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YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules

YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules Background: YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. Results: We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin- treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes. Conclusion: These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation. box binding transcription factor, where it activates tran- Background YB-1 is a multifunctional protein known to interact with scription of various cellular genes, including those impli- nucleic acids, and as such, YB-1 is involved in a wide vari- cated in cell growth, differentiation and apoptosis ety of cellular processes in eukaryotic cells (reviewed in (reviewed in [7]). Translocation of YB-1 from cytoplasm [1]). In the cytoplasm, YB-1 participates in the formation to the cell nucleus can occur at certain steps of the cell of mRNPs and in the regulation of mRNA translation and cycle [8] and in response to stress-induced DNA damages degradation [2-6]. In the nucleus, YB-1 functions as a Y- [9,10]. The association of YB-1 with nucleic acids causes Page 1 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 global changes in their structures by melting short or confirmed by Western blotting using anti-alpha and anti- imperfect duplexes and acceleration of annealing and beta tubulin antibodies. As shown on Fig. 1B, α and β strand exchange reactions between complementary tubulin subunits were detected in all tissue extracts and strands of RNA and DNA [11,12]. YB-1 may thus partici- high salt eluates. It is interesting to note that, in the case pate in DNA recombination and replication [13,14], and of brain and testis, some tubulin remained in the flow- in the case of damaged DNA, YB-1 may assist in its repa- through fraction. This could be either due to the satura- ration. tion of the affinity column or linked to the sequestration of tubulin with partner proteins. In the control experi- Molecular and structural investigations showed that YB-1 ments performed with BSA-Sepharose, all tissue proteins interacts with RNA and DNA through two non-homolo- were collected in the flow-through fractions and were gous domains: the cold-shock domain (CSD), which con- undetectable in high salt eluates (data not shown). These sists of five anti-parallel beta-strands [15,16], and the C- results strongly suggested that tubulin binds specifically to terminal domain, which contains a series of alternating YB-1. clusters of positively and negatively charged amino acid residues. We further investigated YB-1:tubulin interaction and eval- uated the stability of the YB-1-tubulin complex by YB-1 In addition to its interaction with nucleic acids, YB-1 affinity chromatography using pure tubulin as a prey. interplays with different protein partners within the cell. Tubulin was totally adsorbed on the YB-1-Sepharose col- It has been noticed that the interaction of YB-1 with p53 umn in the presence of 150 mM NaCl, while poorly increases the affinity of p53 for DNA promoters that could retained in similar conditions by a casein-Sepharose con- stimulate transcription of p53-controled genes [17,18]. trol column (Fig. 2). Tubulin started to elute from the YB- Similarly, YB-1 interacts with the T-antigen of polyomavi- 1 column at 300 mM NaCl forming a peak around 600 rus JC and triggers transcription of viral genes [19]. YB-1 mM NaCl (Fig. 2). These results indicate that the YB- can also catalyze splicing of pre-mRNA via interaction 1:tubulin interaction is not inhibited at physiological and with RNA polymerase II, EWS (Ewing's sarcoma protein) moderate ionic strength (up to 300 mM NaCl). We finally and TLS (translocation liposarcoma protein) proteins probed the presence of YB-1 and of α and β tubulin subu- [20]. nits in YB-1-tubulin complexes by cross-linking using the zero length cross-linker EDC/Sulfo NHS. As displayed on The objective of the present work was to identify new YB- Fig. 3, cross-linker stabilized dimeric forms of YB-1 and α 1 protein partners to better understand the functions of β-tubulin heterodimers, as well as higher molecular this protein. We discovered, using a series of biochemical weight aggregates (compare lanes 3 and 4 with 1 and 2). in vitro experiments, that YB-1 strongly interacts with It is worthy to note that under these conditions, both YB- tubulin, both soluble and polymerized into microtubules. 1 and tubulin cross-linked samples still contained non- We demonstrated that YB-1 stimulates microtubule cross-linked products (lanes 3 and 4). When the cross- assembly, and in addition, that tubulin competes with linking reaction was performed in the presence of both mRNA for interaction with YB-1. In light of these results, proteins at a YB-1-tubulin molar ratio of 0.5, most YB-1 we propose that YB-1 may contribute to coordination of was found in high-molecular weight complexes contain- regulation of mRNA translation and dynamics of microtu- ing also both tubulin subunits (compare lanes 1 and 5). bule cytoskeleton. Tubulin, via its interaction with YB-1, An increase of the YB-1:tubulin molar ratio over 0.5 may indirectly influence the translational regulation of increased the yield of these high molecular weight com- mRNP complexes. plexes (lanes 6 to 8) with a slight increase of the free YB-1 band. Our results demonstrate that YB-1 interacts directly Results with tubulin and suggest that YB-1 contacts the both tubu- Tubulin is a new YB-1 interacting protein lin subunits in solution. A search for new YB-1 partners from different rabbit tissue extracts was performed by affinity chromatography using The binding of YB-1 to tubulin was then investigated by YB-1-Sepharose as bait. Though eluates varied in protein AFM. YB-1 appeared as discrete particles on the mica sur- composition, two prominent bands migrating as 45 kDa face with an average height of 0.7 nm (Fig. 4, upper and 50 kDa were detected in the eluates of most of the tis- panel). This value appeared lower than that reported by sues (Fig 1A, right panel, marked with an asterisk and a Skabkin et al [22] (about 4 nm for monomeric YB-1 in dot, respectively). The 45 kDa protein was identified by solution of high ionic strength) and probably resulted MALDI-TOF mass spectrometry as actin, a well-character- from YB-1 flattening on the mica surface. Tubulin ized YB-1 partner [21] and the 50 kDa band was identified appeared as particles with an average height of 3.7 nm by similar means as tubulin (see Additional file 1). The (Fig. 4, middle panel) in agreement with previous reports presence of tubulin in extract and eluate fractions was also [23]. In the YB-1-tubulin sample, in contrast to isolated Page 2 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 YB-1 affin Figure 1 ity chromatography of rabbit tissue extracts YB-1 affinity chromatography of rabbit tissue extracts. A, Rabbit tissue extracts (50 μg of total protein) were loaded onto YB-1-Sepharose. After washing with buffer containing 100 mM KCl, bound proteins were eluted with buffer containing 1 M KCl. Extracts and eluates were analyzed by 12% SDS-PAGE followed by Coomassie blue staining. Two prominent bands migrating as 45 kDa and 50 kDa were detected in the eluates of most of the tissues (marked with an asterisk and a dot) and were identified by mass spectrometry as actin and tubulin respectively. B, Western blot of rabbit tissue extracts and YB-1- Sepharose fractions. Fractions were collected as described in A and probed with anti-alpha and anti-beta tubulin antibodies (fl- th, flow through). proteins, particles were less homogenous with a size dis- tubulin complexes probably made of several molecules of tribution ranging from about 3 to 8 nm (Fig. 4, bottom both tubulin and YB-1. panel). A novel class of particles was clearly distinguisha- ble, centered around 7 nm. It was attributed to the YB-1- Page 3 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 close to cellular, we performed a series of experiments with MAPs-tubulin. This preparation contained approxi- mately 15% of MAPs (w/w) as estimated by Coomassie staining of proteins separated on SDS-PAGE. The addition of YB-1 to MAPs-tubulin at a total YB-1-tubulin ratio of 0.3 decreased the lag-time similarly to pure tubulin, increased the rate of polymerization and slightly increased the final microtubule mass as estimated from the steady state plateau value (Fig. 6). Higher amounts of YB-1 fur- YB-1 interacts dir Figure 2 ectly with pure tubulin ther reduced the lag-time of polymerization but did not YB-1 interacts directly with pure tubulin. Affinity chro- change significantly the mass of polymerized tubulin. matography analysis of YB-1-tubulin interaction. Tubulin (20 These data indicate that the presence of MAPs does not μg) was applied onto 50 μl YB-1- or casein-Sepharose (used abrogate the positive effect of YB-1 on the overall kinetics as a control) in buffer containing 150 mM NaCl at 4°C and incubated for 10 min. After washing, bound proteins were of microtubule assembly. eluted stepwise using buffer containing 150 mM, 300 mM, 600 mM and 1 M NaCl as described under Materials and YB-1 accelerates tubulin S assembly in vitro Methods. Fractions were analyzed by 15% SDS PAGE fol- The C-termini of alpha and beta tubulin subunits are lowed by Coomassie blue staining. highly negatively charged at physiological pH and involved in the regulation of tubulin function [24]. On the other hand, YB-1 is highly positively charged at neu- YB-1 strongly favors tubulin assembly in vitro tral pH, so it was reasonable to think that YB-1 accelerates The influence of YB-1 on in vitro microtubule assembly microtubule assembly via interaction with tubulin C-ter- was first assessed by turbidimetry. Compared to tubulin mini. It was therefore critical to investigate whether the control, YB-1 induced a dramatic shortening of the lag- effects of YB-1 on microtubule assembly could result only time in a dose dependant manner. It also dose-depend- from a charge effect or could also be partly due to more ently increased both the apparent rate of microtubule specific molecular recognition mechanisms between YB-1 assembly and the steady state plateau value (Fig. 5A and and tubulin. For this purpose, we treated tubulin with Additional file 4). We next examined the distribution of subtilisin in such conditions as to cleave the charged C- YB-1 in the soluble tubulin or microtubule fractions in termini of the both tubulin subunits and investigated the steady-state samples using microtubule sedimentation effect of YB-1 on the assembly of the cleaved tubulin prod- assay. In agreement with turbidimetry data, the presence uct (tubulin S, Fig. 7A). We observed that YB-1 was still of YB-1 strongly increased the total amount of tubulin in able to promote the assembly of tubulin S (Fig. 7B). A the pellet (Fig. 5B). In the presence of 5 μM YB-1 and 20 strong stimulation of assembly was observed when YB-1 μM tubulin, most of YB-1 was associated with microtu- was added at a total YB-1-tubulin S molar ratio as low as bules at steady state. In these conditions, a visual estima- 0.13. At this ratio, YB-1 significantly decreased the lag- tion on Coomassie stained gels of the YB-1:tubulin time and increased the velocity of the polymerization stoichiometry indicated a ratio of about 1 mole of YB-1 (Fig. 7B, curve b). Higher concentrations of YB-1 further per 3 moles of tubulin heterodimer in the pellet. When 20 reduced the lag-time and increased the rate of tubulin S μM tubulin was assembled in the presence of 10 μM YB-1, assembly but did not produce any significant additional we noted a further increase of the amount of tubulin in effect on microtubule mass at steady-state (Fig. 7B, curves the pellet. YB-1 was again found mainly in the pellet with c and d). Together, these data suggest that the promotion however a small amount remaining in the supernatant. In of microtubule assembly by YB-1 involves interaction these conditions, the YB-1-tubulin stoichiometry in the between specific sites of the partners, although non-spe- pellet rose to about 1 mole of YB-1 per 2 moles of tubulin cific electrostatic interactions may also play an important heterodimer. It is worthy to note that YB-1 alone, when role. centrifuged in the same conditions remained totally in the YB-1 promotes the formation of normal microtubules and supernatant fraction (data not shown). probably coats the microtubule wall YB-1 favors MAPs-tubulin assembly in vitro High resolution microscopies like AFM or TEM could pro- It is well documented that the presence of MAPs strongly vide interesting information about microtubule morphol- influences the kinetics of microtubule assembly. MAPs ogy in the presence of YB-1 and about the localization of favor the nucleation of microtubules, increase the rate of YB-1 on/in microtubules. Under control conditions, i.e., assembly, extent of polymerization and stabilize microtu- without YB-1, in AFM images microtubules appeared as bules against disassembly. To investigate whether YB-1 straight rods with an apparent height of 10 nm. This indi- may also influence tubulin polymerization in conditions cates that they were flattened on the surface due to the dry- Page 4 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 YB-1:tubulin cross-linking Figure 3 YB-1:tubulin cross-linking. YB-1 and tubulin were cross-linked with EDC and sulfo-NHS, resolved by 9% SDS-PAGE, blot- ted and probed with anti-alpha, anti-beta tubulin and anti-YB-1 antibodies as described under Materials and Methods. Gels were either stained with Coomassie blue (top) or processed for Western blotting with the indicated antibodies. Page 5 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 A Figure 4 FM images of YB-1, tubulin and YB-1-tubulin complexes AFM images of YB-1, tubulin and YB-1-tubulin complexes. Isolated YB-1 and tubulin form discrete particles of 0.7 and 3.7 nm heights, respectively. YB-1-tubulin complex samples formed at a 1:1 molar ratio show the presence of an additional population of particles with a height of about 7.9 nm. Histograms illustrating the height distribution of particles are displayed on right panels. Page 6 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 YB-1 fa Figure 6 vors MAPs-tubulin assembly YB-1 favors MAPs-tubulin assembly. Turbidimetry plot of MAPs-tubulin (1 mg/ml, ~9 μM) assembly in buffer M with 20% glycerol in the absence (a) or presence of 2.5 μM (b), or 5 μM YB-1 (c). a single layer of tubulin forming their wall and an outer diameter comparable to control (Fig. 9, compare D' with C'). In addition to this, the number of protofilaments was YB-1 promotes m Figure 5 icrotubule pellets microtubule assembly and co-sediments with not significantly different from that of control (see Addi- YB-1 promotes microtubule assembly and co-sedi- tional file 2). This supported further the suggestion that ments with microtubule pellets. A, Turbidimetry plot of YB-1 coats the outer surface of the microtubule. It is also 20 μM tubulin assembly in the absence (a) or presence of worth noting that, under control conditions, microtu- increasing concentrations of YB-1 [2.5 μM (b), 5 μM (c) and bules could often be found in close contact with each 10 μM (d)] in buffer M with 10% glycerol. B, 20 μM tubulin other (Fig. 9A, C and 9C'), whereas in the presence of YB- was polymerized in buffer M with 10% glycerol in the absence (ctrl) or presence of 5 μM or 10 μM YB-1. After polymeriza- 1, microtubules appeared regularly distributed and spaced tion until steady-state, the samples were centrifuged, then from each other (Fig. 9B, D and 9D'). Due to its coating supernatants and pellets were analyzed by SDS-PAGE. (S, the microtubule surface, YB-1 could induce a change in supernatant; P, pellet). (Additional file 4). rigidity of microtubule or steric hindrance on its outer sur- face, which may lead to a larger inter-microtubule spac- ing. ing procedure (Fig. 8, upper panel) and the height measurement thus corresponded to two tubulin layers in Tubulin interferes with mRNP formation close contact (see [23] and Fig. 8, upper schema). Micro- YB-1 is a major mRNA binding protein that forms com- tubules formed in the presence of YB-1 were significantly plexes with mRNA and regulates its translational activity. higher than the control ones with an average height of In this context, it is necessary to explore whether tubulin about 17 nm. The increase in height could correspond to can interfere with formation of RNP and induce some YB-1 coating of the microtubule outer wall (Fig. 8, bottom modifications of RNP structure. Since RNP differs from panel and schema). However, such morphology can also naked RNA in net charge and molecular weight, we result from microtubules with double walls or with a decided to investigate the effect of tubulin on YB-1-RNA higher number of protofilaments. complexes by electrophoretic mobility gel-shift assay. It is known that RNP saturated with YB-1 contains about one To distinguish between these scenarios, we investigated molecule of YB-1 per 25 RNA bases [25]. Compared to the effect of YB-1 on the ultrastructure of microtubules by RNA alone, these saturated YB-1:RNA complexes demon- TEM with thin sections of EPON-embedded microtu- strate a significant reduction of mobility in native agarose bules. In control conditions, TEM analyses showed regular gel due to either partial RNA discharging or the increased microtubules with a diameter of about 25 nm. With YB-1, mass of the formed YB-1:RNA complex, or both (Fig. 10, the ultrastructure of microtubules remained normal with compare lane 1 and 3). The presence of tubulin did not Page 7 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 opment and in cell proliferation and transformation. Here, we report that YB-1 interacts with tubulin and microtubules in vitro and demonstrate that this protein promotes the assembly of microtubules. This new func- tional link of cellular proteins seems to be important, since, on the one hand, the regulation of many vital proc- esses, such as cell division, motility and intracellular traf- fic depends on the fine tuning of the intrinsic dynamics of microtubules by protein partners. The inventory of tubu- lin partners is not complete and the comprehension of their mechanisms of regulation of microtubule dynamics is still under investigation. On the other hand, with YB-1 being an RNA-binding protein, the study of YB-1:tubulin interaction is highly relevant to further address the com- petition between tubulin and mRNA for YB-1 binding. The conformational changes of RNP containing YB-1 induced by tubulin may be of particular interest to inves- tigate RNP accessibility for translation and degradation. YB-1 strongly interacts with tubulin The strong binding between YB-1 and tubulin was sup- ported by a series of in vitro biochemical data which also demonstrated that the YB-1:tubulin interaction was inde- pendent of the presence of other tissue extract compo- nents. Since YB-1 is positively charged at neutral pH (pI ~ 9.5) [2] while tubulin is negatively charged (pI ~ 5.6) [26], we assayed if YB-1 interacts with tubulin in a manner sim- YB-1 favors tubulin S assembly Figure 7 ilar to that of many other positively charged nucleic acid YB-1 favors tubulin S assembly. A, Tubulin and tubulin S (5 μM each) were analyzed by native agarose gel (left panel) binding proteins. As tubulin was still retained on a YB-1 and 8% SDS-PAGE (right panel). In our conditions, complete column at physiological ionic strength, we concluded that digestion of both α and β tubulin were total. B, Turbidimetry the attraction between tubulin and YB-1 was the result of plot of tubulin S (5 μM) assembly in the absence (a) or pres- a strong, short-ranged, electrostatic interactions and (or) ence of 0.625, 1.25, 2.5 μM YB-1 (b, c, and d, respectively) in other more specific mechanisms like hydrophobic inter- buffer M with 20% glycerol. actions. Additionally, we found that YB-1 did not provoke the formation of huge aggregates containing tubulin, in contrast to other highly cationic proteins like histones [27]. The YB-1-tubulin oligomeric complexes formed in change mobility of RNA that excluded the possibility of "non-assembly conditions" had their heights about 7 nm direct interaction between these two molecules (compare (Fig. 4), which indicated the involvement of a few YB-1 lanes 1 and 2). The addition of tubulin to YB-1:RNA com- and tubulin molecules in formation of these complexes. plexes clearly increased the mobility of RNP (compare lane 3 with 4 and 5). The presence of tubulin may thus Microtubule polymerization is a two-step process in induce structural changes of RNP complexes and/or a which tubulin first forms nucleation templates and then change in the RNP charge via YB-1 withdrawal mediated adds to and elongates them [28,29]. The templates were by tubulin. Further investigations are required for a better modelled either in equilibrium with tubulin dimers [30- description of the interaction between mRNA:YB-1 com- 32] or as persistent structures [33]. MAPs facilitate the for- plexes and tubulin, which may play an important role in mation of templates and are thought to clamp them. The transition of mRNA from silenced to translationally active YB-1-tubulin complexes we observed here were function- state. ally reminiscent of MAPs-tubulin complexes [29]. The YB- 1-tubulin complexes could represent nucleation tem- Discussion plates, and thus, YB-1 could act as a nucleation factor for YB-1 is a member of the Y-box protein family, a family microtubule assembly in vitro in a manner similar to that highly conserved among prokaryotes and eukaryotes. In of classical MAPs [34]. eukaryotes, Y-box proteins are regulators of transcription and translation and play important roles during the devel- Page 8 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 A Figure 8 FM images of microtubules assembled with or without YB-1 AFM images of microtubules assembled with or without YB-1. Microtubules were assembled with (bottom panel) or without YB-1 (top panel), fixed and analyzed by AFM as described under Materials and Methods. Histograms of the microtu- bule height distribution are shown on the right. Schemas on the left illustrate the proposed structure of microtubules. YB-1 favours microtubule assembly and coats the sin. This observation suggests that the promotion of microtubule wall microtubule assembly by YB-1 results not only from YB-1 strongly favoured polymerization of pure tubulin purely electrostatic interactions but also from a more spe- into microtubules. YB-1 was found to accelerate the cific molecular recognition mechanism. This suggestion is apparent rate of microtubule assembly, to increase the supported by the fact that most positively charged mole- microtubule mass at steady state and to be associated with cules which interact in a non-specific manner with tubu- microtubules. lin promote the assembly of tubulin into aberrant structures instead of microtubules. For example, polyca- The ability of MAPs or other non-specific basic molecules tions induce the formation of double-walled microtu- to promote microtubule assembly depends largely upon bules [37,38], and aminoacyl-tRNA synthetases promote the presence of the negative charges of tubulin C-termini microtubule bundling [39]. In the present work, electron [35,36]. In contrast to these positively charged molecules, microscopy analysis showed that microtubules formed in we found that YB-1, which is also a basic protein, still the presence of YB-1 possess a normal morphology with a stimulated polymerization of tubulin cleaved by subtili- single wall of circa 25 nm diameter and parallel AFM Page 9 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 YB- Figure 9 1 induces the formation of single wall microtubules YB-1 induces the formation of single wall microtubules. Tubulin (30 μM) was polymerized without (A, C, C') or with 10 μM YB-1 (B, D, D') at 37°C. Transmission electron microscopy of longitudinal ultrathin sections revealed that YB-1 contrib- utes to the formation of normal microtubules and that, in addition, the microtubules remain regularly spaced from one another (compare B with the control A). Transversal sections of microtubules (C, D) confirmed this observation. B, C, D are at the same magnification as A. D' is at the same magnification as C'. Page 10 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 bly because of multiple tubulin-binding sites and high molecular mass of these proteins [42]. YB-1 promotes polymerization of MAPs-tubulin In the presence of MAPs-tubulin, YB-1 also accelerated microtubule assembly, although it didn't promote an increase of the total mass of polymerized tubulin. This was most probably due to the fact that our MAPs-tubulin preparation contained around 15% of MAPs (w/w), which makes microtubules nearly saturated with MAPs. Interestingly, some high molecular weight MAPs were par- tially displaced by YB-1 from MAPs-microtubules stabi- lized with taxol when the total YB-1-tubulin molar ratio was above 0.5 (see Additional file 3). As mentioned above, classical MAPs, such as tau and MAP2, interact mostly with the negatively charged C-termini of tubulin. In addition, tau binds to the amino terminal region of alpha tubulin subunit [43]. Since YB-1 strongly promoted assembly of tubulin lacking C-termini, we predict that YB- 1 interacts with microtubules not only via non-specific binding to the negatively charged C-terminal tail. Finally, in this context, it is interesting to note that YB-1 doesn't display any homology with classical MAPs and may repre- sent the generic element of a novel class of microtubule interacting proteins. YB-1 may shuttle between mRNA and the microtubule cytoskeleton in vivo Microtubules play a critical role in mRNA translation and localization in vivo (reviewed in [44]). Numerous plant proteins known to regulate translation were recently iden- tified by affinity chromatography as tubulin-binding pro- teins [45]. For example, the plant initiation factor eIF- (iso)4F was found associated with the microtubule Tu Figure 10 bulin modifies the structure of YB-1-RNA complexes cytoskeleton in the cell and is able to induces microtubule Tubulin modifies the structure of YB-1-RNA com- bundling in vitro [46]. plexes. 30 nM Luciferase RNA was incubated in buffer M with 5% glycerol alone (lanes 1 and 2) or in the presence of Since YB-1 is a major component of cellular RNPs, inter- 2.1 μM YB-1 (lanes 3, 4 and 5) for 10 min at 25°C. After action of tubulin with YB-1 could potentially regulate the incubation, 2.1 μM tubulin (lane 4) or 4.2 μM tubulin (lanes 2 translation state of mRNAs. Indeed, YB-1 protects mRNAs and 5) were added and mixtures were analyzed by native aga- from degradation and packs them in non-translatable rose gel electrophoresis followed by staining with ethidium RNPs. We have shown here that tubulin changes the elec- bromide. trophoretic mobility of YB-1-RNA complexes. This change most probably occurs due to the partial dissociation of YB-1 from RNA, which could be a signal to trigger mRNA investigations showed that YB-1 can coat the outer surface translation and/or degradation. Interestingly, it has of the microtubule wall. recently been shown that the phosphorylation of YB-1 by Akt kinase may regulate its binding to mRNA [47]. It is In agreement with these microscopic data, YB-1 was thus expected that the phosphorylated form of YB-1, found to co-sediment with microtubules (Fig. 5). The sto- which possesses a lower affinity for the cap-structure of ichiometry corresponds well to some other microtubule- mRNA, could be more easily released from RNA than its binding proteins with single tubulin dimer-binding site non-phosphorylated form. This release could occur due to [40,41]. Many canonic MAPs were reported to bind interaction of mRNA with other RNA-binding proteins or microtubules with a stoichiometry ranging from 1:15 to through the interaction of YB-1 with protein partners, 1:8 (MAPs:tubulin ratio in the microtubule wall) proba- such as tubulin. Our results thus provide new prospects Page 11 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 on the role of protein-protein interaction in the regulation Tubulin and microtubule proteins preparation of mRNA translation. Furthermore, these results could Tubulin was purified from sheep brain using the method also help to unravel situations where tubulin is involved of Castoldi and Popov [53] and stored at -80°C in 50 mM in the overall processes of mRNA translation and degrada- MES-KOH, pH 6.8, 0.5 mM dithiothreitol, 0.5 mM EGTA, tion. 0.25 mM MgCl , 3.4 M glycerol, 0.1 mM GTP. Before use, tubulin stock was thawed and an additional cycle of Conclusion polymerization was performed. Microtubule proteins Thus, we have first demonstrated that YB-1 stimulates (tubulin + microtubule-associated proteins, MAPs, herein microtubule assembly in vitro. By all in vitro criteria, YB-1 referred to as MAPs-tubulin) were purified from sheep represents a novel microtubule-interacting protein related brain through two cycles of assembly/disassembly as to the function of MAPs but clearly with different proper- described by Mitchison and Kirschner [54] aliquoted and ties. The YB-1 properties described here may contribute to stored at -80°C. Before use, MAPs-tubulin preparation the understanding of its role in the cell division and was rapidly thawed and centrifuged at 25 000 × g 10 min embryogenesis and shed light on its oncogenic and anti- to remove aggregated material. oncogenic activities [48-51]. Finally, these results could provide a framework to bridge different aspects of regula- Tubulin S preparation tion of mRNA translation and the function of the micro- Tubulin S was prepared as described by Knipling et al. tubule cytoskeleton. [55]. Briefly, 250 μM tubulin in the stock buffer was diluted five times with 1 mM GTP in water. Subtilisin was then added to reach a subtilisin/tubulin ratio of 1/200 (w/ Methods Unless stated otherwise, chemicals were purchased from w). The mixture was incubated for 40 min at 25°C, and Sigma-Aldrich (Milwaukee, WI, USA). PMSF was added to 0.5 mM to stop cleavage. We then added MES-KOH, pH 6.8, MgCl and EGTA to reach a YB-1 purification final concentration of 50 mM, 1 mM and 1 mM of these YB-1 was expressed in the Escherichia coli BL21(DE3) compounds, respectively. The mixture was incubated on strain transformed with the pET 3-1-YB-1 construct [12]. ice for 30 min and centrifuged at 100 000 × g for 10 min. Bacteria were cultured at 37°C to mid-log exponential Supernatant was collected and used immediately for phase, then protein synthesis was induced by 0.5 mM polymerization assays. IPTG. After 3 h induction, bacteria were pelleted by cen- trifugation (4000 × g, 10 min) and the pellet was resus- Rabbit tissue extracts preparation pended in 10 volumes of 40 mM Tris-HCl, pH 7.6, 2 M Tissue extracts from adult rabbit were prepared as NaCl, 1 mM PMSF and disrupted by ultrasonication. Cell described by Miwa et al [56]. Briefly, tissues were homog- debris was removed by centrifugation at 140 000 × g for 2 enized in 3 volumes of 50 mM Tris-HCl, pH 7.6, 50 mM h. Supernatant was diluted with four volumes of 10 mM KCl, 5 mM MgCl , 0.25 M sucrose, 1 mM DTT, 1 mM Tris-HCl, pH 7.6, and loaded onto a heparin-Sepharose PMSF in motor-driven homogenizer, and homogenates column (GE Healthcare, UK) equilibrated with 20 mM were centrifuged at 10 000 × g for 20 min to remove cell Tris-HCl, pH 7.6, 500 mM NaCl. The column was washed debris. Supernatants were collected and protein concen- with 5 volumes of 20 mM Tris-HCl, pH 7.6, 500 mM tration was determined as described above. NaCl, after that bound YB-1 was eluted with 20 mM Tris- HCl, pH 7.6, 2 M NaCl. Eluted protein was concentrated Affinity chromatography by centrifugation using a Centriprep 10 concentrator YB-1, BSA and casein were coupled to Sepharose 4B using (Amicon Corporation, Danvers, MA) and purified by size 5 mg of protein per 1 ml of CNBr-activated Sepharose 4B exclusion chromatography on a Sephacryl S-200 column (GE Healthcare) according to the manufacturer's instruc- (GE Healthcare) pre-equilibrated with 20 mM Tris-HCl, tions. pH 7.6, 2 M NaCl. Fractions containing YB-1 were pooled, dialyzed against 20 mM Tris-HCl, pH 7.6, 250 mM NaCl Rabbit tissue extracts (50 μg of total protein) were incu- and concentrated again using the same procedure. Protein bated with 10 μl of YB-1- or BSA-coupled Sepharose in 50 concentration was determined by comparison with a μl of low salt buffer (20 mM Tris-HCl, pH 7.6, 100 mM standard BSA curve using the Bio-Rad protein assay kit KCl, 1 mM MgCl , 1 mM DTT, 1 mM PMSF) for 10 min at (Bio-Rad Laboratories, Richmond, CA). Anti-YB-1 anti- room temperature. Reaction mixtures were centrifuged at bodies were produced in rabbit as described by Davydova 1 500 × g for 1 min, supernatants were discarded, and the et al. [52]. resin pellets were resuspended in 250 μl of incubation buffer and pelleted again. This washing step was repeated twice. Bound proteins were eluted with 250 μl of high-salt buffer (20 mM Tris-HCl, pH 7.6, 1 M KCl, pH 7.6, 1 mM Page 12 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 MgCl , 1 mM DTT, 1 mM PMSF) and precipitated by addi- were precipitated with 75% acetone, dissolved in SDS tion of 75% acetone (v/v). Dried pellets were dissolved in sample buffer and resolved on 9% SDS-PAGE. Protein SDS sample buffer and analyzed by electrophoresis on detection was performed using rabbit anti-YB-1 antibod- 12% SDS-PAGE. For the identification of tubulin, pro- ies [52], anti-alpha tubulin antibodies and anti-beta tubu- teins were separated similarly by SDS-PAGE and then lin antibodies as described above. blotted on nitrocellulose as described [57]. Nitrocellulose Atomic Force Microscopy (AFM) membranes were blocked with 1% nonfat milk in TBST buffer (10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 0.1% Tri- To study YB-1:tubulin complexes, we prepared reaction ton X-100) and probed for tubulin with mouse anti-alpha mixtures with either both or separately taken tubulin and (clone B-5-1-2) and anti-beta tubulin antibodies (clone YB-1 (1 μM each) in 50 mM MES-KOH, pH 6.8, 1 mM Tub 2.1) diluted at 1/5000 in blocking solution. Primary MgCl , 1 mM EGTA, 0.5 mM GTP. These mixtures were antibodies were detected using goat anti-mouse horserad- incubated for 10 min at 37°C, then fixed with 0.2% glu- ish peroxidase conjugated secondary antibodies followed taraldehyde. by development using 3,3',5,5'-tetramethylbenzidine (TMB). Microtubules were prepared for AFM imaging as follows: 5 μM tubulin was assembled with or without (control) 5 Analysis of YB-1:tubulin complex stability μM YB-1 in 50 mM MES-KOH, pH 6.8, 1 mM EGTA, 5 Tubulin (20 μg) was loaded onto 50 μl of YB-1- or casein- mM MgCl , 1 mM GTP, and 20 μM taxol for 15 min at coupled Sepharose 4B columns equilibrated with 10 mM 37°C, pelleted as described above, gently resuspended in Tris-HCl, pH 7.6, 150 mM NaCl and incubated for 10 min a starting volume of polymerization buffer with 0.2% glu- at 4°C. Flow-through was collected, and the resins were taraldehyde and fixed for 15 min at 37°C. washed with five column volumes of equilibrium buffer. 2+ Bound tubulin was eluted stepwise using five column vol- All AFM samples were deposited on Ni pretreated freshly umes of 10 mM Tris-HCl, pH 7.6, containing either 300 cleaved mica as described by Pastre et al. [58]. AFM imag- mM, 600 mM or 1 M NaCl. Proteins from all fractions ing was performed in tapping mode with a multimode were precipitated by addition of trichloroacetic acid to AFM instrument (Digital Instruments, Veeco, Santa Bar- 10% and analyzed by SDS-PAGE. bara, CA) operating with a Nanoscope IIIa controller. We used AC160TS silicon cantilevers (Olympus, Hamburg, In vitro tubulin polymerization assays Germany) with a resonance frequency of 300 kHz. Images Tubulin or MAPs-tubulin assembly was followed turbidi- were collected at a scan frequency of 1.5 Hz and a resolu- metrically at 340 nm (1 cm light path) in an Ultrospec tion of 512 × 512 pixels. 3000 spectrophotometer (GE Healthcare) equipped with a temperature controller. Experiments were carried out in Transmission Electron Microscopy (TEM) buffer M (50 mM MES-KOH pH 6.8, 1 mM EGTA, 5 mM For ultrathin sectioning, microtubules were prepared with MgCl and 1 mM GTP) with either 10 or 20% glycerol (v/ 30 μM tubulin with or without 10 μM YB-1 in 50 mM v). MES-KOH, pH 6.8, 1 mM EGTA, 5 mM MgCl and 1 mM GTP, 10% glycerol. Microtubules were pelleted at 40 000 Microtubule sedimentation assays × g for 30 min at 37°C. The pellets were gently resus- 50 μl of 25 μM tubulin in buffer M containing 10% glyc- pended in 50 mM MES-KOH, pH 6.8, 1 mM EGTA, 5 mM erol were assembled at 37°C with increasing concentra- MgCl , 1 mM GTP, 1% glutaraldehyde and incubated for tions of YB-1 for 30 min to reach the steady-state. fixation for 1 hour at room temperature. Samples were Microtubules were pelleted at 300 000 × g for 5 min at then post-fixed with 1% osmium tetraoxide (OsO ) for 1 37°C and resuspended in 50 μl of SDS sample buffer. To h. After gradual dehydratation in ethanol series, the pel- determine the amounts of tubulin and YB-1 in the micro- lets were embedded in EPON mixture. Ultrathin sections tubule or supernatant fractions, 3 μl of supernatants and were stained with 2% uranyl acetate and examined with a resuspended pellets were analyzed by SDS-PAGE. Tecnai F20 S-Twin transmission electron microscope (FEI company, Hillsboro, OR, USA) operating at 200 kV. Cross-linking of YB-1:tubulin complexes Cross-linking reactions were performed at tubulin con- Electrophoretic mobility shift assay centration far below the critical concentration. To prepare Luciferase RNA (1500 base length) was synthesized in YB-1:tubulin complexes, 1 μM tubulin and 0.3, 0.5, 0.8 or vitro as described by Svetlov et al [59]. RNA (0.6 pmoles) 1.2 μM YB-1 were incubated in 50 mM MES-KOH, pH 6.8, was incubated in 20 μl of buffer M containing 5% glycerol 1 mM MgCl , 1 mM EGTA, 0,5 mM GTP, 5 mM EDC, 12 alone or in the presence of 42 pmoles YB-1 for 10 min at mM sulfo-NHS for 1 h at 30°C. Cross-linking reactions room temperature. After incubation, either 42 pmoles or were quenched by the addition of 50 mM glycin. Proteins 84 pmoles tubulin were added to preformed RNP. The Page 13 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 reaction products were separated at 80V for 2 hours on Additional file 3 0.6% agarose gel prepared in buffer M. After migration, YB-1 partially displaces MAPs from taxol-stabilized microtubules. MAPs- the gel was stained with ethidium bromide. tubulin (0.5 mg/ml, ~4μM) was polymerized in the absence (control) or presence of increasing concentrations of YB-1 (from 1.25 μM to 10 μM, Abbreviations as indicated) in buffer M with 10% glycerol and 20 μM taxol. After MT: microtubules; MAPs: microtubule associated pro- polymerization, the samples were pelleted, and equal volumes of superna- teins; RNP: ribonucleoprotein; Tubulin S: subtilisin- tants and resuspended pellets were analyzed by SDS-PAGE. (S, superna- treated tubulin; TEML: transmission electron microscopy; tant; P, pellet). Click here for file EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; [http://www.biomedcentral.com/content/supplementary/1471- Sulfo-NHS: N-hydroxysulfosuccinimide. 2091-9-23-S3.jpeg] Authors' contributions Additional file 4 KGC: Designed and performed experiments. Participated Plot of the tangent at the microtubule assembly slope versus YB-1 concen- to the analysis of data, to the writing of the manuscript tration observed on figure 5. We can notice that rate of microtubule assembly reaches a maximum plateau value from about 8 μM YB-1. and revision. AM: Performed experiments, participated to Click here for file the analysis of data and critical revision. NVP: carried out [http://www.biomedcentral.com/content/supplementary/1471- initial experiments on affinity chromatography of cell 2091-9-23-S4.pdf] extracts and tubulin on YB-1-Sepharose, participated in experiments on YB-1 co-sedimentation with microtubules and in manuscript drafting. DP: Performed AFM investiga- tion, participated to the revision of the manuscript. ESN: Acknowledgements was involved in the study design and drafting the manu- Authors thank INSERM, The Conseil Régional d'Ile de France; the "Service script and in revising it critically for important intellectual pour la Science, la Technologie et l'Espace (SSTE)" from French Embassy at content. OV: carried out the molecular genetic studies and Moscow, Russia; Genopole Evry, and the AFM. This study was partly sup- YB-1 purification and affinity chromatography, coordi- ported by the Programs on "Molecular and Cellular Biology" and on "Basic Sciences to Medicine" from the Presidium of Russian Academy of Sciences. nated studies on YB-1-tubulin interaction and on analysis of stability of this complex. NAS: isolated tubulin and References obtained microtubules, participated in experiments on 1. Kohno K, Izumi H, Uchiumi T, Ashizuka M, Kuwano M: The pleio- YB-1-microtubule interaction. 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YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules

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Copyright © 2008 by Chernov et al; licensee BioMed Central Ltd.
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Life Sciences; Biochemistry, general; Life Sciences, general
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Abstract

Background: YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. Results: We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin- treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes. Conclusion: These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation. box binding transcription factor, where it activates tran- Background YB-1 is a multifunctional protein known to interact with scription of various cellular genes, including those impli- nucleic acids, and as such, YB-1 is involved in a wide vari- cated in cell growth, differentiation and apoptosis ety of cellular processes in eukaryotic cells (reviewed in (reviewed in [7]). Translocation of YB-1 from cytoplasm [1]). In the cytoplasm, YB-1 participates in the formation to the cell nucleus can occur at certain steps of the cell of mRNPs and in the regulation of mRNA translation and cycle [8] and in response to stress-induced DNA damages degradation [2-6]. In the nucleus, YB-1 functions as a Y- [9,10]. The association of YB-1 with nucleic acids causes Page 1 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 global changes in their structures by melting short or confirmed by Western blotting using anti-alpha and anti- imperfect duplexes and acceleration of annealing and beta tubulin antibodies. As shown on Fig. 1B, α and β strand exchange reactions between complementary tubulin subunits were detected in all tissue extracts and strands of RNA and DNA [11,12]. YB-1 may thus partici- high salt eluates. It is interesting to note that, in the case pate in DNA recombination and replication [13,14], and of brain and testis, some tubulin remained in the flow- in the case of damaged DNA, YB-1 may assist in its repa- through fraction. This could be either due to the satura- ration. tion of the affinity column or linked to the sequestration of tubulin with partner proteins. In the control experi- Molecular and structural investigations showed that YB-1 ments performed with BSA-Sepharose, all tissue proteins interacts with RNA and DNA through two non-homolo- were collected in the flow-through fractions and were gous domains: the cold-shock domain (CSD), which con- undetectable in high salt eluates (data not shown). These sists of five anti-parallel beta-strands [15,16], and the C- results strongly suggested that tubulin binds specifically to terminal domain, which contains a series of alternating YB-1. clusters of positively and negatively charged amino acid residues. We further investigated YB-1:tubulin interaction and eval- uated the stability of the YB-1-tubulin complex by YB-1 In addition to its interaction with nucleic acids, YB-1 affinity chromatography using pure tubulin as a prey. interplays with different protein partners within the cell. Tubulin was totally adsorbed on the YB-1-Sepharose col- It has been noticed that the interaction of YB-1 with p53 umn in the presence of 150 mM NaCl, while poorly increases the affinity of p53 for DNA promoters that could retained in similar conditions by a casein-Sepharose con- stimulate transcription of p53-controled genes [17,18]. trol column (Fig. 2). Tubulin started to elute from the YB- Similarly, YB-1 interacts with the T-antigen of polyomavi- 1 column at 300 mM NaCl forming a peak around 600 rus JC and triggers transcription of viral genes [19]. YB-1 mM NaCl (Fig. 2). These results indicate that the YB- can also catalyze splicing of pre-mRNA via interaction 1:tubulin interaction is not inhibited at physiological and with RNA polymerase II, EWS (Ewing's sarcoma protein) moderate ionic strength (up to 300 mM NaCl). We finally and TLS (translocation liposarcoma protein) proteins probed the presence of YB-1 and of α and β tubulin subu- [20]. nits in YB-1-tubulin complexes by cross-linking using the zero length cross-linker EDC/Sulfo NHS. As displayed on The objective of the present work was to identify new YB- Fig. 3, cross-linker stabilized dimeric forms of YB-1 and α 1 protein partners to better understand the functions of β-tubulin heterodimers, as well as higher molecular this protein. We discovered, using a series of biochemical weight aggregates (compare lanes 3 and 4 with 1 and 2). in vitro experiments, that YB-1 strongly interacts with It is worthy to note that under these conditions, both YB- tubulin, both soluble and polymerized into microtubules. 1 and tubulin cross-linked samples still contained non- We demonstrated that YB-1 stimulates microtubule cross-linked products (lanes 3 and 4). When the cross- assembly, and in addition, that tubulin competes with linking reaction was performed in the presence of both mRNA for interaction with YB-1. In light of these results, proteins at a YB-1-tubulin molar ratio of 0.5, most YB-1 we propose that YB-1 may contribute to coordination of was found in high-molecular weight complexes contain- regulation of mRNA translation and dynamics of microtu- ing also both tubulin subunits (compare lanes 1 and 5). bule cytoskeleton. Tubulin, via its interaction with YB-1, An increase of the YB-1:tubulin molar ratio over 0.5 may indirectly influence the translational regulation of increased the yield of these high molecular weight com- mRNP complexes. plexes (lanes 6 to 8) with a slight increase of the free YB-1 band. Our results demonstrate that YB-1 interacts directly Results with tubulin and suggest that YB-1 contacts the both tubu- Tubulin is a new YB-1 interacting protein lin subunits in solution. A search for new YB-1 partners from different rabbit tissue extracts was performed by affinity chromatography using The binding of YB-1 to tubulin was then investigated by YB-1-Sepharose as bait. Though eluates varied in protein AFM. YB-1 appeared as discrete particles on the mica sur- composition, two prominent bands migrating as 45 kDa face with an average height of 0.7 nm (Fig. 4, upper and 50 kDa were detected in the eluates of most of the tis- panel). This value appeared lower than that reported by sues (Fig 1A, right panel, marked with an asterisk and a Skabkin et al [22] (about 4 nm for monomeric YB-1 in dot, respectively). The 45 kDa protein was identified by solution of high ionic strength) and probably resulted MALDI-TOF mass spectrometry as actin, a well-character- from YB-1 flattening on the mica surface. Tubulin ized YB-1 partner [21] and the 50 kDa band was identified appeared as particles with an average height of 3.7 nm by similar means as tubulin (see Additional file 1). The (Fig. 4, middle panel) in agreement with previous reports presence of tubulin in extract and eluate fractions was also [23]. In the YB-1-tubulin sample, in contrast to isolated Page 2 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 YB-1 affin Figure 1 ity chromatography of rabbit tissue extracts YB-1 affinity chromatography of rabbit tissue extracts. A, Rabbit tissue extracts (50 μg of total protein) were loaded onto YB-1-Sepharose. After washing with buffer containing 100 mM KCl, bound proteins were eluted with buffer containing 1 M KCl. Extracts and eluates were analyzed by 12% SDS-PAGE followed by Coomassie blue staining. Two prominent bands migrating as 45 kDa and 50 kDa were detected in the eluates of most of the tissues (marked with an asterisk and a dot) and were identified by mass spectrometry as actin and tubulin respectively. B, Western blot of rabbit tissue extracts and YB-1- Sepharose fractions. Fractions were collected as described in A and probed with anti-alpha and anti-beta tubulin antibodies (fl- th, flow through). proteins, particles were less homogenous with a size dis- tubulin complexes probably made of several molecules of tribution ranging from about 3 to 8 nm (Fig. 4, bottom both tubulin and YB-1. panel). A novel class of particles was clearly distinguisha- ble, centered around 7 nm. It was attributed to the YB-1- Page 3 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 close to cellular, we performed a series of experiments with MAPs-tubulin. This preparation contained approxi- mately 15% of MAPs (w/w) as estimated by Coomassie staining of proteins separated on SDS-PAGE. The addition of YB-1 to MAPs-tubulin at a total YB-1-tubulin ratio of 0.3 decreased the lag-time similarly to pure tubulin, increased the rate of polymerization and slightly increased the final microtubule mass as estimated from the steady state plateau value (Fig. 6). Higher amounts of YB-1 fur- YB-1 interacts dir Figure 2 ectly with pure tubulin ther reduced the lag-time of polymerization but did not YB-1 interacts directly with pure tubulin. Affinity chro- change significantly the mass of polymerized tubulin. matography analysis of YB-1-tubulin interaction. Tubulin (20 These data indicate that the presence of MAPs does not μg) was applied onto 50 μl YB-1- or casein-Sepharose (used abrogate the positive effect of YB-1 on the overall kinetics as a control) in buffer containing 150 mM NaCl at 4°C and incubated for 10 min. After washing, bound proteins were of microtubule assembly. eluted stepwise using buffer containing 150 mM, 300 mM, 600 mM and 1 M NaCl as described under Materials and YB-1 accelerates tubulin S assembly in vitro Methods. Fractions were analyzed by 15% SDS PAGE fol- The C-termini of alpha and beta tubulin subunits are lowed by Coomassie blue staining. highly negatively charged at physiological pH and involved in the regulation of tubulin function [24]. On the other hand, YB-1 is highly positively charged at neu- YB-1 strongly favors tubulin assembly in vitro tral pH, so it was reasonable to think that YB-1 accelerates The influence of YB-1 on in vitro microtubule assembly microtubule assembly via interaction with tubulin C-ter- was first assessed by turbidimetry. Compared to tubulin mini. It was therefore critical to investigate whether the control, YB-1 induced a dramatic shortening of the lag- effects of YB-1 on microtubule assembly could result only time in a dose dependant manner. It also dose-depend- from a charge effect or could also be partly due to more ently increased both the apparent rate of microtubule specific molecular recognition mechanisms between YB-1 assembly and the steady state plateau value (Fig. 5A and and tubulin. For this purpose, we treated tubulin with Additional file 4). We next examined the distribution of subtilisin in such conditions as to cleave the charged C- YB-1 in the soluble tubulin or microtubule fractions in termini of the both tubulin subunits and investigated the steady-state samples using microtubule sedimentation effect of YB-1 on the assembly of the cleaved tubulin prod- assay. In agreement with turbidimetry data, the presence uct (tubulin S, Fig. 7A). We observed that YB-1 was still of YB-1 strongly increased the total amount of tubulin in able to promote the assembly of tubulin S (Fig. 7B). A the pellet (Fig. 5B). In the presence of 5 μM YB-1 and 20 strong stimulation of assembly was observed when YB-1 μM tubulin, most of YB-1 was associated with microtu- was added at a total YB-1-tubulin S molar ratio as low as bules at steady state. In these conditions, a visual estima- 0.13. At this ratio, YB-1 significantly decreased the lag- tion on Coomassie stained gels of the YB-1:tubulin time and increased the velocity of the polymerization stoichiometry indicated a ratio of about 1 mole of YB-1 (Fig. 7B, curve b). Higher concentrations of YB-1 further per 3 moles of tubulin heterodimer in the pellet. When 20 reduced the lag-time and increased the rate of tubulin S μM tubulin was assembled in the presence of 10 μM YB-1, assembly but did not produce any significant additional we noted a further increase of the amount of tubulin in effect on microtubule mass at steady-state (Fig. 7B, curves the pellet. YB-1 was again found mainly in the pellet with c and d). Together, these data suggest that the promotion however a small amount remaining in the supernatant. In of microtubule assembly by YB-1 involves interaction these conditions, the YB-1-tubulin stoichiometry in the between specific sites of the partners, although non-spe- pellet rose to about 1 mole of YB-1 per 2 moles of tubulin cific electrostatic interactions may also play an important heterodimer. It is worthy to note that YB-1 alone, when role. centrifuged in the same conditions remained totally in the YB-1 promotes the formation of normal microtubules and supernatant fraction (data not shown). probably coats the microtubule wall YB-1 favors MAPs-tubulin assembly in vitro High resolution microscopies like AFM or TEM could pro- It is well documented that the presence of MAPs strongly vide interesting information about microtubule morphol- influences the kinetics of microtubule assembly. MAPs ogy in the presence of YB-1 and about the localization of favor the nucleation of microtubules, increase the rate of YB-1 on/in microtubules. Under control conditions, i.e., assembly, extent of polymerization and stabilize microtu- without YB-1, in AFM images microtubules appeared as bules against disassembly. To investigate whether YB-1 straight rods with an apparent height of 10 nm. This indi- may also influence tubulin polymerization in conditions cates that they were flattened on the surface due to the dry- Page 4 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 YB-1:tubulin cross-linking Figure 3 YB-1:tubulin cross-linking. YB-1 and tubulin were cross-linked with EDC and sulfo-NHS, resolved by 9% SDS-PAGE, blot- ted and probed with anti-alpha, anti-beta tubulin and anti-YB-1 antibodies as described under Materials and Methods. Gels were either stained with Coomassie blue (top) or processed for Western blotting with the indicated antibodies. Page 5 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 A Figure 4 FM images of YB-1, tubulin and YB-1-tubulin complexes AFM images of YB-1, tubulin and YB-1-tubulin complexes. Isolated YB-1 and tubulin form discrete particles of 0.7 and 3.7 nm heights, respectively. YB-1-tubulin complex samples formed at a 1:1 molar ratio show the presence of an additional population of particles with a height of about 7.9 nm. Histograms illustrating the height distribution of particles are displayed on right panels. Page 6 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 YB-1 fa Figure 6 vors MAPs-tubulin assembly YB-1 favors MAPs-tubulin assembly. Turbidimetry plot of MAPs-tubulin (1 mg/ml, ~9 μM) assembly in buffer M with 20% glycerol in the absence (a) or presence of 2.5 μM (b), or 5 μM YB-1 (c). a single layer of tubulin forming their wall and an outer diameter comparable to control (Fig. 9, compare D' with C'). In addition to this, the number of protofilaments was YB-1 promotes m Figure 5 icrotubule pellets microtubule assembly and co-sediments with not significantly different from that of control (see Addi- YB-1 promotes microtubule assembly and co-sedi- tional file 2). This supported further the suggestion that ments with microtubule pellets. A, Turbidimetry plot of YB-1 coats the outer surface of the microtubule. It is also 20 μM tubulin assembly in the absence (a) or presence of worth noting that, under control conditions, microtu- increasing concentrations of YB-1 [2.5 μM (b), 5 μM (c) and bules could often be found in close contact with each 10 μM (d)] in buffer M with 10% glycerol. B, 20 μM tubulin other (Fig. 9A, C and 9C'), whereas in the presence of YB- was polymerized in buffer M with 10% glycerol in the absence (ctrl) or presence of 5 μM or 10 μM YB-1. After polymeriza- 1, microtubules appeared regularly distributed and spaced tion until steady-state, the samples were centrifuged, then from each other (Fig. 9B, D and 9D'). Due to its coating supernatants and pellets were analyzed by SDS-PAGE. (S, the microtubule surface, YB-1 could induce a change in supernatant; P, pellet). (Additional file 4). rigidity of microtubule or steric hindrance on its outer sur- face, which may lead to a larger inter-microtubule spac- ing. ing procedure (Fig. 8, upper panel) and the height measurement thus corresponded to two tubulin layers in Tubulin interferes with mRNP formation close contact (see [23] and Fig. 8, upper schema). Micro- YB-1 is a major mRNA binding protein that forms com- tubules formed in the presence of YB-1 were significantly plexes with mRNA and regulates its translational activity. higher than the control ones with an average height of In this context, it is necessary to explore whether tubulin about 17 nm. The increase in height could correspond to can interfere with formation of RNP and induce some YB-1 coating of the microtubule outer wall (Fig. 8, bottom modifications of RNP structure. Since RNP differs from panel and schema). However, such morphology can also naked RNA in net charge and molecular weight, we result from microtubules with double walls or with a decided to investigate the effect of tubulin on YB-1-RNA higher number of protofilaments. complexes by electrophoretic mobility gel-shift assay. It is known that RNP saturated with YB-1 contains about one To distinguish between these scenarios, we investigated molecule of YB-1 per 25 RNA bases [25]. Compared to the effect of YB-1 on the ultrastructure of microtubules by RNA alone, these saturated YB-1:RNA complexes demon- TEM with thin sections of EPON-embedded microtu- strate a significant reduction of mobility in native agarose bules. In control conditions, TEM analyses showed regular gel due to either partial RNA discharging or the increased microtubules with a diameter of about 25 nm. With YB-1, mass of the formed YB-1:RNA complex, or both (Fig. 10, the ultrastructure of microtubules remained normal with compare lane 1 and 3). The presence of tubulin did not Page 7 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 opment and in cell proliferation and transformation. Here, we report that YB-1 interacts with tubulin and microtubules in vitro and demonstrate that this protein promotes the assembly of microtubules. This new func- tional link of cellular proteins seems to be important, since, on the one hand, the regulation of many vital proc- esses, such as cell division, motility and intracellular traf- fic depends on the fine tuning of the intrinsic dynamics of microtubules by protein partners. The inventory of tubu- lin partners is not complete and the comprehension of their mechanisms of regulation of microtubule dynamics is still under investigation. On the other hand, with YB-1 being an RNA-binding protein, the study of YB-1:tubulin interaction is highly relevant to further address the com- petition between tubulin and mRNA for YB-1 binding. The conformational changes of RNP containing YB-1 induced by tubulin may be of particular interest to inves- tigate RNP accessibility for translation and degradation. YB-1 strongly interacts with tubulin The strong binding between YB-1 and tubulin was sup- ported by a series of in vitro biochemical data which also demonstrated that the YB-1:tubulin interaction was inde- pendent of the presence of other tissue extract compo- nents. Since YB-1 is positively charged at neutral pH (pI ~ 9.5) [2] while tubulin is negatively charged (pI ~ 5.6) [26], we assayed if YB-1 interacts with tubulin in a manner sim- YB-1 favors tubulin S assembly Figure 7 ilar to that of many other positively charged nucleic acid YB-1 favors tubulin S assembly. A, Tubulin and tubulin S (5 μM each) were analyzed by native agarose gel (left panel) binding proteins. As tubulin was still retained on a YB-1 and 8% SDS-PAGE (right panel). In our conditions, complete column at physiological ionic strength, we concluded that digestion of both α and β tubulin were total. B, Turbidimetry the attraction between tubulin and YB-1 was the result of plot of tubulin S (5 μM) assembly in the absence (a) or pres- a strong, short-ranged, electrostatic interactions and (or) ence of 0.625, 1.25, 2.5 μM YB-1 (b, c, and d, respectively) in other more specific mechanisms like hydrophobic inter- buffer M with 20% glycerol. actions. Additionally, we found that YB-1 did not provoke the formation of huge aggregates containing tubulin, in contrast to other highly cationic proteins like histones [27]. The YB-1-tubulin oligomeric complexes formed in change mobility of RNA that excluded the possibility of "non-assembly conditions" had their heights about 7 nm direct interaction between these two molecules (compare (Fig. 4), which indicated the involvement of a few YB-1 lanes 1 and 2). The addition of tubulin to YB-1:RNA com- and tubulin molecules in formation of these complexes. plexes clearly increased the mobility of RNP (compare lane 3 with 4 and 5). The presence of tubulin may thus Microtubule polymerization is a two-step process in induce structural changes of RNP complexes and/or a which tubulin first forms nucleation templates and then change in the RNP charge via YB-1 withdrawal mediated adds to and elongates them [28,29]. The templates were by tubulin. Further investigations are required for a better modelled either in equilibrium with tubulin dimers [30- description of the interaction between mRNA:YB-1 com- 32] or as persistent structures [33]. MAPs facilitate the for- plexes and tubulin, which may play an important role in mation of templates and are thought to clamp them. The transition of mRNA from silenced to translationally active YB-1-tubulin complexes we observed here were function- state. ally reminiscent of MAPs-tubulin complexes [29]. The YB- 1-tubulin complexes could represent nucleation tem- Discussion plates, and thus, YB-1 could act as a nucleation factor for YB-1 is a member of the Y-box protein family, a family microtubule assembly in vitro in a manner similar to that highly conserved among prokaryotes and eukaryotes. In of classical MAPs [34]. eukaryotes, Y-box proteins are regulators of transcription and translation and play important roles during the devel- Page 8 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 A Figure 8 FM images of microtubules assembled with or without YB-1 AFM images of microtubules assembled with or without YB-1. Microtubules were assembled with (bottom panel) or without YB-1 (top panel), fixed and analyzed by AFM as described under Materials and Methods. Histograms of the microtu- bule height distribution are shown on the right. Schemas on the left illustrate the proposed structure of microtubules. YB-1 favours microtubule assembly and coats the sin. This observation suggests that the promotion of microtubule wall microtubule assembly by YB-1 results not only from YB-1 strongly favoured polymerization of pure tubulin purely electrostatic interactions but also from a more spe- into microtubules. YB-1 was found to accelerate the cific molecular recognition mechanism. This suggestion is apparent rate of microtubule assembly, to increase the supported by the fact that most positively charged mole- microtubule mass at steady state and to be associated with cules which interact in a non-specific manner with tubu- microtubules. lin promote the assembly of tubulin into aberrant structures instead of microtubules. For example, polyca- The ability of MAPs or other non-specific basic molecules tions induce the formation of double-walled microtu- to promote microtubule assembly depends largely upon bules [37,38], and aminoacyl-tRNA synthetases promote the presence of the negative charges of tubulin C-termini microtubule bundling [39]. In the present work, electron [35,36]. In contrast to these positively charged molecules, microscopy analysis showed that microtubules formed in we found that YB-1, which is also a basic protein, still the presence of YB-1 possess a normal morphology with a stimulated polymerization of tubulin cleaved by subtili- single wall of circa 25 nm diameter and parallel AFM Page 9 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 YB- Figure 9 1 induces the formation of single wall microtubules YB-1 induces the formation of single wall microtubules. Tubulin (30 μM) was polymerized without (A, C, C') or with 10 μM YB-1 (B, D, D') at 37°C. Transmission electron microscopy of longitudinal ultrathin sections revealed that YB-1 contrib- utes to the formation of normal microtubules and that, in addition, the microtubules remain regularly spaced from one another (compare B with the control A). Transversal sections of microtubules (C, D) confirmed this observation. B, C, D are at the same magnification as A. D' is at the same magnification as C'. Page 10 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 bly because of multiple tubulin-binding sites and high molecular mass of these proteins [42]. YB-1 promotes polymerization of MAPs-tubulin In the presence of MAPs-tubulin, YB-1 also accelerated microtubule assembly, although it didn't promote an increase of the total mass of polymerized tubulin. This was most probably due to the fact that our MAPs-tubulin preparation contained around 15% of MAPs (w/w), which makes microtubules nearly saturated with MAPs. Interestingly, some high molecular weight MAPs were par- tially displaced by YB-1 from MAPs-microtubules stabi- lized with taxol when the total YB-1-tubulin molar ratio was above 0.5 (see Additional file 3). As mentioned above, classical MAPs, such as tau and MAP2, interact mostly with the negatively charged C-termini of tubulin. In addition, tau binds to the amino terminal region of alpha tubulin subunit [43]. Since YB-1 strongly promoted assembly of tubulin lacking C-termini, we predict that YB- 1 interacts with microtubules not only via non-specific binding to the negatively charged C-terminal tail. Finally, in this context, it is interesting to note that YB-1 doesn't display any homology with classical MAPs and may repre- sent the generic element of a novel class of microtubule interacting proteins. YB-1 may shuttle between mRNA and the microtubule cytoskeleton in vivo Microtubules play a critical role in mRNA translation and localization in vivo (reviewed in [44]). Numerous plant proteins known to regulate translation were recently iden- tified by affinity chromatography as tubulin-binding pro- teins [45]. For example, the plant initiation factor eIF- (iso)4F was found associated with the microtubule Tu Figure 10 bulin modifies the structure of YB-1-RNA complexes cytoskeleton in the cell and is able to induces microtubule Tubulin modifies the structure of YB-1-RNA com- bundling in vitro [46]. plexes. 30 nM Luciferase RNA was incubated in buffer M with 5% glycerol alone (lanes 1 and 2) or in the presence of Since YB-1 is a major component of cellular RNPs, inter- 2.1 μM YB-1 (lanes 3, 4 and 5) for 10 min at 25°C. After action of tubulin with YB-1 could potentially regulate the incubation, 2.1 μM tubulin (lane 4) or 4.2 μM tubulin (lanes 2 translation state of mRNAs. Indeed, YB-1 protects mRNAs and 5) were added and mixtures were analyzed by native aga- from degradation and packs them in non-translatable rose gel electrophoresis followed by staining with ethidium RNPs. We have shown here that tubulin changes the elec- bromide. trophoretic mobility of YB-1-RNA complexes. This change most probably occurs due to the partial dissociation of YB-1 from RNA, which could be a signal to trigger mRNA investigations showed that YB-1 can coat the outer surface translation and/or degradation. Interestingly, it has of the microtubule wall. recently been shown that the phosphorylation of YB-1 by Akt kinase may regulate its binding to mRNA [47]. It is In agreement with these microscopic data, YB-1 was thus expected that the phosphorylated form of YB-1, found to co-sediment with microtubules (Fig. 5). The sto- which possesses a lower affinity for the cap-structure of ichiometry corresponds well to some other microtubule- mRNA, could be more easily released from RNA than its binding proteins with single tubulin dimer-binding site non-phosphorylated form. This release could occur due to [40,41]. Many canonic MAPs were reported to bind interaction of mRNA with other RNA-binding proteins or microtubules with a stoichiometry ranging from 1:15 to through the interaction of YB-1 with protein partners, 1:8 (MAPs:tubulin ratio in the microtubule wall) proba- such as tubulin. Our results thus provide new prospects Page 11 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 on the role of protein-protein interaction in the regulation Tubulin and microtubule proteins preparation of mRNA translation. Furthermore, these results could Tubulin was purified from sheep brain using the method also help to unravel situations where tubulin is involved of Castoldi and Popov [53] and stored at -80°C in 50 mM in the overall processes of mRNA translation and degrada- MES-KOH, pH 6.8, 0.5 mM dithiothreitol, 0.5 mM EGTA, tion. 0.25 mM MgCl , 3.4 M glycerol, 0.1 mM GTP. Before use, tubulin stock was thawed and an additional cycle of Conclusion polymerization was performed. Microtubule proteins Thus, we have first demonstrated that YB-1 stimulates (tubulin + microtubule-associated proteins, MAPs, herein microtubule assembly in vitro. By all in vitro criteria, YB-1 referred to as MAPs-tubulin) were purified from sheep represents a novel microtubule-interacting protein related brain through two cycles of assembly/disassembly as to the function of MAPs but clearly with different proper- described by Mitchison and Kirschner [54] aliquoted and ties. The YB-1 properties described here may contribute to stored at -80°C. Before use, MAPs-tubulin preparation the understanding of its role in the cell division and was rapidly thawed and centrifuged at 25 000 × g 10 min embryogenesis and shed light on its oncogenic and anti- to remove aggregated material. oncogenic activities [48-51]. Finally, these results could provide a framework to bridge different aspects of regula- Tubulin S preparation tion of mRNA translation and the function of the micro- Tubulin S was prepared as described by Knipling et al. tubule cytoskeleton. [55]. Briefly, 250 μM tubulin in the stock buffer was diluted five times with 1 mM GTP in water. Subtilisin was then added to reach a subtilisin/tubulin ratio of 1/200 (w/ Methods Unless stated otherwise, chemicals were purchased from w). The mixture was incubated for 40 min at 25°C, and Sigma-Aldrich (Milwaukee, WI, USA). PMSF was added to 0.5 mM to stop cleavage. We then added MES-KOH, pH 6.8, MgCl and EGTA to reach a YB-1 purification final concentration of 50 mM, 1 mM and 1 mM of these YB-1 was expressed in the Escherichia coli BL21(DE3) compounds, respectively. The mixture was incubated on strain transformed with the pET 3-1-YB-1 construct [12]. ice for 30 min and centrifuged at 100 000 × g for 10 min. Bacteria were cultured at 37°C to mid-log exponential Supernatant was collected and used immediately for phase, then protein synthesis was induced by 0.5 mM polymerization assays. IPTG. After 3 h induction, bacteria were pelleted by cen- trifugation (4000 × g, 10 min) and the pellet was resus- Rabbit tissue extracts preparation pended in 10 volumes of 40 mM Tris-HCl, pH 7.6, 2 M Tissue extracts from adult rabbit were prepared as NaCl, 1 mM PMSF and disrupted by ultrasonication. Cell described by Miwa et al [56]. Briefly, tissues were homog- debris was removed by centrifugation at 140 000 × g for 2 enized in 3 volumes of 50 mM Tris-HCl, pH 7.6, 50 mM h. Supernatant was diluted with four volumes of 10 mM KCl, 5 mM MgCl , 0.25 M sucrose, 1 mM DTT, 1 mM Tris-HCl, pH 7.6, and loaded onto a heparin-Sepharose PMSF in motor-driven homogenizer, and homogenates column (GE Healthcare, UK) equilibrated with 20 mM were centrifuged at 10 000 × g for 20 min to remove cell Tris-HCl, pH 7.6, 500 mM NaCl. The column was washed debris. Supernatants were collected and protein concen- with 5 volumes of 20 mM Tris-HCl, pH 7.6, 500 mM tration was determined as described above. NaCl, after that bound YB-1 was eluted with 20 mM Tris- HCl, pH 7.6, 2 M NaCl. Eluted protein was concentrated Affinity chromatography by centrifugation using a Centriprep 10 concentrator YB-1, BSA and casein were coupled to Sepharose 4B using (Amicon Corporation, Danvers, MA) and purified by size 5 mg of protein per 1 ml of CNBr-activated Sepharose 4B exclusion chromatography on a Sephacryl S-200 column (GE Healthcare) according to the manufacturer's instruc- (GE Healthcare) pre-equilibrated with 20 mM Tris-HCl, tions. pH 7.6, 2 M NaCl. Fractions containing YB-1 were pooled, dialyzed against 20 mM Tris-HCl, pH 7.6, 250 mM NaCl Rabbit tissue extracts (50 μg of total protein) were incu- and concentrated again using the same procedure. Protein bated with 10 μl of YB-1- or BSA-coupled Sepharose in 50 concentration was determined by comparison with a μl of low salt buffer (20 mM Tris-HCl, pH 7.6, 100 mM standard BSA curve using the Bio-Rad protein assay kit KCl, 1 mM MgCl , 1 mM DTT, 1 mM PMSF) for 10 min at (Bio-Rad Laboratories, Richmond, CA). Anti-YB-1 anti- room temperature. Reaction mixtures were centrifuged at bodies were produced in rabbit as described by Davydova 1 500 × g for 1 min, supernatants were discarded, and the et al. [52]. resin pellets were resuspended in 250 μl of incubation buffer and pelleted again. This washing step was repeated twice. Bound proteins were eluted with 250 μl of high-salt buffer (20 mM Tris-HCl, pH 7.6, 1 M KCl, pH 7.6, 1 mM Page 12 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 MgCl , 1 mM DTT, 1 mM PMSF) and precipitated by addi- were precipitated with 75% acetone, dissolved in SDS tion of 75% acetone (v/v). Dried pellets were dissolved in sample buffer and resolved on 9% SDS-PAGE. Protein SDS sample buffer and analyzed by electrophoresis on detection was performed using rabbit anti-YB-1 antibod- 12% SDS-PAGE. For the identification of tubulin, pro- ies [52], anti-alpha tubulin antibodies and anti-beta tubu- teins were separated similarly by SDS-PAGE and then lin antibodies as described above. blotted on nitrocellulose as described [57]. Nitrocellulose Atomic Force Microscopy (AFM) membranes were blocked with 1% nonfat milk in TBST buffer (10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 0.1% Tri- To study YB-1:tubulin complexes, we prepared reaction ton X-100) and probed for tubulin with mouse anti-alpha mixtures with either both or separately taken tubulin and (clone B-5-1-2) and anti-beta tubulin antibodies (clone YB-1 (1 μM each) in 50 mM MES-KOH, pH 6.8, 1 mM Tub 2.1) diluted at 1/5000 in blocking solution. Primary MgCl , 1 mM EGTA, 0.5 mM GTP. These mixtures were antibodies were detected using goat anti-mouse horserad- incubated for 10 min at 37°C, then fixed with 0.2% glu- ish peroxidase conjugated secondary antibodies followed taraldehyde. by development using 3,3',5,5'-tetramethylbenzidine (TMB). Microtubules were prepared for AFM imaging as follows: 5 μM tubulin was assembled with or without (control) 5 Analysis of YB-1:tubulin complex stability μM YB-1 in 50 mM MES-KOH, pH 6.8, 1 mM EGTA, 5 Tubulin (20 μg) was loaded onto 50 μl of YB-1- or casein- mM MgCl , 1 mM GTP, and 20 μM taxol for 15 min at coupled Sepharose 4B columns equilibrated with 10 mM 37°C, pelleted as described above, gently resuspended in Tris-HCl, pH 7.6, 150 mM NaCl and incubated for 10 min a starting volume of polymerization buffer with 0.2% glu- at 4°C. Flow-through was collected, and the resins were taraldehyde and fixed for 15 min at 37°C. washed with five column volumes of equilibrium buffer. 2+ Bound tubulin was eluted stepwise using five column vol- All AFM samples were deposited on Ni pretreated freshly umes of 10 mM Tris-HCl, pH 7.6, containing either 300 cleaved mica as described by Pastre et al. [58]. AFM imag- mM, 600 mM or 1 M NaCl. Proteins from all fractions ing was performed in tapping mode with a multimode were precipitated by addition of trichloroacetic acid to AFM instrument (Digital Instruments, Veeco, Santa Bar- 10% and analyzed by SDS-PAGE. bara, CA) operating with a Nanoscope IIIa controller. We used AC160TS silicon cantilevers (Olympus, Hamburg, In vitro tubulin polymerization assays Germany) with a resonance frequency of 300 kHz. Images Tubulin or MAPs-tubulin assembly was followed turbidi- were collected at a scan frequency of 1.5 Hz and a resolu- metrically at 340 nm (1 cm light path) in an Ultrospec tion of 512 × 512 pixels. 3000 spectrophotometer (GE Healthcare) equipped with a temperature controller. Experiments were carried out in Transmission Electron Microscopy (TEM) buffer M (50 mM MES-KOH pH 6.8, 1 mM EGTA, 5 mM For ultrathin sectioning, microtubules were prepared with MgCl and 1 mM GTP) with either 10 or 20% glycerol (v/ 30 μM tubulin with or without 10 μM YB-1 in 50 mM v). MES-KOH, pH 6.8, 1 mM EGTA, 5 mM MgCl and 1 mM GTP, 10% glycerol. Microtubules were pelleted at 40 000 Microtubule sedimentation assays × g for 30 min at 37°C. The pellets were gently resus- 50 μl of 25 μM tubulin in buffer M containing 10% glyc- pended in 50 mM MES-KOH, pH 6.8, 1 mM EGTA, 5 mM erol were assembled at 37°C with increasing concentra- MgCl , 1 mM GTP, 1% glutaraldehyde and incubated for tions of YB-1 for 30 min to reach the steady-state. fixation for 1 hour at room temperature. Samples were Microtubules were pelleted at 300 000 × g for 5 min at then post-fixed with 1% osmium tetraoxide (OsO ) for 1 37°C and resuspended in 50 μl of SDS sample buffer. To h. After gradual dehydratation in ethanol series, the pel- determine the amounts of tubulin and YB-1 in the micro- lets were embedded in EPON mixture. Ultrathin sections tubule or supernatant fractions, 3 μl of supernatants and were stained with 2% uranyl acetate and examined with a resuspended pellets were analyzed by SDS-PAGE. Tecnai F20 S-Twin transmission electron microscope (FEI company, Hillsboro, OR, USA) operating at 200 kV. Cross-linking of YB-1:tubulin complexes Cross-linking reactions were performed at tubulin con- Electrophoretic mobility shift assay centration far below the critical concentration. To prepare Luciferase RNA (1500 base length) was synthesized in YB-1:tubulin complexes, 1 μM tubulin and 0.3, 0.5, 0.8 or vitro as described by Svetlov et al [59]. RNA (0.6 pmoles) 1.2 μM YB-1 were incubated in 50 mM MES-KOH, pH 6.8, was incubated in 20 μl of buffer M containing 5% glycerol 1 mM MgCl , 1 mM EGTA, 0,5 mM GTP, 5 mM EDC, 12 alone or in the presence of 42 pmoles YB-1 for 10 min at mM sulfo-NHS for 1 h at 30°C. Cross-linking reactions room temperature. After incubation, either 42 pmoles or were quenched by the addition of 50 mM glycin. Proteins 84 pmoles tubulin were added to preformed RNP. The Page 13 of 16 (page number not for citation purposes) BMC Biochemistry 2008, 9:23 http://www.biomedcentral.com/1471-2091/9/23 reaction products were separated at 80V for 2 hours on Additional file 3 0.6% agarose gel prepared in buffer M. After migration, YB-1 partially displaces MAPs from taxol-stabilized microtubules. MAPs- the gel was stained with ethidium bromide. tubulin (0.5 mg/ml, ~4μM) was polymerized in the absence (control) or presence of increasing concentrations of YB-1 (from 1.25 μM to 10 μM, Abbreviations as indicated) in buffer M with 10% glycerol and 20 μM taxol. After MT: microtubules; MAPs: microtubule associated pro- polymerization, the samples were pelleted, and equal volumes of superna- teins; RNP: ribonucleoprotein; Tubulin S: subtilisin- tants and resuspended pellets were analyzed by SDS-PAGE. (S, superna- treated tubulin; TEML: transmission electron microscopy; tant; P, pellet). Click here for file EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; [http://www.biomedcentral.com/content/supplementary/1471- Sulfo-NHS: N-hydroxysulfosuccinimide. 2091-9-23-S3.jpeg] Authors' contributions Additional file 4 KGC: Designed and performed experiments. Participated Plot of the tangent at the microtubule assembly slope versus YB-1 concen- to the analysis of data, to the writing of the manuscript tration observed on figure 5. We can notice that rate of microtubule assembly reaches a maximum plateau value from about 8 μM YB-1. and revision. AM: Performed experiments, participated to Click here for file the analysis of data and critical revision. NVP: carried out [http://www.biomedcentral.com/content/supplementary/1471- initial experiments on affinity chromatography of cell 2091-9-23-S4.pdf] extracts and tubulin on YB-1-Sepharose, participated in experiments on YB-1 co-sedimentation with microtubules and in manuscript drafting. DP: Performed AFM investiga- tion, participated to the revision of the manuscript. ESN: Acknowledgements was involved in the study design and drafting the manu- Authors thank INSERM, The Conseil Régional d'Ile de France; the "Service script and in revising it critically for important intellectual pour la Science, la Technologie et l'Espace (SSTE)" from French Embassy at content. OV: carried out the molecular genetic studies and Moscow, Russia; Genopole Evry, and the AFM. This study was partly sup- YB-1 purification and affinity chromatography, coordi- ported by the Programs on "Molecular and Cellular Biology" and on "Basic Sciences to Medicine" from the Presidium of Russian Academy of Sciences. nated studies on YB-1-tubulin interaction and on analysis of stability of this complex. NAS: isolated tubulin and References obtained microtubules, participated in experiments on 1. Kohno K, Izumi H, Uchiumi T, Ashizuka M, Kuwano M: The pleio- YB-1-microtubule interaction. 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