Tau RNP granules and synthesis in axons - Journal of Cell Science

IntroductionAsymmetric localization of mRNAs as a regulatory mechanismfor the determination of cellular asymmetry has been shown inyeast, Xenopusoocytes, Drosophilaembryos and somatic cells(Bassell et al., 1999; St Johnston, 1995). In neuronal cells,mRNA localization is involved in the development of neuronalpolarity and neuronal function (Kiebler and DesGroseillers,2000; Steward, 1997; Tiedge et al., 1999). Restriction ofmRNAs to a particular microdomain of the polarized cell andtheir local translation facilitates the rapid supply of proteinswhere they are needed (Wilhelm and Vale, 1993). In neuronalcells, several localized mRNAs are upregulated by neuronalactivity or following application of physiological stimuli(Akalu et al., 2001; Kuhl and Skehel, 1998; Rook et al., 2000;Schuman, 1999; Zhang et al., 2001). As a result of specificneuronal stimulation, local translation of a green fluorescenceprotein (GFP) reporter in dendrites of hippocampal neurons,and even in isolated dendrites, has been observed (Akalu et al.,2001; Eberwine et al., 2001; Job and Eberwine, 2001).

mRNA localization is initiated by association of the mRNAmolecule, through a targeting signal most commonly locatedin the 3′ untranslated region (3′ UTR), with RNA-bindingproteins (RBPs). Together they form ribonucleo-protein (RNP)granules that can translocate along the microtubules (MTs)(Ainger et al., 1993; Aronov et al., 2001; Ferrandon et al.,1994; Knowles et al., 1996; Wang and Hazelrigg, 1994).Although the granules have been directly visualized inDrosophilaembryos, oligodendrocytes and neurons, relatively

little is known about their components or about their functionalrole in mRNA transport to the dendrites and axons (Ainger etal., 1993; Ainger et al., 1997; Deshler et al., 1998; Hoek et al.,1998; Kiebler et al., 1999; Knowles et al., 1996; Kohrmann etal., 1999; Ross et al., 1997; Theurkauf and Hazelrigg, 1998).Biochemical analysis of granules in Drosophila oocytes hasrecently yielded information on the presence of at least sevenproteins in the oskar mRNA granule (Wilhelm et al., 2000).Granules in neuronal cells contain translational componentsincluding ribosomes that are packed in clusters and that are nottranslationally competent; instead, they serve as local storagesites for mRNA molecules (Bassell et al., 1998; Krichevskyand Kosik, 2001). The identities and functions of the multipleproteins composing the granules, motor proteins and RNA-binding proteins are just emerging (Gu et al., 2002; Hirokawa,2000; Kikkawa et al., 2001; Ross et al., 1997; Tang et al.,2001).

Tau, a neuronal cytoskeletal protein, is a MT-associatedprotein (MAP) that stabilizes MTs and promotes theirassembly. In neuronal cells, tau is found primarily in the cellbody and axon. Axonal localization of tau mRNA to theproximal segment of the axon is dependent on 3′ UTR cis-acting signals, neuronal proteins and assembled MTs (Aranda-Abreu et al., 1999; Aronov et al., 2001; Behar et al., 1995;Litman et al., 1993; Litman et al., 1994).

In a recent study to identify the minimal tau axonallocalization signal (ALS), differentiating P19 embryoniccarcinoma (EC) cell lines stably transfected with GFP-tagged

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Localization of tau mRNA to the axon requires the axonallocalization cis signal (ALS), which is located within the 3′untranslated region, and trans-acting binding proteins,which are part of the observed granular structures inneuronal cells. In this study, using both biochemical andmorphological methods, we show that the granules containtau mRNA, HuD RNA-binding protein, which stabilizesmRNA, and KIF3A, a member of the kinesin microtubule-associated motor protein family involved in anterogradetransport. The granules are detected along the axon andaccumulate in the growth cone. Inhibition of KIF3Aexpression caused neurite retraction and inhibited taumRNA axonal targeting. Taken together, these results

suggest that HuD and KIF3A proteins are present in thetau mRNA axonal granules and suggest an additionalfunction for the kinesin motor family in the microtubule-dependent translocation of RNA granules. Localized tau-GFP expression was blocked by a protein synthesisinhibitor, and upon release from inhibition, nascent tau-GFP ‘hot spots’ were directly observed in the axon andgrowth cones. These observations are consistent with localprotein synthesis in the axon resulting from the transportedtau mRNA.

Key words: Tau mRNA, Tau protein, RNA axonal localization, RNPgranules, KIF3A, HuD, Axonal local translation, P19 EC cells

Summary

Visualization of translated tau protein in the axons ofneuronal P19 cells and characterization of tau RNPgranules Stella Aronov, Gonzalo Aranda, Leah Behar and Irith Ginzburg*Department of Neurobiology, The Weizmann Institute of Science, Rehovot, 76100 Israel*Author for correspondence (e-mail: [email protected])

Accepted 17 July 2002Journal of Cell Science 115, 3817-3827 © 2002 The Company of Biologists Ltddoi:10.1242/jcs.00058

Research Article

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tau constructs linked to fragments derived from the 3′ UTRregion of tau mRNA were employed. The minimal tau ALS,which is required and sufficient for axonal localization, wasidentified. This region includes the stabilization sequence oftau mRNA, which binds to the HuD stabilization protein(Aranda-Abreu et al., 1999; Good, 1997). We observed that taumRNA was non-randomly distributed in the cells; instead itwas localized as discrete granules along the axon and in thegrowth cone and it colocalized on the MTs with ribosomalproteins, which indicated the presence of protein synthesismachinery in the axon (Aronov et al., 2001). RNA granuleshave been observed in fibroblasts, oligodendrocytes andprimary neuronal cell cultures, suggesting that they mayconsist of RNA-protein complexes that contribute to theformation of cellular microdomains (Bassell et al., 1999; StJohnston, 1995; Wilhelm and Vale, 1993).

In this study, we identify HuD and KIF3A as components ofthe tau RNP granules in the neuronal axon and growth cone.We suggest that these proteins are involved in the movementof the granules and the attachment of the granules to the MTtracks. Following targeting of tau mRNA to the axon, weobserved local translation of GFP-tau protein as ‘hot spots’along the axon and in the growth cone, thus indicatingsimultaneous local translation, coinciding with tau mRNAlocalization in the distal processes. To our knowledge, this isthe first axon-targeted mRNA to be shown to be locallytranslated in the axon.

Materials and MethodsP19 stable cell culturesA P19 stable cell line expressing a GFP-tau coding-ALS construct(axonal localization signal of 240 bp from tau 3′ UTR 2529-2760)was used in this study (Aronov et al., 2001). P19 cells were grownand differentiated as previously described (Falconer et al., 1992) inMEM medium containing 10% fetal calf serum, 100 units/mlpenicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine in anincubator with 5% CO2.

In situ hybridization analysis, RNA probes andimmunohistochemical stainingDifferentiated P19 cells were fixed with 4% paraformaldehyde in thepresence of 4% sucrose (Litman et al., 1993). GFP (452 bp), tubulin(400 bp) and tau (400 bp) single-stranded RNA probes (Aronov et al.,2001) were synthesized in the sense and antisense orientations, usingthe appropriate polymerase (T3 or T7 RNA polymerase) in thepresence of digoxigenin UTP (RNA transcription kit, BoehringerMannheim Biochemicals). In situ hybridization was performed aspreviously described (Aranda-Abreu et al., 1999). For visualization ofthe in situ hybridization signals together with immunostaining, theslides were incubated overnight at 4°C with HRP-conjugatedmonoclonal anti-Dig conjugated to CY5 (1:500) (JacksonImmunoResearch) together with the specific antibodies: monoclonaltubulin (1:200) (Sigma), KIF3A (1:30) monoclonal antibodies, KIF1A(1:100) polyclonal [kindly provided by N. Hirokawa, Japan(Kondo et al., 1994)], and HuD (1:1000) monoclonal antibodies16A11 or human purified HuD antibodies [kindly provided by H.Furneaux, USA (Marusich et al., 1994)]. The slides were washed withPBS and then incubated for 2 hours at room temperature with theappropriate secondary antibodies, that is, goat anti-mouse (1:500)(Jackson), goat anti-human (Jackson), and goat anti-rabbit (Jackson)for the polyclonal and monoclonal antibodies, respectively. Thecoverslips were mounted with mowoil and visualized with an LSM

confocal laser scanning imaging system equipped with a 40×objective, using the following laser wavelengths: for GFP, excitation488 nm, emission 505-550 nm; for CY3-goat anti human, excitation545 nm, emission 560−580 nm; and for tau mRNA labeled with CY5-digoxigenin, excitation 650 nm, emission 680 nm. For detection ofendogenous tau mRNA, an RNA probe of tau repeats was used (Fig.3) (Aranda-Abreu et al., 1999). KIF3A was detected by FITC-goatanti-mouse secondary antibodies, excitation 488 nm, emission 505-550 nm. Control experiments for in situ hybridization showed nobackground with the sense probe. In analysis of the fluorescencestaining, no signal was observed in the absence of primary antibodies,and no penetration of signals between specific laser was detected.

Image analysisImage analysis of in situ hybridization experiments was performedusing a Zeiss LSM confocal microscope with LSM 510 softwareequipped with a 40× (1.0 numerical aperture) and 100× (1.4 numericalaperture) oil immersion lenses. The axon showing the in situhybridization signal was divided into 20 µm segments, and thenumber of granules was counted and their size measured. Forcolocalization experiments, images of the same axon region observedby the GFP or rhodamine specific filter set and marked by the programcoordinate system were analyzed. To determine whether a tau mRNAhybridization signal colocalized with HuD and KIF3A proteins, thecoordinates of the particle and the proteins were compared. Onlywhen the coordinates were identical were the particles considered tocolocalize.

For quantitative analysis, 25-45 cells were analyzed on onecoverslip for each experiment. Experiments were done with threecoverslips for each variable, and each experiment was repeated at leastthree times. The data were analyzed by ANOVA and Student’s t test,and statistical significance was determined for the experimentalconditions.

Protein synthesis inhibition in P19 cellsDifferentiated P19 cells (8-10 days) were treated with 50 µg/mlemetine (Sigma), a protein translation inhibitor, for 3 to 4 hours. Atthe end of the treatment, the medium was replaced with fresh medium,and the cells were left to recover for the specified time periods. Cellswere visualized by confocal microscopy with GFP-adapted filters asdescribed above. The quantitative analysis of mRNA and proteinlocalization within the axon was measured by fluorescence (pixel)intensities. Regions of interest (ROIs) were marked, and the meanfluorescence intensity per pixel (±s.e.m.) was measured over time,relative to t0 (Job and Lagnado, 1998). Care was taken to minimize‘background pixels’ within ROIs, and background fluorescence wassubtracted from the mean fluorescence. The data were analyzed byANOVA and Student’s t-test to find the statistical significance underthe various experimental conditions. The data were displayed by usingAdobe Systems (Mountain View, CA).

To monitor tau protein synthesis during the time-lapse experiments,cells grown on coverslips were transferred to growth chambersmaintained at 37°C on a heated stage. The cell medium was changedto Ham’s F12 medium without phenol-red indicator. Fluorescenceimages were captured at the indicated time points with a cooled CCDcamera and processed with LSM 510 software (Zeiss Germany). Tominimize photobleaching and phototoxicity of the living cells, acomputer-driven automatic shutter was used to achieve minimumillumination.

Antisense oligodeoxynucleotide (ODN) treatment ofdifferentiated P19 cellsDifferentiated P19 cells (8-10 days) were treated for 30 hours with 50µM unmodified sense or antisense KIF3A ODN, essentially as

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previously described (Aranda-Abreu et al., 1999). The mediumof the treated cells was replaced every 10 hours, with freshmedium and antisense ODN. The sequence of the antisenseKIF3A ODN was 5′-TCCTACTTATTGATCGGCAT-3′(1-20)(Kondo et al., 1994). No significant homology was foundbetween the antisense KIF3A ODN and other sequences in thedatabase. The morphological appearance of the treated P19cells was observed by light microscopy. Following removal ofthe antisense ODN and replacement with fresh medium, thecells resumed normal morphology.

Immunoblot analysis of P19 protein extractsProteins were extracted from P19 cells (treated with KIF3Asense or antisense ODN) in 1 volume of lysis buffer consistingof 50 mM Tris pH 8.5, 1% Triton X-100, 5 mM EDTA, 0.15M NaCl and 50 µg/ml PMSF. Cell debris was removed bycentrifugation for 10 minutes at 16,000 g at 4°C. Proteinsamples (25 µg) were resolved by sodium dodecyl sulfate(SDS) polyacrylamide-gel electrophoresis, transferred tonitrocellulose filters and reacted with the specified monoclonalantibodies at 4°C for 16 hours. Following incubation for 1 hourat room temperature with HRP anti-mouse secondaryantibodies (Jackson ImmunoResearch), the blots weredeveloped using the ECL chemiluminescence technique.

Immunoprecipitation analysis of P19 cell extractsThe preparation of cell extracts and immunoprecipitations wasperformed as previously described (Aranda-Abreu et al.,1999). Anti-HuD sera, anti-KIF3A and anti-β tubulin (Sigma)were added and the mixture incubated for hour at 4°C,followed by incubation with protein A-sepharose (Pharmacia,10% final concentration). The immunocomplex wasprecipitated by centrifugation, washed, analyzed by SDS-PAGE and processed as already described.

ResultsIdentification of HuD and kinesin proteins in thetau mRNA granulesIn a previous study using differentiated PC12 cells, wedetected an in vivo association between HuD RBP andtau mRNA (Aranda-Abreu et al., 1999). This bindingstabilizes the tau message and is dependent on an AU-rich element located in the tau ALS. The binding of theHuD protein is mediated by association with the MTs,which may anchor the message prior to its localtranslation (Aronov et al., 2001).

To further characterize tau mRNA granules indifferentiated P19 cells, we tested the colocalization oftau mRNA and HuD protein by in situ hybridization andimmunostaining using confocal microscopy (Fig. 1Aa).GFP-tau protein was seen in the cell body andaxon (Fig. 1Ab), which is similar to the location oftransfected GFP-tau mRNA detected using theGFP probe in differentiated cell lines (Fig. 1Ad).Immunohistochemical analysis using HuD-specificantibodies demonstrated that HuD protein is highlyenriched in the neuronal cell body and in the axon ofdifferentiated P19 cells, whereas only a faint stainingwas observed in the dendrites (Fig. 1Ac). The mergedimage, presented at a higher magnification (×2500), oftau mRNA (red signal) and HuD protein (cyan signal)

Fig. 1.HuD protein colocalizes with tau mRNA in RNP granules.(A) Confocal image analysis of HuD protein localization in differentiatedP19 cells. (a) Phase image of a differentiated P19 cell. (b) Expression ofGFP-tau protein. (c) Staining with HuD antibodies. (d) In situhybridization analysis with the GFP probe (which detects the transfectedtau mRNA). (e) Merged confocal image of b, c and d (×2500magnification). The solid arrowheads denote axons; empty arrowheadsdenote dendrites; and small solid curved arrowheads denote neuronal cellbodies. Bar, 10 µm. (B) Confocal merged image of a growth cone. Anenlarged merged image from the growth cone of the cell shown in A. Themerged staining includes the GFP-tau fluorescence (green), HuDimmunostaining (cyan) and GFP in situ hybridization (red) to yield a whitearea of colocalization. Aggregation of tau granules is marked by a solidwhite arrowhead; free HuD protein (cyan) is marked by an asterisk. Bar,1 µm (×20000 magnification).

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coincided with that of GFP-tagged tau protein (green signal),all of which overlapped to yield white-pink granules, which aredistributed along the axon (filled arrowhead) (Fig. 1Ae).

A merged image at high magnification of the axon and thegrowth cone is shown in Fig. 1B. The size of the small granulesis between 0.3-0.5 µm (20 granules were measured in eachaxon of 18 different cells, P<0.01), and the size of the largergranules is between 0.7-1.5 µm (10 granules were measured ineach axon of 20 different cells). The variation in the size ofthese granules, which include tau mRNA, suggests thepresence of two size groups, which may represent the smallgranules and aggregates of granules attached to the MTs thatwere previously detected in neuronal and oligodendrocyte cells(Knowles et al., 1996; Bassel et al., 1998; Ainger et al., 1997).The previously described small granules contain other mRNAsand exhibit dynamic behavior, whereas the larger granuleswere hypothesized to represent the translationally inhibitedgranules that were recently identified in neuronal primarycultures (Krichevsky and Kosik, 2001).

Free HuD protein (Fig. 1B), seen as cyan dots (marked byan asterisk), and not associated with MTs, may represent anoligomeric structure consisting of the protein alone or boundto other mRNAs that are not detectable by the GFP in situhybridization probe (Tenenbaum et al., 2000). Similaraggregates have been observed in embryonic cortical neuroncell cultures and HeLa cells stained with HelN1 and HuRantibodies, both of which are HuD homologues (Brennan et al.,2000; Gao and Keene, 1996).

The kinesin motor family is involved in the directionalmovement of RNP granules along the MTs (Carson et al.,1997; Severt et al., 1999). To determine whether kinesinexpression affects axon outgrowth and tau mRNA sorting indifferentiated P19 cells, we focused on one member of thekinesin motor family, KIF3A, which is localized primarily inthe axons and is involved in anterograde movement (Kondo etal., 1994). Our experimental data showed increased levels ofKIF3A protein in the differentiated P19 cells, as detected byKIF3A-specific antibodies (data not shown). Immunostainingof differentiated P19 cells with KIF3A-specific antibodiesshowed intense staining in the cell body and axon and fainterstaining in the dendrites. The staining of kinesin proteinshowed colocalization with the tau mRNA (Fig. 2Ad), whichwas detected as pink granules in the axon of the enlargedconfocal image, while no colocalization was seen in thedendrites (Fig. 2Ae). A high magnification image of the axonand growth cone (Fig. 2B) shows granules that contain taumRNA, kinesin and GFP-tau protein distributed along the axonand in the growth cone (white-pink granules). Free kinesinprotein, which did not colocalize with either tau mRNA orGFP-tau protein, is seen as cyan dots along the axon.

The above experiments indicated that tau RNP granules inthe P19 cell line expressing GFP-tau ALS (Aronov et al., 2001)included HuD and KIF3A proteins and prompted us to test thecolocalization of both proteins in the tau mRNA granule. Forthat experiment, non-transfected cells were used (owingto technical limitations of visualizing more than threechromophores). Endogenous tau mRNA was visualized by insitu hybridization using an RNA probe derived from the regionof tau 4 repeats (Fig. 3b) together with HuD (Fig. 3c) andKIF3A (Fig. 3d) antibodies. Colocalization of KIF3A, HuDand endogenous tau mRNA is shown in Fig. 3e and is


Fig. 2.KIF3A protein colocalizes with tau mRNA in RNP granules.(A) Confocal image analysis of KIF3A protein localization indifferentiated P19 cells. (a) Phase image of differentiated P19 cell.(b) Expression of GFP-tau protein. (c) Staining with KIF3Aantibodies. (d) In situ hybridization analysis with the GFP probe(which detects the transfected tau mRNA). (e) Merged confocalimage of b, c and d (×2500 magnification). The solid arrowheadsdenote axons, empty arrowheads denote dendrites and small solidcurved arrowheads denote neuronal cell bodies. Bar, 10 µm.(B) Confocal merged image of a growth cone. The enlarged mergedimage from the cell shown in A. The merged staining includes theGFP-tau fluorescence (green), KIF3A immunostaining (cyan) andGFP in situ hybridization (red), yielding a white colocalization.Aggregation of tau granules is marked by a solid arrowhead. A non-localized KIF3A signal (cyan) is marked by an asterisk. Bar, 1 µm(×20000 magnification).


magnified twice compared with the magnifications shown inFig. 1B or Fig. 2B for HuD and KIF3A, respectively. HuD andKIF3A proteins, which colocalize with tau mRNA, are presentin the axon and growth cone. Quantitative analysis wasperformed on 10-12 fields from four separate experiments,analyzing the size and proportion of tau mRNA granules thatcolocalize with KIF3A or HuD proteins. The size of thegranules ranged between 0.5-0.7 µm (P<0.01), and 57.5% ofthe granules exhibited tau mRNA and HuD (P<0.01), whereas15-22% of the granules exhibited tau mRNA, HuD and KIF3A(P<0.001). These results demonstrated that only a fraction oftau mRNA granules colocalized with the KIF3A motor protein;this fraction may represent the granules that are capable ofmoving in the anterograde direction. Previous experimentsusing live cells imaging have shown that mRNA granules maymove in both the retrograde and anterograde directions (Rooket al., 2000; Zhang et al., 2001). The higher proportion of taumRNA granules colocalized with HuD protein may indicatethe multiplicity of functions that HuD is involved in; thus it isin the core of the granule. In the analysis obtained fromthis experiment, granules containing endogenous tau werevisualized. These comprise about 15-20% of total axonalgranules, as tested by uniform SYTO14 staining (S.A. and I.G.,unpublished). Taken together, these results suggest that bothHuD and KIF3A are components of the tau granules.

The effect of KIF3A antisense ODN treatment ondifferentiated P19 cellsThe effect of KIF3A antisense ODN treatment on thedistribution of tau mRNA was tested in differentiated P19 cells.Differentiated P19 cells were treated with ODN for a period ofup to 30 hours, and the effect of the treatment on the axons isshown in the field view (Fig. 4A) and at the single cell level(Fig. 5). The treatment with kinesin antisense ODN causedaxonal retraction, whereas in control sense ODN-treated cells

Fig. 3.HuD, KIF3A and tau mRNA colocalize in RNP granules.(a) Phase image: the growth cone is shown on the right side of thepanel. Colocalization of tau mRNA (b, red), HuD protein (c, green)and KIF3A (d, cyan) to yield the merged image presented in e of theaxon and growth cone of differentiated P19 cells. The curvedarrowhead denotes colocalization of the three components (white).The asterisk denotes colocalization of HuD and KIF (light green).The straight arrowhead denotes colocalization of HuD and taumRNA (yellow). Bar, 1 µm (×40,000 magnification).

Fig. 4. (A) A field view of differentiated P19 cells treated withKIF3A antisense ODN, analyzed using a confocal micoscope. (a,b)Control untreated cells; (c,d) treated cells; (a,c) GFP-taufluorescence. (b,d) KIF3A protein localization visualized byimmunohistochemical staining with KIF3A antibodies. Bar, 20 µm.(B) Immunoblot analysis of cell extracts prepared from control-differentiated P19 cells or from cells treated with KIF3A antisenseODN and KIF3A sense ODN. The blots were reacted with KIF3A,KIF1A, tau, tubulin, MAP2, synaptophysin and neurofilamentantibodies. (This is a representative blot from three experiments.)

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no axonal retraction was observed,and both GFP-tau and kinesin wereobserved throughout the axons andreached the growth cones (Fig. 4Aa,b).(Growth cones are seen in the upperright hand corners and marked by solidarrowheads; Fig. 4.) The average axonlength of antisense-treated P19 cellsdropped by 3.5-fold to 91.5 µm±13.91,whereas sense-treated cells exhibitedaxon lengths of 346.18 µm±38.15,P<0.05 (20-30 cells were measuredper treatment, in four separateexperiments).

The level of KIF3A protein wasmeasured in total cell extracts preparedfrom untreated differentiated P19 cellsand from cells treated with KIF3Aantisense or sense ODN. The results ofthe western blot analysis (Fig. 4B)demonstrate the effectiveness of theantisense ODN treatment. The level ofKIF3A protein was 35% of controlcells, whereas no effect was observedfollowing treatment with KIF3Asense ODN. The level of KIF1A,neurofilament synaptophysin andMAP2 proteins remained unaffected,suggesting that the treatment is specificto KIF3A. The levels of endogenoustubulin and tau proteins dropped to 70%and 80%, respectively, which mayreflect the retraction of neurites in thetreated cells.

Closer analysis of a single cell treatedwith KIF3A antisense (Fig. 5f-j) orsense (Fig. 5k-o) ODN compared to thecontrol cell (Fig. 5a-e) is shown inFig. 5. Antisense-treated cells haddiminished GFP-tau fluorescent signals(Fig. 5g) and tau mRNA levels (Fig. 5h)as well as axonal retraction. The levelsof GFP-tau protein and tau mRNAlevels reached 50% and 55%,respectively, of sense-treated cells (Fig.5l,m), measured in pixels per unit area,as described in Materials and Methods.Measurements were taken from fourdifferent experiments, and 30 cells wereanalyzed. The in situ hybridizationanalysis with the GFP probe revealedthat the tau mRNA signal is lower in theaxon and in the cell body (Fig. 5h)compared with control cells (Fig. 5c).Moreover, its distribution is altered andis seen in the cell body concentratedclose to the outer membrane (Fig. 5h).The perturbed distribution of tau mRNAcould have been caused by theinhibition of mRNA transport from thecell body to the axon. Previously, we


Fig. 5. Confocal image analysis of a single differentiated P19 cell treated with KIF3Aantisense ODN. (a-e) Differentiated untreated P19 cells. (f-j) KIF3A antisese ODN-treatedcell. (k-o) KIF3A sense ODN-treated cell. (a,f,k) Localization of KIF3A protein, asvisualized by staining with anti-KIF3A antibodies. (b,g,l) Expression of GFP-tau protein.(c,h,m) In situ hybridization with the GFP probe (which detects the transfected taumRNA). (d,i,n) Localization of tubulin protein, as visualized by staining with tubulinantibodies. (e,j,o) In situ hybridization with tubulin. Solid arrowheads denote axons, emptyarrowheads denote dendrites and small solid curved arrowheads denote neuronal cellbodies. Bar, 10 µm.


demonstrated that when tau is not targeted to the axon, reducedlevels of tau mRNA and protein are observed (Aronov et al.,2001). Similar findings have been reported for myelin basicprotein and α-CAMKII mRNAs, when translocation wasdisrupted by treatment with kinesin antisense ODNs inoligodendrocytes and hippocampal neurons, respectively(Carson et al., 1997; Severt et al., 1999). We wondered whetherreduced tau mRNA targeting results in lower tau protein levelsand whether it affects MT stability and/or assembly, leading toreduced tubulin levels. To examine the specificity of the KIF3AODN treatment, cells were stained with tubulin, and in situhybridization was performed using the tubulin probe. The

staining of tubulin shows the retraction of the axon inantisense-treated cell in comparison with sense (Fig. 5n) andcontrol untreated cells (Fig. 5d), although the distributionpattern of tubulin mRNA is retained in the cell body (Litmanet al., 1993), and its intensity is not markedly reduced (Fig.5e,j,o). These results indicate that the reduced level of kinesinmotor protein can disrupt axonal sorting of tau mRNA, whichaffects the distribution and levels of microtubule proteinsinvolved in axonal growth.

To test for the physical association between HuD, kinesinand tubulin proteins, co-immunoprecipitation (IP) experimentswere performed (Fig. 6). Cell extracts were prepared fromdifferentiated P19 cells and immunoprecipitated with HuD,kinesin and tubulin antibodies. The immunoprecipitates wereseparated on SDS gels and analyzed by immunoblotting withthe specified antibodies. The results using the HuDantiobodies (Fig. 6A) demonstrate that kinesin coprecipitateswith HuD protein (IP KIF lane), as indicated by the strongsignal observed. When the presence of HuD protein wastested following immunoprecipitation with tubulin antibodies(IP Tub), a lighter HuD signal was observed, suggesting thatHuD is associated with tubulin but at a lower affinity. Thisconclusion is supported by our previous studies, whichshowed an association between HuD protein and MTs, astested by biochemical assays and by their colocalizationas viewed by confocal microscopy (Aranda-Abreu et al.,1999).

Fig. 6B shows the reciprocal assay in which theimmunoprecipitated complex was immunoblotted withKIF3A antibodies. The results indicate co-immunoprecipitation

of HuD protein with kinesin,suggesting an interactionbetween the two proteins. Whenthe immunoprecipitation wasperformed with anti-tubulinantibodies (IP Tub) and tested forthe presence of kinesin, a strongsignal was observed, suggesting thata strong affinity exists betweenKIF3A and the tubulin. Thissupports previous studies thatdemonstrated kinesin movement on

Fig. 6.Co-immunoprecipitation of HuD, KIF3A and tubulin fromextracts prepared from P19 differentiated cells. (A)Immunoprecipitated complexes analyzed by immunoblotting withHuD antibody. (B) Immunoprecipitated complexes analyzed byimmunoblotting with KIF3A antibody. Non-immune serum (NIS)was used as a control for specific immunoprecipitation.

Fig. 7.Confocal image analysis ofdifferentiated living P19 cell linestreated with emetine, a protein-synthesis inhibitor. (Aa,b) A controldifferentiated cell. The insert showsthe enlargement of the growth coneregion. (c,d) GFP-tau proteinfluorescence in an emetine-treated cell.(e,f) GFP-tau protein fluorescence in acell following a recovery period of 3hours in fresh media. (B) Tau mRNAis found in emitine treated cells.(a) Phase view of a treated cell, whichshows no GFP fluorescence (b). (c) Insitu hybridization with GFP probe.Solid arrowheads denote axons, emptyarrowheads denote dendrites and smallsolid curved arrowheads denoteneuronal cell bodies. Bar, 10 µm.

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microtubules (Hirokawa, 1998). The cumulative results suggestthat an association exists between HuD and kinesin proteins.Furthermore, they indicate that the complex associates withMTs. Our previous results, demonstrating an associationbetween tau mRNA, HuD protein and MTs, together with theresults presented in this study, add KIF3A to the proteincomponents present in the tau RNP granules.

Local translation of tau protein in the axons of living P19neuronal cellsAccording to the multistep localization hypothesis, followingthe targeting of the mRNAs, local translation should occur,which may respond to external signals (Wilhelm and Vale,1993; Zhang et al., 2001). In a previous study we demonstratedthe presence of GFP-tau mRNA, tau proteins and ribosomes inthe axons of differentiated P19 cells (Aronov et al., 2001). Toexamine the local translation of GFP-tau protein, emetine, aninhibitor of the translocation step in protein synthesis, wasadded to the differentiated P19 cells stably expressing a GFP-tau-coding construct, which included the ALS at its 3′ UTR(Fig. 7). Application of emetine for 3-4 hours caused thedisappearance of the GFP-tau fluorescent signal in the cellbody and axon (Fig. 7Ac, Fig. 7Ad). After a recovery periodof 3 to 4 hours, GFP-tau protein fluorescence appeared as ‘hotspots’ along the axon and in the growth cone (Fig. 7Ae, Fig.7Af). After 3 hours of recovery, the intensity ratio, expressedas pixels per unit area and measured in the cell body and theaxon, was 1:2.5. The quantitative analysis was based on a totalof 18 to 30 cells measured at each time point in four separateexperiments.

To exclude the possibility that the GFP-tau protein observedin the axon originated from transport out of the cell body,time-course analysis of the local synthesis was visualized (Fig.8). GFP-tau protein was initially observed along the axon after1.5 hours of recovery, whereas no signal was yet seen in thecell body. The signal was observed in the cell body only 2.5hours later. (The cell bodies are circled in Fig. 8.) Moreover,the intensity of the GFP-tau ‘hot spots’ was stronger in theaxon than in the cell body. The increase in intensity of theaxonal ‘hot spots’, measured as fluorescence pixels per areaunit, was highest (3.5 fold) during the first 30 minutes ofobservation. From 1.5-2 hours and 2-3 hours of incubation,the intensity increased by two- and 1.3-fold, respectively.Recently, using a similar approach in transiently transfectedhippocampal neurons, local translation of dendriticallytargeted GFP-reporter protein was observed. It was suggestedthat the rate of GFP translation was exponential in thedendrites but linear in the cell bodies (Job and Eberwine,2001).

Since emetine affects protein synthesis, we checked whethertau mRNA was still present in cells after 3 hours of treatmentwith emetine (Fig. 7B). Tau mRNA was observed in the cellbody and axon (Fig. 7Bc) when no GFP-tau signal was visible(Fig. 7Bb), which is similar to the localization of tau mRNAin differentiated cells (Fig. 1c).

On the basis of the time frame during which the GFP-tau‘hot spots’ were visualized along the axon and no signal wasobserved in the cell body, these observations are consistentwith local protein synthesis in the axon being dependent ontransported tau mRNA.


Fig. 8. Local GFP tau synthesis in the axon of P19 differentiated cellduring the recovery from emetine treatment. (a) Phase image. (b,e)Recovery periods of 1.5, 2, 2.5 and 3 hours. The cell bodies arecircled. Bar, 10 µm.


Discussion In this study, employing differentiated living P19 cells, weidentified two proteins contained in tau mRNA granules thatare highly concentrated in the cell body, axons and growthcones. The advantage of the P19 system is that it is nottransiently transfected and therefore does not overexpress thestudied mRNA. As such, it may mimic the regulation ofendogenous tau mRNA (Aronov et al., 2001). The identity ofthese granules was indicated by their specific in situhybridization with tau probe, immunostaining with HuD andKIF3A antibodies and colocalization with MTs (Figs 1 and 2).These results are in agreement with our previous demonstrationof in vivo binding of HuD to a specific sequence located in the3′ UTR of tau mRNA and its colocalization with the MTsystem (Aranda-Abreu et al., 1999; Litman et al., 1994).

The identification of HuD protein as a component of the tauRNP granule suggests multiple functions for this protein. It canact as an mRNA-stabilizing protein (Aranda-Abreu et al.,1999) and can also serve as a linker protein to the MT tracksgoing down the axon. Interestingly, recent experiments havesuggested that Hel-N1, a homologue of HuD, controls thetranslation of neurofilament M mRNA in human embryonicterato carcinoma cells through the recruitment of neurofilamentmRNA into the heavy polysome fraction; this recruitment isdependent on sequences located in the 3′ UTR of neurofilamentM mRNA (Antic et al., 1999). This activity fits with the recentdata demonstrating that RNA granules include mRNAs andclusters of ribosomes in a non-translated form, which uponactivation move to the polysome fraction (Krichevsky andKosik, 2001). The Elav protein family was originally identifiedin Drosophilaas being involved in neurogenesis and thus mayfunction in an MT-dependent manner (Antic and Keene, 1998;Aranda-Abreu et al., 1999; Aronov et al., 2001). As wedemonstrated that HuD protein binds directly to a specific cissignal located in the tau 3′ UTR, this may suggest that HuDbinding is among the initial events in mRNA granule assemblyand is also involved in mRNA shuttling from the nucleus to thecytoplasm (Campos et al., 1985; Ma et al., 1997). Moreover,recent data demonstrating that the Elav protein family binds toadditional protein ligands, which do not bind directly to thetargeted mRNA, adds additional insight into their function indiverse signaling cascades in vivo (Brennan et al., 2000).

Studies on β-actin axonal localization in neuronal primarycultures have demonstrated that the accumulation of β-actinmRNA and protein in the axonal growth cones can be inducedby dibutyryl cAMP treatment or stimulated by application ofneurotrophins. This axonal localization depends on theformation of an RNP complex between β-actin mRNA withtwo proteins, ZBP1 and ZBP2, that have been identifiedthrough their specific binding to the axonal localizationsequence within the 3′ UTR of β-actin mRNA. Both proteinshave a role in β-actin mRNA localization, and ZPB2 mostprobably shuttles between the nucleus and the cytoplasm(Bassell et al., 1998; Gu et al., 2002; Zhang et al., 1999; Zhanget al., 2001).

Deciphering which of the motor proteins, belonging to thekinesin or dynein family, translocate the granules along theirspecific tracks – either MTs or microfilaments – is a crucialand unresolved issue (Hirokawa, 1998; Kikkawa et al., 2001;Schnapp, 1999). The specific interaction between these motorproteins and mRNAs may explain the asymmetric mRNA

localization within the neuronal microdomains. These motorproteins use the cytoskeleton for active subcellular mRNAlocalization. Using HuD antibodies as bait for co-immunoprecipitation experiments, we found that KIF3A ispresent in the complex that associates with MTs (Fig. 6).Moreover, as shown previously by RT-PCR analysis, taumRNA was identified in the pellet immunoprecipated by HuDantibodies (Aranda-Abreu et al., 1999). Similarly, tau-mRNAwas identified in the KIF3A immunoprecipitate (data notshown). KIF3A belongs to the kinesin superfamily and hasbeen characterized as an MT-based anterograde motor enrichedin neuronal axons (Hirokawa, 1998; Hirokawa, 2000; Kondoet al., 1994). A complex between the tau 3′ ALS region and aprotein of a similar size to KIF3A was previously detected bya UV crosslinking assay using brain or neuronal cell extracts(Behar et al., 1995).

To examine the possible functional association of tau withkinesin in vivo, we tested the effect of treatment with antisenseODN specific for KIF3A on neuronally differentiated P19cells. The treatment caused retraction of neurites, with a moresevere effect on the axons, where a higher concentration ofKIF3A protein was detected. This treatment caused a specificdecrease of KIF3A protein level in the cell, whereas the levelof KIF1A protein was not affected. There was a concomitantdecrease in tau mRNA levels, as tested by in situ hybridization,and a change in its distribution in the cell body, probablybecause of the inhibition of tau mRNA targeting. Previousstudies have shown that translocation of myelin basic proteinin oligodendrocytes requires MTs and kinesin (Carson et al.,1997; Carson et al., 1998). In another study, inhibition ofkinesin heavy chain expression by ODN treatment of neonatalrat hippocampal neurons inhibited dendritic localization of α-CAMKII. Although we cannot disprove completely the ideathat the effects on tau mRNA and protein localization arecaused by the secondary effect of axonal retraction, thispossibility is less favorable since our preliminary results usingSYTO14 staining of total granules (Knowles et al., 1996) showthat following anti-kinesin ODN treatment, the velocity of thegranules is reduced, whereas their density per unit lengthremains the same (S.A. and I.G., unpublished). Therefore,the current results and the previous observations inoligodendrocytes and neurons indicate the function of kinesinmotor proteins in targeting of mRNA granules to specificcellular microdomains (Carson et al., 1997; Severt et al., 1999).The interaction of the kinesin proteins with MTs may link thegranules to the MT-assisted ATP movement in the plus-end-oriented MTs present in the axons (Kikkawa et al., 2001).Previous studies have shown that transport by the KIF3Acomplex exhibits a velocity of 0.3 µm/second and that itsassociation with vesicles in rat axons is involved in fast axonaltransport (Hirokawa, 2000). This may imply its involvement inaxonal sprouting events by supplying the required material(Takeda et al., 2000).

We observed two major classes of granules in our studies,which may represent small granules and aggregates ofgranules, both of which are mostly concentrated toward thelower region of the axon and in the growth cones. Thecalculated size of the observed small granules was between 0.3and 0.5 µm in diameter, similar to the size observed in neuronalcells and in lamellopodia of fibroblasts (Kohrmann et al., 1999;Krichevsky and Kosik, 2001; Latham et al., 1994). These

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granules are smaller than the calculated size of localizedparticles in oligodendrocytes [0.7 µm in diameter (Barbareseet al., 1995)], suggesting that the size may vary among differentcells and that these particles can accommodate many proteinsand mRNA molecules. The big granules, which may reach asize of 0.7-1.1 µm, may represent the densely packed RNAgranules that have recently been characterized in primaryneurons. It was suggested that they include components of theprotein translational apparatus and clusters of ribosomes,which are in a translationally inhibited state and can beactivated by external signals, leading to local protein synthesis(Krichevsky and Kosik, 2001).

Following the targeting of mRNA granules to their specificmicrodomain, local translation can ensue, as predicted in themultistep localization pathway hypothesis (Wilhelm and Vale,1993). We showed the presence of ribosomal proteins in P19axons, and the presence of additional protein synthesiscomponents of the translational machinery has beendemonstrated in the dendrites and axons of developing neuronalcells (Bassell et al., 1998; Crino and Eberwine, 1996; Zhang etal., 1999). In addition, focal centers of local translationalactivity have recently been shown along mammalian axons(Koenig and Giuditta, 1999; Koenig et al., 2000).

In this work, blockage of protein synthesis by emetine, aninhibitor of the translocation step of protein synthesis, causedthe disappearance of GFP-tau protein from the axon, althoughtau mRNA was detected in the cell body and axon (Fig. 7B).After releasing the cells from inhibition and following arecovery period, the presence of GFP-tau protein in the axonin the form of ‘hot spots’ – indicating local protein synthesis– was observed. The initial presence of the GFP signal in theaxon after 1.5 hours was later followed, after 2.5 hour interval,by its appearance in the cell body, demonstrating that GFP-taulocal synthesis in the axon occurs independently from the cellbody.

Local protein synthesis has recently been demonstrated indendrites of neuronal cells and in isolated dendriticpreparations, following recovery from inhibition of translationor after stimulation by pharmacological or neurotrophicfactors. In all these experiments, the appearance of newlysynthesized proteins has been on a similar time scale to theresults observed in this study, which represent local mRNAtranslation (Akalu et al., 2001; Eberwine et al., 2001; Job andEberwine, 2001; Kacharmina et al., 2000). A recent study,which lends further support to the idea that growth conesexhibit a remarkable amount of cellular autonomy, showed thatisolated Xenopus retinal neurons contain the protein synthesismachinery required for local protein synthesis in the growthcones in response to chemotrophic stimuli (Campbell and Holt,2001; Eberwine, 2001). The translocation of both tau and β-actin mRNAs to the axon, a movement which is MT dependent,suggests that growth factors can exert their effects by signalingthrough the MT system and further supports the involvementof the dynamic cytoskeleton in neuronal function.

Identification of the protein components present in the RNPgranules may lead to a better understanding of their functionin the movement of the granules and their regulation duringneuronal differentiation and growth cone outgrowth. Some ofthe identified proteins may belong to conserved proteinfamilies or, alternatively, may share conserved elements in theirstructure that are involved in the localization of diverse mRNA

species. The granules may contain additional trans-actingprotein factors, which are less abundant, and may contribute tothe specificity of movement in various cell systems, and/or atdifferent stages of neuronal development. Further studies onthe specificity of KIF3A motor protein in mRNP translocationwill lead to identification of their multiple cargoes inanterograde microtubule-based function in the axon.Additional characterization of tau RNP granule componentsand the kinetics of their movement is currently underway inour laboratory.

This work was supported by grants from the Minerva Foundation,Germany, the Nella and Leon Benoziyo Center for Neuroscience,WIS, Grant No1999149 from the United States-Israel BinationalScience Foundation (BSF), Jerusalem, Israel. I.G. is the incumbent ofthe Sophie and Richard S. Richards Professorial Chair in CancerResearch. The authors wish to thank I. Nevo for editing themanuscript.

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Tau RNP granules and synthesis in axons - Journal of Cell Science

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