heterotrimeric g-proteins associate with microtubules during differentiation in pc12...

Heterotrimeric G-proteins associate with microtubulesduring differentiation in PC12 pheochromocytomacells

TULIKA SARMA,* TATYANA VOYNO-YASENETSKAYA,† THOMAS J. HOPE,‡

AND MARK M. RASENICK*,§,1

Departments of *Physiology, *Biophysics and *,§Psychiatry, Department of †Pharmacology,Department of ‡Microbiology and Immunology, University of Illinois at Chicago, College ofMedicine, Chicago, Illinois, USA

ABSTRACT Tubulin modifies G-protein signalingand heterotrimeric G-proteins regulate microtubuleassembly. Here we report an interplay among G-pro-tein-coupled receptor and receptor tyrosine kinase(such as nerve growth factor–NGF) signaling systems inPC12 pheochromocytoma cells that resulted in a trans-location of G�s, G�i1, and G�o from cell bodies tocellular processes where they appear to localize withtubulin-containing structures. This relocation appearedto depend on the integrity of microtubules, as it wasblocked and reversed by nocodazole. Latrunculin,which promotes actin filament depolymerization, hadno effect. Both deconvolution microscopy and immu-noprecipitation showed a significant increase of G�

association with microtubules that was coincident withthe extension of “neurites.” There were distinctionsamong the G� subtypes, with G�s showing the mostprofound NGF-induced colocalization with tubulin.Translocation of G� was blocked by agents that inhibitthe MAP kinases required for neuronal differentiation,suggesting that G-protein relocation is triggered by theintracellular signals for differentiation. Consistent withthis, G� in Neuro-2A cells, which spontaneously differ-entiate, showed a similar translocation coincident withdifferentiation. Thus, diverse signals that promote neu-ronal differentiation and changes in cell morphologymay use specific G-proteins to evoke cytoskeletal re-arrangement.—Sarma, T., Voyno-Yasenetskaya, T.,Hope, T. J., Rasenick, M. M. Heterotrimeric G-proteinsassociate with microtubules during differentiation inPC12 pheochromocytoma cells. FASEB J. 17, 848–859(2003)

Key Words: tubulin � G-protein � NGF � cytoskeleton � growthcone

G-proteins act to transfer signals from cell surfacereceptors to intracellular effector molecules. Activatedreceptors catalyze the exchange of GTP for GDP on theG-protein � subunit. Subsequently the activated G� andG�� are able to activate intracellular effector molecules,such as adenylyl cyclase or phospholipase C. There is aconvergence of G-protein-coupled receptors and ty-

rosine kinase receptors on mitogenic signaling path-ways (1–5).

Nerve growth factor (NGF) acts on receptors withtyrosine kinase activity to differentiate PC12 cells into a“neuronal” phenotype (6–10). Binding of NGF to thetyrosine kinase receptors in PC12 cells is known tostimulate three main signaling pathways: MAP kinase(ERK1/2), PI3-kinase/Akt, and PLC-�1 (11, 12).

Purinergic P2Y2 receptors, which are G-protein-cou-pled, stimulate PC12 cell MAP kinase activity through apathway distinct from the classical RTK-Ras-Raf-MAPKcascade (13, 14). Related adhesion focal tyrosine kinase(RAFTK, also called PYK2 and CAK�) and proteinkinase C � are involved in mediating the MAP kinaseactivation by UTP. UTP causes increased tyrosine phos-phorylation of multiple proteins in PC12 cells that arecommon for growth factor and G-protein receptor-mediated signaling. This is sensitive to pertussis toxinand thought to be mediated by G�i (14).

Although the generation of intracellular signals bytyrosine kinase receptors has been investigated inten-sively, regulation of signaling and trafficking events isstill not well understood. Many studies have revealed afunctional relationship between tubulin and varioussignaling molecules. A series of studies has demon-strated direct binding between various G� and G��

subunits and tubulin (15–18), suggesting new modes ofregulation for microtubule assembly. Other studiesprovide examples of tubulin or microtubules regulatingeither the activity or the localization of signaling mol-ecules such as the �1 adrenergic receptors (19), phos-pholipase C-�1 (20), and Ki-Ras (21).

It has become increasingly clear that G-proteins arefound in cellular compartments other than the plasmamembrane. Heterotrimeric G-proteins have been im-plemented in membrane trafficking (22) and havebeen seen in the nucleus (23). Some have been seen inthe cytosol, and this appears to be increased uponG-protein activation (24–27). The relationship of het-

1 Correspondence: Department of Physiology and Biophys-ics, University of Illinois at Chicago, 835 S. Wolcott M/C 901,Chicago, IL 60612-7342, USA. E-mail: [email protected]

848 0892-6638/03/0017-0848 © FASEB

erotrimeric G-proteins to cell growth and differentia-tion is not well established.

This study compares the effects of activation ofreceptor tyrosine kinases with G-protein-coupled recep-tors with regard to altered distribution of G� subunitsand the association of those G-proteins with tubulin ormicrotubules. The role of G-protein relocalization tothe newly forming processes and growth cone-likestructures was investigated. Stimulation of tyrosine ki-nase and P2Y2 receptors and the interaction betweenG-proteins and microtubules may serve to direct theformation of cellular processes. The G-protein associa-tion with the microtubule cytoskeleton may regulatelocalization of those signaling proteins, thereby provid-ing a putative link between receptor tyrosine kinase andG-protein-mediated signaling pathways.

MATERIALS AND METHODS

Cell cultures

PC12 cells and SK-N-SH (ATCC) were grown in 75 cm2 tissueculture flasks (Falcon, Becton Dickinson, Oxnard, CA, USA)at 37°C in a 5% CO2 humidified atmosphere in Dulbecco'smodified Eagle's medium (DMEM; Cellgro, Mediatech, Hern-don, VA, USA) supplemented with 4.5 g/L glucose, 10%bovine calf serum (Hyclone, Logan, UT, USA), and 100�g/mL penicillin/streptomycin (Gibco BRL, Life Technolo-gies, Gaithersburg, MD, USA). Neuro-2A cells (ATCC) werepropagated in Dulbecco’s minimal essential medium (Cell-gro, Mediatech) containing 10% fetal bovine serum (Hy-clone) and amino acid. For immunofluorescence studies,cells were plated on coverslips and cultured overnight in theabove media. Cells were plated at a density of 2 � 106

cells/cm2. Where indicated, cells were starved overnight inDulbecco’s minimal essential medium containing 0.5% fetalbovine serum, then stimulated with medium supplementedwith 50 ng/mL 7S NGF (Alomone Laboratories, Jerusalem)or 100 �� UTP (Sigma, St. Louis, MO, USA) for 3 days andresupplemented every other day. NGF stimulated cells wereadditionally treated with 8 �g/mL concanavalin A (Sigma) inorder to flatten the cellular processes. For nocodazole(Sigma) treatment (10 �M), the drug was added 30 or 90 minbefore fixation or cell extraction. For PD (Sigma) treatment(10 �M), cells were treated with PD98059 for 1 h beforeaddition of NGF to the growth medium. Cells were incubatedwith NGF-PD98059 for 3 days before fixation or cell extrac-tion. Cells were treated with 2.5 �M latrunculin B (BIOMOLResearch Laboratories, Plymouth Meeting, PA, USA) for 30min before fixation.

Immunocytochemistry

PC12 cells were plated on 12 mm coverslips in 12-well tissueculture plates. They were washed twice with PBS (180 mMNaCl, 10 mM sodium phosphate buffer, pH 7.4) before andafter permeabilization. For cell surface staining, NGF-treatedcells were first surface-labeled for 30 min at 4°C by addition oftetramethylrhodamine isothiocyanate (TRITC) -conjugatedwheat germ agglutinin (WGA) (1:100, Molecular Probes,Eugene, OR, USA) in PBS containing 0.2% gelatin). FreeWGA was removed by five washes with ice-cold buffer. Cellswere fixed in methanol for 3 min at –20°C. After washing 3 �10 min in PBS with 0.1% Triton X-100 cells were incubated

for 20 min with blocking buffer (5% nonfat dry milk in PBS),followed by 3 � 10 min washes in PBS. The coverslips wereincubated for 40 min with monoclonal anti-�-tubulin, cloneDM 1A, or phalloidin-TRITC (Sigma), washed 3 � 10 min inPBS with 0.1% Triton X-100, and incubated for 30 min with10 �g/mL goat anti-mouse rhodamine-conjugated antibody(Pierce, Rockford, IL, USA) in PBS. Unbound antibody waswashed out with 0.1% Triton X-100 in PBS 3 � 10 min. Todemonstrate colocalization of �-tubulin or F-actin with G-protein subunits, anti-G�s, G�i1, and G�� rabbit polyclonalantibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA)were used. After the above �-tubulin/anti-mouse rhodamineincubations, the cells were incubated for 1 h with the secondprimary antibody at the appropriate concentration (15 �g/mL, 15 �g/mL, 10 �g/mL) in PBS. After 3 � 10 min washeswith 0.1% Triton X-100 in PBS, the cells were incubated for30 min with 10 �g/mL donkey anti-rabbit fluorescein-conju-gated antibody (Pierce), washed three times with 0.1% TritonX-100 in PBS, and finally once in PBS. Stained cells weremounted onto slides with Vectashield (Vector Laboratories,Burlingame, CA, USA) and viewed under a Zeiss LSM510laser-scanning confocal microscope (Zeiss, Oberkochen, Ger-many).

Deconvolution microscopy

Images were captured with the Applied Precision (Seattle,WA, USA) DeltaVision system built on an Olympus IX-70base. Z-stacks were deconvolved using the softworx software.Sections were captured every 200 nm. Typically, 15 iterationsbased on a measured point spread function, calculated from1 �M fluorescent beads, were used; 24 images from controland NGF-stimulated cells were counted by three differentobservers blind to the experimental conditions. The totalcontent of G� was measured by counting the number of green“dots” in each randomly selected image on a monitor. Thenumber of G� clusters associated with microtubules wasdetermined in a separate count. G� immunofluorescence“touching” microtubules was carefully revealed by computer-driven rotation of the images. Agreement among the threeobservers varied by 6% or less.

Immunoblot analysis

Tissue culture medium was removed and nonadherent cellswere washed off with PBS. Cells were solubilized by scrapingwith ice-cold solubilization buffer (1 mM EDTA, pH 7.4, 20mM HEPES, pH 7.4, 2 mM MgCl2) containing proteaseinhibitors. Cells were passed through a 26-gauge needlesyringe 15 times on ice to lyse and homogenize the cells. Thelysate was centrifuged at 1000 � g for 5 min at 4°C to pelletunbroken cells and nuclei. Protein concentration was deter-mined by the method of Bradford (Bio-Rad Laboratoriesprotein assay, Hercules, CA, U SA). Whole cell extractscontaining equal proteins were separated by SDS-PAGE(PAGErTM Gold Precast Gels, BioWhittaker Molecular Appli-cations, Rockland, ME, USA) using 10% gels and transferredto PVDF membranes (Millipore; Bedford, MA, USA) asdescribed previously (28). Filters were blocked for 1 h atroom temperature with 5% nonfat dry milk in Tris-bufferedsaline (50 mM Tris-HCl, pH 7.4, 200 mM NaCl) and incu-bated overnight with primary antibodies (polyclonal rabbitantisera against the various G-protein subunits from SantaCruz Biotechnology) G�s, G�i1 , and G�o or �III-tubulin(Sigma) at a 1:1000 dilution. The PVDF membranes werewashed five times for 5 min with Tris-buffered saline supple-mented with 0.1% Tween-20, followed by an 1 h incubationwith the appropriate peroxidase-conjugated secondary anti-

849G-PROTEINS AND THE CYTOSKELETON

body (1:10,000), (Jackson ImmunoResearch, West Grove, PA,USA). The filters were washed five times for 5 min withTris-buffered saline supplemented with the same detergent,and developed by chemiluminescence using ECL (AmershamPharmacia Biotech, Uppsala, Sweden). Bands on X-ray filmwere quantified by laser densitometry. Filters were occasion-ally subjected to stripping and reprobing according to themanufacturer's instructions.

Coimmunoprecipitation of G� subunits and �-tubulin fromPC12 cells

PC12 cells were treated with 10 �M nocodazole for 30 minand with 50 ng/mL NGF for 3 days in DMEM supplementedwith 4.5 g/L glucose, 10% bovine calf serum, and 100 �g/mLpenicillin/streptomycin. Cells were washed twice before lysisin 500 �L of lysis buffer (50 mM HEPES, pH 8.0, 50 mMNaCl, 0.5% lubrol, 1 mM EDTA, 5 mM MgCl2 , 1 mM DTT)containing protease inhibitors. Cells were passed through a27-gauge needle 10 times. After centrifugation for 10 min at14,000 rpm in a bench-top centrifuge, the supernatant wasmixed with 5 �L of rabbit polyclonal anti-G� subunit/anti-body and 40 �L of 50% slurry of protein A/agarose (GibcoBRL, Life Technologies, Gaithersburg, MD, USA), followedby an overnight incubation at 4°C. The next day agarose waspelleted and washed three times with 500 �L of lysis buffereach time. The agarose was then resuspended in 30 �L of 2�sample buffer and separated by SDS-PAGE, followed byimmunoblot using monoclonal anti-�-tubulin antibody(Sigma) with 1:1000 dilution.

Online supplemental material

Representative examples of G�i1 association with microtu-bules and the induction of this interaction to NGF stimula-tion are viewable as Quicktime movies. Videos 1 and 2accompany Fig. 3Aa and Fig. 3Ba. Microtubules are shown inred, G�i1 in green; 3D rotational analysis was performed withDeltavision system software.

Figure 1 shows the distribution of a surface-labeled TRITC-conjugated wheat germ agglutinin that does not associatewith tubulin. NGF-treated cells were costained using TRITC-conjugated wheat germ agglutinin (red) and anti-�-tubulinantibody (green) and processed for deconvolution micros-copy. Insert indicates higher magnification of the cell processhighlighted by square to show no colocalization of themembrane and fluorescein-labeled tubulin. Scale bar repre-sents 15 �m.

RESULTS

Subcellular distribution of endogenous G�i1 , G�s,and G�o subunits in resting PC12 cells

To explore the subcellular distribution of G-proteins,immunofluorescence confocal microscopy was per-formed (Fig. 1A). In untreated PC12 cells, G�i1 , G�s,and G�o appeared to be distributed throughout thecytoplasm (Fig. 1Ab, e, h). G�s and G�o showed anintense plasma membrane localization, with uniformstaining across the cell surface (Fig. 1Ae–f, h–I, arrow-head). G� protein staining was most pronounced in theintracellular perinuclear region and coincided withregions of high microtubule density (Fig. 1B). G�i1 and

G�s, but not G��, were present in nuclei of the restingPC12 cells. Z-stack analysis of the fluorescence pattern(Fig. 1B) confirmed this.

Given the evidence that tubulin and G-proteins,particularly G�i1 and G�s, have a functional interactionand the observation that G-protein subunits affect theregulation of microtubule assembly (16, 18), we lookedfor colocalization of all three G� subunits with tubulinin PC12 cells. Filamentous cytoplasmic tubulin distrib-uted throughout the untreated PC12 cell cytoplasm wasobserved (Fig. 1Aa, d, g). Images in Fig. 1A represent 1� optical sections. Double labeling revealed a signifi-cant overlap in the distribution of G�i1 subunit withmicrotubules, whereas G�s and G�o showed a spottyregional codistribution with �-tubulin (Fig. 1A, com-pare c with f, i). Note the intense colocalization at aperinuclear ring-like structure (Fig. 1B) and no colo-calization at nucleus and membrane.

Stimulation with nerve growth factor results in adifferential redistribution of G� proteins

PC12 cells were treated with 50 ng/mL NGF for 3 days.Cytoplasmic tubulin redistribution was observedthroughout the cells (Fig. 2a, d, g). Treatment withNGF for 3 days translocated each of the G� subunits tothe newly formed cellular processes and their termi-nals. G� subunits appeared to be more concentrated inmultiple filopodia as well as at the very tip of the growthcone-like extensions (Fig. 2b–c, e–f, h–i, arrow). �-Tu-bulin and G� proteins revealed an increased colocaliza-tion primarily in the processes along the microtubulenetwork (Fig. 2c, f, i). There was no noticeable changein G� distribution after 1 day of treatment with NGF.

NGF promotes association of G� subunits withmicrotubules in PC12 cells

Although confocal observations and results of immuno-precipitation indicated that G-proteins are associatedwith tubulin in PC12 cells, it was not clear whetherG-proteins were bound to microtubules. To test this, weexamined deconvolved images. Three-dimensional(3D) deconvolution microscopy improves experimentperformance and image appearance by mathematicallyremoving the out-of-focus effects common to opticallight microscopy. We examined large, well-spread PC12cells for possible colocalization of G-protein with mi-crotubules (Fig. 3A). As expected, G� proteins werelocalized in the cell body and on the cell surface or atthe border of microtubules in control cells. However,some G-proteins were randomly distributed along thelength of microtubules. We focused our attention onthose optical cell sections where microtubule networkscan be neatly visualized. We tested the localization ofG-proteins after 3 days of NGF exposure (Fig. 3B).Colocalization between tubulin and G� was diminishedin the perinuclear region, since G� translocated sub-stantially to the growth cone tip (Fig. 2, Fig. 3D).However, G� proteins showed an apparently high de-

850 Vol. 17 May 2003 SARMA ET AL.The FASEB Journal

gree of colocalization with tubulin in the cellularprocesses induced after NGF treatment (Fig. 3). Thisappeared to be similar for each G-protein examined.Analysis of the microtubules at higher magnificationfrom the box region in Fig. 3B showed direct associa-tion of G-proteins with microtubules in the cellularprocesses. Images of 0.2 �m optical, planar sectionscontaining microtubule networks taken from four ran-domly selected control and NGF-treated intact cellswere quantified. Total G-protein was measured by

counting the number of “dots” in the specified region.The percentage of G� protein juxtaposed with micro-tubules of control vs. NGF-treated cells was compared.G�i1 showed the greatest degree of microtubule associ-ation in the resting state and NGF increased this by73%. This is a marked contrast to G�s and G��, forwhich NGF increased colocalization by 140% and 163%compared with the respective controls (Fig. 3C); 3Drotational analysis was used to confirm an increase inassociation of G� with microtubules subsequent to NGFtreatment (Fig. 3video1.mov; Fig. 3video2.mov).

Microtubule depolymerization decreases G�–tubulinassociation in resting PC12 cells

Microtubules are important for the extension of cellu-lar processes (29–31); thus, we wondered whethermicrotubule depolymerization would effect G� proteinlocalization and its association with microtubules. Cellswere treated with nocodazole for 30 and 90 min andcostained with monoclonal anti-�-tubulin and G� sub-units before processing for confocal or deconvolutionmicroscopy. Nocodazole caused profound changes inthe distribution of cytoplasmic G� subunits and micro-tubules. After 30 min of nocodazole treatment, the G�

subunits were dispersed in the cytoplasm and redistrib-

Figure 1. Subcellular distribution of endogenous G�i1, G�s,and G�� subunits and their association with �-tubulin in PC12cells. Aa–i) PC12 cells were costained using anti-�-tubulin (a,d, g) and anti-G� antibodies (b, e, h) and processed forconfocal microscopy. G�i1 is microtubule-associated, whereasG�s and G�� are colocalized with �-tubulin in the perinuclearregion. 5 experiments were done for each G� antibodystaining. Panels e, f, h, i show the membrane localization ofG�s and G�� respectively (arrowheads). Filamentous cytoplas-mic tubulin distribution in panel g represents a 1 � opticalsection (#4) through the cell, whereas panels a, d showsection #5. c, f, i) Yellow, areas of overlap in merged images.Ba– c) A 1 � optical slice through the nucleus that demon-strates intranuclear localization of G�i1 and G�s and theabsence of G�o in the nucleus. Several 1 �-thick optical slicesfrom 17 cells were examined. 99% of nuclei containedpunctate staining of G�i1 and G�s. All three G� subunitscolocalize with tubulin in the perinuclear region. Scale bars,10 �m.

Figure 2. Nerve growth factor alters the subcellular localiza-tion of G� subunits. PC12 cells were treated with 50 ng/mLNGF. Cells were costained using anti-�-tubulin (a, d, g) andanti-G�i1 (b– c), anti-G�s (e–f ) or anti-G�o (h–i) antibodies andprocessed for confocal microscopy. NGF stimulates the trans-location of G� subunits to the newly formed neurites. Strongimmunofluorescent staining is observed with G� subunits atthe cell growth cones (b– c, e–f, h–i, arrow). c, f, i) G-proteinsare colocalized with �-tubulin in the processes along themicrotubule network. Yellow indicates areas of overlap inmerged images. 5 experiments were done for each G-proteinantibody staining. Scale bars, 10 �m.


uted to specific sites around the nucleus. After removalof the drug, scattered G-proteins moved back to thecytoplasmic region (data not shown) and reunited intoa continuous system similar to the images presented inFig. 1Aa, d, g. When cells were treated for 90 min, G�

subunits became more tightly clustered near the nu-cleus (Fig. 4Ab, f). One or multiple bright immunoflu-orescent foci staining were observed around the nu-cleus in some cells (Fig. 4Aj). The localization oftubulin was seen to change from a filamentous patternto punctate structures by 90 min (cf. Fig. 1A and Fig.4A). Nocodazole treatment led to a strong accumula-tion of G� proteins in both the perinuclear and blebregions, where the depolymerized tubulin was localized(Fig. 4Ac–d, g, h, l). However, the nuclear presence ofG�i1 and G�s was unaffected by nocodazole. There wasdiminished staining of all three G� proteins subsequentto nocodazole treatment.

Microtubule depolymerization reverses NGF-inducedG� translocation to microtubules in cellular processes

As these results support the hypothesis that NGF in-duces association of G-proteins and microtubules incellular processes, we wondered whether microtubuledepolymerization would reverse this process in differ-entiated cells. NGF-treated cells were exposed to 10 �Mnocodazole, and cell morphology was analyzed at dif-ferent times after addition. Before nocodazole treat-ment, 95% of the differentiated cells showed longprocesses. Neurite retraction was time dependent, andthe extended processes were almost completely re-tracted 1–2 h after nocodazole addition. At that time,80–90% of cells were rounded and showed only bluntcytoplasmic extensions. Analysis of the microtubulenetwork during the growth cone collapse and neuriteretraction process showed that the polymerized tubulin

Figure 3. G� subunits are associated with micro-tubules. A, B) Cells were costained using anti-�-tubulin and anti-G� antibodies and processedfor deconvolution microscopy. Images werecaptured with the Applied Precision DeltaVi-sion system built on an Olympus IX-70 base.Insert indicates higher magnification of cellareas highlighted by squares to show codistribu-tion of rhodamine-labeled tubulin and G� flu-orescence (FITC antibody). Panels d–f are anenlarged threefold compared with panels a– c.Aa–f) Deconvolution microscopy of controlPC12 cells shows G-protein � subunit localiza-tion on the surface of the microtubules. Ba–f)Nerve growth factor promotes a dynamic redis-tribution of G� toward the cell processes. PC12cells were treated with 50 ng/mL NGF. Decon-volution microscopy of NGF-treated PC12 cellsshows direct binding of G� and microtubules inthe cell processes. Scale bars, 5 �m. C) The %increase in G� proteins associated to microtu-bules after NGF treatment. The number of G�

protein bound to microtubules is counted sep-arately (see Materials and Methods). The % ofG� protein attached to microtubules of controlvs. NGF-treated cells were compared. Asterisksindicate statistical significance between controland NGF-treated from each G� protein exam-ined (paired Student’s t test, two-tailed P value,*0.05; **0.01). There is no significant dif-ference among the 3 controls or the 3 NGF-treated samples compared with each other(Bonferroni). Da– c) G� subunits are concen-trated at the growth cones in differentiatedcells. PC12 cells were treated with 50 ng/mLNGF. Nerve growth factor promotes a dynamicredistribution of G-protein subunits to the tipsof the extending neurites, growth cones. Scalebar represents 15 �m.


was disorganized 30 min after nocodazole addition(Fig. 4Ba, e, i); 90 min after nocodazole addition,tubulin was no longer in the axon-like extension buthad accumulated in the base of the retracted processes,correlating with the morphological retraction shown inFig. 4A. Thus, microtubule depolymerization effectivelyeliminated the formation of filamentous outgrowths inPC12 cells after NGF pretreatment. G� subunits movedback toward the perinuclear region. Confocal immuno-fluorescence examination of all three G� proteinssuggested a colocalization with �-tubulin in the regionaround the nucleus and at the tips of the growth cones(Fig. 4Bc, g, k). Analysis by deconvolution microscopy ofthe cells from five different experiments confirmed theassociation of G-proteins with tubulin in these regions(Fig. 4Bd, h, l). Thus, depolymerization of microtubulesin NGF-treated cells dispersed G� in a manner similarto that of control cells.

G� subunits show minimal interaction with actin

To confirm the specificity of microtubules and G-protein interaction, we performed immunostaining forfluorescent phalloidin. The actin cytoskeleton visual-ized at the cell bottom formed well-organized, parallelfilaments that extended into the cell cortex with uni-form staining across the cell surface (Fig. 5Ad). Athigher planar sections, F-actin filaments were markedlyreduced, being more diffuse and decreasing in boththickness and length. G-proteins were mostly diffuselydistributed in the cell body at the higher sections of thecell and did not localize to actin (Fig. 5). Unlike thesituation with G� subunits and tubulin, minimal colo-calization between three of the G� subunits and actinwas observed in F-actin-rich lamellopodia (Fig. 5A,arrow). As these observations suggested that heterotri-meric G-proteins have little interaction with the actincytoskeleton, we treated the cells with latrunculin B, acompound altering F-actin polymerization. PC12 cellstreated with latrunculin B for 30 min acquired anelongated, polarized shape with narrow, finger-likeprojections along the cell edge. G-proteins were oftenenriched in those cell-edge projections (Fig. 5B).Changes in overall cell morphology induced by latrun-culin B were accompanied by a decrease in the intensityof actin staining. Actin fibers became small patches ofpunctate actin clustered at certain focal adhesion-likesites. Treatment with latrunculin had no noticeableeffects on both overall distribution of G-protein and itsassociation with microtubules (Fig. 5B).

NFG increases tubulin-G� complex formation inPC12 cells

To confirm that NGF increased the interaction betweenG� and tubulin, biochemical evidence was also needed.The expression of G� was not altered by NGF treatmentand the amount of tubulin remained constant (Fig. 6A,B). Anti-G� antibodies coprecipitated G� and �-tubulinfrom PC12 cell extracts treated with either NGF, no-

Figure 4. Microtubule depolymerization alters the subcellularlocalization of G� subunits. Aa–l) PC12 cells were treated with10 �M nocodazole for 90 min. Cells were costained usingmonoclonal anti-�-tubulin (a, e, i) and anti-G� antibodies (b,f, j) and processed for confocal microscopy (3 columns onleft). Nocodazole causes the cells to assume a rounded shape.Nocodazole redistributes G� proteins toward the nucleus (b, f,i). G� subunits colocalize with �-tubulin at the centrosomalregion and form blebs at the cell periphery. Yellow indicatesareas of overlap in merged images (c, f, k). d, h, l) Deconvo-lution microscopy of nocodazole-treated cells. G� proteinsremain associated with tubulin in both perinuclear and blebformations. B, a–l) PC12 cells were first treated with NGF for3 days, then with nocodazole. Nocodazole truncates theNGF-induced cellular processes and causes a perinucleardistribution of tubulin. a, e, I) G� subunits move toward theperinuclear region and the collapsing growth cones (b, f, j). c,g, k) NGF-induced processes retract; G� subunits are colocal-ized with �-tubulin at the growth cones and in the perinuclearregion but not in the truncated cellular processes. Yellow,areas of overlap in merged images. d, h, l) Deconvolutionmicroscopy of nocodazole-treated differentiated cells showsthat G� proteins remain associated with tubulin in bothperinuclear and growth cone areas. These staining patternsare representative of 6 individual experiments. Scale barrepresents 5 �m in panels A, Bd, h, l; 10 �m in panels A, Ba– c,e–g, I–k.


codazole or both agents (Fig. 6C). Tubulin coimmuno-precipitates with all three G� subunits in cells whetheror not they are NGF treated (Fig. 6C, left). Nocodazoledid not disrupt the association of tubulin with G�

proteins (Fig. 6C, lanes 2 and 4). Actin was not coim-

munoprecipitated with G� antibodies under these sameconditions. These results were consistent with the colo-calization data (Figs. 1, 2). NGF treatment increasedcoimmunoprecipitation of tubulin with G-proteins, sug-gesting stabilization of G�–tubulin interaction. The

Figure 5. Actin depolymerizationdoes not alter G� distribution. Con-trol (A) or latrunculin-treated (B)PC12 cells were immunostainedwith G� antibodies and phalloidin-TRITC for actin and processed fordeconvolution microscopy. A) Co-localization between G� subunitsand F-actin was observed only inthe cell membrane. B) Treatmentwith latrunculin B did not changethe distribution of G� subunits.

Figure 6. NGF does not alter the expression ofG� subunits or tubulin, but NGF does increasethe formation of G�-tubulin complex. A) Ex-pression of G� and tubulin. Western blots wereperformed with antibodies against the indi-cated G� or against �-tubulin. Antibodies ap-pear specific and show little difference amongthe treatment groups. B) Quantitation of G� ortubulin expression and the effects of NGF.Western blots as in panel A were quantified byPhosphorImaging. NGF (3 days) and controlcells were composed from 7 experiments. NGFshows no significant effect on G� or tubulinexpression (column statistics). C) Coimmuno-precipitation of G� and tubulin. Cells werelysed and immunoprecipitated using G�i1 , G�o,and G�s antibodies. Immunoprecipitated pro-teins were subjected to SDS-PAGE and immu-noblotting with �-tubulin antibody to detectcoimmunoprecipitation of G� subunits with tu-bulin. The results are representative of 7 indi-vidual experiments for each protein.


NGF-induced increase in coimmunoprecipitation oftubulin and G� ranges from 31 to 69%.

UTP stimulation alters the subcellular localization ofG-proteins and increases its association withmicrotubules in the cell

Treatment with UTP for 5 days enhanced the propor-tion of cells displaying neurite-like processes and, incontrast to NGF, evoked slowly developing processeswith multiple branching at the tips (Fig. 7). The shapesshown were those that represent the transition in formfrom flattened to a monopolar morphology. Continu-ous stimulation of P2Y2 receptor exerted a stronginfluence on cytoskeletal organization and dynamics.PC12 cells displayed large numbers of microtubulesextending into the outgrowths formed by the cells (Fig.7A). G-proteins were preferentially localized on thesurface or edges of microtubule filaments (Fig. 7Ad–f ).An increased association of G-proteins similar to effectsof NGF was observed with microtubules in the cellularprocesses as well as in the cell body (Fig. 7B). Thepercentage of punctate G� “dots” touching microtu-bules of control vs. UTP-stimulated cells was compared.UTP increased G�s and G�o association with microtu-bules by 124% and 146%, in contrast to G�i1 , whereG�–microtubule association increased by 73% (notstatistically significant). These values were quite similar

to those seen for NGF stimulation. Pretreatment withpertussis toxin blocked this association with microtu-bules, suggesting that the effects of UTP are mediatedby Gi or Go rather than Gq. Pertussis toxin blocked thephenotypic changes of the differentiated cells. About50% of cells treated with UTP lost processes in responseto pertussis toxin.

G-protein redistribution is linked to the neuronaldifferentiation process

To test the relationship between G-protein redistribu-tion and neuronal differentiation, we used a proteinkinase inhibitor that blocks the MAP kinase pathway.Cells were incubated with NGF and PD98059 for 3 days,immunostained with G�s and microtubule antibodies,and processed for deconvolution microscopy. As ex-pected, most of the cells did not develop processesunder such conditions. G�s was enriched at the perinu-clear region and was closely associated with the micro-tubules. The tyrosine kinase inhibitor PD98059 blockedthe redistribution of G-protein association with micro-tubules in the cell process (Fig. 8). Whereas NGFinduced a substantial increase in �III-tubulin (an indi-cator of neuronal phenotype), PD98059 blocked this�III-tubulin increase in NGF-treated PC12 cells (Fig. 8C).

Figure 7. UTP alters the subcellular localizationof G-protein subunits. Aa–f) PC12 cells treatedwith 100 �� UTP. Cells were costained usinganti-�-tubulin and anti-G� antibodies and pro-cessed for deconvolution microscopy. Panelsd–f are enlarged 4.7-fold compared with panelsa– c. Activation of the G-protein-coupled P2Y2receptor induces cell differentiation and stimu-lates �-tubulin association with G� subunits inPC12 cell processes. Data are representative of 7individual experiments. Scale bars, 5 �m. B)The % increase in G� proteins associated tomicrotubules after UTP treatment. This wascounted similarly as described in Materials andMethods. *Statistical significance between con-trol and NGF-treated from each G� proteinexamined (paired Student’s t test, two-tailed Pvalue 0.05). There is no significant differenceamong each of the controls or each of theNGF-treated samples (Bonferroni).


Migration of G� protein with tubulin occurs duringthe development and formation of NGF-inducedprocesses

To explore the universality of the migration of tubulinand G-protein into cellular processes, we examined thedistribution and association of G� proteins and tubulinin neuroblastoma SK-N-SH and Neuro-2A cell lines.SK-N-SH cells send out processes spontaneously shortlyafter they become adherent. Double labeling revealed asubstantial overlap in the distribution of G�s and tubu-lin in the developing processes of both cell types (Fig.

9f, i). Process formation in Neuro-2A cells is spontane-ous but does not begin until �48 h after plating.Although there is substantial colocalization in process-bearing Neuro-2A cells, those without processes showedlittle or no colocalization of G� proteins and tubulin(cf. Fig. 9f, c). This suggests that G� protein associationwith microtubules was likely to occur during the forma-tion and development of processes (Fig. 9c) regardlessof the stimulus for process formation.

DISCUSSION

Heterotrimeric G-proteins have been detected at intra-cellular membranes, such as the Golgi complex (22,32–34), and have been implicated in intracellular vesi-cle trafficking (35). In PC12 cells, G�i1, G�s, and G��

display cytoplasmic staining (Fig. 1A). An intense G�sand G�o plasma membrane localization with uniformstaining across the cell surface was observed (Fig. 1A).Optical sectioning of PC12 cells shows all three G�

subunits to be prominent at the perinuclear region(Fig. 1B). G�i1 and G�s, but not G��, are present withinthe nucleus as well, indicating that G�i1 and G�s mightbe selectively imported through the nuclear pores (cf.Fig. 1Ba, b with c).

G-proteins have been seen to associate with cytoskel-etal structures such as microtubules (15–17, 36–39)and actin (40). The immunofluorescence images

Figure 8. G-protein redistribution is coincident to the neuro-nal differentiation process. A) PC12 cells were incubated withNGF. B) PC12 cells were incubated with NGF and PD98059for 3 days, immunostained with G�s and microtubules anti-bodies, and processed for deconvolution microscopy.PD98059 blocked the redistribution of G-protein associationwith microtubules in the cell process. C) Expression of the“neuronal” �III-tubulin in NGF-treated cells. The G� proteinsubunit expression levels were measured in PC12 cells treatedwith either NGF for 2–3 days, UTP for 3 days or both NGF andPD drug treatment regimens. Whole cell lysates (see Materialsand Methods) with equal protein were separated by SDS-PAGE. The relative protein expression levels in NGF- andUTP-treated cells were examined by immunoblotting withantibodies against �III-tubulin. The results are representativeof 5 individual experiments.

Figure 9. G� protein and tubulin migrate during the devel-opment of NGF-induced or spontaneous processes. a–i) Neu-roblastoma Neuro-2A and SK-N-SH cells were costained usinganti-�-tubulin (a, d, g) and anti-G�� antibodies (b, e, h) andprocessed for confocal microscopy. G�i1 and G�s antibodiesshowed similar distribution (data not shown). f, i) Doublelabeling revealed a substantial overlap in the distribution ofG� protein and tubulin in the developing neurites ofNeuro-2A and SK-N-SH cell lines. c) Non-neurite-bearingNeuro-2A cells show very little colocalization of G� proteinsand tubulin. 5 experiments were done for each G-proteinantibody. c, f, i) Yellow, areas of overlap in merged images.Scale bars, 10 �m.


shown in Fig. 1A clearly demonstrate that G�i1 ismicrotubule associated, whereas G�s and G�o have aregional distribution with �-tubulin in the perinuclearregion of resting PC12 cells. Deconvolution microscopyconfirmed that the close association of G� subunits withtubulin in selected regions was indeed with microtu-bules (Fig. 3A). Higher magnification showed that thepercentage of G�i1 localized on the surface or at edgesof microtubules was much greater than colocalizationof microtubules and G�s or G�o in untreated cells (Fig.3C). Studies with dimeric tubulin in rat cerebral cortexsynaptic membrane showed that G�� displayed a muchlower affinity for tubulin compared with G�i1 and G�s(17). However, �-tubulin coimmunoprecipitated withall three G� subunits, raising the possibility that allthree G� subunits are associated with tubulin polymers(41). This association is specific for tubulin and micro-tubules as actin does not colocalize with these G�

subunits (Fig. 5A). Colocalization between tubulin andG� subunits has been observed by coimmunoprecipita-tion (42). Numerous studies have shown that signaltransducing G-proteins can bind synaptic membranetubulin (17, 20, 43–45). G� appears to activate theGTPase activity of tubulin and G�i1, G�s, and G��

subunits increase microtubule polymerization dynam-ics in vitro (16). Note that G�1�2 binds to microtubulesand promotes microtubule assembly in vitro (15).

These results provide the initial evidence for a relo-cation of G-protein subunits in response to cytoskeletalreorganization. Rat pheochromocytoma PC12 cellsused in these studies have been a primary model forstudying mechanisms underlying neuronal differentia-tion (46) and signal transduction. Nerve growth factoracts on receptors with tyrosine kinase activity through aRas-dependent activation of ERKs (47–49). Activationof ERKs causes differentiation in PC12 cells (9) and isassociated with enhanced microtubule dynamics, a statein which process outgrowth is facilitated (50). Wefound that NGF not only stimulates the redistributionof G� subunits in the newly developing neurites alongthe microtubule network, but translocates G�i1 , G�s,and G�o to the tips of neurites, specifically to thegrowth cones (Fig. 3D). Translocation of G�o to thegrowth cones during neurite development has beenreported (51). There is an increased colocalization with�-tubulin in the newly developing neurites along themicrotubule network and a concomitant reduction incolocalization at the perinuclear region.

A closer examination of G� subunits in the cellprocess using deconvolution microscopy revealed thatNGF induced G� subunit association with componentsof cytoskeleton in cell body and especially in tubularextensions of the cellular processes (Fig. 3B). Theinduction of tubulin–G� association by NGF was muchmore dramatic for G�s than for G�i1 (Fig. 3C). Curi-ously, �85% of G�s became associated with microtu-bules after NGF treatment (Fig. 3C). In a previousstudy, coimmunoprecipitation experiments showedthat �85% of the G�s in synaptic membranes from ratcerebral cortex was complexed with tubulin (45).

Taken together, these results suggest that G� subunitsare probably enriched in the tubulin subdomains aswell as intermediate structures involved in the deliveryof G� subunits to the growth cones. Thus, it is plausiblethat G-protein association with microtubules may playan important role in the regulation of microtubuleformation in addition to its regulatory role in cellularsignal transduction.

In contrast to receptor tyrosine kinases, the interme-diate steps linking GPCRs to the activation of ERK arepoorly understood, and significant heterogeneity andcomplexity exist in the signaling pathways used byvarious GPCRs (52). ERK activation elicited by UTPacting on a P2Y2 receptor in HEK-293 cells (53) differsfrom the NGF-mediated ERK activation. The activationof G-protein-coupled purinergic receptors by UTP inPC12 cells stimulates phosphoinositide breakdown, re-lease of intracellular calcium, and influx of externalcalcium but does not stimulate norepinephrine release(54). In addition, recent evidence demonstrates thatpurinergic receptor agonists activated G�q/11 and G�i3in gastric and aortic smooth muscle and heart mem-branes, G�q/11 , G�i1 , and/or G�i2 in liver membranesand G�o and G�i1–3 in brain membranes (55).

In this study, a comparison of UTP and NGF re-sponses reveals that UTP induces a few unbranchedprocesses within 2 days whereas NGF induces multiple,highly branched processes within 24 h (14), (T. Sarmaand M. M. Rasenick, unpublished results). Purinergicreceptor activation results in a different time course ofneurite formation from NGF, and association of micro-tubules with G� subunits follows that same time course(cf. Figs. 2 and 3 with Fig. 5). This idea is supported byrecent findings demonstrating an association of G-protein-coupled receptors and G-protein-coupled re-ceptor kinases with microtubules (56–59). In contrastto NGF-induced association with microtubules, it isinteresting that UTP increases this association signifi-cantly for G�s and G�o but not for G�i1. This may berelated to the fact that the P2Y2 receptors are activatingG�i, but the nature of that relationship is not clear.

It is noteworthy, however, that even in the absence ofactivated receptor (spontaneous differentiation ofNeuro-2A cells; Fig. 6), the association of G� withmicrotubules increases upon neurite formation. Thus,G-protein activation is not likely to be required in orderto increase association between G� and microtubules indifferentiating neuronal cells.

Microtubules are required for the formation of PC12cell outgrowths. For both UTP-induced and NGF-in-duced processes, the outgrowths are retracted afterremoval of the agonist within 30 h. Treatment withPD98059 and subsequent inhibition of the MAP kinasesrequired for NGF-induced neuronal differentiationblocked the G-protein translocation. This suggests thatredistribution of G-protein is linked to the NGF-in-duced cell signaling pathway. This reversibility suggeststhat regulation of process formation is closely related tothe stimulation of signaling pathways associated withcellular differentiation. It is unclear, however, whether


translocation is secondary or is triggered by NGF inde-pendently.

The G-protein translocation appears to depend onthe integrity of microtubules, but not the actin cytoskel-eton, as it was blocked and reversed by nocodazole butunaffected by latrunculin. Furthermore, immunopre-cipitation experiments demonstrated a consistent phys-ical interaction between G� subunits and tubulin (45).Deconvolved images of NGF-treated cells suggest thatG� subunits are associated along the microtubule net-work in the cellular processes and that the formation ofG-protein/microtubule complexes is not dependentupon F-actin.

Two explanations could account for the redistribu-tion of G� subunits. The first would involve localmovement of G� from cell body to the cellular pro-cesses. In fact, we recently observed that an activatedG�s-GFP construct translocates from the membrane inliving cells (27). The second possibly would preservelocal cytoarchitecture but would involve a global fold-ing of G� protein–microtubule complexes. Althoughother explanations are possible, these data support theidea that microtubule organization influences bothmorphology and the distribution of signaling proteinsin the cell. G-protein association with microtubules inneuroblastoma cells lines Neuro-2A and SK-N-SH cellsand NGF-induced PC12 cells demonstrates that micro-tubules play a pivotal role during the neurite formationand development of cell processes.

The discovery of signaling molecules that interactwith microtubules, as well as the multiple effects onsignaling pathways of drugs that reorganize microtu-bules, indicate that microtubules are likely to be criticalto the spatial organization of signal transduction andthat heterotrimeric G-proteins may help dictate theshape and movement of cells.

We would like to thank Dr. M. L. Chen for her expertassistance with confocal microscopy. We are grateful to B.Shah, K. Heretis, S. A. K Chowdhury, and A. Kim for countingthe images. This work was supported in part by grants fromUSPHS AG 15482, MH 39595, and MH 57391 to M.M.R., GM56159 to T.V.Y., and AI 47770–04 to T.J.H.

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Received for publication July 30, 2002.Accepted for publication January 10, 2003.


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