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Interaction between microtubule-associated protein tau and spectrin
BIOCHIMIE, 1984, 66, 305-311
Interaction between microtubule-associated protein tau and spectrin. Marie-France CARLIER*:, Colette SIMON*, Robert CASSOLY** and Lot~ise-Anne PRADEL**. * Laboratoire d'Enzymologie, C.N.R.S., 91190 G(f-sur-Yvette, France ** Laboratoire de Biophysique, lnstitut de Bioiogie Physico-Chimique, rue Pierre et Marie Curie. 75005 Paris France. (Refu le 7-12.1983. acceptd le 7-3-1984).
On ddmontre que le facteur tau, une des protdines associde aux microtubules de cerveau (MAPs) interagit avec la spectrine humaine. Plusieurs approches expdrimentales ont dtd utilisdes comprenant la chromatographie d'affinitd des MAPs sur colonne de sepharose couplde h la spectrine, ie recouvrement des gels d'dlectrophor~se de MAPs avec de la spectrine marqude i'iode 125, l'association du facteur tau ~ la membrane des drythrocytes, et finalement I'dtude de l'inhibition par le facteur tau du pontage par la spectrine de i'aciine F. Ces rdsultats sugg~rent que tau pourrait reprdsenter le facteur protEique impliqud dans la liaison des microtubules avec le rdseau d'actine prdsent all voisinage Je la membrane de nombreuses cellules euca~otes dans lesquelles tau et des analogues de la stTectrine sont largement reprdsentds. R6sum6 --
Mots-cl~s : microtubule / prot~ine tau / spectrine / interaction prot~ine-prot~ine.
Summary -- Tau factor, one o f the microtubule-associated proteins (MAPs), is shown here to bind to spectrin. Evidence for an interaction between these two proteins is provided by spectrin affinity chromatography o f brain MAPs, gel overlay o f electrophoresed MAPs with ;25I-labelled spectrin, incorporation o f tau factor in human erythrocyte ghosts, and demonstration that tau inhibits the F-actin cross-linking activity of tetrameric spectrin. The wide distribution o f both tau and spectrin-like proteins in euka~, otic cells is in favor o f the possible biological significance o f this interaction. The results suggest that tau could be one o f the proteins involved 67 the concerted regulation o f microtubule and actin networks in the membrane vicinity. Key-words : microtubule / tau protein / spectrin / protein-protein interaction.
The identification of proteins mediating the linkage between different components of the cytoskeleton and their attachment to membranes is a central problem in cell motility. Recently a 0 To whom all correspondence should be addressed. Present address : Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, building3, room BI-22, Bethesda, Mao,land 20205 USA.
spectrin-like protein, also named fodrin [!-3], has been isolated from brain membranes and characterized as F-actin cross-linking protein sharing many biochemical and morphological properties with erythrocyte spectrin. Brain spectrin is, therefore, considered as the putative protein responsible for the attachment of some actin network to membranes. However, proteins binding to brain spectrin have not been isolated yet. Microtubules
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M.-F. Carlier, C. Simon, R. Cassoly and L.-A. Pradel
are also known to be underlying the m e m b r a n e along the axons and are present in the marginal band of nucleated erythrocytes. The possibility therefore exists that some of the microtubule-associated proteins might interact with m e m b r a n e components. The present work provides evidence for an interaction between tau factor, one of the microtubule-associated proteins, and erythrocyte spectrin. Tau factor could thus mediate a relationship between microtubules and the actin network beneath the membrane.
Materials and methods Materials Microtubule-associated proteins were isolated from three-time cycled microtubule proteins prepared from pig brain [4] according to Vailee [5], as described elsewhere (Carlier et al.. manuscript submitted for publication). Tau factor was further purified by hydroxylapatite chromatography of this preparation [6] followed by Sepharose 6B chromatography. 70 % of the material thus obtained consisted of the four bands of tau protein on SDS-polyacrylamide gel electrophoresis. Human erythrocyte ghosts were prepared by the method of Dodge et al. [7]. Spectrin dimer and tetramers were prepared according to Ungewickel and Gratzer [8]. The dimeric and tetrameric states of spectrin were further assessed by sedimentation velocity in an analytical ultracentrifuge. G-actin was prepared from an acetone powder of rabbit skeletal muscle~ ~,ccording to Spudich and Watt [9], with one single cycle of polymerization in the presence of 0.6 M I¢C] G-actin was dialyzed against 2 mM Tris-CI-buffer, pH 7.0 containing 0.2mM CaCl,, 0.5 mM ATP, and 0.5 mM 2-mercaptoethanol.
Miscellaneous procedures Dimeric spectrin (50 p,g) was radioactively labelled with 0.5 mCi L~51-Bolton and Hunter reagent (2000 Ci/mmole, NEN) at pH 7.5, chromatographed over Sephadex G25, and stored at - 8 0 " C . The specific radioactivity was 10 l~Ci/l~g. The method of gel overlay with radioactively labelled spectrin for determination of spectrin binding proteins was used as previously described [10-11]. All operations were done at room temperature. Incubation with ! ~tCi/ml 1251-spectrinwas done in l0 mM phosphate buffer, pH 7.6. After two days washing in the same buffer, gels were dried and autoradiographed at - 8 0 " C for two days, using a Kodak XOmat S film and Dupont Cronex intensifying screens. Dimeric spectrin was covalently bound to CNBractivated Sepharose 4B (Pharmacia) in the following way: l0 mg spectrin (3 mg/mi), after dialysis at 4°C
against 0.1 M sodium bicarbonate buffer, pH8.3, containing 0.3 M NaCl, were incubated for two hours at room temperature with 0.85 g activated Sepharose previously washed with I mM HCI. All of the spectrin then appeared bound to Sepharose from the UV spectrum of the filtrate. After several washes involving pH and ionic strength jumps, the gel was equilibrated in l0 mM sodium phosphate buffer, pH 7.6, and used as such for affinity chromatography. Low shear viscosity experiments of F-actin gelation were performed using the falling ball assay described by McLean-Fletcher and Pollard [12]. Muscle G-actin was rapidly diluted at 0°C in polymerization buffer consisting of 20 mM MES, pH 7.0, 0.5 mM dithiothreitol, 2 mM EGTA, 0.1 mM CaCl:, 2 mM Mg(CH3CO.,):, 0.5 mM ATP. Spectrin and tau protein were added when desired. After thorough mixing, the solution was drawn up into 100 l.d capillary pipettes and allowed to stand at 30°C for the time indicated, at which time a stainless steel ball was dropped into the solution and the time taken to fall through 5.5 cm at an angle of 90 ° was measured. Polyacrylamide slab gel electrophoresis in the presence of SDS was carried out according to Laemmli [13] except that the running gel was a 4 % to I I % acrylamide gradient. Gels were fixed in i0 % acetic acid, 5 % trichloroacetic acid and stained with Coomassie blue or using the silver stain method of Morrissey [14l.
Results Microtubule-associated proteins are known to consist of two classes of proteins unrelated to each o t h e r : high molecular weight MAP~ and MAP., (300,000 and 280,000 respectively) and the tau factor which is composed of four proteins migrating in the 60,000-55,000 dalton region on SDS gels. These four proteins have been shown to be isoforms [6, 22, 29]. Figure ! shows that when a solution of microtubule-associated proteins, largely depleted in tubulin, was applied to a spectrin affinity column, essentially tau factor remained b o u n d and was eluted by 0.15 M NaCl. Upon increasing ionic strength to 0.5 M NaCI, no other protein was eluted from the column. On the other hand, high molecular weight MAPs l and 2 did not bind to the column. A small a m o u n t of tubulin contaminating the MAPs solution (yellowish color in the silver staining) did not bind to the c o l u m n and was eiuted in the void volume. The b i n d i n g of tau was i n d e p e n d e n t of the presence of ATP in the buffer. This result was repeated with several preparations of MAPs and with affinity columns which did not have the same a m o u n t of spectrin b o u n d by ml of gel. W h e n
Binding of spectrin to a microtubular protein
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FI(;. I. -- Electrophoretic anahsis o./" the protcins binding It, a spectritt q].fini O" cohonn, arming whoh' MAP~'. One milliliter of a solution of MAPs at I mg/ml x~as deposited on the column of immobilized spectrin. Following elution of the unbound material with 10 mM phosphate. pH 7.6, a 0-0.5 M NaCI gradient in the same buffer was applied to the column. One single peak. representing about 10% of the material deposited, was eluted at 0.15 M NaCI. Lane 1 : Whole MAPs deposited on the column. Lane 2 • Proteins eluted in the void volume showing a total depletion in tau. Lanes 3 and 4: Proteins eluted in the 0.15 M NaCI fraction consisting mainly of tau protein, and lower molecular weight components, presumably actin a n , a 30.000 dalton protein. Lane 5: Pure tubulin. Gels were silver stained. A small amount of tubulin. :ontaminating the MAPs preparation, is not retained on the column and appears in the void volume fraction (yellowish band in silver staining).
whole microtubule protein was chromatographed on the spectrin affinity column, no specific binding of tau was observed. Instead some aspecific retention of all the deposited material was observed. Indeed, under these conditions the MAPs are tightly bound to tubulin oligomers. This result thus indicates that only when it is not bound to tubulin can tau factor interact with spectrin. it can be observed that two other proteins, present in small amounts in the MAPs prepara-
307
tion, appeared to be entirely bound to the spectrin affinity column and were coeluted with tau. One of them was attributed to actin, which always contaminates tubulin preparations, and the other (approximately 30,000 daitons) is unknown. In order to circumvent the possible artefacts of affinity chromatography, binding of tau protein to spectrin was further assessed using the gel overlay technique. The success of this method is dependent on the renaturation of proteins electrophoresed under denaturing conditions, following elimination of SDS from the gel. Whole MAPs were electrophoresed and processed for ~251-spectrin binding. Figure2 shows that ~-~lspectrin binds to a series of polypeptides (65,000-66,000 daltons) characteristic of tau factor. No binding occurred when ~-'51-spectrin had been previously denatured by five minutes boiling. The binding was partially displaced when unlabelled spectrin (0.15 mg/ml) was present in the incubation buffer together with ~-~51-spectrin. It should be noticed that the binding of ~-'-~lspectrin to MAP_~ polypeptide still takes place with boiled spectrin. Since MAP_, is a strongly basic protein [15-161 while spectrin is rather acidic [17], and since no such interaction was observed between native proteins in the spectrin affinity chromatography experiments, this binding was attributed to non-specific charge interactions between these two proteins. Again spectrin bound to a 30,000 dalton compOllCilt pl-C~Cllt ill MAPs a.u"-a ..... tau i-,,ci-,matiu.~.
Binding o f tau factor to erythrocyte ghosts Human erythrocyte ghosts were thoreughly washed, centrifuged and gently resuspended at 0.9 mg/ml in l0 mM phosphate buffer, pH 7.6, containing 50 ~tg/mi PMSF with or without pu-rifled tau factor (0.7m~/ml). Each sample (0.5 ml) was further subdivided in two, according to whether l mM CaCl.~ was present or not. Incubation was carried out at O"C for 15 minutes, then each sample was centrifuged at 50,000 g for 15 minutes at 0~'. The supernatants were saved for protein determination and gel electrophoresis and the pellets were resuspended with 0.5 mi buffer (with or without i mM CaCI2, accordingly). Analysis of the supernatants indicated that the amounts of tau protein used were saturating with regard to the amounts of ghosts present in the suspension. After a second centrifugation, pellets were again resuspended in 0.5 ml buffer and processed as the supernatants above. Figure 3 shows that tau factor was incorporated in the
M.-F. Carlier, C. Simon, R. Cassoly a n d L.-A. Pradel
308
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FIG. 2. -- Overlay of gel electrophoresed MAPs with ~"51-1abelledspectrin. Lane 1 : Coomassie Blue staining of the electrophoresed MAPs. Lanes 2, 3 and 4 : Autoradiogram of parallel gels incubated with : 2/ "Sl-spectrin; 3/ boiled USl-spectrin; 4/ I"Sl-spectrin plus unlabelled spectrin (0.15 mg/ml): 5/ ~:Sl-spectrinwas at I~Ci/ml (0.1 ~tg/ml). Lane 5 : Coomassie blue staining of a partially purified tau preparation. Lane 6 : Autoradiogram of a parallel sample overlayed with ~"5l-spectrin. Lane 7: Gel electrophoresis of ~251-spectrinused for the overlay experiments. Again, a 30,000 dalton component in MAPs and tau preparations appears to bind spectrin. The arrow points to the top of the gels. ghosts whether or not Ca :+ ions were present. The 30,000 dalton component present in tau preparation (migrating below band 6) was also found incorporated in ghosts. From the Coomassie blue staining of the gels shown on figure 3, a rough estimate of 0.65 to 0.! mg tau b o u n d per mg ghost proteins has been deduced, indicating the presence of 2.5 to 5 x 105 tau per ghost. This result was obtained assuming an average molecular weight of 60,000 for tau and using a value of 5.13 x 10-1°mg protein/ghost [18]. It corres-
ponds to stoechiometry o f 1 to 2 tau protein b o u n d per spectrin tq3 dimer. A more precise estimate of the n u m b e r of b i n d i n g sites present on the m e m b r a n e of erythrocyte ghosts should require other independent studies performed preferentially with radiolabelled tau factor. The binding o f tau to ghosts was not a c c o m p a n i e d by the release o f some protein constituent of the ghosts in the supernatant. Further experiments (not shown) demonstrated that when ghosts were incubated with a whole M A P s preparation, only
Binding o f spectrin to a microtubular protein
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Lane / : Tau preparation. Lane 2 : Ghosts. Lane 3: Ghosts incubated with tau and subsequently washed with 10 mM phosphate buffer. Lane 4: Ghosts, washed in the presence of i mM Ca '+. Lane 5: Ghosts incubated with tau in the presence of I mM Ca"*, and washed. Other experimental conditions are given in the text. Note again that the low molecular mass component present in tau preparation (30,000 daltons, below band 6) is also incorporated in ghosts.
tau factor was found bound to the ghosts, and that no binding took place in the presence of 0.2 M NaCl in the incubation b u f f : r : both tau and glyceraldehyde-3-phosphate dehydrogenase (band6) were then found in the supernatant fraction. This last result indicates that the binding of tau to the erythrocyte membrane does not result from an artefactual trapping of tau into the ghosts. As is the case for hemoglobin (68,000 daltons) tau factor appears to diffuse freely through the unique hole present in the ghost membrane consecutively to the hemolytic processes [19]. On the contrary high molecular weight MAPs are probably prevented to enter the ghosts in view of their bigger size.
Inhibition of the F-actin cross-finking activity of spectrin tetramer by tau factor Spectrin tetramer is known to be able to cross-link F-actin filaments in a tridimensional gel network [20-21]. When increasing amounts of speetrin tetramer were added to actin, the visco-
309
sity measured after 30 minutes at 30°C increased very cooperatively, as previously reported, above 20ttg/ml spectrin [20]. When tau protein wz.s present in the actin-spectrin solutions, lower viscosities were observed at the same time (after 30 minutes). The inhibition increased with increasing tau factor concentration. Tau concentrations as low as 0.130 mg/mi inhibited (about 70 %) the enhancement of apparent viscosity induced by 0.042mg/ml spectrin tetramer. At the same concentration, tau factor alone in the absence of spectrin had no effect on the viscosity of actin filaments. However, above 0.25 mg/ml, tau factor induced a slight increase in the viscosity of F-actin, indicating some cross-linking activity of this protein, as previously reported [22]. It is worth pointing out that the time at which the viscosity assay is performed appears to be an important parameter. Figure 4 indeed shows that &t
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FIG. 4. -- Inhibition of F-actin ('ross-linking activity o f tetrameric spectrm by tau protein, as measured by the falling ball assay. At represents the increase in the time taken by the ball to fall 5.5 cm when spectrin (61 I.tg/ml) was present in the solution, as compared to the control without spectrin. • = Actin alone (0.61 mg/ml): (3 = Actin (0.61 mg/ml) + tau protein (0.27 mg/ml). Viscosity measurements were performed at the times indicated on the abscissa. Inset : corresponding semi-logarithmic plots of the time-dependent loss in cross-linking activity of spectrin. In the absence of spectrin, the ball falling time was 25 s for actin alone, independently of the time spent at 30 oC from 15 minutes to 65 minutes. In the absence of spectrin and presence of tau, the falling time was 125 s at time 20 minutes and slowly decreased to 20 s at time 65 minutes, as reported by Griffith and Pollard [22[
M.-F. Carlier, C. Simon, R. Cassoly and L.-A. Pradel
310
independently of the presence of tau the apparent viscosity of acdn-spectrin mixtures (spectrin = 61 llg/ml) decreased with the time spent at 30°C in the capillary pipet. After one hour, spectrin had lost almost all cross-linking activity. The decrease was first-order, with a rate constant of 0.07min-'. In the presence of tau at 0.27 mg/ml, at all corresponding times, lower enhancements of apparent viscosities were measured. However, the decrease in cross-linking activity of spectrin followed the same kinetics, and the first-order rate constant was not changed (Fig. 4, inset), indicating that in no case the effects of tau can be explained by a protease-dependent degradation of spectrin. Assuming that the viscosity enhancement is proportional to the concentration, c, of actively cross-linking spectrin tetramer, the shift in the semi logarithmic plot of the loss of spectdn cross-linking activity in the presence of tau enables calculation, in the presented experiment, of the inhibition due to tau binding to spectrin "c2/c0 = e - k ( t - t 3 where c2 and Co are the concentrations of active spectrin tetramer at zero time in the presence and absence of tau, respectively; t" and t are the times at which the same viscosity was measured, in the presence and absence of tau, respectively. The data indicate that t - t ' = 12 minutes, which gives c2/Co = 0.43. Consequently, tau at the concentration of 0.27 mg/ml inhibits the cross-linking activity of spectrin at 61 gg/ml by 57 %. This result is in agreement with the 70% inhibition recorded q[~kU l . , I ¢ I ~
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spectrin, and roughly indicates that the equilibrium dissociation constant for the tau-spectrin lies in the micromolar range. A possible explanation for the time-dependent loss of spectrin cross-linking activity lies in the dissociation of spectrin tetramer into dimers which are known not to have any cross-linking activity [20]. Indeed under our buffer cond'~tions, at 30°C and at the concentrations of spectrin used, the equilibrium between tetramer and dimer is greatly displaced towards the dimer species [8, 23]. Further, the time-course of the loss in cross-linking activity correlates well with the reported kinetics for the tetramer-dimer conversion under comparable temperature and buffer conditions [8].
Discussion Independent methods show that tau protein, one of the microtubule-associated proteins, binds
to spectrin. For experimental convenience, tau protein and spectrin used in this work were isolated from pig brain microtubules and human erythrocytes, respectively. However, it is now well established that tau is a widespread conserved microtubule-associated protein [24,25] and that spectrin-like proteins have been identified in cultured non-erythroid cells [26-28] and in brain tissue, where spectrin represents 3% of the membrane proteins. Spectrin, too, thus appears widely distributed and is thought to play a general role in the attachment of actin to membranes. Within these considerations, our results suggest that the interaction between tau and spectrin could have a biological significance. One-dimensional peptide mapping and amino acid analysis [31] both reveal striking similarities among the individual tan polypeptides in various species [24]. The fact that all of the polypeptides which compose tau interact with spectrin in the experiments presented here thus argues for the specificity of this interaction. Whether or not small differences exist in the affinities of the different tau polypeptides tbr spectrin remains an open question which needs more refined experimentation in order to be sc,lved. Tau has been shown to bind to calmodulin in the presence of calcium ions [29], and Ca "+calmodulin binding to tau prevents tau from interacting with tubulin, thus inhibiting microtubule assembly [30]. It is also known that brain l~l~'~
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spectrin-calmodulin ternary complex exists is an open question. The data presented here show that only when it dissociates from ,tubulin oligomers can tau interact with spectrin. In addition, spectrin does not bind to microtubu:es [32]. Tau does not seem therefore to be a dir~,ct link between actin oligomers underlying the membrane and microtubu~,~. Our data rather suiggest that it may participate m some regulation p~thway interconnecting the actin and microtubul: dynamics. Further studies would be nec,~,ssary to determine whether tau binds preferentially to dimeric or tetrameric spectrin. In the spectrin affinity chromatography and in the gel overlay experiments, spectrin was in its dimeric, state. On the other hand, tau also associates to erythrocyte ghosts in which the tetrameric state of spectrin is predominant [33], and the experiments of inhibition of F-actin cross-linkking indicate that tau decreases the amount of active spectrin in the gelation phenomenon, without changing the rate of the tetramer -+ dimer conversion. We can
Binding o f spectrin to a microtubular protein
therefore expect that the tau binding site on spectrin is accessible on both tetramer and dimer species. Low shear viscosity experiments indicate that the equilibrium dissociation constant for the !au-spectrin complex would be in the micromolar range. Further experiments are needed to elucidate the biological significance of the interaction between these two proteins. In connection with this work, it is worth noting that in brain extracts, glyceraldehyde-3-phosphatedehydrogenase, which happens to be also a component of ghosts (band 6) has recently been shown to be able to bundle microtubules [34]. These data thus provide further support to a possible link between microtubules and m e m b r a n e proteins.
Acknowledgements We thank Dr. Dominique Pantaloni for his determinant part in the work, through both enlightening scientific discussions and financial support.
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31 1
10. Glenney Jr., J.R. & Weber, K. (19807 J, Biol. Chem., 255, 1055 l- 10554. I I. Carlin, R.K., Grab, D.J. & Siekevitz, P. (1981) jr. Cell Biol., 89, 449-455. 12. Mc Lean-Fletcher, S.D. & Pollard, T.D. (1980) J. Cell Biol., 85, 414-428. 13. Laemmli, U.K. (1970) Nature. 227, 680-685. 14. Morrissey, J.H. (1981) Anal. Biochem.. 117, 307-310. 15. Murphy, D.B. & Borisy, G.G. (1975) Proc. Natl. Acad. Sci. U.S.A., 72, 2696-2700. 16. Erickson, H.P. & Voter, W.A. (1976) Proc. Natl. Acad. Sci. U.S.A.. 73, 28 i 3-28 ! 7. 17. Kirkpatrick, F.H. (1976) Life Sci., 19, 1-18. 18. Lepke, S., Fasoid, H., Pring, M. & Passow, H. (1976) J. Membrane Biol.. 29, 147-177. 19. Lieber., M.R. & Steck, T.L. (1982) J. Biol. Chem.. 257, I ! 65 l- 16659. 20. Brenner, S.L. & Korn, E.D. (1979) J. Biol. Chem., 254, 8620-8627. 21. Cohen, C.M., Tyler, J.M. & Branton, D. (1980), Cell. 21, 875-883. 22. Griffith, L.M. & Pollard, T.D. (1982) J. Biol. Chem.. 257, 9143-9151. 23. Ralston, G., Dunbar, J. & White, M. (1977) Biochim. Biophys. Acta. 491, 345-348. 24. Cleveland, D.W., Spiegelman, B.M. & Kirschner, M.W. (1979) J. Biol. Chem., 254, 12670-12678. 25. Duerr, A., Pallas, D. & Solomon, F. (1981) Cell. 24, 203-2 ! I. 26. Goodman, S.R., Zagon, I.S. & Kulikowski, R.R. (1981) Proc. Natl. Acad. Sci. U.S.A., 78, 7570-7574. 27. Burridge, K., Kelly, T. & Mangeat, P. (1982) J. Cell Biol.. 95, 478-486. 28. Lazarides, E. & Nelson, W.J. (1982) Cell, 31, 505-508. 29. Sobue, K., Fujita, M., Muramoto, Y. & Kakiuchi, S. (1981) FEBS Lett., 132, 137-140. 30. Kakiuchi, S. & Sobue, K. (1981) FEBS Lett.. 132, 141-143. 31. Cleveland, D.W., Hwo, S.-Y. & Kirschner, M.W. (1977) J. Mol. Biol.. 116, 227-247. 32. Davis, J. & Bennett, V. (1982) J. Biol. Chem.. 257, 5816-5820. 33. Middaugh, C.R. & Ji, T.H. (1980) Eur. J. Biochem.. 110, 587-592. 34. Kumagai, H. & Sakai, H. (1983) J. Biochem. (Tokyo), 93, 1259-1270.
Interaction between microtubule-associated protein tau and spectrin. Marie-France CARLIER*:, Colette SIMON*, Robert CASSOLY** and Lot~ise-Anne PRADEL**. * Laboratoire d'Enzymologie, C.N.R.S., 91190 G(f-sur-Yvette, France ** Laboratoire de Biophysique, lnstitut de Bioiogie Physico-Chimique, rue Pierre et Marie Curie. 75005 Paris France. (Refu le 7-12.1983. acceptd le 7-3-1984).
On ddmontre que le facteur tau, une des protdines associde aux microtubules de cerveau (MAPs) interagit avec la spectrine humaine. Plusieurs approches expdrimentales ont dtd utilisdes comprenant la chromatographie d'affinitd des MAPs sur colonne de sepharose couplde h la spectrine, ie recouvrement des gels d'dlectrophor~se de MAPs avec de la spectrine marqude i'iode 125, l'association du facteur tau ~ la membrane des drythrocytes, et finalement I'dtude de l'inhibition par le facteur tau du pontage par la spectrine de i'aciine F. Ces rdsultats sugg~rent que tau pourrait reprdsenter le facteur protEique impliqud dans la liaison des microtubules avec le rdseau d'actine prdsent all voisinage Je la membrane de nombreuses cellules euca~otes dans lesquelles tau et des analogues de la stTectrine sont largement reprdsentds. R6sum6 --
Mots-cl~s : microtubule / prot~ine tau / spectrine / interaction prot~ine-prot~ine.
Summary -- Tau factor, one o f the microtubule-associated proteins (MAPs), is shown here to bind to spectrin. Evidence for an interaction between these two proteins is provided by spectrin affinity chromatography o f brain MAPs, gel overlay o f electrophoresed MAPs with ;25I-labelled spectrin, incorporation o f tau factor in human erythrocyte ghosts, and demonstration that tau inhibits the F-actin cross-linking activity of tetrameric spectrin. The wide distribution o f both tau and spectrin-like proteins in euka~, otic cells is in favor o f the possible biological significance o f this interaction. The results suggest that tau could be one o f the proteins involved 67 the concerted regulation o f microtubule and actin networks in the membrane vicinity. Key-words : microtubule / tau protein / spectrin / protein-protein interaction.
The identification of proteins mediating the linkage between different components of the cytoskeleton and their attachment to membranes is a central problem in cell motility. Recently a 0 To whom all correspondence should be addressed. Present address : Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, building3, room BI-22, Bethesda, Mao,land 20205 USA.
spectrin-like protein, also named fodrin [!-3], has been isolated from brain membranes and characterized as F-actin cross-linking protein sharing many biochemical and morphological properties with erythrocyte spectrin. Brain spectrin is, therefore, considered as the putative protein responsible for the attachment of some actin network to membranes. However, proteins binding to brain spectrin have not been isolated yet. Microtubules
306
M.-F. Carlier, C. Simon, R. Cassoly and L.-A. Pradel
are also known to be underlying the m e m b r a n e along the axons and are present in the marginal band of nucleated erythrocytes. The possibility therefore exists that some of the microtubule-associated proteins might interact with m e m b r a n e components. The present work provides evidence for an interaction between tau factor, one of the microtubule-associated proteins, and erythrocyte spectrin. Tau factor could thus mediate a relationship between microtubules and the actin network beneath the membrane.
Materials and methods Materials Microtubule-associated proteins were isolated from three-time cycled microtubule proteins prepared from pig brain [4] according to Vailee [5], as described elsewhere (Carlier et al.. manuscript submitted for publication). Tau factor was further purified by hydroxylapatite chromatography of this preparation [6] followed by Sepharose 6B chromatography. 70 % of the material thus obtained consisted of the four bands of tau protein on SDS-polyacrylamide gel electrophoresis. Human erythrocyte ghosts were prepared by the method of Dodge et al. [7]. Spectrin dimer and tetramers were prepared according to Ungewickel and Gratzer [8]. The dimeric and tetrameric states of spectrin were further assessed by sedimentation velocity in an analytical ultracentrifuge. G-actin was prepared from an acetone powder of rabbit skeletal muscle~ ~,ccording to Spudich and Watt [9], with one single cycle of polymerization in the presence of 0.6 M I¢C] G-actin was dialyzed against 2 mM Tris-CI-buffer, pH 7.0 containing 0.2mM CaCl,, 0.5 mM ATP, and 0.5 mM 2-mercaptoethanol.
Miscellaneous procedures Dimeric spectrin (50 p,g) was radioactively labelled with 0.5 mCi L~51-Bolton and Hunter reagent (2000 Ci/mmole, NEN) at pH 7.5, chromatographed over Sephadex G25, and stored at - 8 0 " C . The specific radioactivity was 10 l~Ci/l~g. The method of gel overlay with radioactively labelled spectrin for determination of spectrin binding proteins was used as previously described [10-11]. All operations were done at room temperature. Incubation with ! ~tCi/ml 1251-spectrinwas done in l0 mM phosphate buffer, pH 7.6. After two days washing in the same buffer, gels were dried and autoradiographed at - 8 0 " C for two days, using a Kodak XOmat S film and Dupont Cronex intensifying screens. Dimeric spectrin was covalently bound to CNBractivated Sepharose 4B (Pharmacia) in the following way: l0 mg spectrin (3 mg/mi), after dialysis at 4°C
against 0.1 M sodium bicarbonate buffer, pH8.3, containing 0.3 M NaCl, were incubated for two hours at room temperature with 0.85 g activated Sepharose previously washed with I mM HCI. All of the spectrin then appeared bound to Sepharose from the UV spectrum of the filtrate. After several washes involving pH and ionic strength jumps, the gel was equilibrated in l0 mM sodium phosphate buffer, pH 7.6, and used as such for affinity chromatography. Low shear viscosity experiments of F-actin gelation were performed using the falling ball assay described by McLean-Fletcher and Pollard [12]. Muscle G-actin was rapidly diluted at 0°C in polymerization buffer consisting of 20 mM MES, pH 7.0, 0.5 mM dithiothreitol, 2 mM EGTA, 0.1 mM CaCl:, 2 mM Mg(CH3CO.,):, 0.5 mM ATP. Spectrin and tau protein were added when desired. After thorough mixing, the solution was drawn up into 100 l.d capillary pipettes and allowed to stand at 30°C for the time indicated, at which time a stainless steel ball was dropped into the solution and the time taken to fall through 5.5 cm at an angle of 90 ° was measured. Polyacrylamide slab gel electrophoresis in the presence of SDS was carried out according to Laemmli [13] except that the running gel was a 4 % to I I % acrylamide gradient. Gels were fixed in i0 % acetic acid, 5 % trichloroacetic acid and stained with Coomassie blue or using the silver stain method of Morrissey [14l.
Results Microtubule-associated proteins are known to consist of two classes of proteins unrelated to each o t h e r : high molecular weight MAP~ and MAP., (300,000 and 280,000 respectively) and the tau factor which is composed of four proteins migrating in the 60,000-55,000 dalton region on SDS gels. These four proteins have been shown to be isoforms [6, 22, 29]. Figure ! shows that when a solution of microtubule-associated proteins, largely depleted in tubulin, was applied to a spectrin affinity column, essentially tau factor remained b o u n d and was eluted by 0.15 M NaCl. Upon increasing ionic strength to 0.5 M NaCI, no other protein was eluted from the column. On the other hand, high molecular weight MAPs l and 2 did not bind to the column. A small a m o u n t of tubulin contaminating the MAPs solution (yellowish color in the silver staining) did not bind to the c o l u m n and was eiuted in the void volume. The b i n d i n g of tau was i n d e p e n d e n t of the presence of ATP in the buffer. This result was repeated with several preparations of MAPs and with affinity columns which did not have the same a m o u n t of spectrin b o u n d by ml of gel. W h e n
Binding of spectrin to a microtubular protein
MAP11 MAP21
I'
Tau{
Tubulin
1
2
3
4
5
FI(;. I. -- Electrophoretic anahsis o./" the protcins binding It, a spectritt q].fini O" cohonn, arming whoh' MAP~'. One milliliter of a solution of MAPs at I mg/ml x~as deposited on the column of immobilized spectrin. Following elution of the unbound material with 10 mM phosphate. pH 7.6, a 0-0.5 M NaCI gradient in the same buffer was applied to the column. One single peak. representing about 10% of the material deposited, was eluted at 0.15 M NaCI. Lane 1 : Whole MAPs deposited on the column. Lane 2 • Proteins eluted in the void volume showing a total depletion in tau. Lanes 3 and 4: Proteins eluted in the 0.15 M NaCI fraction consisting mainly of tau protein, and lower molecular weight components, presumably actin a n , a 30.000 dalton protein. Lane 5: Pure tubulin. Gels were silver stained. A small amount of tubulin. :ontaminating the MAPs preparation, is not retained on the column and appears in the void volume fraction (yellowish band in silver staining).
whole microtubule protein was chromatographed on the spectrin affinity column, no specific binding of tau was observed. Instead some aspecific retention of all the deposited material was observed. Indeed, under these conditions the MAPs are tightly bound to tubulin oligomers. This result thus indicates that only when it is not bound to tubulin can tau factor interact with spectrin. it can be observed that two other proteins, present in small amounts in the MAPs prepara-
307
tion, appeared to be entirely bound to the spectrin affinity column and were coeluted with tau. One of them was attributed to actin, which always contaminates tubulin preparations, and the other (approximately 30,000 daitons) is unknown. In order to circumvent the possible artefacts of affinity chromatography, binding of tau protein to spectrin was further assessed using the gel overlay technique. The success of this method is dependent on the renaturation of proteins electrophoresed under denaturing conditions, following elimination of SDS from the gel. Whole MAPs were electrophoresed and processed for ~251-spectrin binding. Figure2 shows that ~-~lspectrin binds to a series of polypeptides (65,000-66,000 daltons) characteristic of tau factor. No binding occurred when ~-'51-spectrin had been previously denatured by five minutes boiling. The binding was partially displaced when unlabelled spectrin (0.15 mg/ml) was present in the incubation buffer together with ~-~51-spectrin. It should be noticed that the binding of ~-'-~lspectrin to MAP_~ polypeptide still takes place with boiled spectrin. Since MAP_, is a strongly basic protein [15-161 while spectrin is rather acidic [17], and since no such interaction was observed between native proteins in the spectrin affinity chromatography experiments, this binding was attributed to non-specific charge interactions between these two proteins. Again spectrin bound to a 30,000 dalton compOllCilt pl-C~Cllt ill MAPs a.u"-a ..... tau i-,,ci-,matiu.~.
Binding o f tau factor to erythrocyte ghosts Human erythrocyte ghosts were thoreughly washed, centrifuged and gently resuspended at 0.9 mg/ml in l0 mM phosphate buffer, pH 7.6, containing 50 ~tg/mi PMSF with or without pu-rifled tau factor (0.7m~/ml). Each sample (0.5 ml) was further subdivided in two, according to whether l mM CaCl.~ was present or not. Incubation was carried out at O"C for 15 minutes, then each sample was centrifuged at 50,000 g for 15 minutes at 0~'. The supernatants were saved for protein determination and gel electrophoresis and the pellets were resuspended with 0.5 mi buffer (with or without i mM CaCI2, accordingly). Analysis of the supernatants indicated that the amounts of tau protein used were saturating with regard to the amounts of ghosts present in the suspension. After a second centrifugation, pellets were again resuspended in 0.5 ml buffer and processed as the supernatants above. Figure 3 shows that tau factor was incorporated in the
M.-F. Carlier, C. Simon, R. Cassoly a n d L.-A. Pradel
308
J
~
MAP1{ MAP21
W ~
~
...........
Tau
2
1
2
3
4
j
5
6
7
FIG. 2. -- Overlay of gel electrophoresed MAPs with ~"51-1abelledspectrin. Lane 1 : Coomassie Blue staining of the electrophoresed MAPs. Lanes 2, 3 and 4 : Autoradiogram of parallel gels incubated with : 2/ "Sl-spectrin; 3/ boiled USl-spectrin; 4/ I"Sl-spectrin plus unlabelled spectrin (0.15 mg/ml): 5/ ~:Sl-spectrinwas at I~Ci/ml (0.1 ~tg/ml). Lane 5 : Coomassie blue staining of a partially purified tau preparation. Lane 6 : Autoradiogram of a parallel sample overlayed with ~"5l-spectrin. Lane 7: Gel electrophoresis of ~251-spectrinused for the overlay experiments. Again, a 30,000 dalton component in MAPs and tau preparations appears to bind spectrin. The arrow points to the top of the gels. ghosts whether or not Ca :+ ions were present. The 30,000 dalton component present in tau preparation (migrating below band 6) was also found incorporated in ghosts. From the Coomassie blue staining of the gels shown on figure 3, a rough estimate of 0.65 to 0.! mg tau b o u n d per mg ghost proteins has been deduced, indicating the presence of 2.5 to 5 x 105 tau per ghost. This result was obtained assuming an average molecular weight of 60,000 for tau and using a value of 5.13 x 10-1°mg protein/ghost [18]. It corres-
ponds to stoechiometry o f 1 to 2 tau protein b o u n d per spectrin tq3 dimer. A more precise estimate of the n u m b e r of b i n d i n g sites present on the m e m b r a n e of erythrocyte ghosts should require other independent studies performed preferentially with radiolabelled tau factor. The binding o f tau to ghosts was not a c c o m p a n i e d by the release o f some protein constituent of the ghosts in the supernatant. Further experiments (not shown) demonstrated that when ghosts were incubated with a whole M A P s preparation, only
Binding o f spectrin to a microtubular protein
•/
W
lSoectrin
:&"
i:i~ ~
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1
~
2
....
i:,
3
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Ba nd
~
~
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~
~
4
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FIG. 3. -- Binding o f tau protein to eo'throc.rte ghosts.
Lane / : Tau preparation. Lane 2 : Ghosts. Lane 3: Ghosts incubated with tau and subsequently washed with 10 mM phosphate buffer. Lane 4: Ghosts, washed in the presence of i mM Ca '+. Lane 5: Ghosts incubated with tau in the presence of I mM Ca"*, and washed. Other experimental conditions are given in the text. Note again that the low molecular mass component present in tau preparation (30,000 daltons, below band 6) is also incorporated in ghosts.
tau factor was found bound to the ghosts, and that no binding took place in the presence of 0.2 M NaCl in the incubation b u f f : r : both tau and glyceraldehyde-3-phosphate dehydrogenase (band6) were then found in the supernatant fraction. This last result indicates that the binding of tau to the erythrocyte membrane does not result from an artefactual trapping of tau into the ghosts. As is the case for hemoglobin (68,000 daltons) tau factor appears to diffuse freely through the unique hole present in the ghost membrane consecutively to the hemolytic processes [19]. On the contrary high molecular weight MAPs are probably prevented to enter the ghosts in view of their bigger size.
Inhibition of the F-actin cross-finking activity of spectrin tetramer by tau factor Spectrin tetramer is known to be able to cross-link F-actin filaments in a tridimensional gel network [20-21]. When increasing amounts of speetrin tetramer were added to actin, the visco-
309
sity measured after 30 minutes at 30°C increased very cooperatively, as previously reported, above 20ttg/ml spectrin [20]. When tau protein wz.s present in the actin-spectrin solutions, lower viscosities were observed at the same time (after 30 minutes). The inhibition increased with increasing tau factor concentration. Tau concentrations as low as 0.130 mg/mi inhibited (about 70 %) the enhancement of apparent viscosity induced by 0.042mg/ml spectrin tetramer. At the same concentration, tau factor alone in the absence of spectrin had no effect on the viscosity of actin filaments. However, above 0.25 mg/ml, tau factor induced a slight increase in the viscosity of F-actin, indicating some cross-linking activity of this protein, as previously reported [22]. It is worth pointing out that the time at which the viscosity assay is performed appears to be an important parameter. Figure 4 indeed shows that &t
At
(s)
\
800
600
4OO
,
0
1 ..'2
2~0
I
30
I
40
\,
200
~
\
,
0
10
1
2JO
time
30
40
50
at
30°C
60
70
8~o
rain.
FIG. 4. -- Inhibition of F-actin ('ross-linking activity o f tetrameric spectrm by tau protein, as measured by the falling ball assay. At represents the increase in the time taken by the ball to fall 5.5 cm when spectrin (61 I.tg/ml) was present in the solution, as compared to the control without spectrin. • = Actin alone (0.61 mg/ml): (3 = Actin (0.61 mg/ml) + tau protein (0.27 mg/ml). Viscosity measurements were performed at the times indicated on the abscissa. Inset : corresponding semi-logarithmic plots of the time-dependent loss in cross-linking activity of spectrin. In the absence of spectrin, the ball falling time was 25 s for actin alone, independently of the time spent at 30 oC from 15 minutes to 65 minutes. In the absence of spectrin and presence of tau, the falling time was 125 s at time 20 minutes and slowly decreased to 20 s at time 65 minutes, as reported by Griffith and Pollard [22[
M.-F. Carlier, C. Simon, R. Cassoly and L.-A. Pradel
310
independently of the presence of tau the apparent viscosity of acdn-spectrin mixtures (spectrin = 61 llg/ml) decreased with the time spent at 30°C in the capillary pipet. After one hour, spectrin had lost almost all cross-linking activity. The decrease was first-order, with a rate constant of 0.07min-'. In the presence of tau at 0.27 mg/ml, at all corresponding times, lower enhancements of apparent viscosities were measured. However, the decrease in cross-linking activity of spectrin followed the same kinetics, and the first-order rate constant was not changed (Fig. 4, inset), indicating that in no case the effects of tau can be explained by a protease-dependent degradation of spectrin. Assuming that the viscosity enhancement is proportional to the concentration, c, of actively cross-linking spectrin tetramer, the shift in the semi logarithmic plot of the loss of spectdn cross-linking activity in the presence of tau enables calculation, in the presented experiment, of the inhibition due to tau binding to spectrin "c2/c0 = e - k ( t - t 3 where c2 and Co are the concentrations of active spectrin tetramer at zero time in the presence and absence of tau, respectively; t" and t are the times at which the same viscosity was measured, in the presence and absence of tau, respectively. The data indicate that t - t ' = 12 minutes, which gives c2/Co = 0.43. Consequently, tau at the concentration of 0.27 mg/ml inhibits the cross-linking activity of spectrin at 61 gg/ml by 57 %. This result is in agreement with the 70% inhibition recorded q[~kU l . , I ¢ I ~
~lLIk
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UI
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spectrin, and roughly indicates that the equilibrium dissociation constant for the tau-spectrin lies in the micromolar range. A possible explanation for the time-dependent loss of spectrin cross-linking activity lies in the dissociation of spectrin tetramer into dimers which are known not to have any cross-linking activity [20]. Indeed under our buffer cond'~tions, at 30°C and at the concentrations of spectrin used, the equilibrium between tetramer and dimer is greatly displaced towards the dimer species [8, 23]. Further, the time-course of the loss in cross-linking activity correlates well with the reported kinetics for the tetramer-dimer conversion under comparable temperature and buffer conditions [8].
Discussion Independent methods show that tau protein, one of the microtubule-associated proteins, binds
to spectrin. For experimental convenience, tau protein and spectrin used in this work were isolated from pig brain microtubules and human erythrocytes, respectively. However, it is now well established that tau is a widespread conserved microtubule-associated protein [24,25] and that spectrin-like proteins have been identified in cultured non-erythroid cells [26-28] and in brain tissue, where spectrin represents 3% of the membrane proteins. Spectrin, too, thus appears widely distributed and is thought to play a general role in the attachment of actin to membranes. Within these considerations, our results suggest that the interaction between tau and spectrin could have a biological significance. One-dimensional peptide mapping and amino acid analysis [31] both reveal striking similarities among the individual tan polypeptides in various species [24]. The fact that all of the polypeptides which compose tau interact with spectrin in the experiments presented here thus argues for the specificity of this interaction. Whether or not small differences exist in the affinities of the different tau polypeptides tbr spectrin remains an open question which needs more refined experimentation in order to be sc,lved. Tau has been shown to bind to calmodulin in the presence of calcium ions [29], and Ca "+calmodulin binding to tau prevents tau from interacting with tubulin, thus inhibiting microtubule assembly [30]. It is also known that brain l~l~'~
~idli, l a l l
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~
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spectrin-calmodulin ternary complex exists is an open question. The data presented here show that only when it dissociates from ,tubulin oligomers can tau interact with spectrin. In addition, spectrin does not bind to microtubu:es [32]. Tau does not seem therefore to be a dir~,ct link between actin oligomers underlying the membrane and microtubu~,~. Our data rather suiggest that it may participate m some regulation p~thway interconnecting the actin and microtubul: dynamics. Further studies would be nec,~,ssary to determine whether tau binds preferentially to dimeric or tetrameric spectrin. In the spectrin affinity chromatography and in the gel overlay experiments, spectrin was in its dimeric, state. On the other hand, tau also associates to erythrocyte ghosts in which the tetrameric state of spectrin is predominant [33], and the experiments of inhibition of F-actin cross-linkking indicate that tau decreases the amount of active spectrin in the gelation phenomenon, without changing the rate of the tetramer -+ dimer conversion. We can
Binding o f spectrin to a microtubular protein
therefore expect that the tau binding site on spectrin is accessible on both tetramer and dimer species. Low shear viscosity experiments indicate that the equilibrium dissociation constant for the !au-spectrin complex would be in the micromolar range. Further experiments are needed to elucidate the biological significance of the interaction between these two proteins. In connection with this work, it is worth noting that in brain extracts, glyceraldehyde-3-phosphatedehydrogenase, which happens to be also a component of ghosts (band 6) has recently been shown to be able to bundle microtubules [34]. These data thus provide further support to a possible link between microtubules and m e m b r a n e proteins.
Acknowledgements We thank Dr. Dominique Pantaloni for his determinant part in the work, through both enlightening scientific discussions and financial support.
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