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(The FASEB Journal. 2006;20:1452-1461.)
© 2006 FASEB

Tau associates with actin in differentiating PC12 cells

Jiang-Zhou Yu* and Mark M. Rasenick*,{dagger},1

Department of
* Physiology and Biophysics and

{dagger} Psychiatry, University of Illinois at Chicago, College of Medicine, Chicago, Illinois, USA

1 Correspondence: Department of Physiology and Biophysics, University of Illinois at Chicago, College of Medicine, 835 S. Wolcott Ave. M/C 901, Chicago, IL 60612-7342, USA. E-mail: raz{at}uic.edu or yujz64{at}uic.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The microtubule-associated protein tau may be involved in cell morphogenesis and axonal maintenance. In addition to microtubules, tau has been shown to interact with actin in vitro. In the present study interaction of tau and actin was investigated in PC12 cells. No interaction between tau and actin was observed without NGF treatment. Under NGF stimulation, tau distributed at ends of cellular extensions, where it associated with actin in a microtubule-independent manner. F-actin disruption revealed that relocalization and assembly of F-actin at the ends of cellular extensions were necessary for NGF-induced tau reorganization and association with actin. A truncated tau-GFP (tau(1–186)-GFP, N-terminal of tau) did not associate with actin. However, tau23(174–352)-GFP (carboxyl-terminal of Tau23) did associate with actin and the requirement for NGF was lost. Nevertheless, NGF boosted tau23(174–352)-GFP interaction with actin and promoted colocalization at the ends of cellular extensions. This suggests that the C-terminal of tau is required for associating with actin and the tau N-terminal may play a regulatory role in this process. A possible role for tau-actin interaction in neurite outgrowth is postulated—Yu, J.-Z., Rasenick, M. M. Tau associates with actin in differentiating PC12 cells.


Key Words: microtubule-associated protein • cytoskeleton • growth cone • nerve growth factor • neurite outgrowth and tubulin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE MICROTUBULE-ASSOCIATED PROTEIN tau is necessary for establishment of neuronal cell polarity and axon outgrowth, for axonal transport, and for the maintenance of axonal morphology. Observation in living cells revealed that the interaction of tau with microtubules was very dynamic, with a half-time of fluorescence recovery on the order of 3 s (1) . During neuronal differentiation, tau appears to undergo a transition from immature to mature form (2) and three- and four-repeat tau differed in their abilities to affect microtubule dynamics (3) . Furthermore, overexpression of tau in neurons and non-neuronal cells resulted in the promotion of cellular processes (4 , 5) . Initial studies of tau-deficient mice suggested the absence of a critical role for tau on neuronal growth and survival, as the mice are phenotypically normal (6) . However, maturation of hippocampal neurons from these mice was inhibited (7) . Thus, tau’s specific functions for the development and maintenance of neuronal morphology remain unknown.

Some evidence shows that tau’s function in neurons likely extends well beyond its ability to regulate microtubule dynamics. Tau may interact with plasma membrane (8) and with several proteins involved in signal transduction (9 10 11) . In addition, in vitro studies demonstrated that tau protein might bind to actin and affect actin polymerization (12 13 14) , and might orchestrate the interaction of microtubules and actin polymers in the organization of the cytoskeletal network (15) . Immunocytochemistry studies in primary cultured neuronal cells also suggested that tau extended to growth cones (16) , and actin-disrupting drugs altered the localization of dephosphorylated tau there (17) . Nevertheless, a recent study demonstrated that tau protein did not directly bind to actin in vitro (18) . Thus, the question of actin association with tau remains open.

Nerve growth factor (NGF) induces differentiation of PC12 pheochromocytoma cells from a chromaffin-like to a sympathetic neuron-like phenotype (19) . Prior reports from different laboratories have demonstrated that NGF treatment triggered a rapid redistribution of actin cytoskeleton and lamellipodia formation (20 , 21) . Thus, we used PC12 cells, which also harbor endogenous tau, to examine interaction between tau and actin during neurite outgrowth.

In this study, we observed interaction between tau and actin. While in unstimulated PC12 cells, no interaction between tau with actin was seen, upon stimulation with NGF some tau was distributed to the ends of cellular extensions, where it colocalized with actin. This interaction between tau and actin occurred at an early stage during differentiation. Expression of truncated tau constructs revealed that the C-terminal of tau is necessary for association between tau and actin, and the N-terminal of tau may partially govern this phenomenon. Tau may thus serve to modulate dynamics of actin at the growth cone.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
PC12 cells and PcDNA3 vector were obtained from the American Type Culture Collection and Invitrogen Corporation (Carlsbad, CA, USA), respectively. The monoclonal antibody (mAb) against actin, actin1 and antibody (Ab) against tau, tau5 were generous gifts from James Lessard (Cincinnati) (22) and Lester Binder (23) (Northwestern). Monoclonal anti-{alpha} tubulin and anti-GFP Ab were purchased from ICN Biomedicals (Costa Mesa, CA, USA) and Clontech (Palo Alto, CA, USA), respectively. Polyclonal antibody (pAb) against the C-terminal of human tau was from DakeCytomation (Carpenteria, CA, USA). pAb against actin, phalloidin-TRITC, nocodazole, and cytochalasin B were from Sigma (St. Louis, MO, USA). Latrunculin B and 7S NGF were purchased from Biomol Research Laboratories (Plymouth, PA, USA) and Alomone Laboratories (Jerusalem), respectively. All other biochemicals used were of the highest purity available. Eukaryotic expression plasmids coding GFP and tau fusion proteins were produced as described previously (4) . Tau23-GFP represents the construct coding the fusion protein that the C-terminal of GFP was fused with the N-terminal of full-length tau23. Tau(1–186)-GFP delineates the construct coding the fusion protein where GFP was spliced to N-end of residues 1–186 of tau23 (containing proline-rich domain). Tau23(174–352)-GFP indicated the construct coding fusion protein where GFP was attached to the N-terminal of the residues 174–352 of tau23 (containing microtubule binding domains). These constructs are depicted in Fig. 6 .


Figure 1
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  		Figure 1. Yau appears at ends of cellular extensions in living cells. PC12 cells were transfected with YFP-actin, GFP, and tau23-GFP constructs, respectively. A) Cell expressing YFP-actin, monitored with confocal microscopy at various time points. B) Cells expressing GFP or tau23-GFP were observed with fluorescence microscopy. Scale bar = 10 µm. Arrows refer to the ends of cellular extensions with concentrations of YFP-actin or Tau23-GFP. Cells are representative of 6 individual experiments.


Figure 2
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  		Figure 2. NGF-induced movement of tau23-GFP, but not tubulin or Rdsred fluorescent protein, to the ends of cellular extensions. A) Microtubules and tau do not colocalize at the tips of NGF-induced cellular extensions. Tau23-GFP-transfected PC12 cells were stimulated by NGF for 18 or 30 h, then extracted and fixed with cold methanol. Microtubules were stained with anti-{alpha}-tubulin Ab. Arrowheads indicate minor membrane extensions and arrows delineate the tips of long extensions containing tau23-GFP. Scale bar = 10 µm. B) NGF treatment did not redistribute the cytosolic protein Rdsred fluorescent protein to the ends of cellular extensions. PC12 cells were cotransfected with tau23-GFP and Rdsred, and imaged with confocal microscopy after 18 h NGF treatment. The blue arrows indicate tau23-GFP located in ends of cellular extensions. Results are representative of 4 individual experiments. Images were acquired with confocal microscopy.


Figure 3
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  		Figure 3. Tau colocalization with actin in lamellipodia-like structures is induced by NGF treatment. Cells were stimulated with NGF for 0, 18 or 40 h, respectively, then fixed with cold methanol. The cells were stained with a mAb against actin (red). Green indicates expression of Tau23-GFP or endogenous tau (as noted). The arrowheads indicate the lamellipodia-like structures with colocalization of tau23-GFP and actin. A) Prior to NGF treatment, the actin immunoreactivity was distributed around the cell periphery while Tau23-GFP was seen primary in the cytoplasm. There is no apparent overlap in the distribution of actin and tau23-GFP. B) Low-power images of cells expressing tau23-GFP after 18 h NGF treatment show olocalization of tau23-GFP and actin at the edges of lamellipodia-like structures. C) High magnification images from a cell as in panel B. D) Magnification of lamellipodia-like structures. E) PC12 cells were treated with NGF for 40 h and fixed and stained with tau 5 Ab. Arrowheads showed endogenous tau (green) presented at ends of lamellipodia-like structures and colocalized with actin (red) in a manner identical to tau23-GFP. F) Transfected PC12 cells extracted with Triton X-100. Arrows indicated tau23-GFP (green) and actin (red) colocalized at lamellipodia-like structures. The insert is a magnification of the indicated area. At least 9 individual experiments were carried out. A–D) Images are from confocal microscopy. E, F) Epifluorescent images.


Figure 4
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  		Figure 4. NGF promotes the interaction of tau with actin as seen by coimmunoprecipitation. A) NGF induces coimmunoprecipitation of tau23-GFP with actin. PC12 cells expressing either GFP or tau23-GFP were exposed to NGF (as indicated) for 18 h, lysed, and immunoprecipitated with anti-GFP Ab. Blots were detected with actin Ab (middle panel). The lower panel shows expression of tubulin or actin in each cell. The upper panel is a densitometric quantification of coimmunoprecipitated actin with tau. One of 3 identical experiments is shown. B) Endogenous tau coimmunoprecipitation with actin in NGF-treated PC12 cells. PC12 cells were treated with NGF for the indicated time, lysed, and subjected to immunoprecipitation with tau Ab. Actin coimmunoprecipitation with tau is seen in the middle panel, while overall tubulin expression is in the lower panel. The upper panel represents quantification of actin coimmunoprecipitated with tau. The blot represents 2 similar experiments. Data are represented as mean ± SE.


Figure 5
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  		Figure 5. Actin disruption blocks the colocalization between tau23-GFP and actin whereas microtubule disruption has no effect. Arrowheads delineate tau at ends of lamellipodia-like structures, which is colocalized with actin. A) Nocodazole was added 10 min before application of NGF, which was then incubated with cells expressing tau23-GFP for ~18 h. Cells were fixed with cold methanol and stained with monoclonal anti-actin Ab. B) PC12 cells stimulated with NGF (18 h) were treated with nocodazole for 60 min. F-actin is stained with phalloidin-TRITC. C) Cytochalasin B was applied 10 min prior to treatment with NGF and incubated with tau23-GFP-transfected cells for 18 h. F-actin stained with phalloidin-TRITC is red. D) Cells treated with NGF for 18 h were exposed to cytochalasin B for 60 min. E) Same as for panel C, but F-actin was disrupted with latruculin B. F) Same as panel D, except with latrunculin B. More than 9 individual experiments were carried out for each parameter. All images were captured with confocal microscopy.


Figure 6
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  		Figure 6. The C-terminal of tau is essential for interaction between tau and actin. A) Diagram of truncated tau fusion proteins. B) PC12 cells transfected with indicated constructs and stimulated with NGF for 0 or 18 h. Cells were stained with anti-actin Ab (red) and observed with confocal microscopy. Green represents the truncated tau fusion protein with GFP. Arrowheads indicate colocalization between truncated tau fusion proteins and actin. The same results were obtained in 8 individual experiments.

Cell culture and transfection
PC12 cells were cultured in Dulbecco’s modified Eagle medium (Life Technologies, Rockville, MD, USA) containing 10% bovine calf serum (HyClone® Laboratories, Inc., Logan, UT, USA) and antibiotic (penicillin and streptomycin) at 37°C with 5% CO2. Cells in 12-well culture dishes (40–60% confluent) were transfected with 5 µg of plasmids containing tau-GFP, GFP, or yellow fluorescent protein (YFP) -actin cDNA using GenePORTERTM Transfection reagent (Gene Therapy System, Inc., San Diego, CA, USA). 0.5 milliliters of plasmid DNA (5 µg) and GenePORTERTM Transfection reagent (10 µl) in Opti-MEM I medium were applied to each well of 12-well culture plates. For transfection in 60 mm tissue culture dish, 2.5 ml of plasmid DNA (15 µg) and GenePORTERTM Transfection reagent (50 µl) in Opti-MEM I medium were added. Six hours after incubating, cells were cultivated in complete medium. All experiments were performed in transiently transfected cells. Prior to experiments, cells in 12-well culture plates were observed by fluorescence microscopy to check the percentage of transfected cells. Wells with 20 ± 3% transfected cells were used for experiments.

Immunocytochemistry
Before staining, PC12 cells grown on coverslips in 12-well plates were washed twice with PBS (58 mM Na2HPO4, 17 mM NaH2PO4, 68 mM NaCl), fixed with cold 100% methanol (–20°C) for 4 min, and washed twice with PBS. The coverslips were then incubated with PBSS buffer (PBS+0.01% saponin) containing 10% BSA for 20 min, then incubated in 1:60 dilution of anti-{alpha}-tubulin, 1:10,000 antiactin antibodies, or 1:500 tau5 Ab in PBSS buffer for 3 h. Subsequently, the coverslips were washed with PBSS four times and incubated with 1:180 dilution of secondary antibodies labeled with tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) in PBSS buffer for 40 min. For phalloidin-TRITC staining, the coverslips were first blocked for 20 min in PBS containing 5% (w/v) BSA., then incubated for 40 min with 20 nM phalloidin-TRITC diluted in PBS. Finally, these coverslips were washed with PBSS buffer four times and mounted on the slide with mounting medium. The slides were air dried and examined by digital fluorescence microscope or confocal microscopy.

For saponin extraction of PC12 cells, cells were treated for 20 s with extraction buffer (80 mM PIPES/KOH, PH 6.8, 1 mM MgCl2, 1 mM EGTA, 30% (v/v) glycerol, 1 mM GTP, and 0.2% (w/v) saponin) at room temperature. For Triton X-100 extraction of PC12 cells, extraction buffer (10 mM PIPE, PH 6.8, 50 mM KCl, 10 mM EGTA, 3 mM MgCl2, 2 M glycerol, 0.5% Triton X-100) was applied to cells for 2 min at room temperature (42) Cells were then washed twice with extraction buffer without detergent and fixed with freezing cold 100% methanol in –20°C for 4 min.

Microscopy
Living cells were observed using a digital fluorescence microscope equipped with a 100-W mercury arc lamp. Cells were maintained at 37°C during the entire period of observation (4) . Images were acquired with an interline charge-coupled device camera (1300 YHS; Roper Scientific, Trenton, NJ, USA) driven by IP Lab imaging software (Scanalytics, Inc., Suitland, VA, USA) and processed with IP Lab and Adobe Photoshop 5.0 (Adobe Systems, Mountain View, CA, USA). For confocal microscopy, images were collected with a Zeiss laser scanning confocal microscope (LSM-510 MTS). Equipped with a 60x immersion objective. A single 488 nM beam from an argon-krypton laser was used for GFP excitation, whereas a 543 nM beam was used for Texas Red excitation. Emission from GFP was detected via an LP505 filter, whereas emission from Texas Red was detected through a LP 560 filter. A 514 nM beam was used to excite YFP; emission from YFP was detected via an LP530 filter. Images were processed using Adobe Photoshop 5.0.

Immunoprecipitation and Western blot
PC12 cells transfected with plasmids coding tau-GFP fusion proteins were stimulated by NGF for 18 h, then washed twice in PBS, and cells were lysed in 500 µl lysis buffer (PBS, 0.5% Triton X-100, 5 mM EDTA, protease inhibitors) on ice for 30 min. The lysate was collected and cleared by centrifuging at 12,000 g for 20 min at 4°C. Protein concentration of supernatants was determined by the method of Bradford (Bio-Rad Laboratories protein assay, Hercules, CA, USA). After adjusting protein concentration to equal amounts for each sample, the supernatant (450 µl) was transferred to 1.5 ml microcentrifuge tubes and incubated with agarose beads coated with antimouse lgG for 1 h at 4°C with continuous gentle inversion. The agarose beads were pulled down by centrifuging at room temperature and discarded. The lysate was then incubated with 5 µl mAb against GFP for 14 h at 4°C, then the Ab/lysate mixture was incubated with agarose beads coated with anti-mouse lgG for 2 h at 4°C with continuous gentle inversion. The agarose beads were washed with lysis buffer three times. After that, the 50 µl 2xSDS-PAGE sample buffer was added to the agarose beads. Twenty µl supernatant was applied to 7–14% gradient SDS-PAGE, and the resolved proteins were analyzed on a Western blot using the pAb against actin. Western blot was done as described previously (4) . Briefly, blots were incubated for 1 h in TBST (10 mM Tris-HCl, pH 8.0, 159 mM NaCl, and 0.1% Tween 20) containing 5% powdered skim milk and 1% BSA. After three washes with TBST, membranes were incubated for 2 h with the primary Ab and for 1 h with horseradish peroxidase-conjugated goat anti-rabbit lgG. Proteins were detected using the enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Arlington Heights, IL, USA).

Cell fractionation
For cell fractionation, PC12 cells expressing tau-GFP constructs were harvested and resuspended in HEPES-sucrose buffer (15 mM HEPES, 0.5 mM sucrose, 1 mM DTT) containing CompleteTM protease inhibitor cocktail tablets (Roche Molecular Biochemicals, Indianapolis, IN, USA). Cell suspensions were homogenized with a Potter-Elvehjem homogenizer. After a low-speed centrifugation to remove unbroken cells and a nuclear pellet, samples were centrifuged for 15 min at 35,000 g at 4°C in centrifuge (TL 100; Beckman Coulter, Fullerton, CA, USA). Protein concentrations of supernatant (cytosol fraction) and pellet (membrane fraction) were determined by the method of Bradford (Bio-Rad Laboratories protein assay). The membrane fraction and cytosol fraction were separated by 12% SDS-PAGE and analyzed by Western blot.

Quantification of frequency of colocalization between tau and actin
The colocalization of tau protein and actin was quantitated in > seven independent experiments on fixed PC12 cells. For transfected PC12 cells treated with NGF, cellular extensions were classified into two categories: lamellipodia-like structures (actin-rich extensions from cell body shorter than the cellular diameter in length) and cellular processes (slender projections that exceeded the cellular diameter in length). These structures were counted individually and "colocalization bearing" was scored if yellow pixels accounted for at least half of the area of actin staining at end of a cellular extension. An individual blinded to experimental conditions did all observations leading to quantification.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NGF stimulation alters the distribution of tau23-GFP in PC12 cells
About 20% of the cells expressed GFP, tau23-GFP, or YFP-actin after transfection with appropriate plasmids. Fluorescence microscopy showed that GFP was distributed through the cytoplasm whereas tau23-GFP showed a filamentous distribution, in agreement with a previous report (4) . YFP-actin distributed around the cell periphery and was enriched at several discrete areas; this distribution was comparable to that of endogenous actin (see Fig. 1 A). Observations in living cells showed a rapid reorganization of YFP-actin after NGF treatment. About 5 min after NGF stimulation, an apparent actin relocalization to lamellipodia was observed (Fig. 1A ). These results coordinate with previous studies (21 , 24) . NGF did not alter distribution of either GFP or Tau23-GFP throughout a 1 h period of observation in living cells (data not shown). Cells treated with NGF for 18 or 30 h increasingly sprouted cellular extensions. Tau23-GFP filled in cellular extensions and presented at their ends (Fig 1B ), but GFP did not.

Tau is a microtubule-associated protein. To determine whether the NGF-induced distribution of tau23-GFP was accompanied by a similar altered localization of microtubules, cells were extracted by saponin and stained with anti-{alpha}-tubulin mAb. Filamentous microtubules were seen in both cell bodies and cellular extensions, and these were colocalized with tau23-GFP. At ends of extensions, tau23-GFP was not complexed with microtubules (Fig. 2 A).

To further test whether tau23-GFP is specifically enriched at some ends of NGF-induced cellular extensions, we cotransfected PC12 cells with Rdsred and tau23-GFP. Although Rdsred was present in the cell body and cellular extensions (Fig. 2B ), NGF treatment did not drive the cytosolic Rdsred to the ends of cellular extensions.

NGF promotes the interaction between tau and actin
Though some in vitro studies suggested that tau interacts with actin (12 13 14 15 , 25) , in this study, PC12 cells did not show colocalization between tau and actin unless they were exposed to NGF (Fig 3 A). Eighteen hours after NGF treatment, many minor cellular extensions (actin-containing lamellipodia-like structures) appeared. Overlapping images of actin and tau23-GFP suggested that actin colocalized with tau23-GFP at ends of lamellipodia-like structures (Fig 3B, C ). A confocal z-stack image further implied colocalization between tau23-GFP and actin at lamellipodia-like structures (supplementary Fig. 1 ). Structures stained with Ab against actin at these lamellipodia-like structures were also recognized by phalloidin-TRITC. Figure 3D is a magnified lamellipodia-like structure that displays colocalization between tau23-GFP and actin in more detail. Tau colocalized with F-actin at the inner 3/4 of lamellipodia-like structures, but not at the distal edge. In contrast, this colocalization was not seen in cells expressing GFP after18 h NGF treatment. To further confirm this finding, we applied tau protein Ab (tau5) to stain the endogenous tau in PC12 cells. Control PC12 cells (non-NGF treated) show very weak tau staining. After 40 h treatment with NGF, endogenous tau expression increased markedly and tau presented at ends of lamellipodia-like structures, where it colocalized with actin (Fig. 3E ). When transfected cells were extracted with 0.5% triton X-100, the colocalization between tau23-GFP and actin was still clearly seen at lamellipodia-like structure (Fig. 3F ).

To further establish tau-actin association, coimmunoprecipitation experiments were carried out in PC12 cells transfected with tau23-GFP. Figure 4 A showed that without NGF stimulation, tau23-GFP interaction with actin is faint. When PC12 cells were exposed to NGF for 18 h, the interaction of tau23-GFP with actin increased (Fig. 4A ). NGF treatment (18 h) did not alter the total amount of actin and tubulin (Fig. 4A , lower panel) or expression of Tau23-GFP. Colocalization between tau and actin occurred at ~80% of the lamellipodia-like structures that appeared after treatment with NGF for 18 h (Table 1 ). Using coimmunoprecipitation, we also found that interaction between actin and endogenous tau occurred after 24 h NGF treatment, and long-term NGF exposure (48 h) further increased this interaction (Fig. 4B ).


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  		Table 1. Colocalization between tau protein and actin in PC12 cells under NGF treatment

A longer exposure to NGF (40 h) was also performed in transfected PC12 cells. Colocalization between tau with actin still existed at tips of cellular processes and lamellipodia-like structures even after 40 h exposure to NGF (supplementary Fig. 2A ). These cells possessed more cellular processes and fewer lamellipodia-like structures than the cells treated with NGF for 18 h (Table 1) . The frequency of colocalization of tau23-GFP with actin was 63%±9.5 at tips of cellular processes in cells observed after 40 h NGF treatment (Table 1) . The percentage of interaction between tau23-GFP and actin at tips of cellular processes was comparable after 18 or 40 h of NGF treatment (Table 1) .

Microtubules do not contribute to the NGF-induced interaction between tau and actin
The data described above suggest that microtubules might not be involved in the tau distribution changes promoted by NGF. To confirm this notion, microtubules were disrupted with nocodazole before NGF treatment or after 18 h NGF treatment, respectively. The results showed that nocodazole did not alter the distribution of tau and actin at the ends of lamellipodia-like structures, and the colocalization between tau23-GFP and actin was unaltered even though the filamentous (microtubule-associated) distribution of tau23-GFP in cells disappeared (Fig. 5 A, B). Depolymerization of microtubules by nocodazole was verified by tubulin staining using an anti-{alpha}-tubulin Ab.

Depolymerization of F-actin interrupts tau23-GFP-actin colocalization
NGF induced the rapid reorganization of actin in PC12 cells, which accumulated at lamellipodia-like structures, where it colocalized with tau. Thus, it was possible that F-actin influenced tau distribution. To test this, actin-disrupting agents were used. Transfected PC12 cells were exposed to cytochalasin B (1.5 µM) before NGF treatment. Confocal images revealed that cytochalasin B prevented the NGF-induced redistribution of F-actin. Small particles of F-actin stained with Phalloidin-TRITC could be seen in the cells, however, no lamellipodia-like structures or colocalization between tau23-GFP and actin could be found on these cells (Fig. 5C ). In addition, these cells had short cellular extensions filled with tau23-GFP, but tau was not present at ends of cellular extensions (Fig. 5C ). In subsequent experiments, cells were treated with cytochalasin B (4 µM) for 60 min after 18 h of NGF stimulation. Punctate actin staining with phalloidin-TRITC can be found in these cells, mostly in the cell body. The filamentous distribution of tau23-GFP in these cells did not change (Fig. 5D ). Tau23-GFP remained at the ends of cellular extensions and colocalized with punctate F-actin stained with phalloidin-TRITC. Meanwhile, some tau-actin colocalization could be seen in the cell body (Fig. 5D ). Another F-actin depolymerizing drug, latrunculin, showed results similar to those with cytochalasin B (Fig. 5E, F ).

Colocalization of tau and actin is dependent on the C-terminal of tau
Previous studies suggested that while the C-terminal domain of tau was necessary and sufficient for microtubule binding, the N-terminal region played a regulatory role. To determine the domain of tau that contributes to interaction with actin, tau23(174–352)-GFP and tau23(1–186)-GFP were transfected into PC12 cells. Untreated cells expressing truncated tau fusion proteins were round. The distribution of tau23(1–186)-GFP is diffuse in PC12 cells and similar to GFP alone. Tau23(174–352)-GFP showed a filamentous distribution similar to the full-length protein. Some colocalization between tau23(174–352)-GFP and actin can be seen at cortical actin of the transfected cells independent of NGF treatment (Fig. 6 B). About 30% of the transfected cells showed colocalization of tau23(174–352)-GFP with cortical actin (30%±7.9). If the cells were stimulated for 18 h with NGF, the cellular shape changed as described above. Lamellipodia-like structures and a few cellular processes were formed. Interaction between tau23(174–352)-GFP and actin was predominantly found at lamellipodia-like structures (Fig. 6B ). No tau23(1–186)-GFP was seen at the ends of lamellipodia-like structures (Fig. 6B ). The frequency of colocalization of actin with tau23(174–352)-GFP at lamellipodia-like structures was 80%±16.2, which is almost the same as that of cells expressing full-length tau23-GFP (Table 1) . Furthermore, longer term exposure to NGF (40 h) showed tau23(174–352)-GFP at some tips of cellular processes where it colocalized with actin (supplementary Fig. 2B). Cell fractionation revealed that tau23-GFP, tau23(174–352)-GFP and tau23(1–186)-GFP fusion proteins were enriched in the cytosolic fraction, while tau23(1–186)-GFP and tau23-GFP were also present in membrane fractions. Tau23(174–352) GFP did not associate with plasma membrane (Fig. 7 A). This coordinates with a previous report (26) , which suggested that GFP fused with these truncated fragments of tau did not alter their distribution in cells. Coimmunoprecipitation also revealed that tau23(174–352)-GFP associated with actin and NGF treatment profoundly increased this interaction. Tau23(1–186)-GFP failed to associate with actin under either condition (Fig. 7B ).


Figure 7
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  		Figure 7. Truncated tau-GFP fusion proteins interacted with actin in PC12 cells. A) Subcellular distribution of tau-GFP. PC12 cells were transfected with the indicated tau-GFP construct. Subcellular fractions of 24 h post-transfected PC12 cells were prepared as described in Materials and Methods. Particulate fractions (membrane) and soluble fractions (cytosol) of cells transfected with the indicated constructs were examined for the presence of tau-GFP by Western blot using an Ab against GFP. Blotting was repeated twice with the same results. B) Immunoprecipitation of tau-GFP truncations with actin. PC12 cells were transfected with indicated constructs and treated (as indicated) with NGF for 18 h. Actin and tubulin expression is seen in the lower panel. Cells were lysed and immunoprecipitated with GFP Ab. Quantification of actin coimmunoprecipitated with truncated tau23-GFP is seen in the upper panel. Three experiments were carried out with similar results, and mean ± SE. is shown in upper panel. C) Western blots to show the expression of truncated tau in transfected PC12 cells. Lane 1 shows cells transfected with tau23(1–186)-GFP, lane 2 represents cells expressing tau23(174–352)-GFP. Results are representative of 3 individual experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tau is classically defined as a microtubule-associated protein that plays an important role in promotion and stabilization of axonal microtubules. However, a growing body of evidence suggests that tau has functions other than regulation of microtubule dynamics (8 9 10 11 12 , 14 , 15) . Although tau knockout mice appear to be relatively normal, neuronal development is aberrant (6 , 7) . In this study, interaction of tau with actin was observed. The results from both biochemical and imaging experiments indicated that NGF treatment could induce interaction of tau with actin at the ends of some cellular extensions. We suggest that the interaction of tau with actin might modulate neurite outgrowth.

Without NGF treatment, the tau23-GFP protein was distributed throughout the cytosol, and no colocalization was found between tau and actin in PC12 cells (Fig. 3A ). In addition, a coimmunoprecipitation experiment revealed that actin association with tau23-GFP was very faint in cells not exposed to NGF (Fig. 4A ). NGF treatment initiated tau redistribution such that some tau23-GFP was located at ends of some cellular extensions where actin was enriched (Fig 1B , Fig. 2 ) and interaction of tau23-GFP with actin was significantly increased (Fig. 4A ). Confocal images also revealed colocalization between tau23-GFP and actin at the ends of lamellipodia-like structures in NGF-treated PC12 cells (Fig. 3) . The amount of actin or tubulin was not altered by 18 h NGF treatment (Fig. 4A ). These experiments indicated that interaction between tau23-GFP and actin is dependent on NGF treatment. Note that endogenous tau expression is low in PC12 cells without NGF treatment, and increases after NGF stimulation (27 , 28) . In this study, endogenous tau, identified by staining with tau Ab (tau 5), was not observed in untreated PC12 cells. After NGF treatment, endogenous tau could be readily seen throughout the cells and at ends of lamellipodia-like structures, where it colocalized with actin in a manner similar to tau23-GFP (Fig. 3E ). Furthermore, coimmunoprecipitation indicated that 24 h of NGF treatment promoted the interaction of endogenous tau with actin and long-term stimulation with NGF (48 h) significantly increased this interaction (Fig. 4B ). In the case of the tau-GFP constructs whereas NGF was not required for expression, the movement of tau into cellular processes, where it associates with actin, was dependent on NGF. These studies suggest that NGF stimulation of PC12 cells might induce the reorganization of tau and promote tau interaction with actin at lamellipodia-like structures. Interaction of purified tau protein with actin in vitro has been reported from several labs (14 , 25 , 29 30 31 32) . Nevertheless, one recent paper suggests that tau does not directly bind with actin in vitro (18) . Since tau-GFP expression is high, even in the absence of NGF, the reliance on NGF treatment to induce association between tau and actin also suggests either a post-translational modification of one or both proteins, or the expression of a new protein linking tau and actin. Previous studies have suggested that the tau phosphorylation status governs the association between tau and actin in vitro (13 , 33) ; this post-translational modification might well contribute to NGF-induced tau-actin interaction in PC12 cells.

In addition, disruption of actin with latruculin D or cytochalasin B before administration of NGF revealed that lamellipodia-like structures did not form and tau was not present at the ends of cellular extensions, even though latruculin D or cytochalasin B allowed cellular extensions to form (4 , 34) . However, when cellular F-actin was depolymerized with cytochalasin B or latrunculin after 18 h treatment with NGF, tau could still be seen at the ends of cellular extensions, and there was some sporadic colocalization with punctate actin, which might suggest a possible role of tau in the regulation of actin dynamics. Compared to the reorganization of F-actin induced by NGF, relocalization of tau initiated by NGF is slower. Perhaps the NGF induced migration of F-actin to lamellipodia-like structures is required to recruit tau.

Recent studies have shown that some microtubules can stretch to the growth cone and colocalize with actin, even though these structures are not enriched in either protein (35) . Microtubules may play a role in the reorganization of tau induced by NGF. However, confocal images of microtubules stained with anti-{alpha}-tubulin Ab in NGF-treated cells expressing tau23-GFP revealed that no microtubules colocalized with tau at the ends of cellular extensions (Fig. 2) . Furthermore, microtubule-disrupting drugs did not alter NGF-induced reorganization of tau or tau colocalization with actin (Fig. 5A, B ). This suggests that interaction between tau and actin induced by NGF is a microtubule-independent process. Previous studies in cultured neurons found that tau is enriched at the distal end of growing axons and that the gradient in tau content of microtubules does not generate corresponding gradients in the extent of microtubule assembly or microtubule stability (16 , 36) . Tau may distribute to the central and peripheral regions of growth cones, where tau appears to interact with elements of the subcortical cytoskeleton (26 , 37 , 38) . Our suggestion is consistent with these findings.

The frequency of interaction between tau23-GFP and actin at lamellipodia-like structures is higher than that at tips of the cellular processes (Table 1) . Cells exposed to NGF for 40 h did not increase the percentage of colocalization between tau and actin at lamellipodia-like structures or at tips of cellular processes. This is true even though the number of cellular processes increased significantly and lamellipodia-like structures decreased compared to cells exposed to NGF for 18 h (Table 1) . These data coordinate with a prior study that suggested tau distribution to the distal axons occurred early in development of polarity of cultured neurons (36) . Thus, NGF may promote interaction of tau with actin at an early stage of PC12 cell differentiation. These data also showed that tau23-GFP did not distribute to all lamellipodia-like structures or tips of cellular processes.

In this study, two truncated tau-GFP fusion proteins were expressed in PC12 cells. Some colocalization between tau23(174–352)-GFP and actin can be seen at cortical actin of the transfected cells independent of NGF treatment (Fig. 6B ). NGF treatment promoted the colocalization of tau23(174–352)-GFP with actin at the ends of lamellipodia-like structures (Fig. 5B ). However tau23(1–186)-GFP did not colocalize with actin after exposure to NGF (Fig. 5B ). These results suggested the N-terminal of tau might not be required for interaction between tau and actin but could prevent the interaction of tau and actin in the absence of a differentiation signal. Furthermore, subsequent to NGF stimulation, tau(172–352)-GFP binding to actin was boosted in a manner similar to the full-length protein (Figs. 6 , 7) . The frequency of colocalization of actin with C-terminal tau at lamellipodia-like structures was 80%±16.2, which is also almost the same as that seen in cells expressing the full-length construct (Table 1) . In vitro studies suggest that the C-terminal of tau containing the microtubule binding domains is necessary for actin binding (12 , 31) . Taken together with results in this report, it appears that the C-terminal is required for NGF-induced tau reorganization and interaction with actin, whereas the N-terminal of tau plays a regulatory role in this process.

In vitro studies have also shown that tau phosphorylation status altered its ability to interact with actin; treatment of tau with alkaline phosphatase or acid to remove phosphate increases MAPS association with actin (13) . Phosphorylation of tau by PKC reduced its ability to promote tubulin polymerization and link actin filaments (33) . It is possible that certain kinases or phosphatases evoked by NGF might affect the phosphorylation status of tau (39) . One such kinase, GSK3, is a downstream element of PI3-kinase/Akt pathway, and an activated form of GSK3ß is enriched in growth cones (40 , 41) . Since tau23(174–352) interacts with actin in the absent of NGF, altered tau protein phosphorylation might occur at residues in this region. It will be necessary to study the concentration and nature of tau phosphorylation after NGF treatment to understand the mechanism of NGF-induced interaction between tau and actin.

In summary, NGF-induced microtubule independent relocalization of tau and colocalization with F-actin at the ends of cellular extensions has been observed. The presence of polymerized actin at cellular extensions is necessary for tau relocalization. The C-terminal of tau is required for interaction between tau and actin promoted by NGF, but the N-terminal of tau may play a regulatory role. These data raise the novel possibility that the interaction between tau and actin evoked by NGF treatment may be a part of a complex mechanism for regulation of neurite outgrowth and neuron development.


   ACKNOWLEDGMENTS
 
This work was supported by National Institute of Health Grants AG 15482, MH57391, and MH 39595. We thank Dr. S. Brady (University of Illinois at Chicago) for advice and critical review of this manuscript, Dr. M. L. Chen (University of Illinois at Chicago) for expert assistance with confocal microscopy, and Dr. T. Meyer (Stanford University Medical School) for providing the YFP-actin construct.

Received for publication September 29, 2005. Accepted for publication February 21, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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