RESEARCH ARTICLE

Kinesin-1 promotes post-Golgi trafficking of NCAM140 andNCAM180 to the cell surfaceHilke Wobst1,2, Brigitte Schmitz2, Melitta Schachner3,4, Simone Diestel2,*, Iryna Leshchyns’ka1,* andVladimir Sytnyk1,*

ABSTRACTThe neural cell adhesionmolecule (NCAM, also known as NCAM1) isimportant during neural development, because it contributes toneurite outgrowth in response to its ligands at the cell surface. In theadult brain, NCAM is involved in regulating synaptic plasticity. Themolecular mechanisms underlying delivery of NCAM to the neuronalcell surface remain poorly understood. We used a protein macroarrayand identified the kinesin light chain 1 (KLC1), a component of thekinesin-1 motor protein, as a binding partner of the intracellulardomains of the two transmembrane isoforms of NCAM, NCAM140and NCAM180. KLC1 binds to amino acids CGKAGPGA within theintracellular domain of NCAM and colocalizes with kinesin-1 in theGolgi compartment. Delivery of NCAM180 to the cell surface isincreased in CHO cells and neurons co-transfected with kinesin-1.We further demonstrate that the p21-activated kinase 1 (PAK1)competes with KLC1 for binding to the intracellular domain of NCAMand contributes to the regulation of the membrane insertion of NCAM.Our results indicate that NCAM is delivered to the cell surface througha kinesin-1-mediated transport mechanism in a PAK1-dependentmanner.

KEYWORDS:Cell adhesionmolecule, Cell surface delivery, Neurons

INTRODUCTIONThe neural cell adhesion molecule (NCAM, also known asNCAM1) belongs to the immunoglobulin superfamily of Ca2+

-independent cell adhesion molecules. Alternative splicing of asingle gene gives rise to three major isoforms named aftertheir apparent molecular masses, NCAM120, NCAM140 andNCAM180. NCAM120 is anchored to the surface membranethrough a glycosylphosphatidylinositol (GPI) anchor, whereasNCAM140 and NCAM180 are transmembrane glycoproteins withintracellular domains of different lengths (Walsh and Doherty,1991).NCAM is highly expressed in the developing central and

peripheral nervous systems, where it plays important functionalroles in cell adhesion, cell migration, axonal outgrowth andfasciculation, as well as synapse formation. In the adult NCAM isinvolved in regulating synaptic plasticity (Chazal et al., 2000;

Chipman et al., 2014; Cremer et al., 1994, 1997; Doherty et al.,1990; Hinsby et al., 2004; Kiryushko et al., 2004; Maness andSchachner, 2007; Muller et al., 1996; Panicker et al., 2003; Sytnyket al., 2004). Being expressed at the cell surface of neurons and glialcells, NCAM induces intracellular signal transduction pathways andcytoskeleton rearrangements in response to interactions with itsextracellular domain (Büttner et al., 2003; Ditlevsen et al., 2008;Leshchyns’ka et al., 2003; Li et al., 2013; Nielsen et al., 2010;Pollerberg et al., 1987; Walmod et al., 2004).

Transport of NCAM from the cell body and through thetranslational machinery in cellular processes is a prerequisite forcell surface delivery. In axons, NCAM is distributed by fast axonaltransport (Garner et al., 1986; Nybroe et al., 1986). In dendrites andglial cell processes, NCAM also needs to be distributed to the cellsurface after its transport to the sites of insertion. The molecularmechanisms of the transport and cell surface delivery of NCAM areincompletely understood. It therefore deemed pertinent to search forthe molecules that bind to the intracellular domain(s) of the twotransmembrane NCAM isoforms and could be involved in NCAMtransport and cell surface delivery. By protein macroarray screeningusing the intracellular domains of NCAM as baits, we identifiedkinesin light chain 1 (KLC1) as a new binding partner of theintracellular domains of NCAM140 and NCAM180.

KLC1 is part of kinesin-1, a motor protein that transports cargoestowards the plus end of microtubules in axons and dendrites(Brady, 1985; Hirokawa et al., 1991; Vale et al., 1985). Kinesin-1 iscomposed of two kinesin heavy chains (KHCs; KIF5A, KIF5B andKIF5C isoforms), which use ATP to energize the movement alongmicrotubules, and two light chains (KLCs), which play a role in cargoattachment to kinesin-1 heavy chains (Cabeza-Arvelaiz et al., 1993;Hirokawa, 1998; Hirokawa et al., 1989; Niclas et al., 1994; Rahmanet al., 1998; Verhey et al., 1998; Xia et al., 1998; Yang et al., 1990).

In the present study, we show that KLC1 binds directly to theeight-amino-acid sequence comprising amino acids 747–754 in theintracellular domains of human NCAM140 and NCAM180, andpromotes cell surface delivery of NCAM.

RESULTSKLC1 binds to the intracellular domain of NCAMTo identify new intracellular interaction partners of the intracellulardomains of human NCAM180 and NCAM140 (hNCAM180-IDand hNCAM140-ID), recombinantly expressed highly purifiedhNCAM180-ID and hNCAM140-ID were individually applied to aprotein macroarray comprising 24,000 expression clones fromhuman fetal brain. In addition to the already identified bindingpartners of NCAM, such as spectrin (Leshchyns’ka et al., 2003;Pollerberg et al., 1986), PLCγ and PP2A (Büttner et al., 2005), weobserved binding of hNCAM180-ID and hNCAM140-ID to KLC1(Q07866; note UniProt accession numbers are given in brackets)(Fig. 1A). hNCAM180-ID and hNCAM140-ID did not bind toReceived 28 January 2015; Accepted 18 June 2015

1School of Biotechnology and Biomolecular Sciences, The University of New SouthWales, Sydney, NSW 2052, Australia. 2Institute of Nutrition and Food Science,Department of Human Metabolomics, University of Bonn, Bonn 53115, Germany.3Keck Center for Collaborative Neuroscience and Department of Cell Biology andNeuroscience, Rutgers University, Piscataway, NJ 08854-8082, USA. 4Center forNeuroscience, Shantou University Medical College, Shantou, Guangdong 515041,China.

*Author for correspondence (s.diestel@uni-bonn.de; iryna.leshchynska@unsw.edu.au; v.sytnyk@unsw.edu.au)

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kinesin light chain 2 (KLC2) (Q9H0B6), kinesin light chain 4(KLC4) (Q9NSK0), kinesin-like protein KIF1A (Q12756), kinesin-like protein KIF2C (Q99661), kinesin-like protein KIF3C(O14782), kinesin-like protein KIF22 (Q14807), kinesin-likeprotein KIF15 (Q9NS87), kinesin-like protein KIF21B (O75037),kinesin-like protein KIFC3 (Q9BVG8), kinesin heavy chainisoform KIF5C (O60282), kinesin heavy chain isoform KIF5A(Q12840), kinesin-like protein KIF21A (Q7Z4S6), or chromosome-associated kinesin KIF4A (O95239).To confirm the association between KLC1 and NCAM, they were

immunoprecipitated from detergent lysate of 1-day-old mousebrains, and immunoprecipitates were subjected to western blotanalysis with antibodies against NCAM and KLC1. NCAMimmunoreactivity appeared as a broad protein band with anapparent molecular mass above 180 kDa (Fig. 1B,C). The broadrange of this NCAM band composed of different molecular massesderives from the very heterogeneous lengths of polysialic acidchains attached to the NCAM protein backbone and has beenobserved to be a prominent phenomenon in immature brains

(Hoffman et al., 1982; Rothbard et al., 1982). NCAM co-immunoprecipitated with KLC1 (Fig. 1B), and KLC1 co-immunoprecipitated with NCAM (Fig. 1C). Hence, KLC1 andNCAM are associated with each other.

Next, we aimed to identify the binding site for KLC1 in NCAM.Given that both hNCAM140-ID and hNCAM180-ID bound toKLC1 in the macroarray assay, we analyzed binding of KLC1 toamino acid sequences of hNCAM140-ID and hNCAM180-ID thatwere close to the plasma membrane. This amino acid sequence andthe position of this stretch relative to the plasma membrane areidentical in both transmembrane NCAM isoforms (Fig. 1D). Toanalyze the binding between KLC1 and this amino acid sequence,KLC1 was immunopurified from brain lysates and immobilized asbait on a plastic surface for ELISA. Chemically synthesizedpeptides corresponding to amino acid sequences 747–762, 763–776and 755–769 of hNCAM140-ID and hNCAM180-ID were thenprobed for their ability to bind to KLC1. Dose-dependent binding ofNCAM-ID-747–762 to KLC1 was detectable, whereas NCAM-ID-763–776 and NCAM-ID-755–769 did not bind (Fig. 1E). Given that

Fig. 1. KLC1 binds to the intracellulardomain of NCAM. (A) Amacroarraymembranecomprising 24,000 different expression clonesof human fetal brain was incubated withfluorescently labeled hNCAM180-ID. An area ofthe macroarray membrane comprising 112protein spots is shown. KLC1 spotted twicearound a guiding spot (black spot, arrowhead)interacted with hNCAM180-ID (fluorescentspots indicated by arrows). (B,C) KLC1 (B) andNCAM (C) immunoprecipitates (IP) and brainlysate used for immunoprecipitation (input)were probed by western blot (WB) analysis withantibodies against KLC1 and NCAM. A mockimmunoprecipitation with non-immuneimmunoglobulins (Ig) served as a control. Notethat NCAM co-immunoprecipitates with KLC1,and KLC1 co-immunoprecipitates with NCAM.Ig-HC, heavy chain of immunoglobulins usedfor immunoprecipitation. (D) Schematicdiagram illustrating full-length hNCAM140.NCAM180 has an identical extracellular domainbut a longer intracellular domain. Peptidescovering membrane-proximal amino acidsequences 747–762, 763–776 and 755–769are present both in NCAM140 and NCAM180and are conserved in mouse, rat and humanNCAM. Ig domain, immunoglobulin-likedomain; FNIII domain, fibronectin type IIIdomain. (E) Binding of biotinylated NCAM-ID-747–762, NCAM-ID-763–776 and NCAM-ID-755–769 to KLC1 analyzed by ELISA.Immunoaffinity purified KLC1 from mouse brainwas immobilized on a plastic surface usingKLC1-specific antibodies. Bound peptides weredetected with neutravidin–HRP. The graphshows absorbance as mean±s.d. from arepresentative experiment. Note that onlyNCAM-ID-747–762 binds to KLC1. (F) Westernblot analysis of lysates and KLC1immunoprecipitates (IP) from CHO cellsco-transfected with GFP-KHC/KLC1 andnon-mutated hNCAM180 (wt) orhNCAM180Δ747-754. A mock IP withnon-immune IgG served as control. Note thathNCAM180 but not hNCAM180-Δ747–754co-immunoprecipitates with KLC1.

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NCAM-ID-747–762 but not NCAM-ID-755–769 binds to KLC1,we conclude that the amino acid sequence which is unique inNCAM-ID-747–762 (encompassing amino acids 747–754,CGKAGPGA) is required for binding to KLC1. To confirm this,CHO cells were co-transfected with KLC1 and KHC (KIF5C),together with non-mutated hNCAM180 or hNCAM180 containinga deletion of the amino acid sequence responsible for binding toKLC1 (hNCAM180-Δ747–754). The interaction between KLC1and hNCAM180 was then analyzed by co-immunoprecipitationand western blot analysis. Non-mutated hNCAM180 but nothNCAM180-Δ747–754 co-immunoprecipitated with KLC1(Fig. 1F).To identify the site of putative interactions between NCAM and

KLC1 in cells, cultured mouse cortical neurons were colabeled withantibodies against KLC1 and antibodies recognizing the intracellulardomains of NCAM140 and NCAM180. Immunofluorescenceanalysis showed that KLC1 and NCAM were distributed alongneurites and in neuronal somata (Fig. 2A). Noticeably, confocal

microscopy showed small intracellular accumulations of NCAMcolocalizing with accumulations of KLC1, particularly in somata(Fig. 2A). Replacement of the primary antibodies with control non-immune immunoglobulins from the same species that the antibodieswere derived from eliminated the signal (Fig. 2B).

To verify that NCAM and KLC1 form complexes, a proximityligation assay (PLA) was performed (Söderberg et al., 2006). In thisanalysis, the primary antibodies are detected with secondaryantibodies conjugated to oligonucleotides, which provideamplifiable DNA strands when they are in close proximity to eachother (<40 nm). Amplified products can then be detected withfluorescence signals. Results showed that the intracellular domainsof NCAM140 and NCAM180 are in close proximity to KLC1(Fig. 2C). In agreement with the immunofluorescence data,proximity ligation signals were observed in somata, along neuritesand in growth cones (Fig. 2C), where both proteins are enriched(Morfini et al., 2001; Sytnyk et al., 2002). Omission of one of theantibodies erased the proximity ligation signal (Fig. 2D). No

Fig. 2. Intracellular accumulations of NCAMcolocalize with KLC1 in cultured corticalneurons. (A) Confocal slice images of acultured cortical neuron show colabeling withantibodies against NCAM and KLC1. Highmagnification images of the soma are shown onthe right. NCAM accumulations colocalizingwith clusters of KLC1 are indicated by arrows.Scale bars: 20 µm (low magnifications); 5 µm(high magnification). An immunofluorescenceintensity profile through one of the intracellularaccumulations of NCAM (dashed arrow) isshown below. (B) Labeling with control non-immune immunoglobulins (negative control).Scale bar: 20 µm. (C–E) Proximity ligationanalysis of cultured cortical neurons withantibodies against the intracellular domain ofNCAM and KLC1 (C) or KLC2 (E), or theintracellular domain of NCAM only (D).Proximity ligation fluorescence signals alone(left) or in combination with differentialinterference contrast images (right) are shown.Note proximity ligation products in somata,growth cones and along neurites (C). Scalebars: 20 µm. (F) Confocal slice images of thesoma of a cultured cortical neuron transfectedwith hNCAM180 and GFP–KHC/KLC1. Cellsurface (NCAMsurf ) and total (NCAMtotal)hNCAM180 were detected with antibodiesagainst human NCAM applied before and afterdetergent permeabilization of membranes andvisualized with different fluorochromes. Noteintracellular accumulations of hNCAM180colocalized with GFP–KHC/KLC1 (arrows).Scale bar: 5 µm.

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proximity ligation signals were detectable in neurons colabeledwith antibodies against KLC2 and antibodies recognizing theintracellular domains of NCAM140 and NCAM180 (Fig. 2E).To confirm that NCAM and kinesin-1 colocalize intracellularly,

colocalization of NCAM with kinesin-1 was analyzed in neuronsoverexpressing human NCAM180 (hNCAM180) with GFP-taggedKLC1 and KHC (KIF5C) (hereafter GFP–KHC/KLC1). Neuronswere labeled with antibodies specific for the extracellular domain ofhuman, but not mouse, NCAM, which were applied to the cellsbefore membrane permeabilization with detergent to detect cellsurface hNCAM180 only. After detergent permeabilization, thetotal pool of hNCAM180 was immunolabeled with a differentfluorochrome. Confocal microscopy showed that the intracellularaccumulations of hNCAM180 in somata colocalized with GFP-tagged KHC and KLC1 (Fig. 2F).

NCAM colocalizes with kinesin-1 in trans-Golgi organellesTo identify the subcellular compartment, inwhichNCAMcolocalizeswith kinesin-1, neurons co-transfected with hNCAM180 and

GFP–KHC/KLC1 were colabeled with antibodies against hNCAMand the Golgi marker protein TGN38 (also known as TGOLN2).Analysis of the confocal sections of somata showed that intracellularaccumulations of NCAM partially colocalized with GFP–KHC/KLC1 and TGN-38, suggesting that they associate in the Golgi andtrans-Golgi compartments (Fig. 3A). In agreement, western blotanalysis showed that both NCAM and KLC1 were present in Golgiand trans-Golgi organelles isolated from early postnatal mouse brainsusing sucrose gradient centrifugation (Fig. 3B). Although NCAMwas detectable at similar levels in the Golgi and trans-Golgi fractions,KLC1 and KIF5A were found at higher levels in the trans-Golgifraction, which was enriched in TGN38 when compared to the Golgi,as described previously (Hossain et al., 2014). These data suggestthat KLC1 contributes to the intracellular transport of NCAMpredominantly in trans-Golgi organelles.

Kinesin-1 promotes cell surface delivery of NCAMGiven that NCAM has crucial functions at the cell surface, weinvestigated whether kinesin-1 is involved in cell surface delivery of

Fig. 3. NCAM and kinesin-1 colocalize in the Golgicompartment. (A) Confocal 3D analysis of the soma of acultured cortical neuron co-transfected with hNCAM180and GFP–KHC/KLC1, maintained for 2 days in cultureand then formaldehyde-fixed and colabeled withantibodies against the Golgi compartment markerTGN38 and human NCAM. The confocal z-stack of thesoma was acquired with serial 0.15 µm steps. A confocalxy slice through the center of the Golgi region in the somaand reconstructed xz and yz sections at the pointindicated by arrows are shown. Note colocalization ofNCAM accumulations with GFP–KHC/KLC1 in theTGN38-positive area. Scale bar: 5 µm. (B) Western blotanalysis of brain homogenates (BH), soluble cytoplasmicproteins (cytosol), TGN organelles, Golgi complexmembranes, ER and other small vesicles includingendosomes isolated from 1- to 3-day-old mouse brain.Note the enrichment of NCAM, KLC1 and KIF5A in theTGN-enriched fraction.

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NCAM. CHO cells were co-transfected with hNCAM180 and eitherGFP–KLC1, GFP–KHC/KLC1 or, for control, GFP alone. Inagreement with our observations on neurons, confocal fluorescenceanalysis of CHO cells co-transfected with hNCAM180 and GFP–KHC/KLC1 and analyzed for cell surface and total pools ofhNCAM180 showed that intracellular accumulations of NCAMcolocalized with GFP–KHC/KLC1 (Fig. 4A). Cell surfacelocalization of hNCAM180 was then assayed using antibodiesagainst the extracellular domain of hNCAM applied to live cells onice to inhibit endocytosis of the antibodies (Fig. 4B). Images of thelabeled cells were acquired by fluorescence microscopy to quantifyhNCAM180 immunofluorescence intensities associated with thesurface membrane of transfected cells. This analysis showed thatlevels of hNCAM at the cell surface of GFP–KLC1 or GFP–KHC/KLC1 co-transfected cells were ∼twofold higher when compared tocell surface hNCAM levels of GFP co-transfected cells (Fig. 4C).Cell surface hNCAM levels were not different between CHO cellsco-transfected with hNCAM and GFP–KLC1 or GFP–KHC/KLC1.Given that KIF5 (KIF5A, KIF5B and KIF5C) is expressedendogenously by CHO cells (Hammond et al., 2012) and canform a functional motor with transfected GFP–KLC1 (Araki et al.,2007), our results suggest that it is sufficient for transport of NCAMpreceding cell surface delivery.In individual cells, levels of cell surface hNCAM180 correlated

with levels of expression of GFP–KLC1 and GFP–KHC/KLC1 asmeasured by GFP fluorescence, further indicating that an increase inthe expression of kinesin-1 results in increased cell surface deliveryof NCAM (Fig. 4B). In contrast, cell surface levels of NCAM180poorly correlated with GFP levels in control GFP co-transfectedcells (Fig. 4B).To analyzewhether the intracellular domain of NCAM is required

for kinesin-1-dependent cell surface delivery of NCAM, CHO cellswere transfected with a construct of hNCAM deleted in itsintracellular domain (hNCAMΔCT) and co-transfected with GFP–KHC/KLC1 or GFP. Surprisingly, deletion of the intracellulardomain of NCAM did not abolish cell surface delivery of NCAM.Levels of cell surface hNCAMΔCT were similar in CHO cells co-transfected with GFP–KHC/KLC1 or GFP (Fig. 4D) and similar tothe levels of full-length hNCAM180 in CHO cells co-transfectedwith GFP–KHC/KLC1 (Fig. 4D). Levels of cell surfacehNCAMΔCT correlated poorly with the expression levels ofGFP–KHC/KLC1 (Fig. 4E). Thus, the intracellular domain ofNCAM is required for kinesin-1-regulated cell surface deliveryof hNCAM180, whereas deletion of this domain results in kinesin-1-independent delivery.The intracellular domain of NCAM interacts with adaptor

protein 2 (AP-2), which recruits cell surface cargo proteins toclathrin-coated endocytotic vesicles (Shetty et al., 2013). Hence, wecompared endocytosis rates of hNCAM180 and hNCAMΔCT byanalyzing the internalization of NCAM antibodies in live CHOcells, an assay used by us previously to analyze endocytosis ofCHL1, another cell adhesion molecule of the immunoglobulinsuperfamily (Tian et al., 2012). Transfected CHO cells wereincubated with antibodies recognizing the extracellular domain ofhNCAM. Cell surface and total pools of NCAM–antibodycomplexes were then labeled with Cy3- and Cy5-coupledsecondary antibodies applied before and after detergentpermeabilization of cells, respectively. The efficiency of NCAMendocytosis was estimated using confocal microscopy by analyzingthe ratio of intensities of Cy5 fluorescence in Cy3-negative areas,representing endocytosed NCAM, and Cy5 fluorescence in Cy3-positive areas, representing surface NCAM. This analysis showed

that endocytosis of hNCAMΔCT is reduced when compared toendocytosis of hNCAM180 (Fig. 4F). Accumulation ofhNCAMΔCT at the cell surface could therefore be due toinhibited endocytosis.

Submembrane sequences in the intracellular domain ofNCAM are required for kinesin-1-dependent cell surfacedelivery of NCAMNext, we analyzed whether cell surface delivery of hNCAM180 isaffected by inhibition of the interaction between its intracellulardomain and kinesin-1 using fragments of the intracellular domain ofNCAM. CHO cells were co-transfected with hNCAM180 andGFP–KHC/KLC1 together with cDNA constructs encoding theentire intracellular domain of rat NCAM140 (NCAM-ID), itsfragments NCAM-ID-729–750, NCAM-ID-748–777 and NCAM-ID-777–810 derived from the membrane proximal region, or theNCAM180-specific amino acid sequence encoded by exon 18(NCAM180-ID-808–1078) (Fig. 5A). Immunofluorescenceanalysis of the cell surface levels of hNCAM180 showed thatlevels of hNCAM180 at the cell surface of cells co-transfected withGFP-KHC/KLC1 together with NCAM-ID are reduced to the levelsobserved in GFP co-transfected cells, indicating that NCAM-IDinterferes with kinesin-1-dependent cell surface delivery ofhNCAM180, probably by competing with hNCAM180 forbinding to KLC1 (Fig. 5B). A similar effect was observed forCHO cells co-transfected with NCAM-ID-748–777 (Fig. 5B),which also contains the binding site for KLC1 (Fig. 1E). This effectwas not seen with cells co-transfected with NCAM180-ID-808–1078 (Fig. 5C). Interestingly, transfection with NCAM-ID-729–750and NCAM-ID-777–810 also reduced cell surface hNCAM180levels (Fig. 5B). Because these amino acid stretches contain bindingsequences for components of the exocyst complex (Chernyshovaet al., 2011), we analyzed whether overexpression of the exocystcomplex Sec8 (also known as EXOC4) subunit mutated in itsphosphorylation sites (Sec8-Y401F,Y616F) interferes with the cellsurface delivery of NCAM. We had previously shown that thismutant interferes with exocyst complex assembly at surfacemembranes (Chernyshova et al., 2011). Kinesin-1-dependent cellsurface delivery of hNCAM180 was inhibited in cells transfectedwith Sec8-Y401F,Y616F (Fig. 5C). Kinesin-1-dependent cellsurface delivery of NCAM was also abolished in cells co-transfected with hNCAM180-Δ747–754 (Fig. 5C). We concludethat amino acids 747–754 and also the submembrane regions ofNCAM involved in the interaction with the exocyst complex arerequired for kinesin-1-dependent cell surface delivery of NCAM.

PAK1 competes with kinesin-1 for binding to NCAM andregulates cell surface NCAM levelsGiven that kinesin-1 is required for intracellular trafficking, it istempting to speculate that the interaction between NCAM andKLC1 is disrupted when NCAM is delivered to the cell surface.Interestingly, the amino acid sequence in NCAM-ID that isrequired for its binding to KLC1 overlaps with a recently identifiedbinding site for p21-activated protein kinase (PAK1), whichinteracts with NCAM at the cell surface (Li et al., 2013). Thisobservation prompted us to analyze whether PAK1 and KLC1competewith each other for binding to NCAM-ID. To test this idea,we performed a pulldown assay with KLC1 immunopurified fromthe mouse brain and peptides from NCAM-ID. Beads withimmobilized KLC1 were incubated with chemically synthesizedNCAM-ID-747–762, NCAM-ID-763–776, and NCAM-ID-755–769. Slot blot analysis of the pulldowns showed that only

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NCAM-ID-747–762 bound to KLC1 (Fig. 6A), confirming theELISA results (Fig. 1E). The binding of NCAM-ID-747–762 toKLC1 in the presence of immunopurified PAK1 was reduced to

7.0±0.3% (mean±s.e.m.) of the signal observed in the absence ofPAK1, verifying that KLC1 and PAK1 compete for the binding toNCAM-ID-747–762.

Fig. 4. GFP–KHC/KLC1 increases targeting of hNCAM180 to the cell surface of CHO cells. (A) Confocal slice images of CHO cells co-transfected withhNCAM180 and GFP–KHC/KLC1. Cells were labeled with antibodies against the extracellular domain of NCAM applied before formaldehyde fixation to labelsurface NCAM and colabeled with NCAM antibodies after fixation and permeabilization to label total NCAM. Arrows show intracellular accumulations of NCAM,which were not labeled with NCAM antibodies applied before fixation, colocalized with accumulations of GFP–KHC/KLC1. An immunofluorescence intensityprofile through one of the intracellular accumulations of NCAM (dashed line in the merged image) is shown on the right. (B) Images of CHO cells co-transfectedwith hNCAM180 and GFP, GFP–KLC1 or GFP–KHC/KLC1 and labeled for cell surface NCAM. Three cells per group are shown. Note higher levels of cell surfaceNCAM in cells co-transfected with GFP–KLC1 or GFP–KHC/KLC1. Graphs show correlation between the levels of GFP–KLC1, GFP–KHC/KLC1 or GFP and cellsurface NCAM in individual cells. Note that the levels of cell surface NCAM correlate with the levels of GFP–KLC1 and GFP–KHC/KLC1, but not with the levels ofGFP. AU, arbitrary units; r, Pearson’s correlation coefficient. (C) Graph shows mean±s.e.m. levels of cell surface NCAM immunofluorescence intensity for cellsshown in B. AU, arbitrary units. ***P<0.001 (one-way ANOVA with Dunnett’s multiple comparisons test, n≥55). (D) Graph shows mean±s.e.m. levels of cellsurface hNCAM immunofluorescence intensities in CHO cells co-transfected with hNCAM180 or hNCAMΔCT and either GFP or GFP–KHC/KLC1. ***P<0.001(unpaired t-test; n≥68). (E) Graph shows correlation between levels of GFP–KHC/KLC1 and cell surface hNCAMΔCT in individual cells. Note that cell surfacelevels of hNCAMΔCT do not depend on the co-expression of GFP–KHC/KLC1. (F) Analysis of NCAM endocytosis in CHO cells transfected with hNCAM180 orhNCAMΔCT. Cells were incubated with antibodies against the extracellular domain of NCAM for 1 h at 37°C to allow for endocytosis and then labeled for cellsurface and total pools of the NCAMantibody. Examples of confocal slice images of CHO cells are shown on the left. Note accumulations of internalized NCAMonthe merged images (arrows). Graph shows the ratio of endocytosed (NCAMend) and cell surface (NCAMsurf ) levels of NCAM (mean±s.e.m.). *P<0.05 (unpairedt-test; n>70). Scale bars: 10 µm.

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To analyze whether PAK1 plays a role in the cell surface deliveryof NCAM, hNCAM180 and GFP–KHC/KLC1 were co-transfectedinto CHO cells together with either full-length PAK1where T423 inthe catalytic domain of PAK1 was replaced with glutamic acidresulting in a constitutively active enzyme (CA-PAK1) (Sells et al.,1997) or with a truncated form of PAK1 consisting of amino acidresidues 67–150, representing the autoinhibitory region of mousePAK1 (DN-PAK1) (Hayashi et al., 2002). Immunofluorescenceanalysis of the transfected cells showed that cell surface levels ofhNCAM were reduced in cells co-transfected with either of the twoPAK1 forms (Fig. 6B). We conclude that overexpressed PAK1, andparticularly its autoinhibitory domain, interferes with cell surfacedelivery of NCAM possibly by competing with KLC1 for bindingto NCAM.

KLC1 promotes cell surface delivery of NCAM in culturedcortical neuronsTo investigate whether KLC1 promotes cell surface delivery ofNCAM in neurons, cortical neurons were transfected with

hNCAM180 together with GFP, GFP–KLC1 or GFP–KHC/KLC1. Surface hNCAM180 was detected with antibodiesrecognizing the extracellular domain of human but not mouseNCAM applied to live neurons and visualized with secondaryantibodies coupled to the Cy3 fluorochrome. Neurons were thencolabeled with antibodies specific for hNCAM180, which wereapplied to neurons after permeabilization of membranes withdetergent, and visualized with the Cy5 fluorochrome to detect cellsurface and internal pools of hNCAM180. The efficiency ofhNCAM180 delivery to the cell surface of neuronal somata wasthen estimated using confocal microscopy and analyzing the ratio ofsurface NCAM levels (NCAMsurf, identified as Cy5 fluorescenceintensity in Cy3-positive areas) and internal NCAM levels(NCAMintern, identified as Cy5 fluorescence intensity in Cy3-negative areas). This analysis showed that the NCAMsurf:NCAMintern ratio is higher in neurons co-transfected with GFP–KHC/KLC1 when compared to neurons co-transfected with GFPonly (Fig. 7A,B), indicating increased transport of hNCAM180 fromthe internal pool to the cell surface. Overexpression of GFP–KLC1

Fig. 5. Submembrane amino acidsequences of the intracellular domain ofNCAM are necessary for kinesin-1-dependent cell surface delivery.(A) Schematic diagram illustrating full-lengthrat NCAM140, NCAM-ID-729–750, NCAM-ID-748–777, and NCAM-ID-777–810 fragmentsand the site of insertion of the NCAM180-specific exon-18-coded amino acid sequence(NCAM180-ID-808–1078, exon 18). Note, thatthe CGKAGPGA sequence is shifted in the ratNCAM. (B,C) Images of CHO cells transfectedwith hNCAM180 or hNCAM180-Δ747–754together with either GFP or GFP–KHC/KLC1and labeled for cell surface NCAM. Whereindicated, cells were also co-transfected withrat NCAM-ID, NCAM-ID-729–750, NCAM-ID-748–777, NCAM-ID-777–810, NCAM180-ID-808–1078 or Sec8Y401F,Y616F. Graphsshow mean±s.e.m. levels of cell surfaceNCAM immunofluorescence intensity inarbitrary units (AU). *P<0.05 (one-wayANOVA with Dunnett’s multiple comparisonstest, n≥50 cells were analyzed). Scale bar:10 µm. Note that NCAM-ID, its submembranefragments, Sec8Y401F,Y616F and theΔ747–754 deletion inhibit kinesin-1-dependent cell surface delivery ofhNCAM180.

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alone resulted in only a slight increase in this ratio (Fig. 7A,B),probably due to the high expression of endogenous KLC1 inneurons.To confirm that the interaction between NCAM and KLC1 is

required for kinesin-1-dependent cell surface delivery of NCAM,the NCAMsurf:NCAMintern ratio was analyzed in neuronstransfected with hNCAM180-Δ747–754. Cell surface delivery ofhNCAM180-Δ747–754 in GFP-transfected neurons was reducedwhen compared to that of non-mutated hNCAM180 (Fig. 7A,B) and

remained unchanged, when GFP–KLC1 or GFP–KHC/KLC1 wereoverexpressed (Fig. 7A,B). To corroborate these data, we alsoanalyzed whether knockdown of KLC1 expression affectedhNCAM180 targeting from the Golgi complex to the cell surface.In neurons transfected with KLC1 small interfering RNA (siRNA),cell surface delivery of hNCAM180 was reduced to the levelobserved for hNCAM180-Δ747–754 in neurons co-transfected withcontrol siRNA (Fig. 7C,D). Cell surface targeting of hNCAM180was not affected by KLC2 siRNA (Fig. 7C,D). Our observationsthus indicate that disruption of the interaction of NCAM180 withendogenous KLC1 reduces cell surface delivery of hNCAM180.

In accordance with our observations in CHO cells (Fig. 4D),complete deletion of the intracellular domain resulted in increasedlevels of hNCAMΔCT at the cell surface as reflected by theincreased NCAMsurf:NCAMintern ratio (Fig. 7A,B). Cell surfacedelivery of hNCAMΔCT was not affected by co-overexpression ofGFP–KLC1 or GFP–KHC/KLC1 (Fig. 7A,B), indicating that cellsurface insertion of hNCAMΔCT is independent of kinesin-1. Toanalyze whether endocytosis of hNCAMΔCT is affected,endocytosis rates were estimated by measuring the internalizationof NCAM antibodies. Neurons transfected with hNCAM constructsand maintained for 2 days in culture were incubated with antibodiesrecognizing the extracellular domain of human but not mouseNCAM, and cell surface as well as total pools of NCAM–antibodycomplexes were visualized using secondary antibodies conjugatedto different fluorochromes, applied before and after membranepermeabilization, respectively. The ratio of endocytosed and cellsurface pools of NCAM in somata showed that internalization ofhNCAMΔCT was slightly reduced when compared to endocytosisof hNCAM180 and hNCAM180-Δ747–754 (Fig. 8). Interestingly,internalization of hNCAM180 was also slightly reduced byco-transfection with GFP–KHC/KLC1 (Fig. 8).

DISCUSSIONKinesin-1 is a motor protein enabling fast transport of a variety ofcargoes along microtubules of neurites (Brady, 1985; Hirokawaet al., 1991; Kamal et al., 2000; Konecna et al., 2006; Lazarov et al.,2005; Vale et al., 1985). Kinesin-1 is known to carry a number ofneuronal transmembrane proteins, including apolipoprotein Ereceptor 2 (ApoER2, also known as LRP8) (Verhey et al., 2001),amyloid precursor protein (Kamal et al., 2000; Lazarov et al., 2005),and calsyntenin-1 (also known as alcadein) (Araki et al., 2007;Konecna et al., 2006), which can associate directly or indirectly withKLC1 (Hirokawa et al., 2009). In this study, we identified KLC1 asa new binding partner of the intracellular domain of NCAM140 andNCAM180. We also show that kinesin-1 increases cell surfacedelivery of NCAM180 both in CHO cells and neurons. Ourobservation that intracellular accumulations of NCAM colocalizewith kinesin-1 in the Golgi complex suggests that kinesin-1promotes trafficking of NCAM-containing organelles from theGolgi compartment to the cell surface.

KLC1 is the main binding site for cargoes in kinesin-1 (Hirokawa,1998; Hirokawa et al., 1989). Using macroarray screening, wedemonstrated that KLC1 binds directly to hNCAM180-ID andhNCAM140-ID. Confirming the physiological role of thisassociation, NCAM could be co-immunoprecipitated with KLC1frommouse brain tissue lysates. The KLC1-binding motif within theintracellular domain of NCAM140 and NCAM180 comprises theamino acid sequence CGKAGPGA and does not contain tyrosine ortryptophan residues such as have been shown to be crucial for theinteraction between KLC1 and other cargoes (Aoyama et al., 2009;Araki et al., 2007; Bracale et al., 2007; Dodding et al., 2011;

Fig. 6. KLC1 and PAK1 compete for binding to NCAM-ID. (A) KLC1 wasimmunopurified frommouse brain, immobilized on agarose beads and used forthe pulldown assay. Beads were incubated with NCAM-ID-747–762, NCAM-ID-763–776 or NCAM-ID-755–769 peptides or NCAM-ID747–762 togetherwith PAK1 purified frommouse brain. Input, unbound and bound peptides wereprobed for by slot blot analysis using neutravidin–HRP. Note the binding ofNCAM-ID-747–762 to KLC1, which is abolished in the presence of PAK1.Similar results were obtained in three independent experiments. (B) Images ofCHO cells co-transfected with hNCAM180 and GFP–KHC/KLC1 with orwithout constitutively active PAK1 (CA-PAK1) or autoinhibitory domain ofPAK1 (DN-PAK1) and labelled for cell surface NCAM. Two cells per group areshown. Graph shows mean±s.e.m. levels of cell surface NCAMimmunofluorescence intensity in arbitrary units (AU). *P<0.05, **P<0.01(one-way ANOVA with Dunnett’s multiple comparisons test, n≥103 cellsanalyzed in each group). Scale bar: 10 µm.

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Hayakawa et al., 2007; Konecna et al., 2006; Rosa-Ferreira andMunro, 2011; Schmidt et al., 2009; Verhey et al., 2001). Therefore,the identified sequence in NCAM represents a new binding motif forKLC1.Interestingly, the complete deletion of the intracellular domain of

NCAM does not abolish cell surface delivery, although it results inelimination of the binding sites for KLC1 and other intracellularbinding partners, such as PAK1. This observation is not surprising,given that NCAMΔCT could be considered to be a functional andstructural equivalent of the NCAM120 isoform of NCAM, which isanchored to the membrane through GPI and lacks an intracellulardomain and, hence, is transported by mechanisms distinct fromthose of NCAM180 and NCAM140 (Garner et al., 1986; Nybroeet al., 1986). Our observation that cell surface levels of NCAMΔCTare similar to or even higher than those for NCAM180 indicates thatKLC1 is not required for cell surface delivery of NCAM, but rathersuggests that this interaction plays a role in regulation of NCAMtransport. In agreement, cell surface delivery of NCAMΔCT isindependent of kinesin-1 in our experiments, indicating thatNCAMΔCT is delivered to the cell surface through another,kinesin-1-independent, pathway. Reduced endocytosis alsocontributes to the accumulation of NCAMΔCT at the cell surface,

probably because the intracellular domains of NCAM140 andNCAM180 are required for their interaction with AP-2 (Shetty et al.,2013), which plays a role in the recruitment of the proteins to theclathrin-coated endocytic vesicles.

Somewhat unexpectedly, overexpression of kinesin-1 alsoreduced endocytosis of NCAM180. NCAM180 associates with themembrane–cytoskeleton linker protein spectrin (Pollerberg et al.,1986, 1987; Leshchyns’ka et al., 2003) and promotes polymerizationof the spectrin cytoskeleton beneath the cell surface membrane(Leshchyns’ka et al., 2003; Sytnyk et al., 2006). The spectrincytoskeleton inhibits endocytosis of transmembrane proteins byinterfering with clathrin-coated pit formation (Puchkov et al., 2011;Kamal et al., 1998). It is therefore conceivable that kinesin-1-dependent cell surface delivery of NCAM180 increases the assemblyof the spectrin cytoskeleton, which could interfere with theendocytosis of NCAM180. We also cannot exclude that otherfactors, such as endosomal transport, could be affected by kinesin-1.

Confirming the role of KLC1 in cell surface delivery ofNCAM180, the dominant-negative fragment of the intracellulardomain of NCAM140 and NCAM180, comprising the KLC1-binding site, reduced cell surface levels of NCAM180. Interestingly,two other fragments derived from the intracellular domain of

Fig. 7. Deletion of the KLC1-bindingregion reduces targeting of NCAM to thecell surface in cultured cortical neurons.(A,B) Cortical neurons were transfected withhNCAM180 (wt), hNCAMΔCT orhNCAM180-Δ747–754 together with GFP,GFP–KLC1 or GFP–KHC/KLC1 and labeledfor cell surface and total hNCAM.(A) Examples of confocal slice images ofsomata of neurons co-transfected withhNCAM180 or hNCAM180-Δ747–754. Notethat overexpression of GFP–KHC/KLC1reduces levels of intracellular NCAM,identified as accumulations of total NCAM(red) not colocalizing with cell surface NCAM(green). This effect is reduced in neuronstransfected with hNCAM180-Δ747–754.(B) The graph shows the ratio of cell surface(NCAMsurf ) and intracellular (NCAMint) levelsof hNCAM immunofluorescence.(C,D) Cortical neurons were transfected withhNCAM180 and either control siRNA orsiRNA for knockdown of KLC1 or KLC2expression. Neurons were labeled for cellsurface and total hNCAM, and colabeled forTGN38. (C) Examples of confocal sliceimages of somata are shown. Note increasedlevels of intracellular NCAM in TGN38-positive organelles in KLC1-siRNA-transfected neurons. (D) The graph showsthe ratio of hNCAM immunofluorescencelevels at the cell surface (NCAMsurf ) and inTGN38-positive accumulations (NCAMGolgi).In A,C, immunofluorescence images areshown in pseudocolor (TGN38 or GFP, blue;Cy5, red; Cy3, green) to better visualize theintracellular pool of NCAM appearing as redaccumulations on the merged images. Scalebar: 10 µm. In B,D, mean±s.e.m. values areshown. *P<0.05 (one-way ANOVA withDunnett’s multiple comparisons test, n≥50cells were analyzed in each group).

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NCAM also inhibited its delivery. These fragments, comprisingamino acids 729–750 and 777–810, interact with the exocystsubunits Exo70 (also known as EXOC7) and Sec8, respectively(Chernyshova et al., 2011), which are essential for exocytosis ofmembranous organelles (Hsu et al., 2004). On the basis of theseresults it is conceivable that NCAM-ID-729–750 and NCAM-ID-777–810 inhibit cell surface delivery of NCAM180 by inhibitingthe binding of the exocyst subunits to NCAM and exocytosis ofNCAM-containing vesicles.The role of PAK1 is interesting in the context of cell surface

delivery of NCAM isoforms. PAK1 interacts with NCAM at the cellsurface of growing neurites and growth cones (Li et al., 2013). Itbinds to the amino acid sequence in the intracellular domain ofNCAM, which we now show also contains the binding site forKLC1 (Li et al., 2013). Indeed, PAK1 and KLC1 compete for thebinding to NCAM. PAK1 contains an autoinhibitory domain thatbinds to the kinase domain of PAK1 to inactivate the enzyme.Although overexpression of constitutively active PAK1 reduces cellsurface delivery of NCAM, suggesting that activated PAK1 inhibitsthis delivery, we consider this possibility unlikely because deliveryis also reduced in cells overexpressing the autoinhibitory domainof PAK1, which inhibits endogenous PAK1 activity. Theseobservations rather suggest that the autoinhibitory domain ofPAK1, which is exposed in its active form enabling its interaction

with other proteins (Say et al., 2010), regulates delivery of NCAM.We thus propose that this domain competes with KLC1 for bindingto NCAM and contributes to the detachment of NCAM from KLC1upon delivery of NCAM to the cell surface. In agreement with thisidea, activation of NCAM functions induces activation of PAK1(Li et al., 2013) and exocytosis in growth cones (Chernyshovaet al., 2011). Interestingly, the proposed model also suggests thatactivation of NCAM by homophilic and heterophilic interactions atsites of contacts between cells can promote exocytosis of NCAM-containing vesicles to stabilize these contacts. A recent studyshows that presynaptically expressed NCAM is required for themaintenance of the neuromuscular junction innervation field, and itis tempting to speculate that cell surface delivery of NCAM bykinesin-1 is important for stabilizing the neuromuscular junction(Chipman et al., 2014).

The functions of NCAM are further regulated by post-translational modifications, such as polysialylation (Gascon et al.,2007, 2010; Kleene and Schachner, 2004; Rønn et al., 2000; Sekiand Rutishauser, 1998), and phosphorylation and ubiquitylation(Cassens et al., 2010; Diestel et al., 2004, 2007; Pollscheit et al.,2012; Sorkin et al., 1984; Wobst et al., 2012). Whetherposttranslational modifications affect transport of NCAM bykinesin-1 is an intriguing question, which will have to befollowed up by future studies.

Fig. 8. Disruption of the interaction betweenhNCAM180 and KLC1 does not affect endocytosisof hNCAM180. Cortical neurons were transfected withhNCAM180 (wt), hNCAMΔCT or hNCAM180-Δ747–754 together with GFP, GFP–KLC1 or GFP–KHC/KLC1. Live neurons were incubated with antibodiesagainst the extracellular domain of hNCAM for 1 h at37°C to allow for endocytosis and then labeled for cellsurface and total pools of the NCAM antibody.(A) Examples of the confocal slice images of somata ofneurons transfected with hNCAM180 and hNCAMΔCT.Immunofluorescence images are shown inpseudocolors (GFP, blue; Cy5, red; Cy3, green) tobetter visualize the internalized pool of NCAM antibodyappearing as red accumulations on the mergedimages. (B) Graph shows the ratio of endocytosed(NCAMend) and cell surface (NCAMsurf ) levels of NCAMimmunofluorescence (mean±s.e.m.). *P<0.05(one-way ANOVA with Dunnett’s multiple comparisonstest, n≥50 cells were analyzed in each group). Scalebar: 10 µm.

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In summary, we reveal a new role for kinesin-1 together withPAK1 in regulating the cell surface expression of the twotransmembrane isoforms of NCAM. This new role sheds light ona different level of regulation of cell surface expression of NCAMat the strategic site to allow it influence cell-to-cell interactions inthe developing and adult brain, particularly in view of itsimplications in nervous system disorders.

MATERIALS AND METHODSAntibodiesRabbit polyclonal antibodies (pAb) against mouse NCAM (Westphal et al.,2010) (for western blot analysis and immunoprecipitation) and mousemonoclonal antibody (mAb) ERIC recognizing human, but not mouse,NCAM (for western blot and immunocytochemical experiments; SantaCruz Biotechnology) react with the extracellular domain of all NCAMisoforms. Mouse mAb 5B8 against the intracellular domain of mouseNCAM140 and NCAM180 (for immunocytochemistry and PLA) was fromthe Developmental Studies Hybridoma Bank (University of Iowa, Iowa).Rabbit pAb against KLC1 (H-75, for immunoprecipitation, westernblotting, immunocytochemistry and PLA), goat pAb against KLC2 (N-14,for PLA), rabbit pAb against TGN38 (M-290, for immunocytochemistryand western blotting), rabbit pAb against KIF5A (for western blotting) andnon-immune immunoglobulins were from Santa Cruz Biotechnology.Mouse mAb against EEA1 (for western blotting) and mouse mAb againstPDI (for western blotting) were from BDBiosciences. Secondary antibodiescoupled to horseradish peroxidase (HRP), Cy2, Cy3 or Cy5 were fromJackson ImmunoResearch Laboratories.

AnimalsC57BL/6J mice (1 to 3 days old) were used for preparation of neuronalcultures and biochemical experiments. All experiments were approved bythe Animal Care and Ethics Committee of the University of New SouthWales (permit 12/135B).

DNA constructs and siRNAsThe cDNAs encoding the intracellular domain of human NCAM180(hNCAM180-ID) and NCAM140 (hNCAM140-ID) used to producerecombinant proteins for the protein macroarray assay have beendescribed (Homrich et al., 2014; Wobst et al., 2012).

For transfection of cells, cDNAs coding for full length non-mutatedhNCAM180 (Diestel et al., 2004); hNCAMΔCT (Boutin et al., 2009); ratNCAM140-ID (Leshchyns’ka et al., 2003); rat NCAM-ID-729–750, ratNCAM-ID-777–810, and Sec8Y401F,Y616F (Chernyshova et al., 2011);rat NCAM-ID-748–777 (Li et al., 2013); GFP–KLC1 and GFP–KHC1(generous gifts of Toshiharu Suzuki, Hokkaido University, Sapporo, Japan)(Araki et al., 2007; Kawano et al., 2012), and GFP (Clontech) were used.To construct cDNA coding for hNCAM180-Δ747–754, site-specificmutagenesis was carried out using the overlap extension PCR method(Ho et al., 1989) with hNCAM180 as a template. The following senseprimers 5′-CGCGGATCCATGCTGCAAACTAAGGATCTC-3′ (pI) and5′-GCGGTCAACCTG- - - - -AAGGGCAAAGAC-3′ (pIII) and antisenseprimers 5′-GTCTTTGCCCTT- - - - -CAGGTTGACCGC-3′ (pII) and 5′-CG-GGATCCCGCATGCTCGAGTCATGCTTTGCTCTC-3′ (pIV) were used(dashed lines indicate regions of deleted amino acids). The mutant constructwas checked for correctness by DNA sequencing (MWGBiotech, Ebersbach,Germany). NCAM180-specific exon18-coded sequence was amplified fromrat NCAM180 cDNA(Leshchyns’ka et al., 2003) using the following primers:5′-CTGCCTGCCGACACCACAGCCA-3′, and 5′-TTACTCGGTCTTTG-CTGGTGCGGGC-3′, and was cloned into the pcDNA3 vector.

Control siRNA and siRNA for knockdown of KLC1 (sc-43881) andKLC2 (sc-146492) expression was from Santa Cruz Biotechnology.

Expression and purification of recombinant proteins for proteinmacroarrayhNCAM180-ID and hNCAM140-ID were expressed in BL21 (DE3)bacteria and purified by Nickel-nitrilotriacetic acid affinity chromatography(hNCAM180-ID) or by glutathione affinity chromatography (hNCAM140-

ID) according to manufacturer’s instructions (Qiagen) as describedpreviously (Homrich et al., 2014; Wobst et al., 2012). Purified proteinswere concentrated to a minimum of 1 mg/ml in phosphate-buffered saline(PBS) and fluorescently labeled with Dyomics DY-633 Fluorophore(Dyomics, Jena, Germany).

Protein macroarrayThe macroarray membrane (Source Bioscience imaGenes, Berlin,Germany) containing 24,000 different His-tagged expression clones ofhuman fetal brain was prepared as described previously (Homrich et al.,2014; Wobst et al., 2012). Purified and fluorescently labeled hNCAM180-ID (15 nM) and NCAM140-ID (13.5 nM), respectively, were incubated inOdyssey blocking buffer (LI-COR Biosciences) with the preparedmembrane for 16 h. Signals were detected using the Licor Odysseyscanner (LI-COR Biosciences). Images were analyzed with Aida ImageAnalyzer version 4.24 (Raytest, Straubenhardt, Germany). Positive spotswere identified using the Scoring Template and the annotation table ofSource Bioscience imaGenes revealing the spotting pattern of the expressionclones on the membrane (Homrich et al., 2014; Wobst et al., 2012).

Co-immunoprecipitationBrains of 1-day-old mice were homogenized using a Potter homogenizer inbuffer containing 5 mM Tris-HCl pH 7.4, 1 mM MgCl2, 1 mM CaCl2,0.1 mM PMSF, EDTA-free protease inhibitor cocktail (Roche) and 0.32 Msucrose. Homogenates containing 1 mg of total protein were lysed for30 min at 4°C with lysis buffer containing 50 mM Tris-HCl pH 7.5,150 mM NaCl, 1% Triton X-100, 1 mM Na4P2O7, 1 mM NaF, 2 mMNa3VO4, 0.1 mMPMSF and EDTA-free protease inhibitor cocktail. Lysateswere centrifuged for 15 min at 20,000 g at 4°C. Supernatants were clearedwith protein-A/G–agarose beads (Santa Cruz Biotechnology) for 3 hat 4°C and incubated with antibodies against KLC1, NCAM or non-immune IgG overnight at 4°C. Complexes were collected with protein-A/G-agarose beads for 3 h at 4°C. Beads were washed twice with lysis buffer andonce with Tris-buffered saline (TBS), pH 7.4. Complexes were eluted frombeads with non-reducing LDS sample buffer (Life Technologies) to detectKLC1 by western blot analysis. For detection of NCAM, 150 mMDTTwasadded to the samples. Samples were heated for 10 min at 70°C followed bythe SDS-PAGE electrophoresis and western blot analysis.

Isolation of the cytoplasmic fraction and Golgi and trans-Golginetwork organellesIsolation of the cytoplasmic fraction, Golgi and trans-Golgi network (TGN)organelles was performed as described previously (Fath and Burgess, 1993;Sytnyk et al., 2002). Brains from 1- to 3-day-old mice were homogenizedusing a T10 homogenizer (IKA) in PKM buffer containing 100 mMKH2PO4 pH 6.5, 5 mM MgCl2, 3 mM KCl, 0.1 mM PMSF, EDTA-freeprotease inhibitor cocktail and 0.5 M sucrose. Brain homogenates werecentrifuged at 600 g for 10 min at 4°C. The supernatant was collected andcentrifuged on a discontinuous density gradient of 0.7 and 1.3 M sucrose inPKM buffer (100 mM KH2PO4 pH 6.5, 5 mMMgCl2, 3 mM KCl, 0.1 mMPMSF, EDTA-free protease inhibitor cocktail) at 105,000 g for 60 min at4°C. The upper layer of the gradient was collected and centrifugedat 200,000 g for 45 min at 4°C. The supernatant containing the cytoplasmicprotein fraction was used as source of endogenous KLC1 for ELISA andpulldown assays. The pellet was positive for EEA1 indicating that itcontains endosomes. Membranous organelles were collected at the 0.7–1.3 M sucrose-PKM interface, adjusted to 1.25 M sucrose in PKM, overlaidwith 1.1 M and 0.5 M sucrose in PKM and centrifuged at 90,000 g for90 min at 4°C. Membranes collected at 1.25–1.1 M sucrose interface wereenriched in PDI indicating that they contain endoplasmic reticulum (ER).Golgi and TGN organelles were collected at the 0.5–1.1 M sucroseinterface, adjusted to 0.7 M sucrose in PKM and centrifuged for 15 min at10,000 g at 4°C. The pellet containing Golgi stacks was collected. Thesupernatant containing TGN organelles was centrifuged at 259,000 g for30 min at 4°C, and the pellet containing TGN organelles was collected.Organelles were resuspended in PEMS buffer containing 10 mM PipespH 7.0, 1 mM EGTA, 2 mM MgCl2, 0.25 M sucrose, 0.1 mM PMSF andEDTA-free protease inhibitor cocktail.

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Western blot analysisProteins were separated by 10% SDS-PAGE (Life Technologies) andelectroblotted onto PVDF membranes (Millipore). Membranes wereblocked with 5% skim milk powder in phosphate-buffered saline (PBS),pH 7.4, and incubated with appropriate primary antibodies followed byincubation with HRP-labeled secondary antibodies, which were visualizedusingWestern LightningUltra reagent (PerkinElmer) or Luminata CrescendoWestern HRP substrate (Millipore). Chemiluminescence images wereobtained using the MicroChemi 4.2 imaging system (DNR Bio-ImagingSystems).

ELISARabbit pAb against KLC1 (1 µg/ml in Na2CO3 buffer, pH 9.6) wereabsorbed to the surface of 96-well MICROLON 600 plates (Greiner)overnight at 4°C. Wells were blocked with 1% BSA in TBS for 1 h at 37°Cand incubated with the cytoplasmic fraction from mouse brain dilutedin TBS with 0.1% Tween 20 (TBST) to purify and immobilize KLC1.Wells were washed with TBST and incubated for 1 h at 37°C withincreasing concentrations of chemically synthesized biotinylated peptidescorresponding to amino acid sequences 747–762, 763–776 and 755–769of human NCAM140 and NCAM180 (Peptide 2.0) diluted in TBST.Wells were washed with TBST, incubated for 1 h at 37°C with HRP-coupled neutrAvidin (Thermo Fisher Scientific) in TBST containing 1%BSA, and washed with TBST. The color reaction was developed with1 mg/ml o-phenylenediamine (Sigma). Absorbance was measured at450 nm.

Purification of PAK1PAK1 was immunopurified from brain lysates (as described in theco-immunoprecipitation section) using agarose beads conjugated to rabbitpAb against PAK1 (Santa Cruz Biotechnology) as described previously(Li et al., 2013). PAK1 was eluted from the beads with 0.2 M glycinepH 4.0. The eluate containing PAK1was immediately neutralizedwith 1 MTris-HCl pH 8.0.

Pulldown assayFor immunopurification and immobilization of KLC1, the cytoplasmicprotein fraction frommouse brain was cleared with protein-A–agarose beads(Santa Cruz Biotechnology) for 5 h at 4°C and incubated with rabbit pAbagainst KLC1 applied overnight at 4°C. KLC1 was precipitated withprotein-A–agarose beads applied for 5 h at 4°C. Beads were washed fivetimes with TBST and incubated with 2.86 nmol of chemically synthesizedbiotinylated peptides corresponding to amino acids sequences 747–762,763–776 or 755–769 of human NCAM140 (Peptide 2.0) in TBST. PurifiedPAK1 (12.8 nmol) was added when indicated. Samples were incubatedovernight at 4°C. Supernatants containing non-bound peptides werecollected, and beads were washed four times with TBST. Peptides boundto the beads were eluted with 0.2 M glycine, pH 2.0, and immediatelytreated with 1 M Tris for adjustment to pH 7.4. Samples containing input,bound and non-bound peptides were immobilized on nitrocellulosemembranes using a slot blot apparatus. Peptides were detected withHRP-coupled neutrAvidin.

Cultures and transfection of cortical neurons and CHO cellsCultures of cortical neurons were prepared as described previously(Andreyeva et al., 2010). Neurons were obtained from 2-day-old mice andmaintained in Neurobasal A medium supplemented with 2% B-27,glutaMAX and bFGF (2 ng/ml) (all from Life Technologies) on glasscoverslips coated with poly-D-lysine (100 μg/ml). Neurons were transfectedbefore plating using a Neon transfection system (Life Technologies).Neurons were analyzed 48 h after plating.

CHO cells were cultured in Dulbecco’s modified Eagle’s medium withHam’s F-12 (PAA Laboratories) containing 10% fetal bovine serum(Sigma) and antibiotics (Gentamicin/Amphotericin B, Life Technologies).At 1 day before transfection, cells were plated into 2-cm2 wells on glasscoverslips in culture medium without antibiotics. CHO cells weretransfected with Lipofectamine 2000 (Life Technologies).

Immunofluorescence analysis of neuronsWhen indicated, cell surface hNCAM180 in transfected neurons was labeledby incubating live neurons with a mouse monoclonal antibody against theextracellular domain of human NCAM (ERIC) applied for 1 h at 37°C in aCO2 incubator. Unless indicated otherwise, other steps were performed atroom temperature. Neurons were fixed with 4% formaldehyde in PBS for15 min, washed with PBS, blocked with 5% donkey serum in PBS for15 min and labeled with anti-mouse-IgG Cy3-conjugated secondaryantibodies applied in 1% donkey serum in PBS for 30 min. To visualizetotal hNCAM180 and other proteins, neurons were post-fixed with 2%formaldehyde in PBS for 5 min, washed with PBS, permeabilized with0.25% Triton X-100 in PBS for 5 min, blocked with 5% donkey serum inPBS for 20 min, incubated with ERIC antibodies and antibodies againstother antigens applied in 1% donkey serum in PBS overnight at 4°C, andlabeled with Cy5-conjugated (for total hNCAM180) and Cy2-conjugated(for other antigens) secondary antibodies applied in 1% donkey serum inPBS for 45 min. Targeting of hNCAM180 to the cell surface was analyzedusing confocal slice images of the somata of neurons. To calculate theNCAMsurf:NCAMint ratio, cell surface and internal pools of hNCAM180were estimated by measuring immunofluorescence intensities of Cy5labeling in manually outlined Cy3-negative intracellular compartments(NCAMint) and at the Cy3-positive cell surface membrane regions(NCAMsurf ) using ImageJ (National Institute of Health, Bethesda, MD).

Immunofluorescence analysis of CHO cellsAll antibodies were applied in 0.1% BSA in PBS. To label cell surfaceNCAM, CHO cells were placed on ice and incubated with ERIC antibodiesapplied for 20 min, followed by corresponding secondary antibodiesapplied for 15 min. CHO cells were then fixed with 4% formaldehyde inPBS for 15 min at room temperature and washed with PBS.When indicated,cells were also colabeled for the total hNCAM180. Cells werepermeabilized with 0.25% Triton X-100 in PBS for 5 min, blocked with1% BSA in PBS for 20 min, incubated with ERIC antibodies appliedovernight at 4°C, and labeled with secondary antibodies for 45 min at roomtemperature. Levels of GFP fluorescence and cell surface hNCAMimmunofluorescence were measured using ImageJ by manually outliningcells.

Analysis of hNCAM180 endocytosisEndocytosis of hNCAM180 was analyzed by estimating NCAM antibodyinternalization from the cell culture medium as described previously(Puchkov et al., 2011; Tian et al., 2012). Transfected cells were incubatedwith ERIC antibody applied for 1 h at 37°C in a CO2 incubator. Unlessindicated otherwise, all further steps were performed at room temperature.Cells were fixed with 4% formaldehyde in PBS for 15 min, washed withPBS, blocked with 5% donkey serum in PBS for 15 min and labeled withCy3-conjugated secondary antibodies applied in 1% donkey serum in PBSfor 30 min to visualize cell surface NCAM. Cells were then post-fixed with2% formaldehyde in PBS for 5 min, washed with PBS, permeabilized with0.25% Triton X-100 in PBS for 5 min, blocked with 5% donkey serum inPBS for 20 min and incubated with Cy5-conjugated secondary antibodiesapplied in 1% donkey serum in PBS for 45 min to visualize cell surface andinternalized pools of NCAM antibodies. NCAM antibody internalizationwas analyzed using confocal slice images of CHO cells and somata ofneurons. Endocytosed NCAM and NCAM–antibody complexes wereidentified as Cy3-negative and Cy5-positive accumulations. To calculatethe NCAMend:NCAMsurf ratio, immunofluorescence intensities of Cy5labeling were measured in manually outlined Cy3-negative intracellularcompartments (NCAMend) and at the Cy3-positive cell surface membraneregions (NCAMsurf ) using ImageJ.

Proximity ligation assayThe PLAwas performed as described previously (Li et al., 2013; Tian et al.,2012). Cultured neurons were fixed in 4% formaldehyde in PBS, washedwith PBS, permeabilized with 0.25% Triton X-100 in PBS for 5 min,blocked with 1% BSA in PBS for 20 min, and incubated with antibodiesagainst the intracellular domain of NCAM and KLC1 or KLC2 applied in

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0.1% BSA in PBS overnight at 4°C. Further steps were performed usingsecondary antibodies conjugated with oligonucleotides (PLA probes, OlinkBioscience, Uppsala, Sweden) and Duolink II fluorescence kit (OlinkBioscience) in accordance with the manufacturer’s instructions.

Fluorescence microscopyCoverslips were embedded in FluorPreserve reagent (Calbiochem).Immunofluorescence images were acquired using a confocal laserscanning microscope C1si, NIS Elements software, and oil Plan Apo VC60× objective (numerical aperture 1.4), all from Nikon Corporation (Tokyo,Japan). Immunofluorescence intensity measurements were performed inImageJ software. Immunofluorescence intensities were measured inarbitrary units defined as pixel values of 16-bit grayscale images.

Statistical analysisAll experiments were independently performed three to five times. Exceland GraphPad Prism 6 were used for statistical analyses. Differencesbetween groups were analyzed using one-way ANOVA with Dunnett’smultiple comparisons test.

AcknowledgementsWe are grateful to Prof. Toshiharu Suzuki (Hokkaido University, Japan) forGFP–KLC1 and GFP–KHC1 constructs, to Dr Ohshima (Laboratory forDevelopmental Neurobiology, Brain Science Institute, Japan) for providing Pak1constructs, and to Prof. Jorg Hohfeld and Dr Michael Dreiseidler for help with theprotein macroarray.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsH.W., S.D., I.L. and V.S. designed the study and analyzed data. H.W. and I.L.conducted experiments, S.D., I.L. and V.S. supervised experiments, andH.W., B.S.,M.S., S.D., I.L. and V.S. discussed and wrote the paper.

FundingThis work was supported by the National Health and Medical Research Council andUniversity of New South Wales [grant numbers APP1011478 to V.S., GoldstarAPP1063435 to V.S. and I.L.] and by a doctoral scholarship from the GermanAcademic Exchange Service (DAAD; to H.W.).

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