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GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ASSOCIATES WITH ACTIN FILAMENTS IN SERUM DEPRIVED NIH 3T3 CELLS ONLY
Cell Biology International 2002, Vol. 26, No. 2, 155–164 doi:10.1006/cbir.2001.0819, available online at http://www.idealibrary.com on
GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ASSOCIATES WITH ACTIN FILAMENTS IN SERUM DEPRIVED NIH 3T3 CELLS ONLY HANS-DIRK SCHMITZ and JU } RGEN BEREITER-HAHN* Kinematic Cell Research Group, Biocentre, Goethe University of Frankfurt/Main, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany Received 31 May 2001; accepted 15 August 2001; published electronically 16 January 2002
The in vitro interaction between the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cytoskeletal elements is well documented. To verify this association within cells, the intracellular distribution of GAPDH under various metabolic conditions has been investigated in immunostained cells or cells expressing GAPDH as a GFP fusion protein. GAPDH was homogeneously distributed in the cytoplasm and no interaction of GAPDH with cytoskeletal elements, neither with microfilaments nor microtubules or intermediate filaments, was detectable. In living cells expressing GFP-GAPDH, stress fibres were excluded from the fluorescence. In contrast to proliferating cells, the cytoplasmic GAPDH of serum-depleted cells was not homogeneously distributed, but colocalised with stress fibres. The mechanism for stimulating this actin-binding affinity was independent of the NO-signalling pathway. The results support the idea of a specialised function for the interaction of GAPDH and cytoskeletal elements, rather than a general function, as e.g. microcompartmentalization of glycolytic 2002 Elsevier Science Ltd. enzymes. K: GAPDH; actin; serum withdrawal; association. A: Br-cAMP, Bromo-adenosine-3 ,5 -cyclic-monophosphate; CLSM, confocal laser scanning microscopy; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; L-NMMA, NG-Monomethyl-L-arginine.
INTRODUCTION Glycolysis is an enzymatic process which takes place within the cytosol. However, some of the glycolytic enzymes, for instance aldolase (EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) and phosphofructokinase (PFK, EC 2.7.1.11) may interact with the cytoskeleton and were found to be associated with microtubules and microfilaments in vitro. The association between glycolytic enzymes and the cytoskeleton was first observed in enzyme purification from muscle tissue (Arnold and Pette, 1970): Some enzymes were not completely soluble but remained bound to the cytoskeleton fraction (actin filaments). Further studies revealed direct binding of some glycolytic enzymes (aldolase, GAPDH and *To whom correspondence should be addressed: E-mail: [email protected] 1065–6995/02/020155+15 $35.00/0
PFK) with cytoskeletal elements (reviewed by Bereiter-Hahn et al., 1997; Srere and Knull, 1998). The kinetics of GAPDH and cytoskeleton interaction are well known from in vitro binding studies (Durrieu et al., 1987a; Durrieu et al., 1987b; Humphreys et al., 1986; Walsh et al., 1989), revealing an affinity constant (KD) of 0.6 M for GAPDH to F-actin and 3.1 M towards microtubules (Knull and Walsh, 1992). In vitro, the actin binding of GAPDH is enhanced upon nitrosylation (Wu et al., 1997), a post-translational modification which occurs upon incubation with nitric oxide (NO) donors and represents an important pathway of signal transduction. Further data about the association of GAPDH and cytoskeletal elements come from permeabilized cells retaining GAPDH enzyme activity in cytoskeleton containing fractions (Cao et al., 1999; Humphreys and Masters, 1986). GAPDH is found in pseudopodia, where it 2002 Elsevier Science Ltd.
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might interact with actin (Nguyen et al., 2000). The physiological relevance of this cytoskeletal interaction is still an enigma, although electron microscopy and polymerisation studies implicate a direct alteration of the filament properties, like bundling of microtubules (Huitorel and Pantaloni, 1985; Somers et al., 1990; Volker et al., 1995). Because most experiments were carried out in vitro, it is of central interest to verify the interaction of GAPDH and cytoskeletal elements within cells. To prove such an interaction we monitored the intracellular distribution of GAPDH either by immunostaining or by overexpression of a GFP-GAPDH fusion protein in NIH 3T3 cells. Whereas our in vitro binding studies showed an association of GAPDH with microfilaments dependent on the availability of substrate/cofactor, immunostaining and GFP-GAPDH overexpression failed to detect such interaction in proliferating cells. A co-localisation of GAPDH with stress fibres was observed only in cells after prolonged serum depletion.
calf serum (FCS; Life Technologies). Human keratinocyte cells (HaCaT, p60–70; Boukamp et al., 1988) were cultured in keratinocyte medium (Life Technologies) with 10% FCS. The Xenopus laevis tadpole heart endothelial cell line XTH2 (Schlage et al., 1981) (p320–360) was cultured in MEM with 10% FCS. For serum withdrawal experiments, cells were washed with serum-free medium and then incubated with serum-free medium as described. The inhibitor of the NOsynthase, NG-mono-methyl-L-arginine (L-NMMA, Sigma), was used at a final concentration of 500 M and freshly added each day. The cells were grown on cover glasses in 35 mm Petri dishes when used for fluorescence microscopy. For monitoring GFP fluorescence, cells were washed twice with physiological saline and fixed with 4% (w/v) freshly prepared formaldehyde in PBS for 20 min with three subsequent washing steps. Living cells were investigated under controlled temperature in the appropriate media buffered with Hepes instead of bicarbonate.
MATERIALS AND METHODS
Immunostaining
Cosedimentation analysis
NIH 3T3 or XTH2 cells cultivated on glass were washed twice in PBS fixed with 4% formaldehyde/ 0.25% glutaraldehyde in PBS for 20 min and permeabilised with 0.2% (w/v) Brij 58 (Sigma) in PBS for 7 min. The cells were blocked with 10% FCS in PBS for 1 h and incubated with a monoclonal anti-GAPDH antibody (Biotrend, Cologne, Germany) at a 1:100 dilution for an additional 2 h. After three washing steps with PBS, an FITCcoupled secondary antibody (FITC anti-mouse IgG, Sigma) was used at a 1:200 dilution for 30 min in PBS and washed five times with PBS. GAPDH staining was then monitored by CLSM. For permeabilisation, the cells were incubated for 7 min in PBS containing 0.2% (w/v) Brij 58 prior to fixation.
G-actin was purified from rabbit muscle and kindly provided as an acetone powder by Prof Dr Hugo Fasold (Department of Biochemistry, Biocentre, University of Frankfurt). G-actin (5 mg/ml in 2 mM Tris-HCl pH 8.0, 0.2 mM ATP, 0.5 mM DTT and 0.2 mM CaCl2) was polymerised by the addition of KCl (final concentration of 50 mM), ATP (1 mM) and MgCl2 (1 mM) at room temperature for 30 min. For co-sedimentation analysis 5 M polymerised actin was incubated for 15 min at room temperature with 2.5 M GAPDH (rabbit muscle, Sigma (Deisenhofen, Germany)) and 1.5 mM glyceraldehyde-3-phosphate (Sigma) with 3 mM NADH or NAD+ according to the protocol of Mejean et al. (1989) and centrifuged at 100,000g at room temperature. The supernatant and pellet fractions were collected, dissolved in Laemmli buffer and the proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie brilliant blue using standard protocols. Cell culture The NIH 3T3 cells (p16 to p60) were cultured in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Karlsruhe, Germany) with 10% fetal
CLSM Confocal laser scanning microscopy (CLSM) was done with a Leica TCS 4D (Leica, Bensheim, Germany) equipped with the appropriate filters and PL Fluotar objectives (100, 1.3; 40, 1.0), controlled by the SCAN Ware 5.10 software (Leica, Wetzlar, Germany). Images were processed with IMARIS and Selima software (Bitplane AG, Zu¨rich, Switzerland) on a Unix workstation. The pictures are ‘maximum intensity’ of several individual CLSM pictures.
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Cloning of GAPDH
Protein extraction
The cDNA from human GAPDH was cloned out of the human keratinocyte cell line HaCaT by RT-PCR. For this purpose total RNA was purified from confluent HaCaT cells grown on a 6 cm Petri dish with Trizol (Life Technologies) according to the manufacturer’s protocol. The reverse transcription of the RNA was done with M-MLV Reverse Transcriptase from Promega (Mannheim, Germany) according to the manufacturer’s protocol, with oligo-dT primers. One tenth of the cDNA was used as a template for PCR and amplified with human GAPDH specific primers (5 -primer: 5 -GAGGATCCATGGGGAAGGTGAAG-3 , 3 -primer: 5 -CTGTCGACTCCTTGGAGGCC ATG-3 ) coding additional restriction enzyme sites (5 BamHI, 3 SalI). The oligonucleotides were synthesised by ARK Scientific GmbH Biosystems (Darmstadt, Germany). The amplification was performed as follows: two min at 94C, three cycles at 94 (30 s)/49 (30 s)/72C (1 min) and 30 cycles at 94 (30 s)/69 (30 s)/72C (1 min) in 1.5 mM MgCl2, 20 mM Tris-HCl pH 8.0, 50 mM KCl, 0.2 mM dNTPs, 0.5 M each primer and 0.5 U Taqpolymerase (Sigma, Deisenhofen, Germany) in a reaction volume of 50 l. The amplification product was excised from agarose gels, digested with the appropriate restriction enzymes, and cloned into the BglII/SalI restriction sites of the expression vector pEGFP-C1 (Clontech, Heidelberg, Germany) and named pEGFPGAPDH. The GAPDH clone obtained was verified by sequencing (Scientific Research & Development GmbH, Oberursel, Germany).
Cells grown on a 6 cm Petri dish were washed twice with PBS and incubated with 0.5 ml ice-cold RIPA buffer (50 mM Tris HCl pH 7.4, 1% Nonidet P-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM NaF, mammalian protease inhibitor cocktail (1:100; Sigma)) for 5 min and scraped with a plastic cell scraper. The lysate was transferred to a centrifuge tube and gently rocked for 15 min at 4C. The lysate was centrifuged at 14,000g for 15 min and the supernatant transferred to a fresh centrifuge tube, aliquoted and stored at 20C. Protein concentration was measured using the method of Bradford with the BioRad Laboratories (Munich, Germany) system. Immunoblot analysis SDS-PAGE was carried out using standard protocols with 15 to 30 g of protein. The gels were blotted onto nitrocellulose, stained with Ponceau S to monitor equivalent probe loading and blocked with 5% milk powder in T-TBS (0.01% Tween 20, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0) for 2 h. The anti-GFP primary antibody (Clontech, Heidelberg, Germany) was used at a 1:100 dilution for two h at room temperature or overnight at 4C. After washing four times with T-TBS, the secondary alkaline phosphatase conjugated antibody (AP coupled anti-mouse IgG; 1:1000; Sigma) was incubated for 1–2 h at room temperature in T-TBS. Immunoblot analysis was carried out with the FAST BCIP/NBT tablet system (Sigma) according to the manufacturer’s protocol.
Transient transfection Cells were transiently transfected by electroporation. Subconfluent cells grown in culture flasks were trypsinised and resuspended in ice-cold culture medium, then centrifuged for 5 min at 500 rpm in a Hermle centrifuge Z360K at 4C. The cells were washed with 10 ml cold PBS, centrifuged again and resuspended in 200 l PBS/Ringer. Cells were incubated in a 200 l electroporation volume with 10 g plasmid DNA and electroporated in a 1 ml electroporation cuvette at 225 F, 300 V and 13 Ohm (Electro Cell Manipulator ECM 600 from BTX Electronic Genetics, San Diego, U.S.A.). The cells were incubated on ice for 15 min, then plated on 3 35 mm Petri dishes (for microscopical analysis) or one 6 cm Petri dish (for protein extraction) with DMEM/10% FCS and further used after one or two days.
GAPDH enzyme assay The assay was performed in a volume of 1 ml 80 mM triethanolamine pH 7.6, 1 mM ATP, 6 mM glycerate-3-phosphate, 0.2 mM NADH, 0.9 mM EDTA (pH 8.0) and 2 mM MgSO4 containing 100 mU 3-phosphoglyceratekinase (Sigma), by measuring the decrease in NADH extinction at 340 nm after the addition of 20–40 g protein extracts (Spectronic 3000 array; Milton Roy, Obertshausen, Germany). F-actin staining F-actin was stained with TRITC-labelled phalloidin (‘mixed isomers’; Sigma). The cells were fixed with 4% formaldehyde and incubated with
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2 3 4 + + + G3P G3P/NAD+ G3P/NADH
5 + –
Actin
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Fig. 1. Interaction of GAPDH and F-actin in vitro: SDS-PAA gel of pellet fractions of the GAPDH co-sedimentated with F-actin. GAPDH did not pellet in the absence of F-actin (lane 1). Co-sedimentation occurs with F-actin in the absence or presence of the substrate G3P (lane 5/2). While the addition of G3P and NADH did not affect co-sedimentation (lane 4), the presence of G3P and NAD + enhanced the interaction (lane 3).
50 M TRITC-phalloidin solution for 20 min, washed 3 times with PBS and investigated by CLSM. RESULTS The association of GAPDH with actin filaments was analysed using 3 different methods: first with co-sedimentation analysis to monitor the binding in vitro; second, indirect immunofluorescence showed the distribution of GAPDH within the cell; and third, the genetic approach of overexpression of GFP-tagged GAPDH allowed direct observation of the intracellular localisation of the fusion protein in cells. GAPDH binds to F-actin in vitro To verify the F-actin binding property of GAPDH in vitro, 5 M G-actin was polymerised, incubated with 2.5 M GAPDH protein and ultracentrifuged. The supernatant and pellet fractions
were separated on an SDS-PAA gel and stained with Coomassie blue. The pellet fractions showed the F-actin associated part of GAPDH (Fig. 1). GAPDH specifically co-sedimentated with F-actin (lane 5); GAPDH without F-actin did not appear in the pellet (lane 1). The binding of GAPDH with actin filaments was dependent on the presence of substrates and cofactors, which were added in excess. The interaction was enhanced by the presence of 3 mM glyceraldehyde-3-phosphate (G3P) and 3 mM NAD + (lane 3), while addition of G3P and NADH (lane 4) or G3P without cofactor (lane 2) reduced or did not affect co-sedimentation. The product DHAP and the cofactor NAD + did not promote the binding activity (data not shown). The relevance of this in vitro observation was proven by immunostaining and overexpression of GFP-tagged GAPDH in cells. Intracellular localisation of endogenous GAPDH NIH 3T3 and XTH2 cells were immunostained for GAPDH and the intracellular distribution of GAPDH was monitored by CLSM (Fig. 2). GAPDH is homogeneously distributed within the cytoplasm and no association with cytoskeletal elements was found. As association of GAPDH with F-actin was enhanced upon addition of G3P and NAD + , to allow for manipulating of metabolite concentration cells were permeabilised and incubated with culture medium containing 3 mM G3P and 3 mM NAD + . Subsequent GAPDH immunostaining revealed no cytoskeletal interaction (data not shown). To prevent fixation artefacts causing disruption of a presumed GAPDH–cytoskeleton complex, the cDNA of human GAPDH was cloned and expressed as a GFP fusion protein. Thus intracellular distribution in living cells could be monitored.
Fig. 2. Immunostaining of GAPDH in NIH 3T3 and XTH2 cells. Immunostaining of GAPDH (A, B: NIH 3T3 cells; C: XTH2 cells) revealed homogeneous distribution within the cytoplasm (A/C: overview (40objective); B: higher magnification (100objective, part)).
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3-fold in cells expressing the GFP–GAPDH fusion protein indicates that GFP-tagging does not interfere with the enzymatic activity. The investigation of the localisation of overexpressed GFP–GAPDH by CLSM showed that the fusion protein is homogeneously distributed within the cytoplasm of living cells (Fig. 4A). GFP–GAPDH is excluded from the mitochondria and vesicles, and an association with cytoskeletal elements was not observed. Instead, CLSM sections of the cell bottom revealed dark cables, likely to be stress fibres, thus excluding the fusion protein (Fig. 4B). This indicates that GFP–GAPDH is not associated with any large filamentous structure in vivo during normal culturing conditions.
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Fig. 3. Immunoblot and enzyme activity of protein extracts of cells expressing GFP-GAPDH. (A) Immunoblot with antiGFP antibodies of protein extracts prepared out of cells transfected with pEGFP (1) or pEGFP-GAPDH (2). (B) Relative GAPDH enzyme activity (mU/mg). (MeansS.D. of three independent experiments).
Expression of GFP-tagged GAPDH The cDNA of GAPDH was cloned from the human keratinocyte cell line HaCaT by RT-PCR and fused 3 to the cDNA of GFP in the mammalian expression vector pEGFP-C1. The plasmid was transiently transfected into NIH 3T3 cells. To confirm the correct expression of the fusion protein, total protein was extracted from cells transfected with pEGFP-GAPDH and analysed by immunoblotting. Protein extracts from cells transiently transfected with pEGFP-C1 were used as control. Using anti-GFP antibody in immunoblot studies, the presence of only one protein band with the expected molecular weight of approximately 67 kD in cells transfected with pEGFP-GAPDH and 27 kD in cells transfected with pEGFP only (Fig. 3A) was detectable. The functional integrity of the GFP-tagged GAPDH was confirmed by enzyme assays. Protein extracts of 3T3 cells transiently transfected with pEGFP-GAPDH had a relative GAPDH activity of 50 mU/mg protein (Fig. 3B), whereas cells transfected with pEGFP had a relative enzyme activity of about 15 mU/mg. The increase in GAPDH enzyme activity of about
Serum deprivation alters the cellular structure of NIH 3T3 cells. They spread more and flatten. Although the actin cytoskeleton is rearranged, stress fibres are still prominent as shown by TRITC-phalloidin staining of cells serum deprived for five days (Fig. 5). Serum starvation not only causes G0-arrest but, in addition, it alters the binding property of microfilaments towards other proteins: e.g. GFP as well as gelsolin becomes associated with F-actin upon serum depletion (Paddenberg et al., 2001; Schmitz and BereiterHahn, 2001). Therefore, the localisation of GAPDH in serum depleted cells has been investigated. Serum depletion induces nuclear targeting of endogenous and GFP-tagged GAPDH (Schmitz, 2001; Shashidharan et al., 1999). GAPDH protein which is left in the cytoplasm is no longer homogeneously distributed, but found associated with stress fibres (Fig. 6). As GFP alone also associates with stress fibres upon serum deprivation (Schmitz and Bereiter-Hahn, 2001), the specificity was investigated by immunostaining of endogenous GAPDH. Cells serum-deprived for five days showed cytoplasmic and nuclear localisation of GAPDH (Fig. 7A); within the cytoplasm the protein was co-localised to stress fibres (Fig. 7B). Thus, GAPDH is specifically associated with microfilaments in serum deprived NIH 3T3 cells. The GAPDH protein can be modified posttranslationally by NO (Mohr et al., 1996), which enhances the binding of GAPDH to F-actin in vitro (Wu et al., 1997). As serum deprivation might induce NO release, which is able to modify the GAPDH protein, the influence of the NOsignalling pathway on the binding of GAPDH
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Fig. 4. Intracellular distribution of GFP-GAPDH in vivo. CLSM images of living cells expressing GFP-GAPDH (A: overview, 100objective). Magnification reveals stress fibres and mitochondria excluding GFP-GAPDH (B, 100objective, part).
Fig. 5. F-actin distribution in proliferative and serum deprived cells. CLSM images of cells cultured in the presence (A) or absence (B: 5 days) of 10% serum and stained with TRITC-phalloidin to visualise F-actin. 100objective.
to stress fibres in serum deprived cells was investigated. To prevent a putative NO production, during the serum starvation cells were incubated with L-NMMA, an inhibitor of the inducible NOsynthase (iNOS). Addition of 0.5 mM L-NMMA for five days did not prevent stress fibres association with immunostained GAPDH upon serum depletion (Fig. 7C). Furthermore, the effect of NO on the intracellular distribution of GAPDH was
studied by inducing the NO-synthase with BrcAMP (Kleinert et al., 1996). 3T3 cells were cultured for 16 h with 0.5% serum and stimulated for 8 h with medium containing 10% serum and 1 mM Br-cAMP to induce NO-synthase II. The homogeneous cytoplasmic distribution was not influenced and neither microfilament nor microtubule binding of GAPDH could be detected (Fig. 7D).
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Fig. 6. Intracellular distribution of GFP-GAPDH upon serum deprivation. CLSM images of cells, serum deprived for five days, expressing GFP-GAPDH. (A) (overview, 20objective) serum depletion triggered nuclear transport of GFP-GAPDH. (B) (100objective) fusion protein left in the cytoplasm is associated with filaments. TRITC-phalloidin staining revealed colocalisation of GFP-GAPDH (C: GFP-fluorescence, 100objective, part) with stress fibres (D: TRITC-fluorescence, 100objective, part).
DISCUSSION GAPDH is not associated with microfilaments in proliferating NIH 3T3 cells In vitro, GAPDH binds to F-actin as demonstrated by co-sedimentation analysis (Fig. 1) and several other studies (Bronstein and Knull, 1981; Clarke and Masters, 1975; Lanzara and Grazi, 1987; Mejean et al., 1989). This interaction is modulated by the presence of substrate and cofactor and is enhanced by addition of G3P and NADH (Fig. 1). As other proteins bind to F-actin in vitro, e.g. histones, lysozyme, bovine serum albumin, and RNaseA (Griffith and Pollard, 1982; Lakatos and
Minton, 1991), it is unlikely that all these proteins bind F-actin in vivo. Therefore, the intracellular distribution of GAPDH was investigated by immunostaining in different cell lines (Fig. 2). Confocal laser scanning microscopy revealed a homogeneous, diffuse distribution of GAPDH within the cytoplasm, whilst a co-localisation to cytoskeletal elements like microfilaments and microtubules was not detectable. Several different fixation procedures (permeabilisation followed by fixation; fixation with paraformaldehyde alone or with methanol; addition of substrates/cofactors during fixation/permeabilisation) proved to have no influence on the F-actin association of GAPDH
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Fig. 7. Intracellular distribution of endogenous GAPDH upon serum deprivation and the participation of the NO-signalling pathway. CLSM images of cells serum deprived for five days and immunostained for GAPDH. (A, 40objective) (overview) serum depletion triggered nuclear transport of endogenous GAPDH. (B) (magnification) GAPDH protein left in the cytoplasm co-localised to stress fibres. (C) inhibition of NO-synthase by L-NMMA did not impair the co-localisation of GAPDH to microfilaments upon serum starvation (5 days). (D) cells cultured with 0.5% serum for 20 h and incubated with Br-cAMP for 8 h in the presence of 10% serum show no cytoskeletal association of GAPDH. (B, C & D, 20objective).
(data not shown). The results are contrary to results obtained previously by Minaschek et al. (1992). This could be due to cross-reaction of the antibody used with actin. As fixation might disturb or impair the detection of microfilament bound GAPDH, we used the powerful tool of GFP-tagging to monitor the distribution of GFPGAPDH in living cells. Therefore, the cDNA of human GAPDH was cloned by RT-PCR and fused to the GFP codon. The protein was expressed in NIH 3T3 cells as a single protein with the expected molecular weight of approximately 67 kDa as revealed by immunoblotting with anti-GFP antibodies (Fig. 3A). The fusion protein was catalytically active, although no enzyme measurements
have been performed with purified GFP-GAPDH: cells transiently transfected with pGFP-GAPDH had a threefold higher relative GAPDH enzyme activity compared to pGFP transfected control cells (Fig. 3B). Within the cells GFP-GAPDH was homogeneously distributed and no binding to F-actin or microtubules was detectable, as already shown by immunostaining (Fig. 4A). Moreover, in living cells GFP-GAPDH was excluded from cablelike structures, likely to be stress fibres (Fig. 4B). Serum deprivation induces association of GAPDH with microfilaments Microfilaments become rearranged upon serum withdrawal, but they are still present within
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cells serum-starved for five days (Fig. 5). Serum deprivation, as well as induction of apoptosis, triggers nuclear localisation of GAPDH (Ishitani et al., 1998; Sawa et al., 1997; Schmitz, 2001; Figs 6A, 7A). Upon serum deprivation, GAPDH protein, which is retained within the cytoplasm, is no longer homogeneously distributed, but associates with stress fibres (Fig. 7B). The NO-signalling pathway is not involved in the induction of the microfilament binding of GAPDH upon serum depletion: inhibition of NO-synthase by L-NMMA during serum deprivation did not prevent stress fibre attachment (Fig. 7C). Moreover, induction of NOsynthase by Br-cAMP treatment of serum starved cells was not sufficient to induce association of GAPDH with microfilaments (Fig. 7D). Microfilaments are the only cytoskeletal proteins found to interact with GAPDH. Though GAPDH binds to microtubules in vitro (Durrieu et al., 1987b; Humphreys et al., 1986), no association was detected within cells. Using CLSM we excluded blurring due to unbound GAPDH, which could impede the detection of GAPDH associated with filaments. We cannot exclude the binding of GAPDH to individual filaments only, which would be below the optical resolution of the techniques used. Binding of microtubules, however, should be detectable, especially as in vitro studies reported bundling activity for GAPDH (Huitorel and Pantaloni, 1985; Somers et al., 1990; Volker et al., 1995). Further experiments using e.g. fluorescence life-time imaging would allow us to distinguish between cytoskeleton associated and ‘free’ GAPDH fractions. In contrast to the in vitro and in vivo cytoskeletal binding properties of GAPDH, no such differences were found for the glycolytic enzyme aldolase (Pagliaro and Taylor, 1992; Schindler et al., 2001; Wang et al., 1997). As GAPDH becomes attached to stress fibres in cells deprived of serum, it is unlikely that the association has a general function, such as to create a glycolytic microcompartment or to allow an enhanced glycolytic flux via substrate channelling (Masters et al., 1987; Ovadi and Srere, 2000; Srivastava and Bernhard, 1986). Instead, the cytoskeletal association of GAPDH upon serum depletion might serve a special purpose for this condition, e.g. initiating cytoskeletal rearrangements during apoptosis which is induced by prolonged serum withdrawal. Our results demonstrate that protein associations detected by in vitro studies or simulated by computer (Ouporov et al., 2001) do not necessarily reflect the situation within cells: the binding of GAPDH to microtubules could not be verified in cells and the interaction with
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microfilaments did not occur constitutively, but was prominent in cells deprived of serum. An explanation might be that actin binding proteins prevent the association of GAPDH with F-actin in proliferating cells. Upon serum starvation these proteins may have decreased actin binding affinities, enabling an association with GAPDH. Another possibility could be the involvement of GAPDH in actin reorganisation like that described for gelsolin upon serum withdrawal (Paddenberg et al., 2001).
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and for actin on the enzymes, glyceraldehydephosphate dehydrogenase and phosphofructokinase. Int J Biochem 18: 445–451. I R, T M, S K, K N, C DM, 1998. Nuclear localization of overexpressed glyceraldehyde-3-phosphate dehydrogenase in cultured cerebellar neurons undergoing apoptosis. Mol Pharmacol 53: 701–707. K H, E C, I-B I, F U, 1996. In murine 3T3 fibroblasts, different second messenger pathways resulting in the induction of NO synthase II (iNOS) converge in the activation of transcription factor NF-kappaB. J Biol Chem 271: 6039–6044. K HR, W JL, 1992. Association of glycolytic enzymes with the cytoskeleton. Curr Top Cell Regul 33: 15–30. L S, M AP, 1991. Interactions between globular proteins and F-actin in isotonic saline solution. J Biol Chem 266: 18707–18713. L V, G E, 1987. On an Mg2+-dependent interaction of actin with glyceraldehyde-phosphate dehydrogenase. The fundamental role of KCl in the organization of F-actin. FEBS Lett 221: 387–390. M CJ, R S, D M, 1987. Glycolysis—new concepts in an old pathway. Mol Cell Biochem 76: 3–14. M C, P F, B Y, R C, 1989. Antigenic probes locate binding sites for the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, aldolase and phosphofructokinase on the actin monomer in microfilaments. Biochem J 264: 671–677. M G, G¨ -S U, B S, BH J, 1992. Microcompartmentation of glycolytic enzymes in cultured cells. Eur J Cell Biol 58: 418–428. M S, S JS, B B, 1996. Posttranslational modification of glyceraldehyde-3-phosphate dehydrogenase by S-nitrosylation and subsequent NADH attachment. J Biol Chem 271: 4209–4214. N TN, W HJ, Z S, N A, N IR, 2000. Purification and characterization of beta-actin-rich tumor cell pseudopodia: role of glycolysis. Exp Cell Res 258: 171–183. O IV, K HR, L SL, T KA, 2001. Interactions of glyceraldehyde-3-phosphate dehydrogenase with G- and F- actin predicted by Brownian dynamics. J Mol Recognit 14: 29–41. O J, S PA, 2000. Macromolecular compartmentation and channeling. Int Rev Cytol 192: 255–280. P R, L S, S H-J, W S, M A, I M, M HG, 2001. Serum withdrawal induces a redistribution of intracellular gelsolin towards
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F-actin in NIH 3T3 fibroblasts preceding apoptotic cell death. Europ J Cell Biology 80: 366–387. P L, T DL, 1992. 2-Deoxyglucose and cytochalasin D modulate aldolase mobility in living 3T3 cells. J Cell Biology 118: 859–863. S A, K AA, H LD, S SH, 1997a. Glyceraldehyde-3-phosphate dehydrogenase: nuclear translocation participates in neuronal and nonneuronal cell death. Proc Natl Acad Sci (U.S.A.) 94: 11669–11674. S R, W O, W E, BH J, 2001. Aldolase-localisation in cultured cells: Celltype and substrate-specific regulation of cytoskeletal associations. Biochem Cell Biol, in press. S WK, K W, B-H J, 1981. Established Xenopus tadpole heart endothelium (XTH) cells exhibiting selected properties of primary cells. Eur J Cell Biol 24: 324. S H-D, 2001. Reversible nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase upon serum depletion. Eur J Cell Biol 80: 419–427. S H-D, B-H J, 2001. GFP associates with microfilaments in fixed cells. Histochem Cell Biol 116: 89–94. S P, C-R RM, C GW, R V, G N, Y T, T WG, S SC, 1999. Nuclear translocation of GAPDH-GFP fusion protein during apoptosis. Neuroreport 10: 1149–1153. S M, E Y, B J, 1990. Analysis of the binding of glyceraldehyde-3-phosphate dehydrogenase to microtubules, the mechanism of bundle formation and the linkage effect. Eur J Biochem 193: 437–444. S PA, K HR, 1998. Location-location-location. Trends Biochem Sci 23: 319–320. S DK, B SA, 1986. Enzyme-enzyme interactions and the regulation of metabolic reaction pathways. Curr Top Cell Regul 28: 1–68. V KW, R CA, K HR, 1995. Glycolytic enzymes and assembly of microtubule networks. Comp Biochem Physiol B Biochem Mol Biol 112: 503–514. W JL, K TJ, K HR, 1989. Glycolytic enzyme interactions with tubulin and microtubules. Biochim Biophys Acta 999: 64–70. W J, T DR, P L, 1997. Metabolic compartmentation in living cells: structural association of aldolase. Exp Cell Res 237: 445–451. W K, A C, E A, R-W AA, S P, 1997. The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide [published erratum appears in Proc Natl Acad Sci U.S.A. 1998 Mar 3;95(5):2714]. Proc Natl Acad Sci (U.S.A.) 94: 13273–13278.
GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ASSOCIATES WITH ACTIN FILAMENTS IN SERUM DEPRIVED NIH 3T3 CELLS ONLY HANS-DIRK SCHMITZ and JU } RGEN BEREITER-HAHN* Kinematic Cell Research Group, Biocentre, Goethe University of Frankfurt/Main, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany Received 31 May 2001; accepted 15 August 2001; published electronically 16 January 2002
The in vitro interaction between the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cytoskeletal elements is well documented. To verify this association within cells, the intracellular distribution of GAPDH under various metabolic conditions has been investigated in immunostained cells or cells expressing GAPDH as a GFP fusion protein. GAPDH was homogeneously distributed in the cytoplasm and no interaction of GAPDH with cytoskeletal elements, neither with microfilaments nor microtubules or intermediate filaments, was detectable. In living cells expressing GFP-GAPDH, stress fibres were excluded from the fluorescence. In contrast to proliferating cells, the cytoplasmic GAPDH of serum-depleted cells was not homogeneously distributed, but colocalised with stress fibres. The mechanism for stimulating this actin-binding affinity was independent of the NO-signalling pathway. The results support the idea of a specialised function for the interaction of GAPDH and cytoskeletal elements, rather than a general function, as e.g. microcompartmentalization of glycolytic 2002 Elsevier Science Ltd. enzymes. K: GAPDH; actin; serum withdrawal; association. A: Br-cAMP, Bromo-adenosine-3 ,5 -cyclic-monophosphate; CLSM, confocal laser scanning microscopy; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; L-NMMA, NG-Monomethyl-L-arginine.
INTRODUCTION Glycolysis is an enzymatic process which takes place within the cytosol. However, some of the glycolytic enzymes, for instance aldolase (EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) and phosphofructokinase (PFK, EC 2.7.1.11) may interact with the cytoskeleton and were found to be associated with microtubules and microfilaments in vitro. The association between glycolytic enzymes and the cytoskeleton was first observed in enzyme purification from muscle tissue (Arnold and Pette, 1970): Some enzymes were not completely soluble but remained bound to the cytoskeleton fraction (actin filaments). Further studies revealed direct binding of some glycolytic enzymes (aldolase, GAPDH and *To whom correspondence should be addressed: E-mail: [email protected] 1065–6995/02/020155+15 $35.00/0
PFK) with cytoskeletal elements (reviewed by Bereiter-Hahn et al., 1997; Srere and Knull, 1998). The kinetics of GAPDH and cytoskeleton interaction are well known from in vitro binding studies (Durrieu et al., 1987a; Durrieu et al., 1987b; Humphreys et al., 1986; Walsh et al., 1989), revealing an affinity constant (KD) of 0.6 M for GAPDH to F-actin and 3.1 M towards microtubules (Knull and Walsh, 1992). In vitro, the actin binding of GAPDH is enhanced upon nitrosylation (Wu et al., 1997), a post-translational modification which occurs upon incubation with nitric oxide (NO) donors and represents an important pathway of signal transduction. Further data about the association of GAPDH and cytoskeletal elements come from permeabilized cells retaining GAPDH enzyme activity in cytoskeleton containing fractions (Cao et al., 1999; Humphreys and Masters, 1986). GAPDH is found in pseudopodia, where it 2002 Elsevier Science Ltd.
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might interact with actin (Nguyen et al., 2000). The physiological relevance of this cytoskeletal interaction is still an enigma, although electron microscopy and polymerisation studies implicate a direct alteration of the filament properties, like bundling of microtubules (Huitorel and Pantaloni, 1985; Somers et al., 1990; Volker et al., 1995). Because most experiments were carried out in vitro, it is of central interest to verify the interaction of GAPDH and cytoskeletal elements within cells. To prove such an interaction we monitored the intracellular distribution of GAPDH either by immunostaining or by overexpression of a GFP-GAPDH fusion protein in NIH 3T3 cells. Whereas our in vitro binding studies showed an association of GAPDH with microfilaments dependent on the availability of substrate/cofactor, immunostaining and GFP-GAPDH overexpression failed to detect such interaction in proliferating cells. A co-localisation of GAPDH with stress fibres was observed only in cells after prolonged serum depletion.
calf serum (FCS; Life Technologies). Human keratinocyte cells (HaCaT, p60–70; Boukamp et al., 1988) were cultured in keratinocyte medium (Life Technologies) with 10% FCS. The Xenopus laevis tadpole heart endothelial cell line XTH2 (Schlage et al., 1981) (p320–360) was cultured in MEM with 10% FCS. For serum withdrawal experiments, cells were washed with serum-free medium and then incubated with serum-free medium as described. The inhibitor of the NOsynthase, NG-mono-methyl-L-arginine (L-NMMA, Sigma), was used at a final concentration of 500 M and freshly added each day. The cells were grown on cover glasses in 35 mm Petri dishes when used for fluorescence microscopy. For monitoring GFP fluorescence, cells were washed twice with physiological saline and fixed with 4% (w/v) freshly prepared formaldehyde in PBS for 20 min with three subsequent washing steps. Living cells were investigated under controlled temperature in the appropriate media buffered with Hepes instead of bicarbonate.
MATERIALS AND METHODS
Immunostaining
Cosedimentation analysis
NIH 3T3 or XTH2 cells cultivated on glass were washed twice in PBS fixed with 4% formaldehyde/ 0.25% glutaraldehyde in PBS for 20 min and permeabilised with 0.2% (w/v) Brij 58 (Sigma) in PBS for 7 min. The cells were blocked with 10% FCS in PBS for 1 h and incubated with a monoclonal anti-GAPDH antibody (Biotrend, Cologne, Germany) at a 1:100 dilution for an additional 2 h. After three washing steps with PBS, an FITCcoupled secondary antibody (FITC anti-mouse IgG, Sigma) was used at a 1:200 dilution for 30 min in PBS and washed five times with PBS. GAPDH staining was then monitored by CLSM. For permeabilisation, the cells were incubated for 7 min in PBS containing 0.2% (w/v) Brij 58 prior to fixation.
G-actin was purified from rabbit muscle and kindly provided as an acetone powder by Prof Dr Hugo Fasold (Department of Biochemistry, Biocentre, University of Frankfurt). G-actin (5 mg/ml in 2 mM Tris-HCl pH 8.0, 0.2 mM ATP, 0.5 mM DTT and 0.2 mM CaCl2) was polymerised by the addition of KCl (final concentration of 50 mM), ATP (1 mM) and MgCl2 (1 mM) at room temperature for 30 min. For co-sedimentation analysis 5 M polymerised actin was incubated for 15 min at room temperature with 2.5 M GAPDH (rabbit muscle, Sigma (Deisenhofen, Germany)) and 1.5 mM glyceraldehyde-3-phosphate (Sigma) with 3 mM NADH or NAD+ according to the protocol of Mejean et al. (1989) and centrifuged at 100,000g at room temperature. The supernatant and pellet fractions were collected, dissolved in Laemmli buffer and the proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie brilliant blue using standard protocols. Cell culture The NIH 3T3 cells (p16 to p60) were cultured in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Karlsruhe, Germany) with 10% fetal
CLSM Confocal laser scanning microscopy (CLSM) was done with a Leica TCS 4D (Leica, Bensheim, Germany) equipped with the appropriate filters and PL Fluotar objectives (100, 1.3; 40, 1.0), controlled by the SCAN Ware 5.10 software (Leica, Wetzlar, Germany). Images were processed with IMARIS and Selima software (Bitplane AG, Zu¨rich, Switzerland) on a Unix workstation. The pictures are ‘maximum intensity’ of several individual CLSM pictures.
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Cloning of GAPDH
Protein extraction
The cDNA from human GAPDH was cloned out of the human keratinocyte cell line HaCaT by RT-PCR. For this purpose total RNA was purified from confluent HaCaT cells grown on a 6 cm Petri dish with Trizol (Life Technologies) according to the manufacturer’s protocol. The reverse transcription of the RNA was done with M-MLV Reverse Transcriptase from Promega (Mannheim, Germany) according to the manufacturer’s protocol, with oligo-dT primers. One tenth of the cDNA was used as a template for PCR and amplified with human GAPDH specific primers (5 -primer: 5 -GAGGATCCATGGGGAAGGTGAAG-3 , 3 -primer: 5 -CTGTCGACTCCTTGGAGGCC ATG-3 ) coding additional restriction enzyme sites (5 BamHI, 3 SalI). The oligonucleotides were synthesised by ARK Scientific GmbH Biosystems (Darmstadt, Germany). The amplification was performed as follows: two min at 94C, three cycles at 94 (30 s)/49 (30 s)/72C (1 min) and 30 cycles at 94 (30 s)/69 (30 s)/72C (1 min) in 1.5 mM MgCl2, 20 mM Tris-HCl pH 8.0, 50 mM KCl, 0.2 mM dNTPs, 0.5 M each primer and 0.5 U Taqpolymerase (Sigma, Deisenhofen, Germany) in a reaction volume of 50 l. The amplification product was excised from agarose gels, digested with the appropriate restriction enzymes, and cloned into the BglII/SalI restriction sites of the expression vector pEGFP-C1 (Clontech, Heidelberg, Germany) and named pEGFPGAPDH. The GAPDH clone obtained was verified by sequencing (Scientific Research & Development GmbH, Oberursel, Germany).
Cells grown on a 6 cm Petri dish were washed twice with PBS and incubated with 0.5 ml ice-cold RIPA buffer (50 mM Tris HCl pH 7.4, 1% Nonidet P-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM NaF, mammalian protease inhibitor cocktail (1:100; Sigma)) for 5 min and scraped with a plastic cell scraper. The lysate was transferred to a centrifuge tube and gently rocked for 15 min at 4C. The lysate was centrifuged at 14,000g for 15 min and the supernatant transferred to a fresh centrifuge tube, aliquoted and stored at 20C. Protein concentration was measured using the method of Bradford with the BioRad Laboratories (Munich, Germany) system. Immunoblot analysis SDS-PAGE was carried out using standard protocols with 15 to 30 g of protein. The gels were blotted onto nitrocellulose, stained with Ponceau S to monitor equivalent probe loading and blocked with 5% milk powder in T-TBS (0.01% Tween 20, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0) for 2 h. The anti-GFP primary antibody (Clontech, Heidelberg, Germany) was used at a 1:100 dilution for two h at room temperature or overnight at 4C. After washing four times with T-TBS, the secondary alkaline phosphatase conjugated antibody (AP coupled anti-mouse IgG; 1:1000; Sigma) was incubated for 1–2 h at room temperature in T-TBS. Immunoblot analysis was carried out with the FAST BCIP/NBT tablet system (Sigma) according to the manufacturer’s protocol.
Transient transfection Cells were transiently transfected by electroporation. Subconfluent cells grown in culture flasks were trypsinised and resuspended in ice-cold culture medium, then centrifuged for 5 min at 500 rpm in a Hermle centrifuge Z360K at 4C. The cells were washed with 10 ml cold PBS, centrifuged again and resuspended in 200 l PBS/Ringer. Cells were incubated in a 200 l electroporation volume with 10 g plasmid DNA and electroporated in a 1 ml electroporation cuvette at 225 F, 300 V and 13 Ohm (Electro Cell Manipulator ECM 600 from BTX Electronic Genetics, San Diego, U.S.A.). The cells were incubated on ice for 15 min, then plated on 3 35 mm Petri dishes (for microscopical analysis) or one 6 cm Petri dish (for protein extraction) with DMEM/10% FCS and further used after one or two days.
GAPDH enzyme assay The assay was performed in a volume of 1 ml 80 mM triethanolamine pH 7.6, 1 mM ATP, 6 mM glycerate-3-phosphate, 0.2 mM NADH, 0.9 mM EDTA (pH 8.0) and 2 mM MgSO4 containing 100 mU 3-phosphoglyceratekinase (Sigma), by measuring the decrease in NADH extinction at 340 nm after the addition of 20–40 g protein extracts (Spectronic 3000 array; Milton Roy, Obertshausen, Germany). F-actin staining F-actin was stained with TRITC-labelled phalloidin (‘mixed isomers’; Sigma). The cells were fixed with 4% formaldehyde and incubated with
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Fig. 1. Interaction of GAPDH and F-actin in vitro: SDS-PAA gel of pellet fractions of the GAPDH co-sedimentated with F-actin. GAPDH did not pellet in the absence of F-actin (lane 1). Co-sedimentation occurs with F-actin in the absence or presence of the substrate G3P (lane 5/2). While the addition of G3P and NADH did not affect co-sedimentation (lane 4), the presence of G3P and NAD + enhanced the interaction (lane 3).
50 M TRITC-phalloidin solution for 20 min, washed 3 times with PBS and investigated by CLSM. RESULTS The association of GAPDH with actin filaments was analysed using 3 different methods: first with co-sedimentation analysis to monitor the binding in vitro; second, indirect immunofluorescence showed the distribution of GAPDH within the cell; and third, the genetic approach of overexpression of GFP-tagged GAPDH allowed direct observation of the intracellular localisation of the fusion protein in cells. GAPDH binds to F-actin in vitro To verify the F-actin binding property of GAPDH in vitro, 5 M G-actin was polymerised, incubated with 2.5 M GAPDH protein and ultracentrifuged. The supernatant and pellet fractions
were separated on an SDS-PAA gel and stained with Coomassie blue. The pellet fractions showed the F-actin associated part of GAPDH (Fig. 1). GAPDH specifically co-sedimentated with F-actin (lane 5); GAPDH without F-actin did not appear in the pellet (lane 1). The binding of GAPDH with actin filaments was dependent on the presence of substrates and cofactors, which were added in excess. The interaction was enhanced by the presence of 3 mM glyceraldehyde-3-phosphate (G3P) and 3 mM NAD + (lane 3), while addition of G3P and NADH (lane 4) or G3P without cofactor (lane 2) reduced or did not affect co-sedimentation. The product DHAP and the cofactor NAD + did not promote the binding activity (data not shown). The relevance of this in vitro observation was proven by immunostaining and overexpression of GFP-tagged GAPDH in cells. Intracellular localisation of endogenous GAPDH NIH 3T3 and XTH2 cells were immunostained for GAPDH and the intracellular distribution of GAPDH was monitored by CLSM (Fig. 2). GAPDH is homogeneously distributed within the cytoplasm and no association with cytoskeletal elements was found. As association of GAPDH with F-actin was enhanced upon addition of G3P and NAD + , to allow for manipulating of metabolite concentration cells were permeabilised and incubated with culture medium containing 3 mM G3P and 3 mM NAD + . Subsequent GAPDH immunostaining revealed no cytoskeletal interaction (data not shown). To prevent fixation artefacts causing disruption of a presumed GAPDH–cytoskeleton complex, the cDNA of human GAPDH was cloned and expressed as a GFP fusion protein. Thus intracellular distribution in living cells could be monitored.
Fig. 2. Immunostaining of GAPDH in NIH 3T3 and XTH2 cells. Immunostaining of GAPDH (A, B: NIH 3T3 cells; C: XTH2 cells) revealed homogeneous distribution within the cytoplasm (A/C: overview (40objective); B: higher magnification (100objective, part)).
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3-fold in cells expressing the GFP–GAPDH fusion protein indicates that GFP-tagging does not interfere with the enzymatic activity. The investigation of the localisation of overexpressed GFP–GAPDH by CLSM showed that the fusion protein is homogeneously distributed within the cytoplasm of living cells (Fig. 4A). GFP–GAPDH is excluded from the mitochondria and vesicles, and an association with cytoskeletal elements was not observed. Instead, CLSM sections of the cell bottom revealed dark cables, likely to be stress fibres, thus excluding the fusion protein (Fig. 4B). This indicates that GFP–GAPDH is not associated with any large filamentous structure in vivo during normal culturing conditions.
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Fig. 3. Immunoblot and enzyme activity of protein extracts of cells expressing GFP-GAPDH. (A) Immunoblot with antiGFP antibodies of protein extracts prepared out of cells transfected with pEGFP (1) or pEGFP-GAPDH (2). (B) Relative GAPDH enzyme activity (mU/mg). (MeansS.D. of three independent experiments).
Expression of GFP-tagged GAPDH The cDNA of GAPDH was cloned from the human keratinocyte cell line HaCaT by RT-PCR and fused 3 to the cDNA of GFP in the mammalian expression vector pEGFP-C1. The plasmid was transiently transfected into NIH 3T3 cells. To confirm the correct expression of the fusion protein, total protein was extracted from cells transfected with pEGFP-GAPDH and analysed by immunoblotting. Protein extracts from cells transiently transfected with pEGFP-C1 were used as control. Using anti-GFP antibody in immunoblot studies, the presence of only one protein band with the expected molecular weight of approximately 67 kD in cells transfected with pEGFP-GAPDH and 27 kD in cells transfected with pEGFP only (Fig. 3A) was detectable. The functional integrity of the GFP-tagged GAPDH was confirmed by enzyme assays. Protein extracts of 3T3 cells transiently transfected with pEGFP-GAPDH had a relative GAPDH activity of 50 mU/mg protein (Fig. 3B), whereas cells transfected with pEGFP had a relative enzyme activity of about 15 mU/mg. The increase in GAPDH enzyme activity of about
Serum deprivation alters the cellular structure of NIH 3T3 cells. They spread more and flatten. Although the actin cytoskeleton is rearranged, stress fibres are still prominent as shown by TRITC-phalloidin staining of cells serum deprived for five days (Fig. 5). Serum starvation not only causes G0-arrest but, in addition, it alters the binding property of microfilaments towards other proteins: e.g. GFP as well as gelsolin becomes associated with F-actin upon serum depletion (Paddenberg et al., 2001; Schmitz and BereiterHahn, 2001). Therefore, the localisation of GAPDH in serum depleted cells has been investigated. Serum depletion induces nuclear targeting of endogenous and GFP-tagged GAPDH (Schmitz, 2001; Shashidharan et al., 1999). GAPDH protein which is left in the cytoplasm is no longer homogeneously distributed, but found associated with stress fibres (Fig. 6). As GFP alone also associates with stress fibres upon serum deprivation (Schmitz and Bereiter-Hahn, 2001), the specificity was investigated by immunostaining of endogenous GAPDH. Cells serum-deprived for five days showed cytoplasmic and nuclear localisation of GAPDH (Fig. 7A); within the cytoplasm the protein was co-localised to stress fibres (Fig. 7B). Thus, GAPDH is specifically associated with microfilaments in serum deprived NIH 3T3 cells. The GAPDH protein can be modified posttranslationally by NO (Mohr et al., 1996), which enhances the binding of GAPDH to F-actin in vitro (Wu et al., 1997). As serum deprivation might induce NO release, which is able to modify the GAPDH protein, the influence of the NOsignalling pathway on the binding of GAPDH
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Fig. 4. Intracellular distribution of GFP-GAPDH in vivo. CLSM images of living cells expressing GFP-GAPDH (A: overview, 100objective). Magnification reveals stress fibres and mitochondria excluding GFP-GAPDH (B, 100objective, part).
Fig. 5. F-actin distribution in proliferative and serum deprived cells. CLSM images of cells cultured in the presence (A) or absence (B: 5 days) of 10% serum and stained with TRITC-phalloidin to visualise F-actin. 100objective.
to stress fibres in serum deprived cells was investigated. To prevent a putative NO production, during the serum starvation cells were incubated with L-NMMA, an inhibitor of the inducible NOsynthase (iNOS). Addition of 0.5 mM L-NMMA for five days did not prevent stress fibres association with immunostained GAPDH upon serum depletion (Fig. 7C). Furthermore, the effect of NO on the intracellular distribution of GAPDH was
studied by inducing the NO-synthase with BrcAMP (Kleinert et al., 1996). 3T3 cells were cultured for 16 h with 0.5% serum and stimulated for 8 h with medium containing 10% serum and 1 mM Br-cAMP to induce NO-synthase II. The homogeneous cytoplasmic distribution was not influenced and neither microfilament nor microtubule binding of GAPDH could be detected (Fig. 7D).
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Fig. 6. Intracellular distribution of GFP-GAPDH upon serum deprivation. CLSM images of cells, serum deprived for five days, expressing GFP-GAPDH. (A) (overview, 20objective) serum depletion triggered nuclear transport of GFP-GAPDH. (B) (100objective) fusion protein left in the cytoplasm is associated with filaments. TRITC-phalloidin staining revealed colocalisation of GFP-GAPDH (C: GFP-fluorescence, 100objective, part) with stress fibres (D: TRITC-fluorescence, 100objective, part).
DISCUSSION GAPDH is not associated with microfilaments in proliferating NIH 3T3 cells In vitro, GAPDH binds to F-actin as demonstrated by co-sedimentation analysis (Fig. 1) and several other studies (Bronstein and Knull, 1981; Clarke and Masters, 1975; Lanzara and Grazi, 1987; Mejean et al., 1989). This interaction is modulated by the presence of substrate and cofactor and is enhanced by addition of G3P and NADH (Fig. 1). As other proteins bind to F-actin in vitro, e.g. histones, lysozyme, bovine serum albumin, and RNaseA (Griffith and Pollard, 1982; Lakatos and
Minton, 1991), it is unlikely that all these proteins bind F-actin in vivo. Therefore, the intracellular distribution of GAPDH was investigated by immunostaining in different cell lines (Fig. 2). Confocal laser scanning microscopy revealed a homogeneous, diffuse distribution of GAPDH within the cytoplasm, whilst a co-localisation to cytoskeletal elements like microfilaments and microtubules was not detectable. Several different fixation procedures (permeabilisation followed by fixation; fixation with paraformaldehyde alone or with methanol; addition of substrates/cofactors during fixation/permeabilisation) proved to have no influence on the F-actin association of GAPDH
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Fig. 7. Intracellular distribution of endogenous GAPDH upon serum deprivation and the participation of the NO-signalling pathway. CLSM images of cells serum deprived for five days and immunostained for GAPDH. (A, 40objective) (overview) serum depletion triggered nuclear transport of endogenous GAPDH. (B) (magnification) GAPDH protein left in the cytoplasm co-localised to stress fibres. (C) inhibition of NO-synthase by L-NMMA did not impair the co-localisation of GAPDH to microfilaments upon serum starvation (5 days). (D) cells cultured with 0.5% serum for 20 h and incubated with Br-cAMP for 8 h in the presence of 10% serum show no cytoskeletal association of GAPDH. (B, C & D, 20objective).
(data not shown). The results are contrary to results obtained previously by Minaschek et al. (1992). This could be due to cross-reaction of the antibody used with actin. As fixation might disturb or impair the detection of microfilament bound GAPDH, we used the powerful tool of GFP-tagging to monitor the distribution of GFPGAPDH in living cells. Therefore, the cDNA of human GAPDH was cloned by RT-PCR and fused to the GFP codon. The protein was expressed in NIH 3T3 cells as a single protein with the expected molecular weight of approximately 67 kDa as revealed by immunoblotting with anti-GFP antibodies (Fig. 3A). The fusion protein was catalytically active, although no enzyme measurements
have been performed with purified GFP-GAPDH: cells transiently transfected with pGFP-GAPDH had a threefold higher relative GAPDH enzyme activity compared to pGFP transfected control cells (Fig. 3B). Within the cells GFP-GAPDH was homogeneously distributed and no binding to F-actin or microtubules was detectable, as already shown by immunostaining (Fig. 4A). Moreover, in living cells GFP-GAPDH was excluded from cablelike structures, likely to be stress fibres (Fig. 4B). Serum deprivation induces association of GAPDH with microfilaments Microfilaments become rearranged upon serum withdrawal, but they are still present within
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cells serum-starved for five days (Fig. 5). Serum deprivation, as well as induction of apoptosis, triggers nuclear localisation of GAPDH (Ishitani et al., 1998; Sawa et al., 1997; Schmitz, 2001; Figs 6A, 7A). Upon serum deprivation, GAPDH protein, which is retained within the cytoplasm, is no longer homogeneously distributed, but associates with stress fibres (Fig. 7B). The NO-signalling pathway is not involved in the induction of the microfilament binding of GAPDH upon serum depletion: inhibition of NO-synthase by L-NMMA during serum deprivation did not prevent stress fibre attachment (Fig. 7C). Moreover, induction of NOsynthase by Br-cAMP treatment of serum starved cells was not sufficient to induce association of GAPDH with microfilaments (Fig. 7D). Microfilaments are the only cytoskeletal proteins found to interact with GAPDH. Though GAPDH binds to microtubules in vitro (Durrieu et al., 1987b; Humphreys et al., 1986), no association was detected within cells. Using CLSM we excluded blurring due to unbound GAPDH, which could impede the detection of GAPDH associated with filaments. We cannot exclude the binding of GAPDH to individual filaments only, which would be below the optical resolution of the techniques used. Binding of microtubules, however, should be detectable, especially as in vitro studies reported bundling activity for GAPDH (Huitorel and Pantaloni, 1985; Somers et al., 1990; Volker et al., 1995). Further experiments using e.g. fluorescence life-time imaging would allow us to distinguish between cytoskeleton associated and ‘free’ GAPDH fractions. In contrast to the in vitro and in vivo cytoskeletal binding properties of GAPDH, no such differences were found for the glycolytic enzyme aldolase (Pagliaro and Taylor, 1992; Schindler et al., 2001; Wang et al., 1997). As GAPDH becomes attached to stress fibres in cells deprived of serum, it is unlikely that the association has a general function, such as to create a glycolytic microcompartment or to allow an enhanced glycolytic flux via substrate channelling (Masters et al., 1987; Ovadi and Srere, 2000; Srivastava and Bernhard, 1986). Instead, the cytoskeletal association of GAPDH upon serum depletion might serve a special purpose for this condition, e.g. initiating cytoskeletal rearrangements during apoptosis which is induced by prolonged serum withdrawal. Our results demonstrate that protein associations detected by in vitro studies or simulated by computer (Ouporov et al., 2001) do not necessarily reflect the situation within cells: the binding of GAPDH to microtubules could not be verified in cells and the interaction with
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microfilaments did not occur constitutively, but was prominent in cells deprived of serum. An explanation might be that actin binding proteins prevent the association of GAPDH with F-actin in proliferating cells. Upon serum starvation these proteins may have decreased actin binding affinities, enabling an association with GAPDH. Another possibility could be the involvement of GAPDH in actin reorganisation like that described for gelsolin upon serum withdrawal (Paddenberg et al., 2001).
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