col 02 anvr Germany. Hannover, 30625 School, xeietlBooy oihAaeyo cecs atua3 203Warsaw, 02-093 3, Pasteura Sciences, of Poland. Academy Polish Biology, Experimental xeietlBooy oihAaeyo cecs atua3 203Warsaw, 02-093 3, Pasteura Sciences, of Poland. Academy Polish Biology, Experimental naSkupien Anna Golgi the tyrosine of Src positioning through kinase-dependent morphogenesis regulates CD44 ARTICLE RESEARCH ß 5038 2014 September 5 Accepted 2014; April 4 Received the molecular is the 1 and environment receptors extracellular Its the (ECM). 2010). of Jan, matrix and part extracellular Jan some 2008; important and al., receptors An et Firestein, their (Urbanska signals proteins, and molecules secreted extrinsic adhesion Kulkarni by to mediated 2010; responsive are Jan, are that and Developing Jan 2012). (Kaufmann 2000; autism syndrome, Moser, and X and syndrome fragile Rett’s syndrome, The syndrome, Down’s Angelman’s 2013). schizophrenia, disorders, and neurodevelopmental as Koleske, and are such psychiatric capabilities (Segev 2005; various functional their in al., integrated and altered trees et are dendritic of Gulledge complexity 2000; individual that London, signals from how determining come implications, have functional and subtypes important neuronal define patterns arborization Dendritic INTRODUCTION Dendritic matrix kinase, Extracellular Src arborization, fragmentation, Golgi CD44, WORDS: KEY both by for in complexity role disease. arbor induced and novel dendritic of health a regulator shortening essential indicate an results as dendritic CD44 our Overall, prevents exposure. of glutamate silencing expression the which during on CD44 conditions, influence toxic protective under a brain trees have dendritic normal might of inhibition during alteration its operates but the development, to mechanism leading The kinase, morphology. tyrosine Golgi Src of activation dendritic the in is by role receptor, inhibitory cellular crucial hyaluronan arborization a its tree main plays and that and the dendrites report in molecule, matrix We localized adhesion enigmatic. extracellular remained CD44 functional has the the a process this of of in formation role receptors the aspect The crucial towards a network. development is morphology neuronal dendrite of proper of acquisition The ABSTRACT erbooy nentoa nttt fMlclradCl ilg,Todn ,02- 4, Trojdena Poland. Biology, Warsaw, Cell and 190 Molecular of Institute International Neurobiology, Ato o orsodne([email protected]) correspondence Poland. for Warsaw, *Author 02-093 3, Pasteura Sciences, of Academy Polish ayd Babraj Matylda reozM Wilczynski M. Grzegorz aoaoyo oeua n ytmcNuoopooy eciIsiueof Institute Nencki Neuromorphology, Systemic and Molecular of Laboratory 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,53–01doi:10.1242/jcs.154542 5038–5051 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 5 2 aoaoyo elBohsc,Nnk nttt fEprmna Biology, Experimental of Institute Nencki Biophysics, Cell of Laboratory ellrNuohsooy etro hsooy anvrMedical Hannover Physiology, of Center Neurophysiology, Cellular nvitro in 1 4 1 naKonopka Anna , aoaoyo erbooy eciIsiueof Institute Nencki Neurobiology, of Laboratory uetDolezyczek Hubert , and nvivo in 1 3 aoaoyo oeua n Cellular and Molecular of Laboratory n onaDzwonek Joanna and hsnvlfnto sexerted is function novel This . 1 ae Trzaskoma PaweI , 1 zbl Figiel Izabela , 1, * 4 1 veiPonimaskin Evgeni , oehn Labus Josephine , huh opa e oei h euaino uo cell tumor of regulation the in is role binding key CD44–hyaluronan a comes which play function in CD44 to describes studies, thought that cancer literature a the 2003). from al., cell– of CD44, et bulk (Ponta and is signaling The and cell–cell hyaluronan migration cell mediates for adhesion, matrix that receptor and glycoprotein major (Novak multifunctional The glycosaminoglycans 2000). of Kaye, polymer linear ubiquitous neuronal poorly still to are processes ECM morphogenetic understood. the in involved from are that transduction cells signal of mechanisms endsrbdt nii lsammrn Ca membrane plasma has 2007; inhibit al., sensory to et in Matzke described CD44 (Sherman Recently, 2002; assembly been 2009). al., molecule al., et et signaling Orian-Rousseau Gorlewicz for 2000; site al., a kinases et tyrosine as receptor for serve co-receptor CD44 al., a Additionally, and as 2007). et act al., to et (Sretavan suggested Ries was 2003; development Chan, and neuronal Lin 1994; during in involved guidance be might CD44 that indicates evidence 2013). granule some although al., and et 2007) control (Naruse al., 2009). respiratory cerebellum et adult central al., (Matzke the the stem in et neurons in brain and (Glezer the found within striatum, cortical been forebrain system the has the hypothalamic, it in of Moreover, certain neurons Recently, regions in and 1997). hippocampal detected al., amygdala been et al., extended has et (Jones (Kaaijk (Sretavan mRNA motoneurons sections neurons CD44 facial brain chiasm neuronal and optic human embryonic the 1994) in 1997), demonstrated molecule al., However, et this 2009). have 2000; al., of al., reports et et expression Sherman Gorlewicz subsequent 1993; 2002; al., several 1991; al., et al., was et Moretto et brain 1992; (Girgrah Bouvier-Labit cells al., the glial et in in Vogel exclusively expression occur CD44 to in reported Originally, CD44 (CNS). of system action established. of well not By mechanisms are behavior. and cells cell neuronal that function changes molecule non- the and key various contrast, in influences cascades a cell, studies the signaling as into intracellular regarded on ECM the be from Based signals can transduces 2008). link CD44 (Martin a Bourguignon, cells, cytoskeleton provides neuronal 2003; actin molecule and al., The membrane et plasma 2003). the al., between et (Ponta migration Mtk ta. 07.I ocuin lhuhteaon fdata of amount the although conclusion, excitation In synaptic 2007). al., glutamatergic et reduce (Matzke CD44 to of family lack shown The also Src 2011). al., of et pre-Bo Ghosh in activation Fyn; Lck, the (SFKs, kinases through (PMCA) triphosphatase h ancmoeto h C ntebani ylrnn a hyaluronan, is brain the in ECM the of component main The h ernlfnto fC4 ean ob elucidated, be to remains CD44 of function neuronal The nervous central and peripheral the both in expressed is CD44 tigrcmlxitrern ihntebanse was stem brain the within complex ¨tzinger 2 dmGorlewicz Adam , 2 au Wlodarczyk Jakub , 1 uazSwiech Lukasz , 5 ae Jaworski Jacek , 2+ adenosine nvivo in 3 , 3 ,

Journal of Cell Science hsooia oe fC4 nnuos eeaie the examined we neurons, CD44 of in knockdown vivo CD44 neuronal of the of consequences into site insights major roles further a gain as physiological To dendrites localization. uncover fine-structural and its brain of postnatal in CD44 of comprehensive. being its from of understanding far our is increasing, function is function CD44 neuronal on ARTICLE RESEARCH odprilsadteecmatet oprdwt an with compared dendritic compartments or (dendrites: these distribution profiles the random and between expected dendritic of association particles in significant analysis or a gold particles confirmed quantitative processes membranes gold plasma The dendritic of areas). other number various (yellow to as apposed very processes closely detected association such sites was in particles at structures, and membrane radiatum gold dendritic areas) the stratum (blue 10-nm with dendrites CA3 with within predominantly the 1C), of (Fig. labeling in extracellular characteristic immunogold of expression pattern ultrastructural or used The hippocampus. CD44 we rat P10 glial protein, the of sections the neuronal, of localization reflects immunoreactivity and cortex, on the peaked in 1B). 10–20 days, (Fig. days postnatal decreased and hippocampus then development first the the in during 7–10 on of days expression points increased the that protein time confirmed P60] CD44 that to different (E18) cortices 18 day cerebral at [embryonic and collected hippocampi rat were of from analysis extracts protein blot acidic protein Western fibrillary a protrusions. glial with in astrocytic colocalize neurons arranged (GFAP)-positive to appear was MAP-2-positive not signal did around but CD44 pattern The honeycomb 1A). (Fig. distinctive on onwards observed reduction 14 after a with days day (P0–P10)], first during 0–10 the day expression during [postnatal expression birth its hippocampal highest the investigated in showed we sections Immunohistochemistry CD44 brain, of development. rat pattern postnatal expression the temporal in and protein spatial the specify To neurons pyramidal of development dendrites throughout in been present is CD44 have arborization RESULTS tree in dendritic disorders neurological in CD44 observed. in target alterations Therefore, therapeutic exposure. dendritic novel which a prevent glutamate be to by might acting that induced conditions, neurons subtoxic neuronal young shortening actin to on of effect exposed development on protective are silencing early significant a depend the the exerts during that cells morphology expression show CD44 we Golgi of Moreover, in dynamics. downstream cytoskeleton a changes kinase the as Additionally, Src cascade. acts observed signaling of CD44-induced kinase mutant the of this active effector that by constitutively blocked demonstrating a are (Y527F), trees of dendritic decreased expression the and on Golgi is the knockdown the CD44 (FAK), both of of kinase and effects morphology c-Src The adhesion both knockdown. of focal CD44 activation upon substrate, the and main brain, interacts the its CD44 in that kinase dendritic found Src mediating We with for maintenance. essential and is growth that polarity, organelle alteration an structural Golgi, in neurons the results of CD44 hippocampal of in knockdown the arbors Moreover, dendritic of cultured complexity the in ntepeetsuy edsrb h eeomna regulation developmental the describe we study, present the In odtriewehrteC4 inlta urud MAP-2 surrounds that signal CD44 the whether determine To u eut hwta h oso D4rslsi nincrease an in results CD44 of loss the that show results Our . nvitro in n otclnuoselectroporated neurons cortical and x 2 5 14.75, nvitro in P , nvivo in 0.0005; and in . yohsz bu h osberl fC4 ndendritic in CD44 of role possible the arborization. to us about led molecule the hypothesize of localization the dendritic the in and expression forebrain CD44 rat postnatal of timecourse characteristic The edii membranes: dendritic agdwt re loecn rti GP n ihrempty The either vectors. and shRNA rCD44 (GFP) CD44 with or protein days (control) fluorescent 3 pSuper for green co-transfected with were tagged (HEK- cells 293 The kidney cells. embryonic human 293) of in short- overexpression (rCD44) the cDNA using against CD44 rat shRNAs knockdown different the neurons two tested of first hippocampal efficiency We constructs. primary (shRNA) downregulated RNA in hairpin we CD44, expression of function its neuronal neurons the mammalian determine in To patterning dendrite regulates CD44 o infcnl ifrfo h admdsrbto (dendrites: distribution random the gold from x of differ distribution significantly did membranes the dendritic not at case, or profiles this dendritic the within In particles (IgG). nonimmune with G replaced immunoglobulin was antibody primary control, negative Fg B dniidtems oetsRA(16) which (#1963), shRNA potent most the expression transfection identified protein of the 2B) densitometry Immunoblotting quantitative (Fig. in efficiency. the and transfection 2A) included for (Fig. control additionally a as was mixture vector coding el.Dy6 subsequent Day the cells. in fusion used CD44–GFP was rat molecule the experiments. particular of This knockdown protein. effective an caused h pcfckokono D4rte hnfo off-target from than rather CD44- CD44 that of from effects. knockdown indicate resulted specific data morphology knockdown- dendritic the These the in rCD44 of 3). changes reversal (Fig. shRNA-induced at a for phenotype CD44 and of 2D) induced re-expression (Fig. region the in levels were could resulted CD44Rescue endogenous neurons of coding that in co-expression mutations shRNA The mRNA shRNA. with silent cDNA into by recognized transcribed be which not was the in which We (CD44Rescue), into construct experiments. DNA rescue introduced performed a we 3D), created neurons, (Fig. in control trees. dendritic phenotype pSuper of the complexity the Sholl with in compared were increase The upward an plot an 3B,C). indicating that the revealed (Fig. knockdown of neurons CD44 plasmid shift upon pSuper with neurons of of the analysis shRNA compared number total with (TNDTs) CD44 and transfected (TDL) tips with length 3A. dendritic dendritic Fig. transfection total the in transfection Specifically, increased that presented morphology arbor revealed 3B–D). of are complexity (Fig. that cells the increased neurons groups the shRNA CD44 of of with of all Tracings peak analysis later. of Morphometric days the 5 representative at them are shRNA/GFP fixed cells CD44 and or the vectors pSuper/GFP transfected with (DIV7) we dendritogenesis morphology, of level arbor the in decreased shown As significantly CD44. neuronal compared. shRNA and endogenous was CD44 immunofluorescence the immunostaining 2C,D, by Fig. post-transfection, of for detected days intensity mixture 5 was the transfection At CD44 the cells. endogenous to transfected that added of plasmid The identification was plasmids. GFP shRNA CD44 encoded with or pSuper empty 2 5 ete etdteefcc fti hN nclue neuronal cultured in shRNA this of efficacy the tested then We ocnimteseiiiyo h bevdCD44-knockdown observed the of specificity the confirm To dendritic on downregulation CD44 of effect the evaluate To 0.038, ora fCl cec 21)17 0855 doi:10.1242/jcs.154542 5038–5051 127, (2014) Science Cell of Journal P . nvitro in .5 edii membranes: dendritic 0.05; DV)nuoswr rnfce ihan with transfected were neurons (DIV6) x 2 5 220.75, P , 6.06 x 6 2 5 10 0.025, b 2 -galactosidase- 50 .Frthe For ). P . 0.05). 5039

Journal of Cell Science EERHARTICLE RESEARCH 5040 1. Fig. e etpg o legend. for page next See ora fCl cec 21)17 0855 doi:10.1242/jcs.154542 5038–5051 127, (2014) Science Cell of Journal

Journal of Cell Science muoeciiyta r rsn ndnrts(,bu vras, overlays), blue (D, dendrites CD44 in indicating present of particles are hippocampus immunogold that the out immunoreactivity of point region Arrows CA3 rat. the 10-day-old immunogold in microscopic CD44 rat Electron of developing (C) detection the cortex. in cerebral 10 expression and bar: CD44 hippocampus Scale of (white). analysis DAPI Immunoblot (green), and (B) anti-CD44 (blue) with of anti-GFAP region immunostained (red), CA3 P0–P60, anti-MAP-2 the at of hippocampus rat images rat Confocal the the (A) of cortex. development cerebral postnatal and during regulated. hippocampus developmentally expression is protein cells CD44 neuronal in (A,B) expression CD44 1. Fig. ARTICLE RESEARCH nml e ru.Saebr 0 nm. 500 overlays). bar: magneta Scale (S, group. spines per dendritic animals and overlays) yellow (AC, aa edie Fg G,tema egho aa dendrites basal of that found length We 4I). (Fig. dendrites mean basal of of the number length the the and total 4E), 4H) 4G), (Fig. the (Fig. (Fig. 4F), length dendrites (Fig. branches dendrite basal dendrite apical apical dendrite of are 4D), apical number Fig. (i.e. there dendrites branches; apical whether of with separately length we examine total dendrites, the basal quantified versus To apical on 4B,C). effects (Fig. differential neurons of control those with shRNA-transfected compared arbors GFP-positive dendritic extended exhibited that neurons TDL revealed The analysis V. Sholl layer and cortical performed from was neurons analysis pyramidal morphometric on The exclusively 4A). (Fig. to cortex performed the neurons in was labeled sections sparsely detected the We cytoarchitectonics. of visualize staining Nissl on Fluorescent analyzed and andP21. collected pups rat were vector) newborn Brains electroporation. of GFP-coding performed cortex cerebral the [ with into agent (TCHD)] permeabilizing together 1,2-diol nuclear shRNA, a DNA in plasmid CD44 solubilized the introduced postmitotic or We 2013). into al., (pSuper DNA et Rossa plasmid la the (De performed neurons deliver we brain, to rat the (iPo) in iontoporation occurs that phenomenon general a is odtriewehrtefnto fC4 ndnrt patterning dendrite in CD44 of function the whether determine To trans -cyclohexane- n 5 m m. 3 u idnssgetta D4rgltsteptenn fdendrites development. in of postnatal patterning throughout the increased regulates CD44 markedly that suggest findings was our Collectively, cells. length control of dendrite that to relative apical neurons shRNA-treated not but basal ece yc-xrsino hN-eitn lsi (CD44Rescue) plasmid shRNA-resistant of the co-expression by the to rescued of Golgi Fragmentation ce the 5D). (Fig. of per CD44-shRNA-transfec area Golgi for surface the controls higher total of of the q volume of total that ratio we the with higher, Additionally, calculating compared was with cell 5B,C). expression cells (Fig. per in reduced CD44 fragments was fragments Golgi diminished of of volume of average number the number whereas increased The fragmentation an 5A). Golgi (Fig. observed suggesting structure. structures, Golgi as we GM130-positive the smaller known knockdown, CD44-shRNA-expressing assess (also CD44 to GM130 or marker After were the protein that cis-Golgi pSuper- with GOLGA2) neurons immunostained either were Hippocampal plasmids Golgi. on with knockdown the CD44 transfected of of effect morphology al., the et the examined Ori-McKenney we 2005; Therefore, al., 2012). et (Horton morphogenesis o dendrite position and structure dispersion proper Golgi The in results knockdown CD44 ifrn eltpsi ciaino h FsLk y n Src and Fyn Lck, SFKs the in of described activation action is CD44 types of cell mechanisms different molecular the of One dendritic regulate to morphology kinase tree Src through acts CD44 that parameters the did those from neurons of differ CD44Rescue-transfected significantly values in not The dispersion Golgi shRNA. describe CD44 with together ora fCl cec 21)17 0855 doi:10.1242/jcs.154542 5038–5051 127, (2014) Science Cell of Journal nt.Dt nBDso h mean the * show B,D in Data units. cells. transfected in signal (IF) immunofluorescence CD44 the of the intensity on average based estimated was knockdown CD44 of 10 bar: Scale cells. indicate transfected arrowheads The (red). antibody CD44 anti- the using immunocytochemistry to subjected with together shRNA plasmids CD44 or pSuper with transfected neurons hippocampal Primary (C) GAPDH. to normalized expression, protein of densitometry plasmids. (3) n #90 and (2) #1963 shRNA CD44 rat with co-transfected CD44–GFP, cells HEK293 of lysates down expression. knocks CD44 effectively rat shRNA CD44 2. Fig. P 5 , needn ace fcls B Quantitative (B) cells. of batches independent 3 .5 *** 0.05; n 5 5nuosprgop U arbitrary AU, group. per neurons 25 og pnC4 ncdw was knockdown CD44 upon Golgi e el oprdwt controls with compared cells ted fcnrlcls(i.5B–D). (Fig. cells control of l h ravlm aiswere ratios area:volume The ll. h og r aslylne to linked causally are Golgi the f P b atfe og ipra by dispersal Golgi uantified , glcoiaeadpue 1 or (1) pSuper and -galactosidase 0.001. A muolto cellular of Immunoblot (A) b atnGPwere -actin–GFP m .()Teeffect The (D) m. 6 s.e.m.; 5041

Journal of Cell Science EERHARTICLE RESEARCH efre sn pcfcat-Y1-r nioy n the and antibody, 5042 anti-pY416-Src specific was using Immunocytochemistry transfection. after performed days plasmids 3 shRNA CD44 at or whether fixed pSuper were with determine c-S DIV5 on of transfected To were activation that 416. the tyrosine alters kinase CD44 at c-Src each of with activity co- phosphorylation catalytic interact These The requires No brain. kinase 6A). rat c-Src developing (Fig. parallel. the and in detected. other CD44 in was that Src indicate analyzed 60-kDa findings the were of control an precipitation with isotype incubated To 6A). homogenates anti-IgG (Fig. of interactions, Src precipitation unspecific total after exclude of kDa co-precipitation 60 indicates at panel). which left band CD44, 6A, a confirmed (Fig. detected input Src we 60-kDa the Furthermore, and of CD44 performed Analysis 80-kDa of cortex. we from expression and homogenates hippocampi brain, by tissue rat using followed rat P10 6A) antibody (Fig. the immunoblot anti-CD44 in anti-c-Src an c-Src with CD44 whether in with immunoprecipitation investigate proteins To with interacts reported. interaction been the directly yet but not 2008), has al., brain et the (Bourguignon Lee cells 2001; tumor al., in CD44 et been with has directly Src interact Moreover, to 2010). shown al., In et c- 2011). (Bourguignon promotes activation al., interaction Src et hyaluronan–CD44 Ghosh the 2008; cells, al., cancer et breast Lee 1998; al., et (Ilangumaran overexpression by rescued is silencing of effect 50 the and bar: neurons, Scale hippocampal vector. of RFP-coding dendrites of CD44. elaboration rat the shRNA-resistant an stimulates of CD44 of Knockdown 3. Fig. nlsso ipcma ern fe rnfcinwt h niae lsis tdsacs25–175 ( distances significant At statistically plasmids. were indicated groups the other with the transfection and after shRNA neurons hippocampal of analysis A rcnso ersnaienuosc-rnfce ihpue,C4 hN rC4 hN/D4ecetgte with together shRNA/CD44Rescue CD44 or shRNA CD44 pSuper, with m co-transfected neurons representative of Tracings (A) .()Ttldnrtclnt TL n C oa ubro edii is(NT o ern rae si .()Sholl (D) A. in as treated neurons for (TNDT) tips dendritic of number total (C) and (TDL) length dendritic Total (B) m. c rmr ipcma neurons hippocampal primary rc, P , .0) aaso h mean the show Data 0.001). n ie hm5dy ae.N nraei edii ro size with arbor together shRNA dendritic CD44 in expressed increase that No cells later. in observed days was 5 by them together fixed vectors rescued co-transfected shRNA and CD44 We be or cells. pSuper with can the with DIV7 into neurons on kinase the neurons Src hippocampal of encodes active We in introducing elaboration Y527F). that trees CD44-shRNA-induced (CASrc, plasmid kinase dendritic the Src a whether of determined applied form Src active we by constitutively driven also activation, is herein kinase described morphology tree significantly dendritic was neurons 6D,E). (Fig. FAK expression CD44-shRNA-transfected CD44 phosphorylated diminished with of cells and in level decreased The we control 6D). FAK, (Fig. in anti-pY576-FAK of specific phosphorylation antibody using Src-dependent immunocytochemistry the performed alters whether determine CD44 To 2003). al., et maximal (Hanks activation knockdown promotes catalytic Tyr577 FAK and Tyr576 Src-mediated CD44 The on 2005). FAK al., of et of phosphorylation the (Mitra 6B). One FAK in (Fig. is 6B,C). substrates Src (Fig. and key Src the membrane neurons phosphorylated the of amount control to the diminished close was in c-Src cytoplasm of form that showed active results the Our quantified. was intensity immunostaining odtriewehrtefnto fC4 nrgltn the regulating in CD44 of function the whether determine To b atnGPadacntttvl ciemtn fSckinase Src of mutant active constitutively a and -actin–GFP 6 ora fCl cec 21)17 0855 doi:10.1242/jcs.154542 5038–5051 127, (2014) Science Cell of Journal ...( s.e.m. n 5 m rmtecl oy h ifrne ewe CD44 between differences the body, cell the from m 0nuosprgop;*** group); per neurons 50 P , 0.001.

Journal of Cell Science 0nuos ih3aiasprgop nteShl nlss h ifrne ewe D4sRAadpue eesaitclysgiiat( significant statistically were pSuper and perfo shRNA was CD44 analysis between Morphometric differences cells. the transfected of analysis, b dendrites Scholl dendrite basal the apical of of In number number at group. (I) (F) shown per and 80–180 length, are dendrites animals dendrite distances apical basal white 3 (E) of in with branches), length outlined neurons, with mean dendrite regions 20 (H) (apical The dendrites, dendrites presented. basal apical of of are length length neurons total cortical (D) 100 of analysis, bars: Sholl images Scale (C) Representative indicated. (A) as P21. magnification on higher sacrificed and plasmids shRNA/GFP EERHARTICLE RESEARCH oeaut hte h feto D4slnigo Golgi on silencing activity, CD44 kinase Src of of inhibition the effect on depends the also morphology whether evaluate tree To polymerization dendritic actin Src and on of activity depends dispersion regulation Golgi CD44-knockdown-induced the in CD44 acts Src morphology. that of indicate results These downstream 6F–I). (Fig. mutant CASrc the morphology dendritic proper of development the regulates CD44 4. Fig. m rmtecl oy aaso h mean the show Data body. cell the from m m mdl ae) 50 panel), (middle m 6 ... * s.e.m.; P m , rgtpnl.As hw sqatttv nlsso B oa edii eghand length dendritic total (B) of analysis quantitative is shown Also panel). (right m .5 ** 0.05; nvivo in ifrsgiiatyfo hto oto el tasetdwith (transfected by cells control the rescued of of that ratio from was not area:volume significantly did kinase cells and differ shRNA/CASrc-transfected Src shRNA CD44 number in of Golgi fragments volume, form Golgi of CD44 GM130- The active 7A). effect constitutively (Fig. by the the The CD44 of plasmids. overexpression caused with of (Y527F) co-transfected CASrc dispersion analysis cells and in shRNA morphometric organelle immunolabeled performed we P . , a uswr electroporated were pups Rat .1 s o significant. not ns, 0.01; ora fCl cec 21)17 0855 doi:10.1242/jcs.154542 5038–5051 127, (2014) Science Cell of Journal nvivo in nP ihpue/F rCD44 or pSuper/GFP with P0 on ace,()total (G) ranches, P , .1 at 0.01) mdwith rmed 5043

Journal of Cell Science ipra fteGliadcntttsaptnilmcaimby CD44-knockdown-induced mechanism morphology. potential Golgi a regulates for activity constitutes actin Src and fragmentation which Therefore, Golgi required the 7A–D). Golgi of (Fig. dispersal is that blocked knockdown revealed CD44 B polymerization Golgi by latrunculin the induced GM130-specific with of with analysis treatment immunostained cells and the Morphometric later, DIV7 antibody. hours on (2.5 24 fixed B At were latrunculin DIV5. with on with incubated by shRNA were transfected CD44 reversed were or Neurons be pSuper polymerization. can actin fragmentation inhibiting Golgi knockdown-induced EERHARTICLE RESEARCH euto ndnrtcotrwhwtotnuoa eldeath cell neuronal 5044 without outgrowth a dendritic to low lead a in to shown with been reduction cultures previously has neuronal glutamate pyramidal of concentration young of treatment The outgrowth of dendritic reduction glutamate-evoked hippocampal from young neurons protects partially silencing CD44 morphology Golgi an of play maintenance dynamics the cytoskeleton in Actin (La 2005). role proteins al., important actin-binding et of phosphorylation (Mitra the through morphology. structure Golgi acts neuronal Src regulating that in indicate CD44 results of These 7B–D). downstream Fig. GFP; and pSuper r n A oma ciecmlxta euae actin ´ regulates that complex active an form FAK and Src zaro-Die ´ uze l,20) etse hte CD44- whether tested We 2006). al., et guez m )fr2 or.Cells hours. 24 for M) espoone rwhihbto fdnrtsfollowing with CD44 kinase. co-transfected dendrites Src were of cells active effect the constitutively of when protective observed not the was inhibition shRNA Moreover, plot in growth treatment. the resulted the CD44 glutamate in pronounced of decrease of silencing upon a The less shift trees. neurons indicating dendritic 8C), glutamate downward of of (Fig. complexity controls a were by analysis with revealed Sholl 8A) compared reduced (Fig. exposure The pSuper- significantly trees glutamate 8B,D). of (Fig. were dendritic TNDT treatment and cells and TDL cells DIV5, transfected The The 1988). analyzed. on al., morphometrically (50 et fixed (Mattson glutamate after previously to were described hours exposed as 2 days were At either 3 cells plasmids. with the shRNA/GFP transfected transfection, 2 of CD44 were day or role neurons On glutamate- pSuper/GFP possible pyramidal shortening. of dendrite seeding, the model preventing after examine this in to knockdown applied CD44 regression We dendritic 1988). induced al., et (Mattson oe hsooia ucino D4–ti rti ly role a plays protein this – CD44 of function CD44 physiological Using expression novel brain. the temporal rat developing of vitro the postnatally in localization the document in dendritic molecule we adhesion ultrastructural study, and present pattern the In DISCUSSION ora fCl cec 21)17 0855 doi:10.1242/jcs.154542 5038–5051 127, (2014) Science Cell of Journal and nvivo in ncdw prahs eas dniya identify also we approaches, knockdown h mean show the each Data performed. for cells were 40 condition of experimental Golgi the of CD44Rescue Analyses or plasmids. shRNA transfected cells CD44 of pSuper, Golgi surface with the total of the volume total of to ratio area The of (D) number fragments. the Golgi Golgi of the Quantification of (C) volume mean fragments. the 10 of bar: Quantification Scale (B) cell. transfected a Arrowheads (red). indicate CD44 and Golgi (blue) the GM130 visualize marker to immunostained the were fixation, cells After vector. plasmids GFP-coding shRNA with CD44 together or pSuper with transfected neurons hippocampal cultured of GM130-labeled reconstructions three-dimensional Golgi and neurons in Golgi knockdown. CD44 the following of Dispersion 5. Fig. 6 ... ** s.e.m.; P , .1 *** 0.01; A ofclimages Confocal (A) P , 0.001. m m m. )for m)

Journal of Cell Science EERHARTICLE RESEARCH i.6. Fig. e etpg o legend. for page next See ora fCl cec 21)17 0855 doi:10.1242/jcs.154542 5038–5051 127, (2014) Science Cell of Journal 5045

Journal of Cell Science ** rnfce ihpue rC4 hN lsistgte with together plasmids shRNA neurons CD44 representative hippocampal or are pSuper Primary with data (B–E) transfected The experiments. blotting. independent for western multiple probed by of then Src were and protein Samples CD44 of control). precipitated to mg (negative specific 1 antibody anti-IgG of an or with total CD44 (IP) A immunoprecipitation (Input). to were subjected expression was cortex Src extract and and hippocampi CD44 rat for P10 analyzed of fraction) its (membrane altering extracts and kinase of Src trees with dendritic activity. interacting of by morphology neurons the hippocampal regulates CD44 6. Fig. ARTICLE RESEARCH Y2F lsistgte ihGPcdn etr rC4 hN-rnfce el rae ihltuclnB(a ) cl a:10 bar: 5046 Scale B). (lat B latrunculin with polymerization. shRNA/CASrc treated actin CD44 cells of and shRNA-transfected inhibition shRNA CD44 and CD44 or kinase pSuper, vector- Src with of GFP-coding transfected activation with neurons the together hippocampal by plasmids cultured prevented (Y527F) GM130-labeled is of fragmentation reconstructions Golgi three-dimensional knockdown-induced CD44 7. Fig. CD44 of upon herein reduction observed effects glutamate-induced the of All the outgrowth. that dendrite decreases that demonstrate suggesting silencing we Finally, Golgi, CD44 positioning. modulating the and by morphology development of the Golgi arbor dendritic structure restricting demonstrate regulate the might clearly by CD44 defines results morphology Our CD44 arbors. neuronal that dendritic of of regulation elaboration the in mean the were show groups data other quantitative the All and shRNA significant. CD44 statistically between differences F. the in indicated body, as the treated with transfection neurons after for neurons plasmids. (TNDT) hippocampal of tips analysis dendritic Sholl of (I) number total 50 (H) bar: and together Scale plasmids vector. pSuper/CASrc GFP-coding units. CD44 or with arbitrary pSuper, (Y527F) AU, with shRNA/CASrc cells. co-transfected CD44 transfected neurons shRNA, in representative p-FAK of (E) Tracings and (F) p-Src (C) of intensity cells. transfected 10 indicate bars: or Arrowheads Scale (red) antibodies. anti-pY416-Src (red) (B) anti-pY576-FAK using (D) immunocytochemistry to subjected were B h ubro og rget,()tema oueo h og rget n D h ai fttlsraeae ottlvlm fteGli Analyse Golgi. the of volume mean total to the area show surface total Data of performed. ratio were the (D) condition and experimental fragments each Golgi the per of cells volume mean 40 the of (C) Golgi fragments, Golgi of number the (B) P , .1 *** 0.01; A oimnpeiiaino noeosC4 n r.Protein Src. and CD44 endogenous of Co-immunoprecipitation (A) n 5 0nuosprgop tdsacs10–150 distances At group. per neurons 50 P , m .Qatttv nlsso muoloecne(IF) immunofluorescence of analysis Quantitative m. 0.001. m .()Ttldnrtclnt (TDL) length dendritic Total (G) m. m 6 rmtecell the from m s.e.m.; b -actin–GFP idnsta D4 h anhauoa eetr inhibits receptor, hyaluronan main our the Therefore, CD44, 2013). Giger, that and findings (Mironova the the of CNS in one molecules mammalian is regeneration-inhibiting excessive Hyaluronan and preventing growth- network. best-known neuronal wiring, the an neuronal of play proper (CELSR-3; development can in 3 mechanisms role Growth-inhibitory receptor important 2007). G-type al., seven-pass et (4) Shima and LAG (3) 2013) al., 2013), EGF et al., Lanoue cadherin (BAI3; et 3 neural (SEZ-6; inhibitor McCall the angiogenesis 6 of (PSA-NCAM; brain form gene molecule polysialylated adhesion seizure-related the proper (2) cell of 2007), with product al., interfere et a growth branching. Gunnersen to and (1) a dendritic growth include shown its Only inhibiting These on 2010). been by Jan, formation effect have and arbor dendritic Jan proteins positive 2008; such al., a few et have Urbanska in to have (reviewed dendritogenesis of shown regulators as been date to described been have that disorders for target pathology. therapeutic neuronal dendritic a proper involve be of acquisition might CD44 the that by in morphology induced involved cellular is Golgi novel activity. that a the filaments uncover mechanism findings of actin kinase our fragmentation together, of Taken tyrosine the knockdown. polymerization for the Src crucial that is on show we dependent Moreover, are knockdown rcsl eae oterplrmrhlg.Drn the During at morphology. localizes first polar Golgi the their neurons, pyramidal to of development related precisely field. this in insights new provide dendritogenesis h aoiyo xrclua rtasebaepoen that proteins transmembrane or extracellular of majority The h hp n oaiaino h og nnuosare neurons in Golgi the of localization and shape The 6 ... *** s.e.m.; ora fCl cec 21)17 0855 doi:10.1242/jcs.154542 5038–5051 127, (2014) Science Cell of Journal P , 0.001. m .Qatfcto of Quantification m. A Golgi (A) fthe of s

Journal of Cell Science EERHARTICLE RESEARCH otosbsldnrt roiaini ae otclneurons example, cortical for V NRP1), layer as in known arborization dendrite (also its basal NP1 through controls PLXNA4) co-receptor Sema3A as 2009; its known of (also al., and Signaling PlexA4 of et dendrites. selectively consisting act complex Tran basal receptor mechanisms or 2001; the of apical al., Some on 2012). et and al., (Bi apical et Srivastava the regulate dendrites. outgrowth apical to enhancing dendrite the shown of basal dendrites, been not have but mechanisms dendrites, Numerous pyramidal basal the of of types branching two CD44 the dendrite. of developing thus loss the the growth, of Interestingly, complexity microtubule exaggerated acentrosomal the can fostering efficient Golgi CD44 the more diminished of provide rearrangement to morphological with attributable We Such neurons was in expression. recently 2012). to Golgi trees the al., dendritic is of al. dispersion elaborated et outposts significant the (Ori-McKenney et that Golgi found elaboration of arbor Ori-McKenney function increasing dendritic directly thereby key However, (Sann microtubules, nucleate processes the acentrosomally 2009). dendritic that of demonstrated al., maintenance dendritic and et proper growth crucial of the is which formation have for trafficking, membrane Golgi the directional the determine through of arbors to most function proposed dendritic longest, and to morphology been the localize The into selectively points. outposts outposts branch sending The side dendrite. 2005), apical complex the al., on positioned et (de subsequently side is (Horton basal It 2005). future al., the et becomes Anda which site, emergence axon the nvivo in a ifrnilefcson effects differential had 00.I esr ern,C4 niistepam membrane al., plasma et the Bourguignon inhibits 2008; CD44 al., Ca neurons, et sensory Lee In 2001; 2010). al., 1998; al., et tumor et Ilangumaran Bourguignon 1996; including al., et types, (Taher lymphocytes cell and be cells many in initiate and reorganization CD44 to of cytoskeleton domain intracellular the with interact remain to shown herein CD44- and a described phenomenon be determined. fragmentation this can The dispersion between stimuli. dependent external Golgi relationships to cells that recentlypossible (Thayer neuronal demonstrating of activity response was neuronal 2013), physiological increased al., arborization. fragmentation upon et neurons dendrite Golgi in basal observed reversible in involved Remarkably, aforementioned pathways the with CD44 interesting molecular of be link also molecular would the It investigate Golgi. to the of function the and modulating localization by arborization dendrite that basal regulate suggest might knockdown CD44 CD44 following Golgi the of data,morphology Our the 2013). al., regulating E3A et obtained (Miao by Golgi the ligase the outgrowth of contrast, distribution dendrite asymmetric ubiquitin-protein By apical 2012). promotes protein al., (Ube3A) the et syndrome modulate Anda (de to N- Angelman’s c-Jun (JNK) the shown of kinase activation were terminal the through dendrites (TAOK2) basal of kinase formation thousand-and-one- 2 and Sema3A amino-acid Additionally, 2009). al., et (Tran h o-eetrtrsn iae y,LkadSchv been have Src and Lck Fyn, kinases tyrosine non-receptor The 2+ upb ciaigtrsn-iaedpnetsignaling tyrosine-kinase-dependent activating by pump ora fCl cec 21)17 0855 doi:10.1242/jcs.154542 5038–5051 127, (2014) Science Cell of Journal nvitro in hwn ipre n ec less-polarized hence and dispersed showing , ru) *** group); mean ( the show Data S3. S1– Tables material in supplementary presented are the analysis of statistical data detailed The plasmids. indicated the with transfection of analysis after Sholl neurons (C) hippocampal A. in as neurons treated for (TNDT) of tips number dendritic total (D) and (TDL) bar: Scale 50 groups. all for neurons representative of DIV5. Tracings on (A) fixed were neurons treated glutamate- and Control (non-treated) days. neurons 3 for (Glu) 50 glutamate with treated and on DIV2 vector GFP-coding with together plasmids pSuper/CASrc or (Y527F) shRNA/CASrc CD44 shRNA, CD44 pSuper, with transfected glutamate. of concentration subtoxic a with treated neurons hippocampal young on of knockdown effect CD44 protective Partial 8. Fig. n 5 m 0nuosper neurons 50 .()Ttldnrtclength dendritic Total (B) m. P ern were Neurons , 0.001. 6 m s.e.m. m 5047

Journal of Cell Science ietyitrcswt D4i h a ri n csa a manner. as Src-dependent a acts in and morphology of regulation Golgi brain influences the CD44 rat that in indicate experiments molecule our the Moreover, morphology. CD44 tree dendritic the in kinase of CD44 Src effector that downstream demonstrate with we study, interacts present in the directly growth In 1994). neurite al., et substrate-induced potentiate cultured neurons to shown have been kinases tyrosine of inhibitors Importantly, 2011). al., et (Ghosh ARTICLE RESEARCH 5048 such by of treatment neurite directed the regulate for prospects are that new signals provide that might extracellular injury. outgrowth brain mechanisms negative in and molecular and positive in 2012) the disorders Firestein, implicated and Unraveling are neurodevelopmental Kulkarni branching, in and dendrite (reviewed of neurodegenerative and loss dendrites several or of fragmentation retraction the the patterns, branching dendrite Golgi. the Src, and CD44, remodeling morphogenetic of actin involvement FAK, sequential novel the a with neurons suggest in pathway data the our of vesiculation Collectively, excessive force preventing organelle. thus tension Golgi, the the and decreases on crosslinking exerted and the filaments in actin reduction of a polymerization to leads consequently process (e.g. substrates a downstream its phosphorylates propose mechanism in turn which kinase, We FAK kinase actin-dependent of Src phosphorylation subsequent induces neurons. kinase) and activation Src to an in binding (by CD44 disassembly knockdown, which by of model Golgi CD44 with existence by associated the induced fragmentation indicating B latrunculin Golgi that actin herein show reverses of we findings, blockade these In with 2014). al., accordance The et DNA-damage-induced (Farber-Katz cells non-neuronal reverses 2009). in dispersion B Golgi al., latrunculin et by polymerization (Dippold recently reported has vesiculation in been results that Golgi the on of force generation tensile the the in F-actin the and Myo18A Golgi Golgi-derived myosin in the GOLPH3, of protein involvement of involved the Moreover, processes) 2006). al., be fission et (Egea vesicles to and budding proposed (i.e. been generation by has generated force polymerization The of membrane 2006). actin al., biogenesis et (Egea the cisternae and flat Golgi unique of in shape the cytoskeleton involved of maintenance are directly and actin carriers The actin transport be Golgi-derived effectors 2005). to the al., the appears downstream et trafficking remodeling (Mitra between its dynamics actin interaction on and regulating in kinase focused involved Src we cytoskeleton. pathway, are that signaling mechanisms CD44–Src pyramidal the cellular through morphology of Golgi regulating the arborization for responsible investigate the further in To Golgi neurons. the a with of associated function and particular cell-type-specific on activity be Src can of morphology effect the Golgi were that suggest also that might cells observed neuronal activation cells materialin Src (supplementary of consequence HeLa cells contradictory a The neuronal at S1). In in expressed Fig. was than cells. Src level of higher these form much active in An non- the CASrc, mutant Golgi versus expression with 2010). transfected differential this neuronal the the al., of to in attributable levels et of expression be might CASrc Weller cisternae cells neuronal of S1; vesiculated effect Fig. induce opposite material to (supplementary shown has have activity kinase Src been that cells, non-neuronal kinase In in non- cells. results, neurons control our Src in to ‘normal’ organelles contrast of in active morphology the the influence Constitutively Golgi not restores does expression. the activity CD44 Src of diminished that morphology found fragmented have We h leaino ediemrhlg,icuigcagsin changes including morphology, dendrite of alteration The nvitro in BxyadJavl,19;Williams 1992; Jhabvala, and (Bixby a atnn.This -actinin). osdrdteaetclyrlvn o ri pathologies brain be for might impairment. of cells tree relevant dendritic inhibition neuronal with associated that in therapeutically indicate pathway considered data signaling al., protective et these CD44–Src Liang exert 2001; the together, al., to et Taken shown (Paul stroke been 2009). after injury have brain on kinases effects Moreover, Src 2002). al., of et observation (Wang inhibitors ischemia the cerebral of Src with model CD44 mouse consistent on of lack are depends the data effect that Our protective inhibition. this kinase and glutamate-induced shortening, prevents partially dendrite shRNA death. CD44 that cell that 1988) neuronal show causing We al., an without et shortening apply many dendrite (Mattson we in results damage study, of glutamate-induced present pathogenesis of the Mattson model In in the 2003). (reviewed Sherman, stroke in and in implicated and disorders is neurodegenerative neurotoxic a that is receptors, glutamate mechanism caused of damage activation neuronal excessive of the by form a Excitotoxicity, diseases. brain ainllw htaei ulareetwt h uoenUnion European the with agreement on full based Warsaw, in in are Research that Animal laws on First the national the Committee of by All established Ethical sexes. rules the both Local to of according rats performed Wistar were in procedures performed were experiments The Animals METHODS AND MATERIALS rvosy Sprvco Bumlape l,2002), al., described et been 2005), al., (Brummelkamp have et (Jaworski vector plasmids pSuper expression previously: mammalian following The constructs DNA for ice on anesthetized 1 were with pups injected P1 and modifications. minutes 5 with Rossa la 2013), (De al., recently described et as performed was iontoporation Postnatal vivo In the to normalized were data densitometry GAPDH. (pSuper) blot control, control western the loading the in 1 of as ImageJ All densitometry designated using was group. level performed The protein was the Signaling). blots and western software, (Cell independent three (MBL anti-Src of anti-GFP analysis and rabbit anti- (Millipore) Systems), GAPDH chicken (R&D anti-CD44 International), antibodies cultures sheep following cell The used: from 2009). extracts al., were or et from P3–P60, (Dzwonek extracts previously or described cortical E18 as on or sacrificed hippocampal were in that rats performed was procedure The was blotting Western labeling of evaluation of statistical Institutes The (National the using MD). ImageJ performed using Bethesda, micrographs were Health, digital compartments ultrastructural in The various control. measured negative within a densities as used particle was gold Pharmingen) Non- (BD Sciences). IgG Microscopy sheep (Electron immune applied to were coupled anti- antibodies particles polyclonal secondary gold by 10-nm sheep followed Systems) immunodetection, R&D For (1:50; CD44 2008). previously described al., as et hippocampi P10 (Wilczynski in performed was procedure The microscopy electron Immunogold experimentation. animal on directive riso i uss(0V 0m uain eiee hog 5-mm through skull. the delivered of side duration) each 50-ms on placed V, two were (80 with that injection pulses paddles the after electrode six minutes 4 of at trains apparatus WY21 Nepagene performed a was in Electroporation permeability. membrane nuclear enhance onsi h ie oprmns(ahwadLcc,2008). Lucocq, and (Mayhew compartments given the in counts h lsi ouinwsmxdwt 1 with mixed was solution plasmid 30- the a using cortex cerebral electroporation ora fCl cec 21)17 0855 doi:10.1242/jcs.154542 5038–5051 127, (2014) Science Cell of Journal x 2 b etwt eadt h bevdadepce gold expected and observed the to regard with test atnRP(ognade l,20) EF 2007), al., et (Hoogenraad -actin-RFP nvivo in m -i ls irppte ro oinjection, to Prior micropipette. glass m-tip b m glcoiae(ba) os anti- mouse (Abcam), -galactosidase fDA(3 DNA of l rtcstebanfo nuyi a in injury from brain the protects m fTH 10m ml mg (100 TCHD of l m g m l 2 1 ietyit the into directly ) b -actin-GFP nvitro in a 2 - b 1 -gal )to

Journal of Cell Science eeae sn h ukhneSt-ietdMtgnssKit Mutagenesis Site-Directed QuikChange the the into (Stratagene). introduced using were next (CD44Rescue) sequence. forms were generated coding shRNA-resistant shRNA shRNAs mRNA CD44-GFP the plasmid. these (CD44 within CD44 pSuper encoded #90) 2075–2097 rat shRNA that (CD44 Oligomers sequences against 202–224 and designed the #1963) were targeting sequences (NM_012924), shRNA CD44 into cloned was CGTCGACTGCACCCCAATCTTCATATC-3 CTGAATTCGACAAGGTTTGGTGGCAC-3 gis r Cl inln Technology). Signaling directed (Cell antibody Src polyclonal rabbit against with blotting western and SDS-PAGE rmterthpoapsa epaewt h rmr 5 primers the with template a as hippocampus GenBank isolated rat was form; that the mRNA (standard total sequence using from (constitutively PCR coding by Y527F amplified full-length was http://www.addgene.org/13660/). src BC127485.1) CD44 13660; chick rat plasmid pLNCX The Addgene and Src; 2005) active al., et (Konopka ARTICLE RESEARCH mgso h muoloecnl aee el eeaqie na on acquired with were microscope the the cells confocal analysis, to 780 labeled Golgi The average normalized LSM immunofluorescently the Zeiss software. For was the the cells. and of cells ImageJ adjacent images transfected of non-transfected Src using in of analysis intensity phosphorylated fluorescence performed of was The CD44, intensity FAK of microscope. Zeiss phosphorylated or intensity SP5 confocal TCS Leica labeled immunofluorescence a 780 Fluorescently under 2011). examined al., were al., LSM et sections et brain (Perycz (Meijering and plug-in NeuronJ cells Sholl with the ImageJ and 20 using 2004) a performed with were with microscope obtained analyses were fluorescence images cell Nikon morphology, a dendritic of analysis the For analysis and acquisition Image control isotype and anti-IgG 4 homogenized, sheep at or and Systems) overnight sheep- rats a was (R&D with Wistar antibody incubated were brain anti-CD44 cortex P10 Lysates Briefly, prepared. were from 2012). fractions and al., membrane isolated et were hippocampus (Renner previously samples described the (Life as performed from protocol Co-immunoprecipitation manufacturer’s the Immunoprecipitation to according staining Technologies). Nissl performed Jackson Fluorescent Labs). 1:500; Vector was diluted (Vectashield DAPI, DAPI with donkey, Medium with from Mounting counterstained anti-sheep-IgG (all were and Nuclei 488 649 ImmunoResearch). DyLight DyLight to to DyLight conjugated conjugated to conjugated anti-chicken-IgG anti-mouse-IgG following (1:200; 546, in used: The were anti-MAP-2 Abcam). used antibodies (1:200; mouse secondary anti-GFAP were R&D), to chicken and (1:200; antibodies (Jackson Sigma-Aldrich) anti-CD44 conjugated CY3 primary sheep to following anti-mouse-IgG anti-pY576- sections: conjugated The anti-rabbit-IgG rabbit donkey donkey ImmunoResearch). and Signaling), Invitrogen), 649 anti-CD44 Bioscience), Cell DyLight sheep (1:100; BD (1:100; cells: (1:500; FAK in anti-GM130 anti-pY416-Src mouse used rabbit R&D), were (1:500; antibodies The antibody 2012). al., as primary et sections Urbanska tissue 2008; following al., or et neurons (Wilczynski cultured previously in described performed were 2000 procedures The Lipofectamine previously using described Immunofluorescence transfected as and brains hippocampal 2011) rat rat al., P0 (Invitrogen). Primary from et 2011). prepared (Michaluk al., were et as performed cultures (Perycz were transfection previously their and described cultures cell HeLa and HEK293 cultures Cell oueadsraeae nlsso h rget,a described as the fragments, and and the reconstruction fragments of Golgi analysis three-dimensional distinct area of surface for number and the used volume of was quantification software ˚ ,floe yicbto ihProtein-A–Sepharose, with incubation by followed C, Eco RI/ Sal etito ie fthe of sites restriction I z 6 sak f0.25 of -stacks 9 9 betv.Morphometric objective. rvre.Teproduct The (reverse). frad n 5 and (forward) b atnGPplasmid. GFP -actin m .Imaris m. 9 9 - - Acknowledgements were analyses statistical statistical (StatSoft). The software the the test. Statistica ANOVA whether post-hoc using For mixed-model Bonferroni performed univariant on met. the a used depending by was we followed results, tests, variance Sholl the post-hoc of of analysis and Sidak homogeneity followed mean of or (ANOVA) the assumption variance C of of error Dunnett’s analysis standard by one-way and The using values neurons. analyzed mean of were as batches expressed independent three are from data obtained were data The At cells 8). analyses the Fig. Statistical and in changed, was indicated medium 50 as cell to CASrc the exposed were transfection, and after shRNA ( hours plasmids 2 CD44 with DIV2 pSuper, on GFP, transfected were neurons Hippocampal shRNA treatment CD44 B Glutamate latrunculin or with incubated pSuper were cells with the DIV5 later, (2.5 hours on 24 At transfected plasmids. were Neurons treatment B Latrunculin orugo,L . og . al,C,Kugr .adSea,C C. C. Spevak, and K. Krueger, C., Earle, G., Wong, Y., L. Bourguignon, W. Y. Chen, and L. Shao, H., Zhu, Y., L. Bourguignon, P. Y. L. Bourguignon, Jhabvala, and L. J. Bixby, M. C. Gall, and J. Zhou, G., Lynch, X., Bi, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.154542/-/DC1 online available material Supplementary material Supplementary DEC-2012/06/M/NZ3/ number [grant Centre Science 00163]. was National J.W. the 01.01.02-00-008/08]. by Program POIG supported Operational number the [grant from under Economy grant Fund Innovative a Development and Regional 7873/B/P01/2011/40]; European number the [grant the Centre by Science Fund supported National Development was Regional G.M.W. PARENT- PARENT-BRIDGE/2011-3/2]. Union Science number European Polish [grant the for by Foundation co-financed a grant, by BRIDGE supported was research This Funding project. the G.M.W. supervised and J.D. J.D. paper. data. new the the contributed analyzed E.P. wrote A.G. G.M.W. and and and I.F. J.J. J.L. J.W., H.D., tools. data. L.S., reagents/analytic the J.J., analyzed A.K., experiments. and A.S., the data. experiments performed the the analyzed performed and M.B. experiments and P.T. the performed and designed J.D. contributions Author interests. competing no declare authors The interests Competing plasmid Addgene the from plasmids the (Harvard sharing Brugge repository. for Joan MA) and Boston, School, Poland) Warsaw, Medical Biology, Pro Tomek Experimental thank of We Institute assistance. Warsaw, Biology, experimental Experimental for of Poland) Institute (Nencki Ganguly Krishnendu thank We h og otettlvlm fteGliprcl a acltda an as calculated of was cell area per surface dispersion. Golgi total Golgi the describe the of to of volume parameter total ratio additional the The to 2013). Golgi al., the et (Thayer previously iaeascae yokltnatvto n rattmrcl invasion. cell tumor Rho- breast to and Chem. leading Biol. activation up-regulation, J. signaling, cytoskeleton RhoA/RhoC twist and kinase-associated c-Src-mediated expression, promotes microRNA-10b interaction Hyaluronan-CD44 hyaluronic and migration. cell function tumor cytoskeleton ovarian acid-dependent cortactin-mediated progression. promotes kinase tumor c-Src promotes function Biol. cytoskeleton Cancer and signaling growth. neurite substrate-induced potentiates 184-193. nerni edie fhpoapladcria neurons. cortical and hippocampal of dendrites in integrin m )fr2 or.Clswr ie nDIV7. on fixed were Cells hours. 24 for M) ora fCl cec 21)17 0855 doi:10.1242/jcs.154542 5038–5051 127, (2014) Science Cell of Journal 18 285 251-259. , 20) ylrnnmdae D4atvto fRhoGTPase of activation CD44 Hyaluronan-mediated (2008). 36721-36735. , m ltmt o as(ato ta. 1988). al., et (Mattson days 3 for glutamate m 19) niiino yoiephosphorylation tyrosine of Inhibition (1992). 20) oaie itiuino alpha5 of distribution Polarized (2001). .Bo.Chem. Biol. J. .Neurobiol. J. 20) D4itrcinwith interaction CD44 (2001). ´szyn .Cm.Neurol. Comp. J. 276 23 ´ k (Nencki ski 7327-7336. , 468-480. , b Semin. (2010). 5049 -actin 435 ,

Journal of Cell Science ulde .T,Kma .M n tat .J. G. Stuart, and M. B. Kampa, T., A. Gulledge, Majczynski, A., Cabaj, M., Gawlak, E., Wilczek, J., Wlodarczyk, A., Gorlewicz, S. Rivest, and C. J. Bittencourt, I., Glezer, Moscarello, and E. Theriault, F., T. Cruz, E., L. Becker, M., Letarte, N., Girgrah, hs,B,L,Y n hyr .A. S. Thayer, and Y. Li, B., Ghosh, M., Xing, C., M. Peterman, D., M. Buschman, C., H. Dippold, E., S. Farber-Katz, La G., Egea, van A., Schellens, A., Conidi, S., Cazzola, O., Preobrazhenska, J., Dzwonek, ipl,H . g .M,Fre-az .E,Le .K,Kr,M . Peterman, L., M. Kerr, K., S. Lee, E., S. Farber-Katz, M., M. Ng, C., Lu H. N., Dippold, Toni, J., Moss, I., Vitali, B., Golding, C., Bellone, A., Rossa, la De ukri .A n ieti,B L. B. Firestein, and A. V. Kulkarni, and J. Jaworski, T., Proszynski, B., Mioduszewska, K., Duniec, W., Konopka, D. J. A. Troost, W. Koleske, H. and Moser, A. and D. E. W. Bosch, Kaufmann, F., Morsink, T., S. Pals, P., G. Raivich, Kaaijk, and W. G. Kreutzberg, L., M. L. Sheng, Jones, and C. C. Hoogenraad, P., D. Seeburg, Y. S., Spangler, L. J., Jan, Jaworski, and N. Y. C. Jan, D. Hoessli, and A. Briol, S., Ilangumaran, eAd,F . oai,A . ua,O,Ta,T,Gra T., Tran, O., Durak, L., A. Rosario, and F. C., F. Feiguin, Anda, G., de P. Camoletto, S., J. Silva, Da G., Pollarolo, C., F. Anda, Figarella- R. de Agami, and and F. R. Bernards, J. R., T. Pellissier, Brummelkamp, G., Monti, A., Liprandi, C., Bouvier-Labit, ARTICLE RESEARCH 5050 otn .C,Ra C., A. Horton, Dunah, D., A. Milstein, A., S. Spangler, I., M. Feliu-Mojer, C., M., C. Hoogenraad, Bra J. and Y. Britto, N. M., Shin, L., Silva, Ryzhova, De K., S. J., Hanks, S. Fuller, H., M. Kim, M., J. 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