma consistent data. are Therefore, neuronal neurons living S3C,D). in using obtained Fig. signals material FRET (supplementary significant had CFP-Kv3.1b, ags o ntne L-eedn n idpnetgroups. -independent of and groups KLC-dependent different instance, of for transport the cargos, disrupt to constructs negative oxrse nhpoaplnuos F-7 u o YFP- not when but Furthermore, YFP-T70 neurons, S2). T70 hippocampal Fig. in and material axons coexpressed binding distal (supplementary both CFP-KLC1 of in not signals soma results FRET but strong YFP-T63, the yield YFP-T70, and with and CFP-KLC1 Consistent 3A), (Fig. axons 5D,E). assays distal (Fig. both highly in soma were signals and and FRET YFP-KLC1 strong tail and yielded KIF5B CFP-Tail and only colocalized neurons. between living interaction the in physical KLC1 be the axons, examined regions. not distal somatodendritic we in may domain into tail microtubules the CFP-Tail trap to to brought mechanism binding YFP-KLC1 YFP-Kv3.1b of suggesting coexpression not only 4G), (Fig. 2010) but Rice, domain and T1 (Wong Kv3 KLC and by tail- disrupted the is Whereas binding neurons. transfected microtubule in axons and dendrites both I5bnigpoen ntedsrbto atrso KIF5B-YFP of KIF5B patterns and distribution the least on proteins at KIF5-binding be the should effect. which eliminated the tail, mutation for KIF5 The responsible to partially 2010). microtubules al., the of of et that binding than (Xu to pattern KIF5B clustered difficult axonal KIF5B pronounced tailless expressing more was a neuron in It is the This of bottom). sizes YFP. dendrites 6A, and various (Fig. soma the axons in find along clusters and formed endings exclusively YFP ln xnltuk,wr bevd upiigy uaigthe mutating top), (R Surprisingly, residues 6A, observed. basic (Fig. were three sometimes trunks, axons and axonal endings distal axonal along was the at KIF5B-YFP in especially KIF5. clusters, concentrated although endogenous KIF5B- of unknown. uniformly of that pattern remains more to distribution similar motor the was neurons, the YFP in cluster expressed KIF5 potentially When affect domain and tail KIF5 the activity to proteins on binding various KLC1 How and Kv3.1 of distribution binding KIF5B the of effects Opposite on htYPT0adCPK31,btntYFP-T70 not but CFP-Kv3.1b, we and imaging, YFP-T70 FRET that Using found S3A,B). Fig. material (supplementary niioyrl fKC nKF ciain ntepeec of presence the an KIF5B In suggest activation. may CFP-Kv3.1b, KIF5 which in KIF5B-YFP, KLC1 coexpressed of of role that inhibitory to similar pattern neetnl,cepesdCPKC opeeyconverted completely 6B,D,E). CFP-KLC1 (Fig. KIF5B axons KIF5B- coexpressed along CFP-Kv3.1b, sizes various Interestingly, coexpressed in of clusters sharp formed presence YFP In the unchanged. in remained contrast, pattern distribution KIF5B-YFP he scmltl hne t itiuinpten KIF5B pattern. distribution its changed completely Ds three sn loecnersnneeeg rnfr(RT imaging, (FRET) transfer energy resonance fluorescence Using sn ornfcin eeaie h fet ffu different four of effects the examined we cotransfection, Using RKR RKR infcnl eue h xnllvlo Kv3.1bHA of level axonal the reduced significantly YPfo ihycutrdptent uniformed a to pattern clustered highly a from -YFP RKR YP ntepeec fcepesdCFP-KLC1, coexpressed of presence the In -YFP. 892 K 893 R 894 nteKFBti oanto domain tail KIF5B the in ) RKR YPrmie highly remained -YFP RKR RKR RKR and - - Journal of Cell Science F gen ln xn lbldfrTu i KIF5B Tau1 of for clusters (labeled axons along (green) YFP cl as 10 bars: Scale ern.YPi ngenadCPi nbu nmre mgs inl r netdin inverted are Signals KIF5B co images. merged arrows, in White blue in images. is single-channel CFP and green in is YFP neurons. (top) CFP-KLC1 either with coexpressed Fg 6E). (Fig. KIF5B and (top) KIF5B-YFP on ( proteins (bottom). KIF5-binding C tagged and (top) B in axons along n F-AP atal ooaie ihKFBYPand KIF5B-YFP with colocalized KIF5B partially CFP-VAMP2 and late days two (red), Tau1 marker, tr axonal clusters were Kv3.1 which (top), of neurons presence hippocampal the of or residues charged three KIF5B-YFP. the Mutating 6. Fig. F-v.b(otm nhpoaplnuos ht rosidct colocalizaing indicate or arrows ( (top) White clusters. CFP-KLC1 neurons. either hippocampal in with (bottom) coexpressed CFP-Kv3.1b when merged) in (green KIF5B-YFP RKR RKR C YPcutr ihu F-v.b ( CFP-Kv3.1b. without clusters -YFP itiuinpten fKIF5B of patterns Distribution ) YP u i o hneterdsrbto patterns distribution their change not did but -YFP, ( m A A;100 (A); m eaieysot itiuino distribution smooth Relatively ) RKR YPaogteao emn.( segment. axon the along -YFP m (B,C). m E oaiaigcutr;bakarrows, black clusters; localizating .Hgl lsee atr fKIF5B of pattern clustered Highly r. umr ftecutrn feto CFP- of effect clustering the of Summary ) rCPK31 bto)i hippocampal in (bottom) CFP-Kv3.1b or nfce t5DV ie n tie for stained and fixed DIV, 5 at ansfected RKR e)(otm.Arwed indicate Arrowheads (bottom). red) n YP(re nmre)when merged) in (green -YFP D rflso loecneintensity fluorescence of Profiles ) I5-F gen ln axons along (green) KIF5B-YFP f B itiuinpten of patterns Distribution ) RKR YP(bottom). -YFP RKR - KIF5B 76.4 alone: 327.3 CFP-Kv3.1b: AP a ocerefc ntecneto KIF5B-YFP of content the CFP- on and effect CFP-SNAP25 clear of no had Coexpression VAMP2 8C). (Fig. puncta nrae oprdt I5-F n h oxrsinof coexpression (KIF5B numbers the those increase and further KIF5B-YFP not did to CFP-Kv3.1b compared increased previously markedly 29.2 187.8 alone: dimers Kv3.1b: with (KIF5B-YFP KIF5B-YFP CFP- anterograde of of for dimers presence number increased the estimated we KIF5B In the 2010). Next, Gu, Kv3.1b, of and measured. (Gu numbers also standards calibrated were the retrograde puncta calculated of stationary intensities and Fluorescence moving 8A,B). (Fig. CFP-KLC1 oi ) h nrae oigfeunyfrKIF5B for frequency moving increased material supplementary The 7D,G,H; 4). (Fig. Movie moving were puncta YFP u infcnl erae h ubro KIF5B of number the decreased 86.8 significantly (anterograde: KIF5B-YFP but of number the increased 167.2 161.3 anterograde: alone YFP h nyoeta a ciaeadcutrKIF5B-YFP. cluster and activate can that is one CFP-Kv3.1b Therefore, only examined, velocity. proteins the in KIF5-binding increase four clear CFP- the no material KIF5B- among was supplementary There containing or 3). 7B,C,G; 2, puncta (Fig. Movies CFP-SNAP25 moving increased markedly of of CFP-KLC1, moving YFP presence frequency the not the not In KIF5B-YFP. VAMP2, few but but a or puncta, 7A,G; CFP-Kv3.1b, one (Fig. only only we KIF5B-YFP with study, vesicles observed present detectable the of high occasionally In quantified puncta 1). in Movie only moving material transfected imaging supplementary alone, were neurons expressed cell KIF5B-YFP When hippocampal live KIF5B-YFP. KIF5B cultured performed with affect on proteins we binding magnification these motility, how motor examine further To KIF5B-YFP puncta of anterograde frequency markedly moving proteins, the KIF5-binding increases other not but Kv3.1b, neord oigpnt otiigete I5-F or KIF5B-YFP either in the containing intensities Using fluorescence puncta KIF5B 2005). the Pollard, YFP-fusion moving measured and first anterograde various Wu we 2010; expressing strategy, Gu, same YFP strains and many (Gu yeast YFP- proteins contain with of measurement microscopy might quantitative calibrated using the puncta transporting they moving axonal reported in Kv1.2 these previously that of We suspected intensity molecules. fluorescence we strong puncta, the of Because KIF5B- vesicles of carrier number on copy YFP the increases significantly Kv3.1 atrgae 84.7 (anterograde: ucawsmil nteatrgaedrcin(i.7H). (Fig. KIF5B of direction puncta CFP-VAMP2 moving of anterograde or frequency the affect the CFP-SNAP25 not did CFP-Kv3.1b, in of mainly Coexpression was puncta rtewl yei h rsneo F-v.bwr actually direction. anterograde were the CFP-Kv3.1b in of were presence them of the most in and type mobile, wild Importantly, KIF5B of the motility. clusters whereas many or motor that showed and KIF5B results Kv3.1 imaging regulating 5), our Thus, in 6). differ Movie Movie KLC1 material frequency supplementary the 7F,H; reduced material (Fig. significantly CFP-KLC1 of supplementary coexpression 7E,H; (Fig. nsapcnrs oKFBYP oeta afo KIF5B of half than more KIF5B-YFP, to contrast sharp In v lsesadatvtsKF oos2033 motors KIF5 activates and clusters Kv3 6 RKR RKR 58 Fg C.Cepeso fCPKC also CFP-KLC1 of Coexpression 8C). (Fig. 45.8) YPdmrnmesi aiu ucamarkedly puncta various in numbers dimer -YFP or CFP-Kv3.1b of presence or absence the in -YFP, 6 6 .;+CPK31:764.8 CFP-Kv3.1b: + 9.5; 73,rtord KFBYPaoe 27.8 alone: (KIF5B-YFP retrograde 47.3), 6 6 55,epcal natrgaemoving anterograde in especially 15.5), 61,o ttoaypnt (KIF5B-YFP puncta stationary or 96.1), 6 58 F-v.banterograde: CFP-Kv3.1b + 45.8; 6 0.)(i.8C). (Fig. 106.1) 6 .;+CFP- + 4.2; RKR RKR RKR RKR 6 6 .;+ 4.9; 21.8), -YFP -YFP -YFP -YFP RKR RKR - - Journal of Cell Science eanducagd(i.9) v hnesaehighly are channels Kv3.1 SNAP25, channels particular, and In Kv3 cerebellum. GRIP1 mouse 9A). (Fig. in KLC1, its expressed and unchanged including KIF5 of remained expression proteins, the but binding Kv3.1b of eliminated, expression the completely the In lysates, was with brain 9A,B). mouse confirmed (Fig. of were genotyping blotting mice western PCR-based KO and The blotting 2009). western al., Sa 1997; et al., mice, Hurlock et KO 2000; (Ho Kv3.1 previously in published pattern were distribution which KIF5B the examined We knockout Kv3.1 reduced from are neurons mice cerebellar in clusters channels KIF5 Kv3.1 of motors deletion KIF5 of Can has clusters the This question. influence transport. intriguing intracellular an during raised motors channels Kv3 KIF5 that cluster indicate can results our Therefore, puncta. moving 2034 ora fCl cec 2 (9) 126 Science Cell of Journal nvivo in ? ´ ce tal., et nchez ihtehpoaplnuo utr,wihfvr h survival the favors which culture, condition neuron similar hippocampal the the using with culture, neuron cerebellar performed axons, from, cells. come non-neuronal clusters even the or where soma determine dendrites, to difficult is it I5 lsessgiiatyrdcd(T 3017 (WT: reduced its mice, KO significantly the in same Kv3.1 clusters the the remained Whereas KIF5B KIF5B 9D). of (Fig. staining Kv3.1b level background mice, and overall to KO close Kv3.1 Kv3.1b reduced, In and cerebellum, 9C). markedly layer (Fig. molecular of layer both cell in sections granule colocalized coronal and present In were the KIF5B (Puente 2010). in neurons cells) Purkinje al., in granule express et from not (axons does 1994; Kv3.1b but fibers layer, Therefore, parallel al., molecular 2001). the et McBain, in and (Weiser present Rudy is cells 2000; granule al., et cerebellar Grigg in expressed are oceryvsaieKF lsesaogidvda xn,we axons, individual along clusters KIF5 visualize clearly To 2 / 2 2545 : NP5(elw,CPVM2(le raoe(black). alone or ( (blue) CFP- CFP-VAMP2 (green), (yellow), CFP-KLC1 SNAP25 (red), CFP-Kv3.1b of presence odto.Saitclrslswr bandfo tlatfive least each at for from transfections. movies obtained independent of were number results the Statistical is shows condition. number which movie ‘n’, The as proteins. given CFP-tagged of presence the L1 re rosidct neord-oigpuncta. anterograde-moving ( indicate arrows Green KLC1. KIF5B ( segment. axonal neord rnpr fKIF5B ( of CFP-KLC1. transport of anterograde axonal presence an the along in puncta segment KIF5B-YFP ( of CFP-Kv3.1b. transport of anterograde axonal presence an the along in puncta segment KIF5B-YFP ( of segment. transport axonal anterograde an along puncta YFP KIF5B-YFP of puncta. mobility and the CFP-SNAP25 increased CFP-KLC1, not CFP-VAMP2, but CFP-Kv3.1b, 7. Fig. rnpr fKIF5B of transport ( CFP-Kv3.1b. of presence H G rqec faoa rnpr fKIF5B of transport axonal of Frequency ) rqec faoa rnpr fKFBYPpnt nthe in puncta KIF5B-YFP of transport axonal of Frequency ) 6 RKR 121; ( A YPpnt ln naoa emn nthe in segment axonal an along puncta -YFP yorp fatrgaetasoto KIF5B- of transport anterograde of Kymograph ) P 5 E .1)(i.9,) nbansections, brain In 9E,F). (Fig. 0.016) RKR yorp fatrgaetasotof transport anterograde of Kymograph ) YPpnt ntepeec fCFP- of presence the in puncta -YFP F yorp fanterograde of Kymograph ) RKR YPpnt ln an along puncta -YFP B D yorp of Kymograph ) C yorp of Kymograph ) RKR yorp of Kymograph ) YPpnt in puncta -YFP 6 137; Journal of Cell Science h I5mtri ifrn as hc slkl rtclfrthe has for study critical this likely Therefore, is transport. ride KIF5- which a KIF5-mediated can ways, of different sites, different on specificity in that binding report motor overlapping indicates KIF5 with first the also even a the study on proteins, number is binding Our motor this the vesicle. of knowledge, carrier material regulation the (supplementary our underlying vesicle To mechanism carrier and S4B). the direct on Fig. via number motors the motors KIF5 increase KIF5 hence of and cluster by binding, high-affinity tetramers KIF5 multimeric Kv3 activate material S4A). (supplementary channels the Fig. binding by Kv3 mediated tail-microtubule are and that that tail-motor mechanisms show inhibitory two we the relieving study, this In Discussion mechanisms and/or clustering. proteins KIF5 to multiple were contribute clusters may suggesting KIF5B mice, present, KO note the to still in important reduction is clear it despite However, and that consistent. sections axons are cerebellum clusters KO from neurons Kv3.1 results cultured along the of Therefore, smoother along 10H,I). much pattern (Fig. clustered reduction became 9E, clear and Fig. a axons, had in clear WT staining method KIF5B quantification that was found same we the Using there still 10E,G). neurons, were (Fig. clusters WT although colocalizing KIF5 In neurons, neurites KO 10C). present, In along (Fig. 10D,F). (Fig. weeks clusters KLC1 with neurons 2 formed before KIF5 KO around not input–output neurons endogenous KO but the The and WT weeks, deletion. the in 3 for Kv3.1 different reduced clearly with were curves of clearly consistent hyperpolarization cerebellar after 10B), was The (Fig. WT frequency 10A). potentials high (Fig. culture, frequency Hz generated action 100 in than neurons, to higher KO weeks up smaller Kv3.1 spiking 3 markedly but not clearly but Around stage, at neurons, top). developmental were potentials 10A, same (Fig. neurons action the These at generated neurons cerebellar 1994). in hippocampal Weiser 2001; al., expressed McBain, and are Rudy 2000; et Kv3.3 al., et and (Grigg cells Kv3.1 granule cells. granule of rnpr n ag-eitdrglto fknsnmotors. kinesin cargo of of regulation specificity cargo-mediated the and into transport insights mechanistic novel provided rca o h idn Fg ) et nteSReprmn,we experiment, SPR the ( in affinity residues Next, binding 70- 2). basic the determined (Fig. three (a binding identified the site and for tail T1-binding crucial KIF5B a Kv3.1 of performed region) first the residue we of tail, KIF5 mutagenesis the and systematical of T1 basis Kv3 KIF5 biochemical the between endogenous understand an interaction better in To raising 1). observed partially (Fig. 2010), be clustering, motors least whose KIF5 al., at for may et one channels responsible (Xu Kv3 only that that is binding possibility the proteins tetramerization intriguing Kv3-KIF5 and known is for tetramerization the Kv3 proper required to all on KIF5, binds relies Among directly with function 2010). that interact al., channel physically ion et identified (Xu first KIF5 the KIF5 is underlying Kv3.1 mechanism novel a is clustering T1 tail Kv3 KIF5 between the binding and multimeric and high-affinity The idn ag famncoa nioyadistarget, its GRIP1 and and KIF5 antibody between monoclonal affinity binding a ( the to of comparable range binding ciaeadcutrKF oos ti motn ont htthe subject that are note have assays to biochemical data important protein is with may biochemical It revealed channels motors. interactions These KIF5 Kv3 cluster how 4). and (Fig. regarding activate by information tail motor critical microtubule KIF5 revealed the KIF5B and to domain the motor His- binding the activate His-31T1 of with inhibition may not the purified relieving Kv3.1 but microtubules that suggesting and KLC1, show His-Motor 3C). with experiments (Fig. tetramer competed chains KIF5 channel heavy competition KIF5 Kv3.1 four and The to a up bind that simultaneously Driver/JIP3 can suggest experiments Sunday pulldown between ( affinity binding nincanladamlclrmtr The motor. molecular which a 3B), and (Fig. between channel strength KIF5B binding ion within the for an site quantification binding first the its provides and T1 Kv3.1 K K d d 5 5 v lsesadatvtsKF oos2035 motors KIF5 activates and clusters Kv3 1.8 1.9 6 6 10 10 2 2 6 8 )(u ta. 01.Frhroe our Furthermore, 2011). al., the et than higher (Sun and 2002) M) al., et (Setou M) ubro xeiet sidctdfrec bar. each for indicated is experiments of number ubr fKFBYPdmr rKIF5B or dimers KIF5B-YFP of numbers ( puncta. moving anterogradely indicate arrowheads (right). White CFP-KLC1 or of presence (middle) the CFP-Kv3.1b in or (left), alone either segment olwdb unsts,** test, Dunn’s by followed ANOVA different One-way under conditions. puncta coexpression various in dimers YFP otiigKIF5B puncta containing moving anterogradely of fluorescence ( puncta. White moving right. anterogradely the indicate on arrowheads is axon 10- the a and crossing left along the profile on intensity is the segment axonal of image (right). The CFP-KLC1 of or presence (middle) the in CFP-Kv3.1b or (left) alone either axonal segment an along KIF5B-YFP puncta containing moving anterogradely of fluorescence a vesicle. in carrier number KIF5B-YFP markedly the channels increase Kv3 Tetrameric 8. Fig. K B uniiaino h YFP the of Quantification ) d 5 6.0 ( C A 6 RKR umr festimated of Summary ) uniiaino h YFP the of Quantification ) 1.4 YPaoga axonal an along -YFP 6 10 K P d 2 , 8 swti the within is .1 * 0.01; m )between M) line m P , 0.05; RKR - Journal of Cell Science h I5 lsesfo h idtp lf)and (left) wild-type the from clusters KIF5B the lsespriae(40 image per clusters ncrnlscin fcrblu rmwl-ye()adK31K D ie ,Prij ellyr L rnl ellyr L oeua ae.( layer. molecular ML, layer; cell granule GL, layer; cell Purkinje P, mice. (D) KO Kv3.1 and (C) wild-type from cerebellum of sections coronal in uca(i.7.KC paetyddntcmeewt Kv3.1 with compete not did moving apparently KIF5B-YFP KLC1 on 7). effects (Fig. different puncta had and dramatic they very were Fourth, patterns distribution clear. of changes the 6, Fig. ipre h lsee atr fKIF5B CFP-Kv3.1b of not pattern but clustered axons CFP-KLC1 the along interestingly, dispersed KIF5B-YFP Third, clustered 6). CFP-KLC1 CFP- (Fig. Second, tail- not 4). First, (Fig. activity. but tail and motor KIF5B Kv3.1b in KIF5 regions binding KLC1 different regulating to and in bind domain they Kv3.1 ways 2010), multiple tail-motor Rice, in differ also and the (Wong was binding release KLC1 microtubule Although to function. KIF5B shown regulating this in in examined study proteins KIF5-binding the among unique is Kv3.1 suggesting differently, transport motors cargo-directed KIF5 ride cargos Different neurons. living using experiments various regulations out carried of variety to mice. knockout Kv3.1 ( of neurons cerebellar in ( reduced knockout clusters KIF5 9. Fig. 2036 B eoyigo v. eeoyoe (+/ heterozygotes Kv3.1 of Genotyping ) 2 ora fCl cec 2 (9) 126 Science Cell of Journal / 2 n idtp ++ ie oul rcin lf)adcuemmrns(ih)aecmae.Mlclrwihs(nka r niae nteleft. the on indicated are kDa) (in weights Molecular compared. are (right) membranes crude and (left) fractions Soluble mice. (+/+) wild-type and ) 6 betv es.Tenme feprmnsi niae ihntebars;* the within indicated is experiments of number The lens). objective nvivo in hrfr,w aefurther have we Therefore, . 2 ,hmzgts( homozygotes ), Kv3.1 RKR 2 YP(i.6.In 6). (Fig. -YFP / 2 rgt etos mgswr hehle n iaie oso ie lses ( clusters. pixel show to binarized and thresholded were images sections, (right) 2 / 2 ,adwl-yemc.DAldesaeo h eti p ( bp. in left the on are ladders DNA mice. wild-type and ), ( A ciae yohrbnigpoen,orsuyhsclearly loading has cargo both cluster study of activation. specificity cargo-regulated uniquely and the our tetramers to channels contributing proteins, motor, Kv3 KIF5 binding the that other be demonstrated can motor by KIF5/kinesin-1 the 6E activated although (Fig. Therefore, motors KIF5 7). be activate Fig. or cluster and to to Kv3 and failed with both appears SNAP25 sites tail, KIF5 binding proteins, in overlapping Kv3.1 have KIF5-binding they of Although other VAMP2. action two the examined We motors. KIF5B also 2007), Rice, cluster and and activate al., to (Wong is which KLC1 straightforward, et of Cai roles 6). sophisticated 2010; (Fig. the KIF5 to clusters nor contrast 4E), In (Fig. KIF5 to binding for channels hssuyhsrvae oe ehns neligthe underlying transporting mechanism a on novel motors KIF5B of a number copy revealed the of has regulation study a on This motors KIF5 of vesicle number transporting copy the of Regulation xrsino v.b I5adKF-idn rtisi riso Kv3.1 of brains in proteins KIF5-binding and KIF5 Kv3.1b, of Expression ) P , .5( 0.05 t ts) cl as 300 bars: Scale -test). C,D v.badKFBsann patterns staining KIF5B and Kv3.1b ) m CD;100 (C,D); m F umr fthe of Summary ) m E m(E). oquantify To ) Journal of Cell Science eeelrnuo xn,iae eetrsoddadbnrzdt hwpxlcutr.( clusters. pixel show * to bars; binarized the and within thresholded indicated were images axons, neuron cerebellar eainhp fclue eeelrnuosfo idtp crls n v. O(rage)mc.2,nuosbfr ek l 1–2DV;3w, DIV); (10–12 old weeks 2 before neurons 2w, mice. (triangles) ( KO DIV). Kv3.1 (21 Kv3.1 and old (circles) weeks wild-type 3 from around neurons cerebellar cultured of relationships L1sann atr nclue v. Ocrbla ern t2 I.( DIV. 20 at neurons cerebellar KO Kv3.1 cultured in pattern staining KLC1 utrdcrbla ern 2 I)o idtp tp n v. ncot(otm ie ( mice. (bottom) knockout Kv3.1 and (top) wild-type of DIV) (21 neurons cerebellar cultured rsneo v. n neord rnprigvscecan vesicle the transporting in anterograde that one suggest Kv3.1 data of microscopy Gu, and (Gu quantitative presence Gazzola caveats the mathematical 2008; our all al., despite and contrast, 2010), et sharp experiments In Shubeita 2009). 2011; by al., et al., to supported et believed is 10, (Erickson kinesins modeling than active of less number be the carrier accepted, one transport is to vesicle motors kinesin multiple Although vesicle. neurons. cerebellar cultured of axons along clusters KIF5B reduces Kv3.1 Kv3.1 of Deletion 10. Fig. 2 2 / / 2 2 ie ( mice. os us(– otaa as o ek.( weeks. 3 for days) postnatal (1–2 pups mouse F noeosKFB(re)adKC rd tiigpteni utrdwl-yecrbla ern t2 I.( DIV. 20 at neurons cerebellar wild-type cultured in pattern staining (red) KLC1 and (green) KIF5B Endogenous ) P , .5 ** 0.05, D , E osann fedgnu I5(re)adK31 rd ln xn fclue eeelrnuosfo idtp and wild-type from neurons cerebellar cultured of axons along (red) Kv3.1b and (green) KIF5 endogenous of Co-staining ) P , .1( 0.01 t ts) cl as 25 bars: Scale -test). A cinptnil nue yasur-us urn neto 1scn uain 4 A from pA) 140 duration; (1-second injection current square-pulse a by induced potentials Action ) m DE;100 (D,E); m H oqatf h I5 lsesfo h idtp lf)and (left) wild-type the from clusters KIF5B the quantify To ) ti osbeta aymtr ntevsced o bind not do vesicle reserve the a as on function may motors they but many time, same the that the actively at microtubules possible question. motors under open these is limit an remains of obtained upper It microtubules many along the is walking How to in vesicle. engage is number close transporting It be this a may 8C). for (Fig. that and dimers condition note motor overexpression 200 to to important up contain astonishingly m n(,) 15 (F,G), in m v lsesadatvtsKF oos2037 motors KIF5 activates and clusters Kv3 I uniiaino eut hw nH h ubro xeiet is experiments of number The H. in shown results of Quantification ) B aeom fte1tato oetasi .( A. in potentials action 1st the of Waveforms ) eeelrnuoswr utrdfo idtp and wild-type from cultured were neurons Cerebellar m (H). m G noeosKFBand KIF5B Endogenous ) Kv3.1 C Input-output ) 2 / 2 neurons (right) Journal of Cell Science eutfo lmnto fat-niiin(i.6.Hwvr it However, KIF5B 6). clusters (Fig. what auto-inhibition unclear of remains elimination from result txawt eeelrarpy(aese l,20) o does How 2006). al., et (Waters atrophy cerebellar with ataxia cargo of specificity KIF5, the ensuring by itself mechanism which transported transport. novel motors, a are be KIF5 also Different clusters that may and motors. channels activates form channels known KIF5 Kv3.1 other and unlikely all channels from least Kv3.1 direct not a between demonstrated will at have studies proteins binding Our channel or motors. kinesin the the of monomeric, cluster loading are complexes, Since multimeric 2012). Gu, adaptors and (Barry these proteins adaptor specific via motors n MArcpos n GABA and (NMDA an receptors receptors) glutamate been of AMPA ion type and major has ligand-gated two as Several compartments such neurobiology. channels, neuronal in right question neuronal outstanding the are for proteins to critical channel is How transported transmission. of location synaptic targeting correct and Precise unique to excitability proteins. a channels motor be ion riding may various cargos KIF5 some clustered for by way mediated motors transport KIF5 Axonal clustering of functions Physiological KIF5B KIF5 of a underlying clustering is mechanisms instance, channels Other motors. for Kv3 (3) KIF5 clustering, of cluster channels, process. can transport Kv3 transient Besides that KIF5-mediated (2) proteins domains. Moreover, unidentified T1 including are conserved KIF5 cerebellum, highly there cluster the also in with can to channels present channels, due These channels. Kv3 are Kv3.4 and to Other domains, Kv3.3 due be (1) T1 may This reasons: conserved 10). Clear 9, following mice. (Figs still neurons were the KO KO clusters Kv3.1 Kv3.1 but observed, in we on was abundant clusters clustering, experiments KIF5B KIF5 of of reduction affects series deletion a Kv3.1 performed how determine To motor KIF5 clustering underlying al., mechanisms et potential Other Yu 2009; Spudich, the and to Sivaramakrishnan analogous 2009). 2002; way (Tomishige within al., proteins a function motor et other in motor embedded of a S4), dimerization the be cargo-mediated Fig. are may changing material number profoundly (supplementary motor that mechanism the general regulating proteins of vesicles) transporting integral nature membrane activate the markedly homo- not 7). on (Fig. do form KIF5 both also cluster depend but and can hetero-oligomers, should VAMP2 or dimers and which SNAP25 oligomerization. cluster motors, also can not JIP3 whether is KIF5 unclear al., remains oligomerization et it (Sun its Currently, transport 2011). KIF5-mediated Kv3, for and unlike sufficient, recent KIF5 nor but necessary, to a membrane motor, binds instance, only directly the For Driver/JIP3 the activates Sunday motors. be that kinesin shows not cluster study may can channels that motor Kv3 On proteins the regulated. hand, effectively which other by be by can the mechanism vesicle regulated transporting novel a Nonetheless, a further on number 2008). revealed be has al., study et can this (Dixit microtubules proteins along microtubule-binding engage actively walking that motors The in processivity. the increase to pool 2038 ial,mttosi v. hne eecuespinocerebellar cause gene channel Kv3.3 in mutations Finally, lgmrzto fknsnbnigpoen epcal those (especially proteins binding kinesin of Oligomerization ora fCl cec 2 (9) 126 Science Cell of Journal A RKR eetr,bn okinesin to bind receptors, -YFP. RKR YPmay -YFP rnpr fohrcro i lseigknsn1 hc san axonal is regulate which kinesin-1, may investigation. clustering further channels for via topic cargos Kv3 interesting may other that has 2007), of work al., possibility transport this et particular, a In Xu question. 2010; raised intriguing this al., on proteins. et ankyrin-G light via (Xu shed other cytoskeletons respectively microtubule KIF5, of and and actin transport Thus, channels microtubule to Kv3 linked showing the of works 2011). are previous behavior affect and current al., the our indirectly Therefore, alter et and can (Sanchez tracks motors KIF5 bundles clustered of shows beating microtubule study cilia-like cause recent active can in A kinesin-1 question. changes clustered open artificially morphological that an to remains lead circuits activity neural channel Kv3 altered 5 MTi-C,p .,10m al %Tio -0,adacomplete a and X-100, Triton 1% NaCl, 4 mM at 150 tablet) 7.4, inhibitor pH protease Tris-HCl, mM (50 S-al S-7 (GST-Tail GST-T70 GST-Tail, constructs cDNA Methods and Materials el ih1m Isopropyl mM 1 with 6 cells or GST- of Expression and 0.8 purification Protein containing Opti-MEM in in neurons incubated transfection, transient were replenished For DIV was volume. 5–7 the medium half culture at replacing The culture then by days medium. days, week 2 2 culture a brief, subsequent normal the twice In for the 2010). growth glial with Gu, inhibit and replaced to Gu medium culture 2012; rat neuronal al., the E18 et 1 from Gu plating, described neuron 2010; after previously al., as et prepared (Barry was embryos culture neuron transfection Hippocampal and culture neuron Hippocampal Hoechst with stained were nuclei F-actin and costaining. an 546 (Invitrogen). in or Fluor 33342 used antibody Alexa was phalloidin marker) for antibody with axonal marker) labeled axons an PBS was dendritic distinguish is a To (Tau1 is proteins. in anti-Tau1 (MAP2 total an anti-MAP2 label sucrose neurons, to conditions of of X100) 4% permeabilized dendrites Triton under and and 0.2% and of antibodies grade procedures presence specified EM the PA) with (in ultrapure stained The Warrington, and 10% the minutes, (from brief, Polysciences, 20 formaldehyde In PA). free; 2006). 4% al., with methonal Grove, et fixed (Roche, (Gu were previously (Jackson neurons West antibody described antibodies were secondary anti-HA conjugated immunocytochemistry Laboratories, Cy5 monoclonal and Cy3, ImmunoResearch rat Cy2, and IN), (Abcam), anti-VAMP2 Indianapolis, anti-SNAP25, (Santa antibodies rabbit antibody CA), Cruz, anti-KIF5B antibody anti-KLC1 Santa polyclonal anti-Kv3.1b Inc., rabbit Biotechnology anti-Tau1 polyclonal Cruz Israel), rabbit and Jerusalem, Labs, CA), (MAP2) (Alomone Temecula, 2 (Chemicon, protein antibodies anti-microtubule-associated MA), Cambridge, polyclonal (Abcam, antibody anti- rabbit anti-GRIP1 mouse Davis/ mouse MA), UC Billerica, Facility), respectively; (Millipore, anti-GST N16B/8, mouse NeuroMab and CA), N100/13 NIH Davis, (clone N144/14, antibodies anti-Kv3.1b (clone and facility NeuroMab Davis/NIH anti-6 UC mouse include used Antibodies immunostaining and Antibodies 37 1.5 and plasmid Tail aeb netn h Ce DAfamnscrepnigt h indicated the to GST-T70 corresponding vector. pGEX4T-2 fragments the cDNA into tail PCRed KIF5B of the regions inserting by made eecnimdb sequencing. between by pEC(Y)FP-C1 confirmed into were into cDNAs BlgII cDNA length between length full CA) full View, KLC1 Mountain B the (OpenBiosystem, pRSET Inc., and inserting into Laboratories, cDNA 399 by (Clontech to full-length made 1 pEC(Y)FP-C1 residues was KLC1 KIF5B C(Y)FP-KLC1 the containing cDNA vector. inserting the His- and and by AL) His-KLC1 Huntsville, made respectively. KIF5B-YFP, were and Motor YFP-T70 GST-T70, on based GST-Tail I5-F eepeiul ecie X ta. 00 ue l,20;X tal., et Xu 2006; al., et Gu 2010; al., GST-Tail et YFP-T63, (Xu 2007). YFP-T70, described previously CFP-Kv3.1bHA, were KIF5B-YFP CFP-Kv3.1aHA, Kv3.1bHA, Kv3.1aHA, KIF5B ˚ .Bceilpleswr ouiie ihsncto ntepldw buffer pulldown the in sonication with solubilized were pellets Bacterial C. 892–912 Hin RKR II ()PSA2 n ()PVM2wr aeb netn the inserting by made were C(Y)FP-VAMP2 and C(Y)FP-SNAP25 dIII. 892–924 GST-Tail , YPwr aeb uaigR mutating by made were -YFP GST-Tail , 865–986 m fLpfcaie00(nirgn o 0mntsa 37 at minutes 20 for (Invitrogen) Lipofectamine2000 of l 875–896 m GST-Tail , yoieaaioe(im,S oi,M)wsaddto added was MO) Louis, St (Sigma, arabinose cytosine M 6 i-agdfso rtiswsidcdi BL21 in induced was proteins fusion His-tagged nvitro in 897–934 GST-Tail , b ˚ D1tiglcoyaoie(PG o or at hours 4 for (IPTG) -D-1-thiogalactopyranoside ,adcnrfgda 50,000 at centrifuged and C, 865–934 892–934 GST-Tail , idn assays binding 6 ,GTT3(GST-Tail GST-T63 ), 913–934 i niois(nirgn albd CA; Carlsbad, (Invitrogen, antibodies His GST-Tail , b tblnadat-I5H antibodies H2 anti-KIF5 and -tubulin 892 K 902–934 GST-Tail , 893 Bgl R 870–896 894 n GST-Tail and , IadEo1 l constructs All EcoR1. and II oDDwt Quickchange with DDD to 856–886 GST-Tail , RKR 6 g 758–820 YFP-T70 , GST-Tail , o 0mntsat minutes 30 for m 865–891 875–919 ,His-31T1, ), fcDNA of g RKR 892–929 GST- , .coli E. ˚ C. ,and were , Journal of Cell Science elhaeBoSine B wdn rwt Co with or Sweden) AB, Bio-Sciences Healthcare ihrftigtecre oobtain to curves the fitting either 4 Fmdu Banis pigil,I)pu %B2,05m ltmn,and glutamine, mM 0.5 B-27, 2% chamber plus imaging (HE- IL) the buffer Springfield, into imaging (Brainbits, with loaded incubated medium were and LF PA) coverslips Downingtown, mm Devices, 25 (Molecular on growing imaging Neurons FRET and cell Live ( unit) major arbitrary the (in along intensity line transfected a fluorescence laid other average we from its J, Image isolated acquire neurons NIH to and Using transfected same axon analysis. axons, Only for the quantification and chosen 2010). The were but dendrites al., cells, experiment. et separated saturation, an (Xu clearly of below upright previously with group were described each Zeiss was axons within procedure used the and was that a dendrites slider for time so 10.0 exposure controlled in Sigmaplot in RT were and intensities J times Image camera MI) pixel Exposure NIH with quantification. CCD analyzed intensity and Heights, 20 fluorescence files, Spot objectives TIFF 16-bit Apo Sterling as a Plan saved using Inc., with Axiophot, microscope, captured Instrument were (Diagnostic images Fluorescence quantification and microscopy Fluorescence MgCl mM 0.5 (N,N0- 6.9, PIPES pH mM acid), 80 containing sulfonic buffer polymerization diethane the piperazine in incubated was microtubule reconstitute assays To binding and assembly Microtubule 25 at performed were experiments (SPR) resonance plasmon of Surface affinities binding measure interactions to protein–protein experiments resonance plasmon Surface i-11i bu : teeoe44,i nlzn P xeietldt we data binding. experimental during SPR effect allosteric analyzing no in assuming 4:4), 1:1, ratio (therefore the 1:1 adopted about is His-31T1 loecneitniywsmaue o ahiaeadsubtracted. and image each for measured was intensity fluorescence yaespraatcnann i-11a 4 bacterial with at incubated His-31T1 further were containing proteins supernatant fusion GST lysate purified with coated oa aisi h ulonbfe ihu rtaeihbtr ttlvolume (total inhibitors protease without buffer pulldown different the in 500 proteins in His-tagged purified ratios with molar incubated further were beads Coated antibody. anti-His an 2.5–5 with blotting 2 western to with subjected eluted and were PAGE, precipitants the washing, ee.Tu,terto( ratio the Thus, level. uui n te rti ntemcouueo uentn rcin,15 fractions, supernatant or microtubule the of in amounts the protein determine other To 50,000 buffer. and sample at in tubulin pelleted resuspended and were temperature proteins room binding and Microtubules 5 (around proteins purified al n a atrdb h nioyi el1a oto,adohrGST other and control, a as the 1 cell GST by in Purified antibody recommended (0.6 the Healthcare). was by proteins as captured (GE fusion antibody was coupled kit and anti-GST NaCl) covalently amine-coupling monoclonal the cells (0.6 of using flow microgram manufacturer on chips One sensor CM5 immobilized with Healthcare). NJ) Piscataway, Healthcare, (GE (GE instrument T100 Biacore lto a ute ilzdwt h ulonbfe t4 at buffer The pulldown imidazole. mM the 150 with or with dialyzed glutathione eluted further mM were was and 20 elution proteins either fusion containing purified buffer elution with the coated were beads the washing, fiiyrsn lnehLbrtre n. t4 at Inc.) Laboratories Clontech resin, affinity gaoie5-rpopae,ad5 lcrl t37 at glycerol, 5% and (guanosine-50-triphosphate), idn uvsuigteeulbimrsos au ttepaeuo l uvs(for curves all of plateau the at value response GST-Tail equilibrium the using curves binding loecneitniyo h oaoedii ein( average region the somatodendritic obtain to the soma of with connect intensity which fluorescence dendrites proximal along lines laid etr ltigo olia lesann.I i.3,atrbnig h beads the Each times. binding, buffer. three after least sample 3C, the ( Fig. with once In incubated washed staining. quickly Blue Colloidal were or blotting 2 western with eluted were uentnsadrsseddpleswr nlzdb D-AE nthe In SDS-PAGE. formation. by microtubule stabilize analyzed and 100 His-31T1, promote were and to GST-T70 pellets of experiment resuspended competition and supernatants ifrn ocnrtoswsijce tafo aeo 20 of rate flow a at injected was concentrations different ih1MNC o 0 eod.Alsnogasso aai hc the which in data show sensorgrams fusion (GST signal All total regenerated the seconds. from fully subtracted 600 was was The 1) proteins). chip for cell in the (GST NaCl signal dissociation, background M of 1 seconds 300 with After seconds. 180 ˚ .Tespraat eeicbtdete ihguahoebas(GE beads glutathione with either incubated were supernatants The C. nbnigasy ompteK31T-idn iei i.2 lttin beads glutathione 2, Fig. in site T1-binding Kv3.1 the map to assays binding In npldw sasuigprfe rtis uiidGTfso rtis(around proteins fusion GST purified proteins, purified using assays pulldown In m m )a omtmeauefr1hu.Atretniewsig h precipitants the washing, extensive After hour. 1 for temperature room at l) )foe vri h unn ufr(0m rsHl H74 5 mM 150 7.4, pH Tris-HCl, mM (50 buffer running the in over flowed g) m )wr is otdot lttin ed basttlvlm:30 volume: total (beads beads glutathione onto coated first were g) 892–934 K .Snew siae h idn tihoer fGTT0and GST-T70 of stoichiometry binding the estimated we Since ). d o i-11bnigt S uinpoen eecluae by calculated were proteins fusion GST to binding His-31T1 for 6 m F )wr moiie ncls24 uiidHs3T at His-31T1 Purified 2–4. cells in immobilized were g) apebfe,rsle nSSPG,adsbetdto subjected and SDS-PAGE, in resolved buffer, sample axon /F m nvitro in )wr de n nuae o nte 0minutes. 30 another for incubated and added were g) , sd elcsterltv xnllvl h background The level. axonal relative the reflects ) 5scns ihteI ufradimmediately and buffer IP the with seconds) 15 K on uiidtbln(im)(20 (Sigma) tubulin purified , and nvitro in K off 6 frGTT0 rpotn saturation plotting or GST-T70) (for ˚ o or.Atrextensive After hours. 2 for C apebfe,rsle nSDS- in resolved buffer, sample ˚ idn sa a efre at performed was assay binding o or.Atrextensive After hours. 3 for C 6 2+ F ˚ o 0mnts Then minutes. 30 for C m sd 07 n 100 and /0.75 altxlwsadded was paclitaxel M ed TLNmetal (TALON beads orpeettetotal the represent to ) ˚ overnight. C 6 g o 0mntsat minutes 30 for 2 MGTP mM 1 , m m n2mg/ml) 2 in l /iuefor l/minute F 6 axon ˚ 14oil, /1.4 Cona ,and ), m lof m l). FRET A A T A A T A GG-3 GAG ATT GAT AAT GTC AAA 5 CAC knockout, and WT both (for 31F775 primer forward used: were ausaecnitn ihtoeotie npeiu tde.Therefore, studies. previous in obtained of those ratio the with ratio measured The study. consistent We this FRET in 2010). used are al., proteins YFP-fusion et and values CFP- (Xu the all previously for crossbleeding 64.7% measured system, were which our each particular In from YFP- subtracted the or 1.47% calculation. were CFP- of either values before express fluorescence characteristic that image Background cells was of alone. use protein crossbleeding the fusion by of determined and and extent the FRET system through optical both The bleeding of fluorescence filter). was three- consisted acceptor a YFP and FRET images by donor and basis FRET (the CFP pixel-by-pixel raw components a between non-FRET The on FRET method. image channels. Falls, ‘microFRET’ entire the filter filter Bellows for three Corp, calculated all single and A Technology with measured nm). (Chroma used 470/ 535/30 brief, was emission emission Chroma nm, In nm, VT)] [86004BS; 430/25 described 430/25 2006). 500/ (excitation mirror (emission al., set (excitation were filter filter et dichroic filter CFP FRET Gu (3) YFP (2) imaging 2001; nm); nm); (1) al., 30 535/20 FRET through et emission sequentially fluorescence Gu nm, of acquired 2010; 20 conventional were al., protocol images et a (Xu three and with detail in strategy cells previously living The time-lapse in microscope. The interactions Devices). frames. 100 were protein–protein (Molecular for filters interval The software 2-second time. with MetaMorph Instrument, performed exposure (Sutter was AZ) by second 10-3 imaging Tucson, Lamda 1 CA) through (Photometrics, with controlled Novato, HQ sets wheels filter Images filter Coolsnap through microscope. YFP camera changed inverted or CCD TE2000 CFP a NY) through with Melville, captured Inc., (Nikon were Nikon a upon 25 ncot 5 knockout, GA-3 CAA GAA CGT CAT CCC ( punctum long), moving minutes one 3.3 (total if movie instance, one For seconds/minute). 60 ie,adte meddi pia utn eprtr ei Skr Finetek (Sakura at media stored temperature and cutting CA) post- optimal Torrance, removed, in Inc., was USA, embedded cerebellum then mouse fixation, and tissue fixed, and perfusion cardiac After cluster and sections brain quantification mouse on staining Immunofluorescence study. immunofluorescence the in ( Kv3.1 some from occasionally observed for be BL6 with can backcrossed were UT PCR-based Sa ( Seizure mice at 1997; KO homozygotes a Kv3.1 al., Joho generations. using The et R. 2009). ten maintained (Ho al., Dr et described been by Hurlock previously 2000; have provided as kindly and procedure was genotyping Center line Medical mouse Southwestern (KO) knockout Kv3.1 genotyping mouse knockout Kv3.1 11. a SigmaPlot in in used were curve largely They regression 2010). were linear Gu, and the system (Gu obtain The microscope numbers to 2010). published our the our Gu, with in and calibrate relationship linear (Gu to measured described 2005) intensities previously Arp2- Pollard, fluorescence as Arc1-mYFP, and expressing system (Wu ones microscope the Spn4-mYFP) quantitative and and type Fim1-mYFP, (wild strains mYFP, yeast five used axonal We in motors KIF5B-YFP estimate puncta to microscopy ‘ quantitative Using and ‘+’ as presented were puncta retrograde values. and movie. Anterograde a respectively. in movie) average seen the was per (or events puncta transport of moving is frequency MetaMorph movie of each the the for time calculated with number total The We made 2010). kymographs Gu, seconds. and 198 on (Gu described out previous as carried program were axons along measurements puncta mobile All of quantification and imaging live-cell Timelapse ntergeso,fu onswt ( 0 with points four regression, the In (40- esrd h siae ubro F-ue oos( motors were YFP-fused puncta of KIF5B-YFP of number intensities estimated Fluorescence point. The zero the measured. to close get to efre speiul ecie Jkoae l,21) h hl images whole The 2012). were al., imaging et and (Jukkola described Immunostaining previously microscope PA). Plus as Superfrost Pittsburgh, performed on collected (FisherScientific, and slides MA) Waltham, Scientific, (Thermo oo ies( dimers motor 2 u RTiaigi sdt eetsniie msin utbefrstudying for suitable emission, sensitized detect to used is imaging FRET Our / m 2 m ltmt)a omtmeaue h ieas mgn eu a built was setup imaging timelapse The temperature. room at glutamate) M hcns)o os eeelmwr u ihaMco M5 cryostat HM550 Microm a with cut were cerebellum mouse of thickness) m n he oto l ieo ihrsxa h g f24mnh eeused were months 2–4 of age the at sex either of mice Bl6 control three and ) corrected corrected v lsesadatvtsKF oos2039 motors KIF5 activates and clusters Kv3 6 .8 ( 0.08% 9 CACTCATTGCAGTCTCAC-3 TGC TCC ACG GTC TTT CCA CTT -CTA 5 a efre ihteMtMrhsoftware. MetaMorph the with performed was FRET n 2 2 eecluae o ahpunctum. each for calculated were /2) n / 2 5 u o eeoyoe (+/ heterozygotes not but ) 2 fYPfursec le hog h RTchannel, FRET the through bleed fluorescence YFP of 12) raw F 2 (0.65 qas06o . f2o oigpnt eeseen, were puncta moving 3 or 2 if 0.9 or 0.6 equals 6 CFP) F 9 ,rvrepie 191(o T 5 WT, (for 31R991 primer reverse ), F 2 qas0.3. equals total (0.015 2 n ( 0 and ) 80 ˚ 6 F ni etoig ooa sections Coronal sectioning. until C 9 ,adrvrepie N28(for PNR278 primer reverse and ), (number/minute) F) h aclto of calculation The YFP). 2 F n ie h olwn primers following The mice. ) qas0i omvn punctum moving no if 0 equals eeaddt oc h curve the force to added were ) 6 .%( 0.5% F total n 5 n h ubrof number the and ) n n 4.95 5 5 9 .TreK31KO Kv3.1 Three ). 8 fCP and CFP, of 18) 9 5 )i bevdin observed is 1) GGCTCAA CTT -GCG n n +61. (9 seconds/ /(198 ´ ce tal., et nchez R 2 5 9 -GGC 0.96. 2 ’ Journal of Cell Science he S. Choe, a,D,Hpe .D,Sasn .A n ehy .J. K. Verhey, and A. J. H. Swanson, D., Z. A. Hoppe, Sheng, D., and Cai, G. C. Banker, Gerwin, and Q., A. Cai, M. Silverman, A., J. M. B. Schnapp, Burack, and S. L. Goldstein, J. M., P. S. Pfaffinger, H., Block, Bellamy, A., Kreusch, V., N. Shen, H., M. Nanao, A., K. Bixby, ar,J n u C. Gu, and J. Barry, i .Q,Mri,R . io . letn .M,Shly .M n Steinhardt, and M. J. Scholey, M., J. Alderton, G., Liao, L., R. Morris, Q., G. Bi, en .P. B. Bean, S. T. Reese, and J. B. Schnapp, D., R. Leapman, E., P. Gallant, B., S. Andrews, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.122234/-/DC1 online available material months. Supplementary 12 after release animal for Use PMC All Animal Stroke in NIH C.G. the Deposited and with Guidelines. accordance to in Disorders conducted of R01NS062720] were Institutes Neurological experiments number National of the [grant from Institute (NIH/NINDS) grant a National by Health, supported was work This C.G. Funding and experiment. experiments, and SPR performed experiments the and paper. the with designed the performed wrote helped project, C.S. A.D. the and data. supervised P.J. the Y.G., analyzed M.X., J.B., contributions Author Kv3.1 for Center technical Medical for Southwestern mice. Li UT KO X. at analysis, Joho statistical R. on and advice assistance for Wei L. thank We Acknowledgements pixels intensity 9–300 highest from size 5% in the ranging 250 puncta (around 20 only Kif5B axons a include image. along with the to binarizing captured images and the images pixels, flattening the All by Metamorph sections. thresholding using cerebellar quantification background, for for processed were that cerebellar lens to cultured objective of similar axons is along clusters axons KIF5B for procedure mice quantification KO The Kv3.1 neurons cerebellar and cultured wild-type of axons from along clusters KIF5B of Quantification Each current cultures. the pups. of via by 6 rounds pA) to three induced 10 4 with of performed were had increments were with round potentials studies pA The 145 are Action pipette. to values recording 5 group. from These duration; age measured. (1000-ms resistance, injections each were membrane 10–12 potentials within Neurons’ DIV; DIV). membrane 14 consistent 19–22 (before resting from 2w and DIV; windows, 21 capacitance, time or (around WT two 3w either in from and recorded cultured DIV) were neurons cerebellar mice, brief, previously KO In was Kv3.1 2012). neurons al., cultured et from (Gu potentials described action of recording clamp mice Current KO wild- from Kv3.1 neurons and cerebellar cultured type on recording clamp clusters were patch Whole-cell pixel puncta all Kif5B count pixels. to image. 9–300 tool the from Particles’ size binarizing ‘Analyze in the and ranging using pixels, only ImageJ include in intensity to quantified images highest the 5% thresholding background, the the 40 flattening by a Metamorph with captured 2040 ar,J,G,Y n u C. Gu, and Y. Gu, J., Barry, raiainadcnomtoa hne eeldb RTsocimtyi live in stoichiometry FRET by revealed changes cells. conformational and organization processes. neuronal along mitochondria of transport sorting. protein neuronal in transport channels. K+ voltage-gated S. Choe, and iei oeue tde ihotcltweezers. optical with studied molecules kinesin exocytosis. regulated A. R. Neurosci. channels. ion of transport intracellular and motors molecular vitro. in USA microtubules crossbridge Sci. molecules kinesin Single (1993). n edii udigi vitro. in bundling dendritic and 10.1177/1073858412456088. doi: print] of ahead .Cl Biol. Cell J. 20) oasu hne structures. channel Potassium (2002). 19) iei-admoi-rvnseso eil erimn o Ca2+- for recruitment vesicle of steps myosin-driven and Kinesin- (1997). 90 8 ora fCl cec 2 (9) 126 Science Cell of Journal 20) h cinptnili amla eta neurons. central mammalian in potential action The (2007). 451-465. , 6503-6507. , 19) n+bnigadmlclrdtriat fttaeiainin tetramerization of determinants molecular and Zn2+-binding (1999). 6 176 .Cl Biol. Cell J. 51-63. , 21) opigmcaia ocst lcrclsignaling: electrical to forces mechanical Coupling (2012). betv eswr rcse o uniiainusing quantification for processed were lens objective m emns eeqatfe nImageJ. in quantified were segments) m a.Src.Biol. Struct. Nat. 21) oaie agtn fL-A euae axonal regulates L1-CAM of targeting Polarized (2010). u.J Neurosci. J. Eur. 138 Neuron 999-1008. , 20) ytblnmdae anterograde Syntabulin-mediated (2005). a.Rv Neurosci. Rev. Nat. 26 6 38-43. , 465-472. , Nature 32 19) edmvmn ysingle by movement Bead (1990). 1618-1631. , .Cl Biol. Cell J. 20) h oeo selective of role The (2000). 20) iei- structural Kinesin-1 (2007). 348 348-352. , Neuroscientist 3 170 115-121. , rc al Acad. Natl. Proc. 959-969. , a.Rev. Nat. [Epub 6 rcsn .P,Ja . rs,S .adY,C C. C. Yu, and P. S. Gross, Z., Jia, P., R. Erickson, u .adG,C. Gu, and Y. Gu, J. Barry, and C. Gu, air .A,Kpa,M,Wn,G . hn . e,L,Sa,J . Koushika, E., J. Shaw, L., Wei, K., Shen, J., G. R. Wang, M., Mackinnon, Kaplan, and A., T. B. Maniar, E. Campbell, B., S. Long, Y. L. Jan, and N. Y. Jan, M., Li, S. Choe, and P. Pfaffinger, T., Pollard, D., H. Kaiser, C., R. Strang, Joho, W., A. and Jahng, G. Pierce, M., Bose, C., E. Hurlock, D. R. Vale, and J. A. Hudspeth, J., Howard, H. R. Joho, and W. R. Grange, S., C. Ho, remn .S n ae .D. R. Vale, and S. D. Friedman, L. E. Holzbaur, and E. Y. Goldman, L., J. Ross, R., L. Dixit, A. Cunningham, and J. P. Armati, P., J. Mackay, J., R. Diefenbach, J. Scholey, J. and Howard, R. and M. Cross, Wagenbach, O., W. Hancock, L., D. Coy, u . hu . uhned,M . u . a,Y .adJn .Y. L. Jan, and N. Y. Jan, M., Xu, A., M. Puthenveedu, W., Zhou, C., Gu, M. D. Cooper, and A. Sorkin, C., Gu, L. B. Tempel, and M. H. Brew, J., J. Grigg, epl,P . coal .W,Pitr .K,Bom .S n rd,S T. S. Brady, and S. G. Bloom, K., K. H. Pfister, W. W., Guilford, A. and McDowall, L., A. P. R. Leopold, Bloodgood, A., J. Marin, A., J. Laib, Copeland, H., J. Resau, D., Stenolen, W., B. Richards, T., S. Brady, D., J. Huang, and S. G. Bloom, K., K. Pfister, N., Kobayashi, R., Sato-Yoshitake, N., Hirokawa, R. Takemura, and N. Hirokawa, ltr .E,Mgah .J,Soes .S n cwr,T L. T. Schwarz, and S. R. Stowers, J., L. Megeath, E., E. Glater, oih,Y n eo,M. Setou, and Y. Konishi, F. M. Stock, and D. D. Hackney, C. Gu, and D. Terman, R., McDougel, J., Barry, Y., Gu, azl,M,Brkad,C . aai . nek,M,Gee,U .and F. U. Greber, M., Engelke, B., Bayati, J., C. Burckhardt, M., Gazzola, azae,L . htahre,A,Dsi . a,L,Sn,P,vnHh,C A., C. Hehn, von P., Song, L., Gan, R., Desai, A., C. Bhattacharjee, Gu, K., L. and Kaczmarek, S. S. Zamvil, E., A. Lovett-Racke, I., P. N. Jukkola, Y. Jan, and Y. L. Jan, .P n agan .I. sorting. axon-dendrite C. polarize 48-56. to Bargmann, kinesin localize and and microtubules P. S. channel. K+ family Shaker voltage-dependent mammalian channel. potassium Shaker the of 1225-1230. domain amino-terminal hydrophilic ragdo ag sipratfrvsclrtransport. vesicular for important is cargo e1002032. a on tau. by arranged proteins chain motor heavy kinesin and kinesin dynein human of chain. regulation ubiquitous light of kinesin for domain site stalk binding the the contains of region C-terminal E121. e11931. channels. potassium sensitive brainstem. mouse the of nuclei auditory in 140 genes channel potassium gated 19) soito fknsnwt hrceie ebaebuddorganelles. Cytoskeleton membrane-bounded characterized Motil. with kinesin of Association (1992). Kcnc3-null in channels Kv3.3 of Kcnc1. restoration requires cell-targeted mice Purkinje by coordination A. motors. N. transport Jenkins, and G. N. molecules. kinesin single but contraction, muscle and skill motor seizures. impaired no weight, body reduced gene: channel vivo. in organelles T. S. Brady, oani nihbtr euao ftemtrdomain. motor the of regulator inhibitory an is domain rnmmrn lsesdcaetetreigadfntoa sebyo adenylyl of assembly functional and targeting the cyclase. dictate clusters transmembrane light is and chain heavy kinesin recruit to independent. milton chain requires mitochondria of transport eirclcodnto n ehnc fmlclrmtr nlvn cells. living USA in motors Sci. molecular Acad. of mechanics and coordination reciprocal axons. to domain neurons. in transport directional release. frequency. ADP microtubule-stimulated spiking maximal adjust to targeting K+ Chem. polarized voltage-gated kv3.1 Kv1 regulates for required is EB1 targeting. axonal protein channel tracking plus-end microtubule nvv yolsi rnpr fhmnadenovirus. human of transport cytoplasmic vivo in P. Koumoutsakos, domain. tail cargo-binding the by regulation reveals hr-emadln-emmdlto fptsimchannels. potassium of modulation long-term B. encephalomyelitis. and Yang, short-term and autoimmune D. M. Whim, experimental of progression Dis. the Neurobiol. in alterations 4.2. channel K voltage-gated the of domain T1 Chem. the of assembly mediates Zinc prokaryotes. 77-90. , 287 277 ur Biol. Curr. 1755-1769. , 47885-47890. , rc al cd c.USA Sci. Acad. Natl. Proc. nu e.Neurosci. Rev. Annu. 19) iei soitswt neordl rnpre membranous transported anterogradely with associates Kinesin (1991). 47 106 21) yaiso v hne rnpr naxons. in transport channel Kv1 of Dynamics (2010). Nature a.Neurosci. Nat. 280-293. , .Cl Biol. Cell J. .Cl Biol. Cell J. 20) tcatcmdlfrmcouuemtr ecie the describes motors microtubule for model stochastic A (2009). 3190-3195. , 21) ucinadmcaimo xnltreigo voltage- of targeting axonal of mechanism and Function (2011). 23 11 .Neurosci. J. 19-33. , 185-190. , 19) iei:teti unfolds. tail the Kinesin: (1999). 397 20) uui yoiainnvgtsteknsn1motor kinesin-1 the navigates tyrosination Tubulin (2009). Nature Neuron 19) lndptsimcanl rmekroe and eukaryotes from channels potassium Cloned (1997). 19) ietitrcino irtbl-adactin-based and microtubule- of interaction Direct (1999). 267-270. , 20) euaino h iigo NBnuosby neurons MNTB of timing the of Regulation (2005). rg Neurobiol. Prog. 19) igemlcl nlsso iei motility kinesin of analysis Single-molecule (1999). 114 173 a.Rv Neurosci. Rev. Nat. 21) N-3(RP n nyi organize ankyrin and (CRMP) UNC-33 (2012). 12 342 20) iei’ A aldmi niisinitial inhibits domain tail IAK Kinesin’s (2000). 52 19) pcfcto fsbntasml ythe by assembly subunit of Specification (1992). 29 20 20) oeua oosadmcaim of mechanisms and motors Molecular (2005). 559-567. , 295-302. , 545-557. , 803-816. , 154-158. , 15735-15744. , 91-123. , 20) essetitrcin ewe h two the between interactions Persistent (2001). a.Cl Biol. Cell Nat. 94 19) litoi fet fadsutdK+ disrupted a of effects Pleiotropic (1997). 20) ifrnilepeso fvoltage- of expression Differential (2000). 1533-1538. , 18) oeeto irtblsby microtubules of Movement (1989). 94 Biochemistry 21) o oeua oosare motors molecular How (2011). 115-132. , LSCmu.Biol. Comput. PLOS a.Cl Biol. Cell Nat. 2 6 20) rsa tutr fa of structure Crystal (2005). 257-260. , 201-214. , a.Cl Biol. Cell Nat. Science 21) lentv splicing Alternative (2012). 20) eceo motor of Rescue (2009). Science a.Cl Biol. Cell Nat. er Res. Hear. LSCmu.Biol. Comput. PLOS 37 19) iei’ tail Kinesin’s (1999). 21) +channel K+ (2012). 20) Differential (2008). 319 16663-16670. , a.Neurosci. Nat. 309 1 1086-1089. , 20) Axonal (2006). 293-297. , 206 LSONE PLoS 897-903. , 1 Science 5 20) The (2009). 288-292. , 19) The (1998). 20) The (2006). e1000623. , rc Natl. Proc. er Res. Hear. 133-145. , 1 .Biol. J. E119- , (2002). .Biol. J. 257 Cell 15 7 5 , , , , Journal of Cell Science oozk .M,Se .M,Plt,C,DlTro . ent,V,Dle,T and T. Deller, V., Bennett, D., Turco, Del C., Politi, M., J. Sie, M., J. Sobotzik, A. J. Spudich, and S. Sivaramakrishnan, hbia .T,Ta,S . u . esii,M,Crel,S,Cto,S . Welte, L., and S. Cotton, M. S., Cermelli, Kawagishi, M., Vershinin, J., Y., Xu, L., Takei, S. Tran, Y., T., G. Kanai, Shubeita, Y., Tanaka, H., D. Seog, M., Setou, Z. Dogic, and D. Nicastro, D., Welch, T., Sanchez, aaa . ia . kd,Y,Prz .adHrkw,N. Hirokawa, and F. Perez, Y., Okada, S., Niwa, T., J. Nakata, R. Lasek, and H. R. Miller, cnte,M .adBok .M. S. Block, and J. M. Schnitzer, H., Sa Moreno, M., Saganich, A., Ozaita, Y., Amarillo, D., Lau, A., Chow, B., Rudy, J. C. McBain, and B. Rudy, une . edzblZbaa . lzaa,I,Rgeo . uea .and I. Buceta, L., Reguero, I., Elezgarai, J., Mendizabal-Zubiaga, N., Puente, nhz .A,H,C . aga,D . aca .C,Gag,R .adJoho, and W. R. Grange, C., M. Garcia, M., D. Vaughan, S., C. Ho, A., J. ´nchez, rc al cd c.USA Sci. Acad. Natl. Proc. C. Schultz, ndrcinlmvmn na -ci network. F-actin an on movement unidirectional droplets. P. lipid S. kinesin-1-driven of Gross, transport intracellular and A. M. dendrites. to kinesin N. Hirokawa, r o u oatrdmsl xiaiiyo ie yebtdpn ntegenetic Nature the on depend but bundles. type microtubule fiber or excitability muscle altered to background. due not are al. H. et R. A. excitability. Hernandez-Cruz, neuronal to R., channels 304-343. Hernandez-Pineda, Kv3 of S., Contributions M. Nadal, 2181-2193. o ihfeunyrpttv firing. repetitive high-frequency for cortex cerebellar rat the method. of immunogold layer pre-embedding molecular the a in by revealed Kv3.3 and Kv3.1b subunits P. polarized Grandes, underlies microtubules GTP-tubulin-rich to motor transport. kinesin-1 vesicle a of binding axoplasm. squid in microtubules along transport vesicle retrograde 20) uceadmtrsildsucini +canldfcetmouse channel-deficient K+ a in dysfunction motor-skill and Muscle (2000). 388 386-390. , 20) nyiGi eurdt ananaodnrtcplrt nvivo. in polarity axo-dendritic maintain to required is AnkyrinG (2009). fuesArch. Pflugers 21) rcs oaiaino h otg-ae oasu channel potassium voltage-gated the of localization Precise (2010). 20) ltmt-eetritrcigpoenGI1drcl steers directly GRIP1 protein Glutamate-receptor-interacting (2002). .Cl Biol. Cell J. Nature Science 20) v hnes otg-ae +canl designed channels K+ voltage-gated channels: Kv3 (2001). 106 20) osqecso oo oynme nthe on number copy motor of Consequences (2008). 440 417 333 194 17564-17569. , 34-41. , 83-87. , 19) iei yrlssoeAPpr8n step. 8-nm per ATP one hydrolyses Kinesin (1997). 456-459. , 245-255. , 18) rs-rde eit neord and anterograde mediate Cross-bridges (1985). rnsNeurosci. Trends 20) ope ysnV oosfacilitate motors VI myosin Coupled (2009). itce.Cl Biol. Cell Histochem. .Cl Biol. Cell J. 21) ii-iebaigo active of beating Cilia-like (2011). 24 Cell 517-526. , n.N .Aa.Sci. Acad. Y. N. Ann. 135 187 21) Preferential (2011). 134 1098-1107. , 53-60. , .Cl Biol. Cell J. 403-409. , (1999). 868 101 , , wlere,A . un .Y,AacbaCrao .L,Mcsil .F., A. MacAskill, L., I. Arancibia-Carcamo, Y., E. Yuen, E., D. A. R. Twelvetrees, Vale, and R. D. Klopfenstein, V. M., Cavalli, Tomishige, and R. Dixit, C., M. M. Zhu, Poo, F., and Sun, S. Duan, J., Luo, Y., Li, G., Chen, D., Wang, H., A. Song, aes .F,Mnsin .A,Seai,G,Fgeo,K . anse,J P., J. Bannister, P., K. Figueroa, G., Stevanin, A., N. P. S. Minassian, Gross, F., and M. J. Waters, S. King, S., D. Razafsky, C., B. Carter, M., Vershinin, u . u . ar,J n u C. Gu, and J. Barry, Y., Gu, M., Xu, D. T. Pollard, and E. Q. L., J. S. Wu, Franzen, Rice, H., and Moreno, L. C., Y. Wong, Kentros, E., Miera, de Vega-Saenz M., Weiser, u . a,R,Xa,R,Zu .X n u C. Gu, and X. M. Zhu, R., Xiao, R., Cao, M. M., Li, Xu, and Y. L. Jan, N., Y. Jan, W., Yu, J., Xu, u . eg . e,Z,Mynii . e,W,Za,Y n hn,M. Zhang, and Y. Zhao, W., Wen, Y., Miyanoiri, Z., Wei, W., Feng, C., Yu, otig . ub .J,Hmet . rle,A,Suo,F,Yn .e al. et Z. Yan, F., Saudou, A., Triller, S., Humbert, J., M. Lumb, P., dimerization. Rostaing, after motor processive a into kinesin KIF1A 1160. ev hi ietyadehne t motility. its enhances and directly chain heavy segment. initial axon the at transport cytoplasmic for filter selective ot,D,Mc,A . vdne .G,Fe .B,Mu B., D. Fee, G., V. Evidente, F., A. Mock, D., Nolte, disrupted and HAP1-KIF5 by mediated huntingtin. is mutant synapses by to GABAARs of Delivery (2010). eae +canl ntertcnrlnrossystem. nervous central B. rat the Rudy, in and channels and K+ H. related Baker, degenerative D., cause Hillman, KCNC3 phenotypes. channel system potassium nervous central voltage-gated developmental in Mutations Tau. by regulation its 104 and transport based Multiple-motor fK3(hw hnesi eemndb agtn oi htascae ihteT1 the with associates that motif G. targeting ankyrin a by and determined domain is channels (Shaw) Kv3 of yeast. fission in tail. its of activity microtubule-binding hnestruhteao nta emn i ietbinding. direct via segment initial 16001. axon the through channels oasu hnes osre yrpii oisdtriesubfamily-specific determine motifs alpha-subunits. hydrophilic the between Conserved interactions channels. potassium ysnV nege ag-eitddimerization. cargo-mediated undergoes VI Myosin 87-92. , v lsesadatvtsKF oos2041 motors KIF5 activates and clusters Kv3 Science Neuron .Neurosci. J. 310 20) onigctknsspoen lblyadlocally and globally proteins cytokinesis Counting (2005). 310-314. , 21) iei’ ih hisihbtteha-and head- the inhibit chains light Kinesin’s (2010). 65 53-65. , 27 21) iei rnprsttaeie Kv3 tetramerized transports I Kinesin (2010). rc al cd c.USA Sci. Acad. Natl. Proc. 14158-14170. , .Bo.Chem. Biol. J. 19) ifrnilepeso fShaw- of expression Differential (1994). 21) udyDie/I3bnskinesin binds Driver/JIP3 Sunday (2011). MOJ. EMBO 20) h xndnrt targeting axon-dendrite The (2007). 19) sebyo voltage-gated of Assembly (1995). a.Genet. Nat. Cell 20) ovrino Unc104/ of Conversion (2002). .Neurosci. J. 270 138 rc al cd c.USA Sci. Acad. Natl. Proc. 30 Science 24761-24768. , 3416-3429. , le,U tal. et U. ¨ller, 537-548. , .Neurosci. J. 38 107 447-451. , 297 14 Cell 11781-11786. , 949-972. , 2263-2267. , 136 30 20) A (2009). 15987- , (2006). (2007). (2009). 1148- ,