niaeta h lsi idnso ukmcouuetranslocation microtubule bulk of in findings classic the that indicate tt nvriy 8 amL om36 atLnig I484 USA. 48824, ([email protected]) MI correspondence Lansing, for East *Author 336, Room Ln Farm 288 University, State om36 atLnig I484 USA. 48824, MI Lansing, East 336, Room oga .Roossien H. Douglas forward elongation meshwork axonal cytoskeletal during the pushes dynein Cytoplasmic ARTICLE RESEARCH ß eevd4Mrh21;Acpe ue2014 June 6 Accepted 2014; March 4 Received and 1 2010a) al., et neurons Lamoureux sensory 2006; chick Sheetz, cultured bulk Drosophila 2008), of and Suter, studies (Miller and as several (Lee such systems, been 2009). cones model Vactor, have multiple Van in and there translocation Lowery Burmeister, however, 2003; and Gertler, Recently, (Goldberg and of Dent transport cone, deposition 1986; growth motor-driven the the by or at material polymerization material new cytoskeleton of be through axon addition new either to the the that by considered thought formed was was was 2002). it cytoskeleton and (Dickson, elongation axonal during cytoskeleton stationary the the molecules, on convention, signaling converge a extracellular By with which through by of synapses guided navigation many is and form tissue extension eventually complex Proper will long targets. out that prescribed send neurons projections which by cellular process the is elongation Axonal INTRODUCTION biophysics Neuronal Dynein, elongation, Axonal cytoskeletal WORDS: the KEY push that data forces these a forward generates together, meshwork find Taken dynein and tension. that disruption axonal indicate dynein in directly after increase then that generation We dramatic find force disruption. we net dynein movements, after measure reduced cytoskeleton is bulk translocation for markers to dynein to fiducial contribute targeted pharmacological forces dynein the whether antibodies bloc ask and to tested. function-blocking D chain ciliobrevin been inhibitor intermediate both not dynein use has the meshwork we microtubules cytoskeletal long Here, the of translocation in bulk embedded the influence whether but to forces axonal transport, axons these ‘stop-and-go’ in through unknown. underlying movement microtubules is their on process power forces mechanism this generates drive dynein forces significant Cytoplasmic cellular how a but that elongation, their is indicate of diameter studies the translocation Recent than longer bodies. times can cell of that processes hundreds axonal lengths out reach send neurons development, During ABSTRACT eladMlclrBooyPorm ihgnSaeUiest,28Fr Ln Farm 288 University, State Michigan Program, Biology Molecular and Cell 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,39–62doi:10.1242/jcs.152611 3593–3602 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. Xenopus yokltntasoain ytakn okdmtcodi as mitochondria docked tracking By translocation. cytoskeleton motoneurons ern Rishe l,19;Cage l,1998) al., et Chang 1991; al., et (Reinsch neurons nmasse en 1 nvivo in hli Lamoureux Phillip , uigaoa elongation. axonal during 2 Rose ta. 03.They 2013). al., et (Roossien eateto olg,Michigan Zoology, of Department nmasse en Aplysia 2 n yeE Miller E. Kyle and microtubule growth en 01 ngte l,20;Glhite l,20;Wn tal., et Wang 2007; al., et Gilchrist al., 2006; et al., (Wang et deformation Knight subcellular bulk 2001; for well-accepted non- are markers of mitochondria fiducial field docked the al., biophysics, in et cell Furthermore, (O’Toole neuronal 2010a). cone al., growth et the to Lamoureux to response 2008; applied in or and in (Miller 2010a; 2013) generated al., al., species et forces et Roossien Lamoureux various 2010b; 2008; al., from al., et Lamoureux et neurons O’Toole cultured 2006; Sheetz, in and 2013), al., et tecigocr uigaoa elongation axonal that axonal shown during and velocity have we (Miller occurs ‘low approach, kymographs stretching this in this Using transport 2006). between axonal Sheetz, fast distinguish and to transport’ of of straightforward stretching movement rate is or this a this Because movement at that meshwork. occurs bulk cytoskeletal indicates the underlying together the points reflects which of branch movement axonal advance 2010a), and forward slow al., axon the the et with of outside paired (Lamoureux the is studies, to previous this bound In that beads 2008). al., shown et have (O’Toole we axon the of length the tart f1–50 forward Along move of slowly rate 2012). mitochondria a 2004; docked at Hollenbeck, axon, Hollenbeck, and the where of Saxton and length time 2004), the 2004; a Chada Sheetz, at Sheetz, 1993; and hours Miller several Hollenbeck, and for and (Miller position (Morris in filaments axon remain neuronal can the mitochondria regular or at along occurs intermediate Docking 2003). intervals and al., et 2010) Wagner 1982; (Hirokawa, al., filaments actin to et 2008) al., (Pathak et Kang 1982; (Hirokawa, syntaphilin ln h xndsiaefre eeae ntegrowth the in generated forces dissipate adhesions 2013; because al., axon that noting et worth the Lu is 2011; It along 2013). Miller, generated al., and forces et (Suter the pushing Roossien motors or of molecular pulling terms through by In either are 2010). apart filaments al., sliding because axon, axon occurs et the the presumably along stretching of (Lamoureux cytoskeleton, the addition stretching thinning mass with cause prevent intercalated paired to by forces is followed This which is cone bulk. which growth in in the advance 2011), of to (C-domain) Miller, domain central and microtubule-rich intercalation’ (Suter and broadly ‘stretch for (SAI) termed model a emerged, new rather has a elongation this, but axonal From elongation. phenomenon, for species-specific mechanism conserved a not were fe oo-rvnfs rnpr Plige l,20)a aeof rate a dyes. at 2006) fluorescent al., et easily using (Pilling , transport be conditions fast can motor-driven low-light After that cytoskeletal organelles under to large visualized docked are mitochondria Mitochondria of elements. movement (Lim the the body monitor whereas 1990). cell axon, Hirokawa, the and distal Okabe near 1990; the stationary al., in et is these only framework 2008), cytoskeletal occur al., cytoskeleton et the (O’Toole cone 3600 otrack To 2, * m /,mtcodi tbydc omcouue through microtubules to dock stably mitochondria m/h, nmasse en m /,wt eoiypoieta nrae along increases that profile velocity a with m/h, , oeeto h yokltlmswr,we meshwork, cytoskeletal the of movement 00tmssoe hnfs rnpr,it transport, fast than slower times 1000 nmasse en nvivo in oeet of movements (Roossien 3593

Journal of Cell Science ergaetasoto ebaeognle ShapadReese, and (Schnapp driving organelles include membrane the axons of of growing transport in maintenance retrograde dynein and for growth roles proper Identified the axon. candidates. for logical essential are is dynein, microtubule- Dynein cytoplasmic but as answered, such motors, been based not and has Silberberg meshwork 2012; cytoskeletal al., et Bell 2008; al., 2013). et Pelling, Alamo del 2007; ARTICLE RESEARCH ois(i.1) sapstv oto ovrf htinjected that verify to control 3594 positive a As cell of 1A). rounded presence (Fig. normal the morphologically bodies on and based marker injection fluorescent the successful for screened in out were carried at were bodies Injections cell neurons. neuronal injected for screening and dynein disrupt to sought in neurons of therefore microinjection and sensory the We 2011) through 2007). chick function al., al., of et et (Yi elongation (Grabham neurons neurite hippocampal laminin-induced rat neurons, in In 2000). transport Wang, and O’Connell a 2000; mitosis during al., positioning et and (Dujardin formation (Faulkner spindle migration and cell 2003), been directed al., 2001), et has al., centre et it microtubule-organizing (Palazzo the cells, of (MTOC) dynein positioning non-neuronal the 74-kDa study In to the used function. of dynein acids targeted ( perturbing amino chain antibodies 63 intermediate N-terminal function-blocking the of against microinjection meshwork cytoskeletal the The by of elongation retraction axonal bulk reduces causing activity dynein of Disruption RESULTS to 2006; contributes al., dynein et whether Myers but 2006; 2007), al., disruption masse et al., dynein Ahmad et have 2000; following Grabham axons al., elongation in et in function (Ahmad dynein Ahmad decrease of 2005; a al., studies et reported Previous for He 2006). 1998; al., required al., individual et actomyosin- et of be resist (Ahmad transport they microtubules ‘stop-and-go’ to axon, short power 2006; the and al., thought along retraction et and, based are (Myers 2009) al., flow cone et actin Vallee retrograde growth resist to the Dynein microtubules 2006). al., in et Dehmelt during 1998; forces bundle al., microtubule et (Ahmad initial initiation the axon of establishment and 1989) nnpcfcmueIGpoen)wr ie t1 gm with mg/ml 10 at mixed were 0.1 proteins) IgG mouse (nonspecific plastic poly-ornithine). on grown with neurons coated (i.e. dishes conditions culture our under grown pushes dynein forward that elongation. meshwork axonal cytoskeletal indicate during bulk the tension. disrupt studies in in neuronal to in embedded these CilD increase microtubules added an together, then found we and Taken response, axons In measured in dynein. we generation forward, axonal material force to pushing net or contributes during pulling dynein meshwork by whether elongation cytoskeletal determine the To drives of activity elongation. dynein translocation that forward found and also the before we meshwork disruption, cytoskeletal dynein as axonal after mitochondria the docked of a using markers in By fiducial resulted elongation. dynein treatments axonal of both in role that decrease found the We study elongation. to axonal (CilD) in D published ciliobrevin recently inhibitor the dynein with disruption pharmacological acute and I a enue opoedni ucini ergaeaxonal retrograde in function dynein probe to used been has DIC h usino htpwr uktasoaino the of translocation bulk powers what of question The noreprmns both experiments, our In antibodies dynein-specific function-blocking used we Here, m /lFT–eta,alwn o iulzto ffudflow fluid of visualization for allowing FITC–dextran, g/ml yokltnmvmn sunknown. is movement cytoskeleton a , I)hsbcm odsadr for standard gold a become has DIC) 82 fe ltn.Atr2h neurons h, 2 After plating. after h 18–20 a I n oto antibodies control and DIC a I neogtn axons elongating in DIC en n hs netdwt oto g Fg B.Aosinjected Axons 1B). (Fig. neurons IgG non-injected control between with with mitochondrial injected direction fast those retrograde in and the the difference in in significant point counting no flux midway by found the measured passed We then with that axon. was mitochondria were Flux labeled of h. number and were 2 the for microinjection Mitochondria recover to following 2006). allowed al., immediately et MitoTracker Hollenbeck, and Pilling (Saxton 2012; transport mitochondrial fast dependent a rwhcnswr ttoaywt naeaevlct of velocity average an with stationary were 2 cones growth ta. 99.Tkntgte,teeeprmnsvrf that verify experiments these together, (Martin decrease disrupted Taken of was microinjection 1999). significant been the dynein also al., where has further systems it et surprising, other a seem in compared of might found reported this that we was Although we with 1B). When flux (Fig. neurons IgG. anterograde neurons, IgG-injected with fast injected in that injected flux 2011), neurons anterograde al., in et reduced after (Yi of organelles previously analysis membrane 5.60 previous other a of those a with 1B; transport (Fig. and agreement axonal in retrograde axons is IgG This non-injected respectively). with both than injected lower significantly 1.15 n olnek 93 atnadHlebc,21)after 2012) (Morris Hollenbeck, mitochondria and and docked IgG Saxton of of 1993; process, microinjection movement this Hollenbeck, Roossien 2008; to the contributes 2010b; and al., dynein al., tracked whether et cytoskeletal et test we To O’Toole Lamoureux the 2013). 2010a; al., 2006; of et al., Sheetz, movement et and Lamoureux forward (Miller bulk meshwork and stretching hsw ne httectseea ehoki advancing is with docked meshwork cytoskeletal addition, injected the cone In that growth unit. masse infer From coherent the we elongation. during one that advanced this axon distal as suggests the the in bulk mitochondria This with in tandem in 1C). that in (Fig. advances docked advanced found cone controls, cone We IgG growth growth with 1E). the (Fig. injected in neurons mitochondria added from been cones color- growth have and transport duplicated mitochondria arrows anterograde been has of coded fast kymograph the populations transport, transport, bulk different retrograde injected and neuron fast the control by show a moving from To axon IgG. an with shows 1C Fig. neurons. iohnra eoiiswr otdbsdo hi initial 25- into their placed on cone, based growth the sorted from retracting were distance velocities was mitochondrial meshwork cytoskeletal the 1D). (Fig. that suggesting h rwhcn.B otat nnuosijce with injected neurons at end in pulled contrast, a and of By body piece cell cone. the a growth the at to end that the similar fixed 2013), a al., stretched, al., with at being et et ‘silly-putty’ is grew Roossien O’Toole meshwork 2010a; 2006; controls al., cytoskeletal Sheetz, et IgG Lamoureux and the 2008; (Miller images, previously phase reported of analysis 24.9 based Likewise, the apart. cone growth on spread the increasingly from from distance becoming the mitochondria decreased, as docked increased of axons control velocities IgG The 1F). (Fig. averaged I nioisdces yenfnto,w nlzddynein- analyzed we function, dynein decrease antibodies DIC I neto Y ta. 01.W lofud sreported as found, also We 2011). al., et (Yi injection DIC 1.5 ehv rvosysonta ern rwb axonal by grow neurons that shown previously have We oqatf oeet olwn microinjection, following movements quantify To 6 6 6 ycnrs,dce iohnrai xn rmneurons from axons in mitochondria docked contrast, By . .9mt/ [mean mito/h 1.39 7.9 a 3.8 ora fCl cec 21)17 5330 doi:10.1242/jcs.152611 3593–3602 127, (2014) Science Cell of Journal I,b otat a vrg ergaefu of flux retrograde average had contrast, by DIC, m m / (mean m/h / (mean m/h a I xiie lwrtord movements, retrograde slow exhibited DIC a 6 I infcnl eue yenactivity. dynein reduces significantly DIC 6 5 I.Ti ugss shsbeen has as suggests, This CI). 95% 5 CI; 95% 6 a 5 ofdneitras(CI)], intervals confidence 95% I nioisit hc sensory chick into antibodies DIC P , 6 .1 nardtwo-tailed unpaired 0.01; .7ad8.31 and 1.87 m isand bins m 6 a a DIC- 2.11, DIC, en

Journal of Cell Science oe erad Fg F.Teedt niaeta dynein the that of indicate translocation forward data bulk meshwork. cytoskeletal and These elongation 1F). axonal (Fig. powers rearwards moved EERHARTICLE RESEARCH efudta t60 of at microinjection following that seen to found that elongation We blocked to that similar concentration extent a in effectively an step find first to most and Our was gliding 2012). CilD to al., using microtubule et shown in (Firestone assays RNA measured was transport as vesicular (CilD) function (i.e. ciliobrevin dynein the reduce of inhibitors, they approaches these form Of that applied. D in systematically previous and knockout) acutely be genetic can over and microinjection interference, advantages inhibitors these time of the use post-power- offers The increasing the 2013). in thus (Kikkawa, microtubule motor, conformation the stroke dynein to attached the spends in dynein that site ATP small-molecule binding with compete of its They 2012). class at al., et discovered (Firestone inhibitors recently dynein cytoskeleton a axonal are the of Ciliobrevins retraction pharmacological causes the CilD with inhibitor dynein of disruption Acute Student’s t ts)addce iohnrai h itlaxon distal the in mitochondria docked and -test) m lnainbgnt lw t100 at slow, to began elongation M a DIC. m M rwhcnst erc rel,atrwihte become they During which 1). fluorescent after Movie material and briefly, advancing supplementary phase retract causes 2C; CilD (Fig. both to images, and stationary phase in to cones the prior addition in growth min seen 20 CilD As for channels. after intervals 1-min min at 60 imaged were axons 2012; 100 between al., results et in similarity (Firestone The and 2013). vesicles al., of membrane et disruption Lu other bidirectional a of reported transport ciliobrevins using studies 100 with decreased n fe h diino 100 of addition the after and function. dynein impairing for appropriate 60 at affected not was flux of 100 Anterograde and microinjection 2B). 60 (Fig. at with controls seen DMSO in those observed to 100 and similar a 60 as effects of transport found concentrations mitochondrial we CilD fast at dynein measured above of we 150 disruption discussed CilD, the at verify by To and function 2A). (Fig. stationary, occurred became retraction cones growth I.Rtord rnpr a eue o6%ad2%o that of 22% and 66% to reduced was transport Retrograde DIC. et etakdtemvmn fdce iohnrabefore mitochondria docked of movement the tracked we Next, a I irijcinsget htti ocnrto is concentration this that suggests microinjection DIC ora fCl cec 21)17 5330 doi:10.1242/jcs.152611 3593–3602 127, (2014) Science Cell of Journal ofre ytepeec fFT–eta hogotthe 25 bar: throughout Scale FITC–dextran neuron. is of entire injection presence Successful the injection. by after confirmed h 2 at morphology neuron A (A) with FITC–dextran. injected with mixed antibodies control ( chain the intermediate against dynein antibodies function-blocking with meshwork. microinjected axonal the inducing of by chain retraction elongation intermediate axonal dynein reduces 74.1 antibodies of Microinjection 1. Fig. ubro iohnraue o ahgroup. each for used mitochondria of number Student’s F iohnra eoiiswr otdbsdo hi initial their on based sorted were retracting. velocities is Mitochondrial meshwork (F) cytoskeletal the that suggesting injected neurons in mitochondria with that 20 showing bar: series Scale image min. transport. 15 fast arrows: retrograde Time blue, transport; red, fast translocation; anterograde mitochondria mitochondrial docked color- of yellow, with pools motion: labeled different and representing E arrows This in coded elongation. duplicated during been advance has cone kymograph growth axon the distal in the in and mitochondria docked color-inverted that The shows antibodies. kymograph control IgG following with tracked injection were mitochondria Docked (C) test). Tukey’s controls. IgG of at injection *significant following observed that by reduced with is axon, mid-way the the of passed that point by mitochondria measured of number direction, the retrograde counting and anterograde the both aaso h mean the show data after velocities a negative is The injection stretching. axonal IgG with The after consistent averaged. gradient and velocity binned increasing cone, distally growth the from distance I neto niaebl ercin C rwhcn.All cone. growth GC, retraction. bulk indicate injection DIC a I nioisudrobl ergaemovement, retrograde bulk undergo antibodies DIC m t ts) ausa h otmo h asso the show bars the of bottom the at Values -test). iD(i.2) ent htprevious that note We 2B). (Fig. CilD M a a I hw omlcl oyadneurite and body cell normal shows DIC 5 .5 *infcn at **significant 0.05, 6 m 5 I * CI. 95% iD nttl 2elongating 32 total, In CilD. M a m I)o oseii os IgG mouse nonspecific or DIC) .()Fs iohnra lxin flux mitochondrial Fast (B) m. a P I neto ij)compared (inj.) injection DIC , m .1(nardtwo-tailed (unpaired 0.01 m iD respectively CilD, M .()Corresponding (D) m. a ern were Neurons 5 .1(post-hoc 0.01 m n above and M m ,btwas but M, m m CilD M ,and M, 3595

Journal of Cell Science marn rgfnto,w esrdteeogto aeof rate or our elongation retraction that the acutely measured possibility causing is we the were dynein function, for drug when conditions control impairing cone imaging To growth fluorescent move 2E). the cone (Fig. growth phase- of disrupted the and mitochondrial in out fluorescent the mitochondria backward the that in shows seen Overlaying series be 2G. image Fig. also can in This (supplementary kymograph arrows) 1). (orange Movie body material cell the towards retracting EERHARTICLE RESEARCH 3596 Upon IgG- cone. growth in docked the 100 with seen moving of tandem as in addition in advance labeled are arrows), mitochondria cone addition, (red growth In mitochondria the 1C,E). elongates (Fig. fluorescently neurons axon These control injected the shows time. as forward over 2D accordion-like Fig. an mitochondria in retracts axon fashion. distal the phases, retraction the m iD oee,dsa iohnrabegin mitochondria distal however, CilD, M fe iDtetet otdbsdo hi itnefo the from distance 25 their and in quantify on placed before based To cone, measured sorted not function. growth treatment, were were CilD drug velocities conditions after impairing imaging motion, or our mitochondrial that retraction suggesting 16.1 causing 2A, Fig. from in elongation 100 Student’s of tailed that rate found 2.8 after the and We before decreased images addition. phase in CilD cones growth individual these xn(e as eoedu)t ercin(rnebr,100 bars, (orange retraction to distal the drug) in before meshwork bars, from cytoskeletal (red the change 2H). the axon significant (Fig. of in and advance axon rapid forward distal a stationary bulk the caused in CilD were with forward Treatment moved mitochondria and axon addition, proximal CilD Before 6 0.9 ora fCl cec 21)17 5330 doi:10.1242/jcs.152611 3593–3602 127, (2014) Science Cell of Journal m / Fg F mean 2F; (Fig. m/h t ts) hsi nareetwt h aashown data the with agreement in is This -test). m oeavnebfr n fe the after and before growth advance of cone rate The (F) post-drug. arrows, orange pre-drug; is arrows, cone Red stationary. growth the when even rearward, move to continue mitochondria docked that indicates (C,D) images phase and fluorescent the Overlaying (E) mitochondria. of docked retraction induces dynein of disruption 100 of application each in axons 0 of group; number the are top at the numbers dose- Orange a manner. in dependent elongation of rate CilD the with reduces dynein of Disruption meshwork. (A) axonal the of retraction induces and slows elongation CilD axonal with dynein of disruption pharmacological Acute 2. Fig. at Student’s two-tailed 100 of addition ierepresents orange line The stationary. are the cones Afterwards, growth images. phase in as seen retraction of induces disruption initially Acute dynein (C) used group. axons each of in numbers the are of bars base the the at white in Numbers dose-dependent manner. a in fast flux reduces mitochondrial dynein of Disruption (B) h mean show data the quantitative All F. cone in growth retraction and GC H between in rate mitochondria in difference the axon. Note per mitochondrion the distal comprises most (GC) bin the cone where growth bin, each in of mitochondria number the the of represent bottom graph the at numbers Color- coded retraction. bulk and contraction induces disruption dynein that demonstrates analysis 20 Quantitative bars: (H) Scale min. 15 Time arrow: treatment. CilD following the axon along contraction and retraction to bulk elongation during stretching from axonal shift the shows kymograph inverted isadaeae Fg 2H). (Fig. averaged and bins m 6 a 5 CI; 95% , .1(othcTkystest). Tukey’s (post-hoc 0.01 m 6 steDS control. DMSO the is M 5 I * CI. 95% m iDsignificantly CilD M P t m 5 iD G h color- The (G) CilD. M , :0frtebath the for 0:00 m .1 ardtwo- paired 0.01; iD D Acute (D) CilD. M t ts) **significant -test); P , 6 .1(unpaired 0.01 5.4 m m / to m/h m. m M

Journal of Cell Science oa irpino yeni h rwhcn n itlaxon distal and cone growth the in dynein of disruption Local cone growth and cytoskeleton elongation. axonal axonal during the of with movement disruption forward dynein after found those These a with bulk. consistent in backward are to pulled due data being is meshwork compaction cytoskeletal accordion-like the the that suggests This CilD). ARTICLE RESEARCH ihCl eetakdatrrmvlo h irpptei ahu xeiet hyrsmdnra opooyadfradeogto.()Focal (C) elongation. forward 20 and bar: morphology Scale normal (mean elongation. resumed cones axonal Growth They on (B) experiment. effect images. phase washout no the a had in in overlay control, red micropipette retraction. a the the axonal by as shown of causes DMSO, is removal axon of application after drug distal application of tracked and extent were cone The retraction. CilD growth axonal with the induced axon, in distal dynein and of cone growth disruption Focal 3. Fig. 30 to CilD distal applied and focally we cone new end, delivering growth this in To the role axon. in their distal separate the from to dynein to cone difficult material growth and is the it kinesin in that activity of been their has role caveat a the elongation, investigating axonal studies transport axonal prior fast In of independently retraction causes oiinadpotddrn h 0mntetetwt iD(le rDS rd.Tikdr asso h engot oepsto tec -i interv 1-min each at position cone growth mean the show bars dark Thick (red). 17.4 DMSO of or rate (blue) at CilD grew with cones treatment 20-min growth the Control during plotted and position 0mnpro hw h oa plcto f150 of over application series local image the an shows 3A, period Fig. 20-min In a chamber. flow a in micropipettes I n,tkntgte,sgetta yenpwr bulk powers dynein that suggest together, taken and, DIC 6 5 I.Got oe rae ihCl eue rwha 13.6 at growth resumed CilD with treated cones Growth CI). 95% m fteao o 0mnusing min 20 for axon the of m 6 7.0 m / (mean m/h 6 m iDa a as CilD M 5 I.Fcldsuto fdni nue ercina naeaert of rate average an at retraction induced dynein of disruption Focal CI). 95% 6 3.2 m / uigwsot ro asso 5 CI. 95% show bars Error washout. during m/h n omlzdte yajsigteiiilpsto o0 to position initial the We adjusting 3C). intervals by (Fig. 1-min them at application contrast, cones normalized growth by and fluid individual of DMSO, during position the with tracked advance treated to distal cones and CilD continued growth cone growth that Control the damaging suggesting axon. by advance, retraction cause forward after not the does that, resumed and found restored, cones was We behavior growth 3B). and (Fig. morphology micropipette normal after washout, the axons of address monitoring removal by To the performed CilD, cone. retracting of were concentrations growth experiments high then of washout the effects off-target at observation, or toxic application potential the (see pre-treatment CilD during direct column advancing of during cone rapid left period growth for account the the to 20-min shows concentration and 3A response in this Fig. cone used growth diffusion. images robust We a phase ensure 2). to Movie the material on supplementary overlay red m .()Idvda rwhcn oeet eenraie o initial for normalized were movements cone growth Individual (D) m. ora fCl cec 21)17 5330 doi:10.1242/jcs.152611 3593–3602 127, (2014) Science Cell of Journal A oa plcto fCl,t irp yeni the in dynein disrupt to CilD, of application Focal (A) 2 22.1 6 6.9 treated m 3597 m/h m m. al.

Journal of Cell Science h ukavneo h rwhcn n htteeaeother are the there of that translocation and forward cone power meshwork. axonal growth that dynein, the together, besides of Taken motors, oppose advance 2H). that bulk forces Fig. generates the and NMII that 1E and suggest Fig. experiments of dynein these disruption (compare when to CilD alone contrast forward in dynein of moved disrupted; grew axon simultaneously presence still pre-treatment were the neurons NMII combined than that along note material along the lower we and interest, in significantly velocities particular Of axon were velocities. mitochondrial the blebbistatin addition, of and length In the test). either with Tukey’s 50 compared CilD or of presence pre-treatment the in lower significantly eutdi euto neogto o13.1 to elongation in reduction a in resulted bulk (mean in 21.9 increase from h cone an growth caused the disruption of NMII translocation that found We bars). rosadbr)adte o ia 5mni h rsneof presence the in min 45 final 100 a and for blebbistatin then both and bars) and arrows ntefnlcag npsto tteedo h xeietand experiment cone the of growth end 17.4 the for be at to position sufficient it in found based change is velocity final cone the axon growth on average average the distal the calculated represent We the and retraction. in 3D disruption Fig. cone dynein in that growth showing graph time-point, the each at on position The lines 3D). (Fig. dark retracted CilD thick with to treated continued those whereas cones advance, growth DMSO-treated together, plotted When ARTICLE RESEARCH hne nntaoa oc eeainbfr n fe the after and 3598 before generation force 100 axonal of short addition net measure in of of to assay disruption rest-tension changes the transport force-calibrated a (Mazel used in that we needles cells is the this, towing prediction test substrate-coated non-neuronal To the in tension. case, increase of will this and dynein cortex In al., 2014). 2006) cell al., et pushing et the al., (He be at transport et could microtubules stop-and-go Ahmad dynein in Alternatively, 2005; observed net axon. as which decrease to the microtubules in predicted 2003); be in would al., dynein tension et of disruption (Dujardin in the migration seen case, and as be and 1997) forward, (Carminati positioning could Stearns, microtubules pole spindle pulling dynein during cells primarily how non-neuronal be meshwork. for could cytoskeletal the explanations It of translocation broad forward the two powering tension axonal are in increase There an causes disruption Dynein for bars), and arrows 50 blue for with 4, treatment axons (Fig. during addition 28 min drug 45 in to tracked prior min In were 15 2003). mitochondria al., et experiments, (Straight these blebbistatin using treatment the II disruption CilD of NMII combined translocation with therefore myosin bulk We in unknown. non-muscle is cooperate cytoskeleton but motors resist 2000), two al., these to et whether (Ahmad retraction required cone growth is (NMII)-induced activity non- Dynein of activity absence II the myosin in muscle translocation decreases D Ciliobrevin and cones growth itlprino h xnidpnetyo t ciiyi the in activity its of the independently in axon elongation axon. the axonal proximal of drive to portion functions distal dynein 13.6 that of growth suggest rate average CilD-treated an the (mean at washout, elongating resumed After cones cones. growth treated 6 5 I upeetr aeilMve2.Teedata These 2). Movie material supplementary CI; 95% 6 5 I i.4.Sbeun niiino dynein of inhibition Subsequent 4). Fig. CI; 95% m iD hsasymaue e ocsbeing forces net measures assay This CilD. M 6 2 7.0 m 22.1 lbittnaoe( alone blebbistatin M m 6 / (mean m/h m 6.9 iD(i.4 rnearw and arrows orange 4, (Fig. CilD M m / ( m/h m lbittn(i.4 yellow 4, (Fig. blebbistatin M 6 5 CI; 95% n 5 20; 6 . o31.2 to 7.4 6 P a 3.2 n , 5 5 .1 nCilD- in 0.01) .1 post-hoc 0.01, m 8 ncontrol in 18) /.Ti is This m/h. 6 6 5.6 5.2 m m m/h m/ two-tailed salse.O vrg,tninicesdfo 37.6 from increased was tension CilD tension 79.8 before average, steady-state new period On a 30-min after established. a period 30-min over 17 measurements a averaged and Force the addition were way. tension of axon same axonal Each each the for in 3). in responded Movie increase assayed material the axons supplementary by the force on graph 5A,B; indicated harder pulls (Fig. initial the in as cone growth images on needle, the the phase added, of towing arrows is the CilD are series When The a which 5B. by red Fig. blue. after represented timespan in In min, the 0 indicate plotted 5A. at Fig. are addition measurements force in CilD individual An before shown axon. measurements the is along and cone profile growth membrane generated the those and both reflect in forces dynamics measured the cytoskeleton activity Furthermore, combined tension. motors, the from cytoskeletal result which of neuron, the in generated itlmtcodi nec rwhcn.Dt hwtemean most the the show GC, Data bin. cone. at each growth *significant in each above mitochondria in Numbers of mitochondria levels. number distal pre-treatment the with represent at compared bar reduction axon subsequent each a the a causes in and CilD points of translocation all Addition cone axon. growth the bulk along in decrease increase an causes bars: blebbistatin Scale blebbistatin+CilD. pre- bar, bar, Blue orange 20 A. blebbistatin; in bar, shown axon yellow the arrows). drug; in yellow mitochondria versus docked of arrows Kymograph (orange (B) subsequent it whereas arrows), reduces yellow addition growth to CilD arrows increases blue blebbistatin (compare that advance show cone mitochondria of 100 images which fluorescent after min, 45 50 for with treated dynein then and min, II 15 advance. myosin cone between growth bulk relationship controls antagonistic An 4. Fig. m .()Qatttv nlsso okdmtcodi lutae that illustrates mitochondria docked of analysis Quantitative (C) m. 6 10.9 ora fCl cec 21)17 5330 doi:10.1242/jcs.152611 3593–3602 127, (2014) Science Cell of Journal t m -test). a ye(i.5;mean 5C; (Fig. dyne 5 .5 *infcn at **significant 0.05, m m iDwsadd ardpaeand phase Paired added. was CilD M lbittn(l odsutmoi Ifunction II myosin disrupt to (bl) blebbistatin M a A ern eealwdt rwfor grow to allowed were Neurons (A) 5 6 .1(othcTkystest). Tukey’s (post-hoc 0.01 5 CI; 95% P , .1 pairwise 0.01; 6 5 CI; 95% 6 07to 10.7

Journal of Cell Science oiino h w ede ttetm fCl diin cl a:25 Data bar: addition. Scale CilD addition. after mean and CilD the before initial of show measurements the time tension show the average to at The series needles (C) the two to the added of been position have lines Vertical addition. EERHARTICLE RESEARCH ena uln ntenel nasre fpaeiae,crepnigto corresponding A. images, phase in of arrows series by a demarcated in is time needle tension the the in axon. increase on each The pulling for (B) as B. tension seen in average presented the time-span the drug calculate represent after to Arrows and used before was balance force which taken steady-state addition, measurements the Note force addition. blue, CilD CilD; after before profile taken force 100 measurements individual when force An increases measure (A) tension to disruption. that used dynein shows are after cones forces growth tension. in to axonal changes attached increases needles CilD towing with calibrated disruption Dynein 5. Fig. 6 5 I * CI. 95% P , .1(ardtotie Student’s two-tailed (paired 0.01 t 5 :0rpeet h ieo CilD of time the represents 0:00 m iDi de at added is CilD M t 5 t i.Red, min. 0 -test). Force- m m. niaeta yendie xnleogto ygenerating observations the by push an These thus elongation and with forward generation meshwork 5). axonal force coupled cytoskeletal (Fig. contractile Using drives oppose is that tension 2). dynein forces this that 1, neuronal that (Figs indicate in find reducing meshwork novel we by increase the the elongation analysis, of report By slows fiducial biophysical 2007). translocation we disruption as al., meshwork, dynein forward mitochondria et cytoskeletal that docked Grabham the finding 2006; of of al., movement markers et the Myers tracking Ahmad 2006; 2000; al., al., decrease et (Ahmad a 2010b; et previously found al., reported and and as et dynein elongation, disrupted Lamoureux in We Miller 2010a; 2013). al., al., 1991; et et Schaefer Roossien 2008; Lamoureux al., al., 2008; meshwork et al., O’Toole et 2008; et cytoskeletal Suter, (Reinsch and mechanism Lee the 2006; molecular elongation Sheetz, of the axonal identify translocation during to bulk sought we underlying paper, this In DISCUSSION ie Niadatbd irijcin os lblyand globally dynein local of so role the do sort to microinjection) axonal difficult it antibody makes fast kinesin. This and chronically. dynein inhibits for disrupting RNAi dynein required for (i.e. of approaches are disruption well-established both Furthermore, particular, that In is transport. elongation axonal dac.Seuaiesbellrmcaim oepanhow explain cone dynein to growth generates whereas mechanisms assist subcellular normally to elongation, Speculative forces our NMII oppose advance. pushing on that counteracting that Based generates is forces cone. that explanation think contractile growth we simplest blebbistatin, the rearwards inhibitor the NMII of needle the using retraction towing tension experiments neuronal the increases induces both pulled disruption and dynein cone Thus, 5). with growth (Fig. force the the increased dynein of with which disruption treatment that after found forces We axonal CilD. net transport measured we bulk elongation, exciting to to an contributing is for kinesin-1 2013) elongation. microtubules axonal al., initiation, long during et axon move (Lu to during ability possibility axon its contributing the on motors Based into additional process. are forward there this that the to suggests disrupted. this counters were us, NMII and and that To transport dynein This inhibit both bulk cone to when Interestingly, alone. continued is growth elongation dynein. neurons by dynein the in driven NMII of of translocation of advance disruption as function decreased net much bulk the the as dynein that nearly during indicates and not observed although NMII transport, was bulk both and 4). of (Fig. elongation NMII increased blebbistatin disruption whether translocation with cone test Subsequent growth neurons To bulk treated that microtubule unknown. found we and is role stop-and-go transport, the transport bulk 2001), of bulk Ahmad, modulates in and context Baas has NMII 2000; of this the al., et Although (Ahmad in accepted. transport well examined cargo is in been elongation that in relationship of an independent has body. cell axon elongation the distal from and for transport we cone growth role Here, the local. This important in 3). and dynein (Fig. elongation acute that axonal blocks indicates is also method it this dynein that advantages; disrupt show to of distinct cone growth two contribution method the Our to offers long-term cone. CilD growth applying the the focally to of from body here cell cone the from growth delivery cargo the at action ao hleg nsuyn h oeo iei n yenin dynein and kinesin of role the studying in challenge major A oudrtn h ipyia otiuinta yenmakes dynein that contribution biophysical the understand To antagonistic an have dynein and NMII that idea The ora fCl cec 21)17 5330 doi:10.1242/jcs.152611 3593–3602 127, (2014) Science Cell of Journal nmasse en . 3599

Journal of Cell Science as nw sPFHB)adCI-7 as nw as 1999; known al., et (also (Vaughan CLIP-170 LIS1 telescoping and slides through microtubule microtubules PAFAH1B1) 2009) drives of as CLIP1) al., Tolic plus-ends known et the and (also (Markus to Num1 bound (Pavin or Dynein IQGAP1 bulk by forward 2002) either possibilities to cortex al., microtubules the cell the contribute include et at These actin that (Fukata to 6. targeted Fig. forces dynein in that generating outlined are be transport might dynein ARTICLE RESEARCH 3600 Vale, 2008. from Desai, modified and Dynein Cheeseman 2000. from al., modified and et Microtubules NMII Lodish 2003. forward. cortical from them of modified move independently and were axon microtubules actin capture the plasma could along the it or in where cone anchored actin, microtubules growth be long the might slide in Dynein to membrane (6) axon filament); the actin along (AF, actin apart; forward cortical telescope to use them might cause Dynein neighboring that T-(5) two forces the to generate in bound could actin Dynein (MT) to (4) microtubules forward; bound microtubules Dynein contractile move (3) generates might stretching; axon zone axon the oppose forward along could oppose cortex that could cell forces the that in turnover T- NMII and the (2) in flow translocation; NMII actin (1) retrograde dynein: drives and the zone microtubules in NMII, function actin, motor between NMII interactions cone. and growth dynein and for axon model Subcellular 6. Fig. in made been these in has regulation dynein progress of a basis Great molecular into the elongation. push) understanding to axonal appear and that others elongation. axonal and for pull model coherent to be Gallo, of appear will (some 2000; challenge mechanisms which force-generating future al., by different important et these An and/or integrate Ahmad 2007). to 2011) al., 1985; et al., Bradke, telescoping Bernal et 2004; oppose al., and that (Joshi et axon Stiess microtubules the forward (Schaefer of 2011; in the cone forces al., contractile to growth generating et barrier the Hur a in NMII as 2002; microtubules contrast, acting By of by 2011). advance elongation Srayko, (Kozlowski and inhibit forward Gusnowski could them the 2007; moves captures al., and actin et microtubules the of cortical to ends of bound plus independently dynein membrane Alternatively, 2006). plasma al., et Kholmanskikh ti eonzdta yeni motn o elmigration cell for important is dynein that recognized is It hsmdlpooe h olwn intracellular following the proposes model This -orlke 2013). ´-Norrelykke, 0Kcnrcndvcs(ilpr;Bleia A pna 10,000 at spun MA) Billerica, 0.5-ml Ultra (Millipore; Amicon 4 devices Millipore centricon using MO), K Louis, 50 St (Sigma; kDa) (134 us inln ahas yenrglto n rwhcone growth and guidance between regulation work chain dynein the Our motility. potentially pathways, in that 2006). signaling link 6) new al., cues, (Fig. important et model an biomechanical Kholmanskikh provides novel 2005; 2004; a al., al., et suggests Watanabe 2002; et al., of et Gomes targets (Fukata downstream signaling are proteins, family which regulatory Rho CLIP-170, other and of IQGAP1 number as a such 2004). with al., interact et these Lansbergen of in 2003; Both roles al., important et play (Dujardin regulation example, dynein for dynactin, and LIS1 contexts. tde Gahme l,20;Y ta. 01,bta 0mg/ml. 10 at but 2011), al., et cultures neuronal Yi sensory chick 2007; in out al., carried previous was in et as Microinjection mouse control as Non-specific (Grabham used antibodies. were MO) studies IgG Louis, of St (Sigma; weight antibodies IgG molecular the to similar tc ( Stock treatments Drug 100 as penicillin, cultured glucose, U/ml 0.6% and 100 with glutamine, isolated supplemented mM were medium 1 L-15 ganglia in root previously dorsal described chick from Neurons culture Cell METHODS AND MATERIALS 10m EE,10m C)mxdwt 1 with mixed KCl) buffer mM injection 140 sterile Millipore; in HEPES, mg/ml (74.1; 10 mM chain to (100 intermediate concentrated were dynein MA) the Billerica, to targeted Antibodies Microinjection oiae o 0mn hnsoe t37 CA), at Carlsbad, stored then (Invitrogen; of min, kDa) 10 that (10 150 for to to identical sonicated Texas-Red–dextran diluted was was mg/ml CilD used. CilD 1 concentrations of all application, for focal handling 1:6 at For The always dilutions DMSO. final up with during made in 50 blebbistatin, was dish mM solution of stock 60 culture concentration MA) to Billerica, the blebbistatin (Millipore; final into D ciliobrevin a directly The giving performed acquisition, were image 1:6 of dilutions h iet.Paeiae eecpue vr 0s n fluorescent and s, exposures. 10 100-ms using every min of captured 1 out every were flow captured fluid images were instead of used images control Phase was tighter for fluorescence, pipette. by allows it the pipette the because of Picospritzer connected out a syringe of comes monitoring it ml with as combination 10 fluid in a the pressure, using Manual tubing. micropipette rubber the through ml/ to cone 0.42 growth applied 20–30 of the adjoining manually rate at the highest flow over was P-97 a away that tapered to 10–20 application and set drug Instruments within in chamber growth resulted flow positioned This the 10-ml Sutter min. a were at within a tips cone application growth micropipette Precision focal on the maximize (World pulled cone, To micropipettes puller. FL) Flaming/Brown glass Sarasota, TW100F-4 Instruments; into back-loaded h eladfrhvn omlyruddcl bodies. cell rounded in normally dextran having fluorescent for of and presence successfully cell the then injection was the by h, After screened This 2 min. were for 30–45 tubing. neurons recovered for were injected rubber dishes fluid bodies, of with cell flow neuronal holder steady into a needle supply to the sufficient to pushing connected manually by syringe as started micropipettes was same the Flow into above. back-loaded were Antibodies plating. after MO ro oisadto oteclueds,sokbebsai was blebbistatin stock dish, culture the to addition its to Prior DMSO. three rinsed then min (Harlan 30 MA) factor for Tewksbury, dH poly-ornithine growth sterile (Corning; with 0.01% dishes times nerve with culture treated 7S (Lamoureux Polystyrene were supplement 2010a). ng/ml growth 25 al., N9 et and serum, IN) Indianapolis, calf Bioproducts, fetal 10% r-amdaddltd60fl nsplmne -5medium, L-15 37 supplemented at incubated in then min, 660-fold 5 for diluted sonicated and pre-warmed ˚ o 0mn h oeua egto eta a hsns st be to as so chosen was dextran of weight molecular The min. 10 for C 2 ora fCl cec 21)17 5330 doi:10.1242/jcs.152611 3593–3602 127, (2014) Science Cell of Journal -lbittn(im;S oi,M)wsmd pt 0m in mM 50 to up made was MO) Louis, St (Sigma; )-blebbistatin 2 O. ˚ ˚ ni s.Sltoswere Solutions use. until C m o tlat3 i.Final min. 30 least at for C m fteao.Pesr was Pressure axon. the of m /lsrpoyi sulfate, streptomycin g/ml , co i noa10-ml a into air of cc 2 m nPScontaining PBS in M m /lFITC–dextran g/ml m fthe of m , 8h 18 m g M. at

Journal of Cell Science iohnra(only and kinesin mitochondria al., by driven et transport (360–3600 fast Roossien of fast- dynein 2006; velocity as the Sheetz, because between performed and brief, In (Miller was Distinguishing 2013). direction. previously mitochondria detail cone. each in docked growth in described and counted the mitochondria their were as from transported well axon as distance measured the were initial mitochondrion of docked images that each point mitochondria for of Velocities phase midway number the the flux, and transport pass fast acquisitions, For s ImageJ. 30 in captured time-lapse stacks every were mitochondrial captured images other min. 1 were every phase all were captured images images whereas For fluorescent fluorescent intervals, min. 5 imaging, 10-s transport every except at 250- fast ms, required 100 2010). For which captured to treatments, al., exposures. CilD set et micro-manager and ms were blebbistatin (Edelstein images combined using Health) Instruments; the mitochondrial for of controlled for (Sutter times Institutes 10-C were Exposure National Lambda (US Acquisitions MitoTracker. a software CA). with of controlled Novato, light controller Fluorescent VMM-D3 was visualization NY). Rochester, a exposure Associates; with (Vincent cube for shutter controlled ET CS25 was and 49008 VT) exposure TexasRed light a Transmitted attenuated Rockingham, through lamp xenon filters (Chroma; 100-W neutral-density a with with 98% illuminated were Cells 40 objective. L Plan MRs(nirgn albd A nL1 o i troom at min 37 5 at h for 1 for L-15 L-15 in fresh in 0.1 CA) recovered with and Carlsbad, temperature labeled (Invitrogen; were CMXRos neurons sensory Chick imaging Mitochondrial ARTICLE RESEARCH rt h aucit ...dvlpdtemdlpeetdi i.6. Fig. in K.E.M. presented and model D.H.R. the data. developed other D.H.R. K.E.M. all 5. manuscript. analyzed Fig. the and in wrote experiments presented other data all the performed analyzed and experiments performed P.L. contributions Author interests. competing no declare authors The interests Competing the on comments helpful for MI) Heidemann Lansing, manuscript. Steve East and University, MI) State Flint, University, (Michigan (Kettering O’Toole Matthew thank We Acknowledgements 40 at intervals 100 observed 20-s was of period with addition baseline focus begun the and of plane stabilizing was before growth better 30-min a the imaging A achieve attachment, time-lapse magnification. to After dish substrate the the needle. the near and off the down lifted to brought and was growth attachment needle cone The coated firm min, min. the 30 30 allow to for for to attached solution MO) A then concanavalin was Louis, been mg/ml cone St 1 has were (Sigma; a towing micropipettes cones by poly-ornithine brief, followed of growth In 0.01% 2011). of calibration with al., micromanipulation coated and et the (Lamoureux preparation previously as given the well above. of as described needles, as description imaging detailed for maintained A and prepared were Cultures assay Tension utr ihswr edi rncbtr Hieane l,20)stto set 2003) al., et (Heidemann ‘ringcubator’ a 38 in held were dishes culture hs w ouain r aiydsenbe(e yorpsin kymographs (see discernable easily 100 conservative, are be To 1C,D). populations Fig. two these h xeiets l eso hne r trbtbet h cellular the to attributable are Pasteur changes long tension a all with through response. unaltered so manually maintained experiment dish was position the the needle to reference The added pipette. and above described atda n ieatrdu diinwsecue rmanalysis. from excluded was addition that drug after transport into time fast any fast-transported undergoing at converting halted mitochondrion CilD any of mitochondria, effects docked To confounding population. docked any the avoid from velocities mitochondrial excluding for off o iohnra nlss yorpswr rdcdfo time-lapse from produced were kymographs analysis, mitochondrial For ˚ naLiaD R netdmcocp n bevdwt nN an with observed and microscope inverted IRB DM Leica a on C 6 05 orh iha dutbecla infinity/0-2/c collar adjustable an with corrPh2 /0.55 m , /)i usatal ihrta hto docked of that than higher substantially is m/h) 25–30 m m / ntehgetprin fteaxon) the of portions highest the in m/h iibei.Cl a rprdas prepared was CilD ciliobrevin. M m / n bv a sda cut- a as used was above and m/h m iorce Red MitoTracker M ˚ .Frimaging, For C. 6 unwk,E .adSak,M. Srayko, and M. E. Gusnowski, e . rni,F,Mes .A,Y,W,Bak .M n as .W. P. Baas, and M. M. Black, W., Yu, A., K. Myers, F., Francis, Y., He, B. R. Vallee, and J. D. Goldberg, M., Bennecib, E., G. G. Seale, G. W., P. Gundersen, Grabham, and S. Jani, R., E. Gomes, W. D. Burmeister, and A. J. L. Setton, D. and Goldberg, F. Guilak, W., S. Witvoet-Braam, L., C. Gilchrist, G. Gallo, enl . ulra,P .adMl,F. Melo, and L. A. P. Pullarkat, S. R., Voytik-Harbin, Bernal, and E. Nauman, J., B. Bell, J. F. Ahmad, and and M. W. M. P. Black, Baas, F., Francis, P., T. Hasaka, A., K. Myers, Y., He, J., F. Ahmad, W. P. Baas, and M. Greaser, A., Hyman, T., Wittmann, J., Hughey, J., F. Ahmad, W. P. Baas, and B. R. Vallee, J., C. Echeverri, J., F. Ahmad, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.152611/-/DC1 online available material Supplementary material Supplementary [grant Foundation Science National K.E.M. the to from IOS_0951019] funding number by supported work This Funding uaa . aaae . oiae . aaaa . aaa . uoa S., Kuroda, M., Yamaga, M., Nakagawa, J., Noritake, T., Watanabe, M., Fukata, D., L. Langston, K., Barlan, M., Maldonado, S., B., J. C. Weinger, O’Connell, J., T., A. K. Firestone, Vaughan, Y., C. Tai, L., D. Dujardin, E., N. Faulkner, hd,S .adHlebc,P J. P. Hollenbeck, and R. S. Chada, T. Stearns, and L. J. Carminati, dlti,A,Aoa,N,Hoe,K,Vl,R n tumn N. Stuurman, and R. Vale, K., Hoover, N., Gundersen, R., Amodaj, E. A., Gomes, Edelstein, A., S. Stehman, E., L. Barnhart, L., D. Dujardin, hn,S,Rdoo,V . oiy .G n oo,S V. S. Popov, and G. G. Borisy, I., V. Rodionov, S., Chang, iko,B J. B. Dickson, A. Desai, and M. I. Cheeseman, et .W n ete,F B. F. Gertler, and W. E. S. Chien, Dent, and C. J. Lasheras, S., Y. Li, N., G. Norwich, C., J. Alamo, S. del Halpain, and W. Steffen, P., Nalbant, L., Dehmelt, 5823-5834. oeo yolsi yeni h xnltasoto irtblsand microtubules of transport axonal the in dynein cytoplasmic neurofilaments. of Role positioning. spindle for implications cortex: cell Biol. the at gliding advance outgrowth. microtubule microtubule axonal for fast and required remodeling MTOC are cone LIS1 establishes growth and flow during dynein actin Cytoplasmic (2007). and myosin, cells. MRCK, migrating in Cdc42, polarization video-enhanced by using regulated culture in axons microscopy. Aplysia 1931. contrast of texture interference growth contrast-differential with of substrates observations deformable on strain intracellular correlation. of Measurement vitro. in nlsso iseeuvlnsuigacso-eindbailtsigdevice. testing biaxial custom-designed a using J. equivalents Biophys. tissue of analysis retraction. and elongation axonal 244-249. of regulator a as proteins microtubules. axonal W. P. and Baas, microtubules between interactions axon. force the in regulate microfilaments proteins Motor (2000). axon. the into microtubules of transport Biol. the Cell for J. required are dynactin and dynein asua . wmtu . ee,F n abci K. Kaibuchi, CLIP-170. and and IQGAP1 F. through Perez, microtubules capture A., Cdc42 Iwamatsu, Y., Matsuura, K. J. dynein. Chen, cytoplasmic and motor ATPase M. 125-129. AAA+ T. the Kapoor, of I., inhibitors V. molecule Gelfand, M., O’Donnell, function. dynein cytoplasmic B. and R. mitosis Vallee, and Y. Wang, es hog yendpnetitrcin ihtecl cortex. cell the with interactions 138 dynein-dependent through yeast Lett. Rev. Phys. optrcnrlo irsoe sn microManager. 14 using Chapter microscopes of control Computer B. R. movement. cell Vallee, directed and G. G. 1959-1964. euae oiiyaddcigo xnlmitochondria. axonal of docking and motility regulates unvro irtblsi rgnuosdpn ntepteno xnlgrowth. axonal of pattern the on Neurosci. depend neurons J. frog in microtubules of turnover rwhcn oiiyadao guidance. axon and motility cone growth directiona cells. and endothelial rheology Anisotropic for Implications protrusions: cell local initiation. induces neurite force dynein-dependent based, interface. kinetochore-microtubule 629-641. , 194 ora fCl cec 21)17 5330 doi:10.1242/jcs.152611 3593–3602 127, (2014) Science Cell of Journal 20) ysnI ciiyi eurdfrsvrn-nue xnretraction axon severing-induced for required is activity II Myosin (2004). x.Neurol. Exp. 377-386. , Unit14.20. , .Biomech. 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Journal of Cell Science i,S . do,K . eoreu .C n oiy .G. G. Borisy, and C. P. Letourneau, J., K. Edson, S., S. Lim, M. D. Suter, and C. A. Lee, C., C. Hoogenraad, C., Wyman, M., Modesti, Y., Komarova, G., Lansbergen, F. Nedelec, and M. Srayko, C., Kozlowski, oih . ek .adZprk,S. Zipursky, and A. Berk, H., Lodish, E. K. Miller, and S. E. Heidemann, P., K. Lamoureux, Miller, and R. E. S. K. Heidemann, Miller, R., M. and O’Toole, R. L., P. N. Lamoureux, Martzke, R., S. Heidemann, P., Lamoureux, Bader, and A. D. Lee, M., A. Sharma, E., Kimmel, Z., Bomzon, M., M. Knight, M. Kikkawa, B. H. Koeller, S., S. Kholmanskikh, ilr .E n hez .P. M. Sheetz, P. and E. M. K. Miller, Sheetz, and E. K. Miller, ag .S,Ta,J . a,P . ad . i . eg .adSeg .H. Z. Sheng, and C. Deng, C., Li, P., Zald, Y., P. Pan, H., J. Tian, S., J. Kang, yr,K . it . aa,C . e . lc,M .adBa,P W. P. Baas, and M. M. Black, Y., He, V., C. Nadar, I., Tint, A., K. Myers, J. P. Hollenbeck, and L. R. Morris, L. Dehmelt, and O. 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