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Mica Surface Promotes the Assembly of Cytoskeletal Proteins Loic Hamon,*,† Dulal Panda,‡ Philippe Savarin,† Vandana Joshi,† Johann Bernhard,† Elodie Mucher,† Alain Mechulam,† Patrick A. Curmi,† and David Pastre´† Laboratoire Structure-ActiVite´ des Biomole´cules Normales et Pathologiques, INSERM/UEVE U829, EVry, 91025 France, and School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai 400076, India ReceiVed October 28, 2008. ReVised Manuscript ReceiVed December 3, 2008 We report the surface-mediated polymerization of FtsZ protein, the prokaryote homologue of tubulin, by AFM. FtsZ protein can form filaments on mica whereas the bulk FtsZ concentration is orders of magnitude lower than the critical concentration. Surface polymerization is favored by a local increase in protein concentration and requires a high mobility of proteins on the surface. To generalize to other cytoskeleton protein, we also show that mica can initiate the formation of tubulin protofilaments. This study is of particular interest for studying cytoskeletal protein dynamics by AFM but also for the surface autoassembly of nanostructures.
Introduction Atomic force microscopy (AFM) is a good tool for imaging DNA and DNA/protein complexes at the single-molecule level,1-5 and it has been used recently to investigate the structure and dynamics of assembly of tubulins6-9 and FtsZ.10-12 The use of AFM to image anionic biomolecules on like-charged anionic mica requires their adsorption on the surface, which is usually achieved via electrostatic interactions using multivalent cations.13 As biomolecules are adsorbed on the mica surface, the surface concentration can be higher than in the bulk solution. Consequently, in the case of cytoskeletal proteins that assemble above a threshold known as the critical concentration (Cc), we hypothesize that the increased concentration in the surface vicinity may yield conditions for polymerization even though the bulk protein concentration is lower than Cc. The possibility of such a surface-driven assembly also necessitates the 2D diffusion of proteins on mica. This possibility has already been demonstrated for DNA molecules14 and was found to depend on buffer composition or pretreatment of the mica surface.15 * Corresponding author. Tel: (33) 1 69 47 01 79. Fax (33) 1 69 47 02 19. E-mail:
[email protected]. † INSERM/UEVE U829. ‡ Indian Institute of Technology Bombay. (1) Abdelhady, H. G.; Allen, S.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Nucleic Acids Res. 2003, 31, 4001–4005. (2) Ristic, D.; Modesti, M.; Kanaar, R.; Wyman, C. Nucleic Acids Res. 2003, 31, 5229–5237. (3) Moreno-Herrero, F.; Perez, M.; Baro, A. M.; Avila, J. Biophys. J. 2004, 86, 517–525. (4) Sorel, I.; Pietrement, O.; Hamon, L.; Baconnais, S.; Le Cam, E.; Pastre, D. Biochemistry 2006, 45, 461–468. (5) Hansma, H. G. Annu. ReV. Phys. Chem. 2001, 52, 71–92. (6) Mukhopadhyay, R.; Hoh, J. H. FEBS Lett. 2001, 505, 374–378. (7) Makrides, V.; Shen, T. E.; Bhatia, R.; Smith, B. L.; Thimm, J.; Lal, R.; Feinstein, S. C. J. Biol. Chem. 2003, 278, 33298–33304. (8) Wagner, O. I.; Ascan˜o, J.; Tokito, M.; Leterrier, J. F.; Janmey, P. A.; Holzbaur, E. L. F. Mol. Biol. Cell 2004, 15, 5092–5100. (9) Elie-Caille, C.; Severin, F.; Helemius, J.; Howard, J.; Muller, D. J.; Hyman, A. A. Curr. Biol. 2007, 17, 1765–1770. (10) Lafontaine, C.; Valleton, J. M.; Orange, N.; Norris, V.; Mileykovskaya, E.; Alexandre, S. Biochim. Biophys. Acta 2007, 1768, 2812–2821. (11) Gonza´lez, J. M.; Ve´lez, M.; Jime´nez, M.; Alfonso, C.; Schuck, P.; Mingorance, J.; Vicente, M.; Minton, A. P.; Rivas, G. Proc. Natl. Acad. Sci. U.S.A 2005, 102, 1895–1900. (12) Mingorance, J.; Tadros, M.; Vicente, M.; Gonzalez, J. M.; Rivas, G.; Velez, M. J. Biol. Chem. 2005, 280, 20909–20914. (13) Pastre´, D.; Hamon, L.; Landousy, F.; Sorel, I.; David, M. O.; Zozime, A.; Le Cam, E.; Pietrement, O. Langmuir 2006, 22, 6651–6660. (14) Pastre´, D.; Pietrement, O.; Zozime, A.; Le Cam, E. Biopolymers 2005, 77, 53–62.
The aim of the present work was thus to investigate the possibility that the mica surface may drive the assembly of cytoskeletal proteins under conditions where the bulk protein concentration is below Cc of assembly. To that end, we first examine the case of FtsZ, a prokaryotic protein homologue of tubulin, which is involved in bacterial division.16-20 FtsZ can polymerize in solution to form filaments of varying shapes and lengths when its concentration is above Cc. We thus analyzed FtsZ polymerization on the mica surface at bulk concentrations far below Cc and its modulation by the pretreatment of the mica surface. In a second experiment, to demonstrate that mica can also promote the assembly of other cytoskeletal proteins, we extend our investigations to tubulin and show the formation of tubulin protofilaments on mica again at bulk concentrations lower than Cc.
Material and Methods FtsZ Expression, Purification, and Polymerization. Recombinant E. coli FtsZ was overexpressed in the E. coli BL21 strain and purified as described previously.21,22 Prior to use, FtsZ was thawed and centrifuged at 225 000g at 4 °C for 15 min to remove insoluble aggregates. FtsZ samples were prepared as follows: FtsZ was incubated at varying concentrations in 300 µL of polymerization buffer (25 mM PIPES-KOH at pH 6.8, 50 mM KCl, and 10 mM MgCl2) for 10 min on ice. Then GTP was added to obtain a 1 mM final concentration, and the mixture was immediately transferred to a 37 °C environment and incubated for 5 min. Tubulin Purification and Polymerization. Tubulin was purified from sheep brain using the method of Castoldi and Popov.23 Before use, tubulin stock was thawed, and an additional cycle of polymerization was performed. Tubulin samples were prepared as follows: tubulin at varying concentrations was incubated in 300 µL of polymerization buffer (50 mM MES-KOH at pH 6.8, 5 mM MgCl2, (15) Bezanilla, M.; Drake, B.; Nudler, E.; Kashlev, M.; Hansma, P. K.; Hansma, H. G. Biophys. J. 1994, 67, 2454–2459. (16) Romberg, L.; Levin, P. A. Annu. ReV. Microbiol. 2003, 57, 125–154. (17) Errington, J.; Daniel, R. A.; Scheffers, D. J. Microbiol. Mol. Biol. ReV. 2003, 67, 52–65. (18) Mitchie, K. A.; Lowe, J. Annu. ReV. Biochem. 2006, 75, 467–492. (19) Surovtsev, I. V.; Morgan, J. J.; Lindahl, P. A. PLOS Comput. Biol. 2008, 4-7, e1000102. (20) Margolin, W. Nat. ReV. Mol. Cell Biol. 2005, 6, 862–871. (21) Beuria, T. K.; Krishnakumar, S. S.; Sahar, S.; Singh, N.; Gupta, K.; Meshram, M.; Panda, D. J. Biol. Chem. 2003, 278, 3735–3741. (22) Santra, M. K.; Panda, D. J. Biol. Chem. 2003, 278, 21336–21343. (23) Castoldi, M.; Popov, A. V. Protein Expr. Purif. 2003, 32, 83–88.
10.1021/la8035743 CCC: $40.75 2009 American Chemical Society Published on Web 02/17/2009
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Figure 1. (I) FtsZ critical concentration plot: Cc under our experimental conditions is equal to 1.0 ( 0.1 µM. (II) The mica surface strongly promotes FtsZ polymerization. Mica was incubated with 80 nM FtsZ in polymerization buffer without glycerol for the indicated periods of time. The scale bar is 500 nm. (III) Enlarged image for the 600 s incubation time. The scale bar is 100 nm.
1 mM EGTA, and 20% glycerol) for 10 min on ice. Then, GTP was added to obtain a 1 mM final concentration, and the mixture was immediately transferred to a 37 °C environment for 5 min. Determination of the FtsZ Critical Concentration of E. coli FtsZ. The reaction mixture of different concentrations (1, 2, 3, 4, 6, 8, 10, and 12 µM) of E. coli FtsZ was prepared in 25 mM Pipes buffer at pH 6.8, 1 mM GTP, 10 mM MgCl2, and 50 mM KCl. The reaction mixtures were immediately transferred to a 37 °C environment for 15 min. After 15 min of assembly, FtsZ polymers were sedimented at 250 000g at 30 °C for 25 min. The protein concentration in the supernatant was determined by the Bradford method, and the polymerized mass was calculated by subtracting the supernatant concentration from the total protein concentration. Each datum point is the average of three independent experiments. AFM Samples. Freshly cleaved mica or pretreated mica (freshly cleaved mica dipped in 10 mM NiCl2 or 10 mM cobalthexamine solution and then thoroughly rinsed with pure water and dried with filter paper) was dipped into the FtsZ or tubulin polymerization buffer at the end of the 5 min, 37 °C incubation period. After various incubation times, mica was removed from the mixture, plunged into
a 0.02% (w/v) uranyl acetate solution to maintain the FtsZ or tubulin structures in their conformation, and then rinsed in pure water and dried with filter paper. AFM Imaging. Imaging was performed in tapping mode with a Multimode AFM (Veeco, Santa Barbara, CA) operating with a Nanoscope IIIa controller. We used Olympus (Hamburg, Germany) AC160TS silicon cantilevers with nominal spring constants between 36 to 75 N/m. The scan frequency was typically 1.5 Hz per line, the modulation amplitude was a few nanometers, and the z scale was 10 nm.
Results and Discussion Under the conditions used here, the critical concentration for E. coli FtsZ assembly was found to be 1.0 ( 0.1 µM (Figure 1.I). This value is in agreement with other work reporting Cc values ranging from 0.7 to 4 µM,24-29 according to the buffer (24) Yu, X. C.; Margolin, W. EMBO J. 1997, 16, 5455–5463. (25) Mukherjee, A.; Lutkenhaus, L. EMBO J. 1998, 17, 462–469.
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Figure 2. (I) Effect of FtsZ bulk concentration and glycerol on FtsZ surface polymerization for an incubation time of 60 s: (a) [FtsZ] ) 80 nM, (b) [FtsZ] ) 40 nM, and (c) [FtsZ] ) 40 nM in the presence of 30% glycerol. The scale bar is 500 nm. (II) Time course of surface polymerization of FtsZ at 40 nM in the presence of 30% glycerol. The scale bar is 500 nm. (III) Distribution of the FtsZ filaments for different incubation times.
composition, pH, or detection technique. Surprisingly, when mica was incubated with FtsZ at 80 nM (i.e., about 12.5 times lower than the present Cc for FtsZ assembly), FtsZ filaments were observed on the mica surface by AFM, and their number and length increased with the incubation time (Figure 1.II). At short incubation times, straight or slightly curved FtsZ filaments were observed whereas longer filaments and rings appeared only after 30 s of incubation. For longer incubation times, the mica surface became more and more crowded because of increases in filament number and length. Interestingly, we noticed that as the filaments grew with time, they rolled up into snail-shaped structures with a parallel decrease in the inner radii of these structures (Figure 1.III). The smallest radii (40 nm minimum) were significantly different than that of rings observed at short incubation time (80 nm minimum), indicating a change in the filament curvature and most probably the FtsZ-FtsZ organization. These observations (26) Gonzalez, J. M.; Jimenez, M.; Velez, M.; Mingorance, J.; Andreu, J. M.; Vicente, M.; Rivas, G. J. Biol. Chem. 2003, 39, 37664–37671. (27) Caplan, M. R.; Erickson, H. P. J. Biol. Chem. 2003, 18, 13784–13788. (28) Romberg, L.; Mitchison, T. J. Biochemistry 2004, 43, 282–288. (29) Dajkovic, A.; Mukherjee, A.; Lutkenhaus, J. J. Bacteriol. 2008, 190, 2513–2526.
present some slight differences compared to previous AFM images of FtsZ filaments obtained after polymerization in solution by Mingorance et al.12 These authors found that FtsZ filaments had a trend toward aligning and aggregating laterally into bundles. These bundles dissociated and evolved toward curved filament structures when the FtsZ-adsorbed mica was incubated in FtsZfree buffer as a result of the depolymerization and reorganization of the filaments. In the present work, linear filaments were observed for short incubation times, and they appeared as isolated structures, whereas curved structures were formed only after a certain delay. However, the early growth of FtsZ filaments was difficult to analyze under our conditions because even at very short time (30 s) the filaments reached nearly the maximum length. One way to limit the polymerization rate is to decrease the FtsZ concentration (here 40 nM, Figure 2.Ib) and to increase the viscosity of the reaction medium (here glycerol 30%, Figure 2.Ic). It is worth pointing out that glycerol is not a crowding agent and thus could not trigger the formation of FtsZ ribbons as observed by Gonzalez et al. in the presence of Ficoll and Dextran, two largely used inert crowders.26 Under such conditions, though the bulk FtsZ concentration was 25 times lower than Cc,
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Figure 3. (I) Evidence for the surface-driven polymerization of FtsZ. The mica surface is dipped in polymerization buffer containing 40 nM FtsZ at 4 °C for 1 min. At 4 °C, the polymerization kinetics is slowed down, and the AFM image shows only a few FtsZ oligomers (a). After this 1 min incubation, the mica surface is transferred in polymerization buffer without FtsZ. Nevertheless, polymerization occurs (b) from proteins previously adsorbed on the surface. The scale bar is 1 µm. (II) Influence of the surface treatment on FtsZ polymerization ([FtsZ] ) 40 nM, glycerol 30%, incubation time 300 s). The NiCl2 and cobalthexamine surface treatments lead to a decrease in the FtsZ polymerization compared to bare mica.
the filaments were still observed on the surface, and as predicted, the polymerization rate was found to decrease (Figure 2.II) and it was possible to analyze the variation of filament length with time (Figure 2.III). It clearly appeared that at short incubation times only a few short filaments were present on the surface. As the incubation time increased, the number of filaments increased, and the distribution of length became broader with a shift toward longer lengths. Two different hypotheses can be advanced to account for the observations of FtsZ filaments on mica at bulk FtsZ concentrations far below Cc: (i) Even at very low bulk concentration of FtsZ, a few filaments of various lengths may be formed in the bulk solution. Consequently, at short deposition times, only monomers and short filaments are adsorbed on mica because their adsorption rate is higher than that of longer filaments, owing to the laws of diffusion. Then, the longer filaments are later adsorbed on the surface for a long deposition time. In addition, adding glycerol to the solution leads to a decrease in filament diffusion. (ii) On the basis of the local increase in the FtsZ concentration on the mica surface, the FtsZ concentration can exceed Cc, allowing polymerization. In this case, only short filaments are produced at short incubation time by protein diffusion on mica. The filament growth here is highly dependent on the adsorption strength between the protein and the surface. For mica, proteins are interacting with the surface via weak electrostatic binding that allows the free diffusion of the protein over a large area as already demonstrated for DNA.14 Furthermore, after the adsorp-
tion of the protein on the surface, if the protein can proceed to a random scan on the surface via surface diffusion, a higher association rate of free FtsZ to the filament ends is expected as compare to 3D diffusion in solution.30 To test these two hypotheses, we incubated mica with 40 nM FtsZ at 4 °C (to prevent polymerization) for 1 min. Under these conditions, we observed only monomers and very short filaments on the surface (Figure 3.Ia). Then, to remove unadsorbed proteins and eventually to allow surface polymerization of protein, this surface was immediately transferred after a rinsing step to polymerization buffer lacking FtsZ at 37 °C for 5 min. The AFM image of this surface revealed the presence of long FtsZ filaments (Figure 3.Ib) that can form here only from preadsorbed proteins. This observation strengthens hypothesis ii given above. To add further credit to this hypothesis, we used a mica surface pretreated with 10 mM NiCl2 or with 10 mM cobalthexamine, both of which increased the strength of protein adsorption.15 The surface pretreatment led to a 3-fold decrease in the surface density of FtsZ filaments along with a decrease in their length (Figure 3.II), in agreement with an inhibition of protein surface diffusion. This line of evidence strongly indicates that FtsZ polymerization occurs on the mica surface. Finally, our investigations are extended to the assembly of tubulin, a major cytoskeleton protein in eukaryotes. At a bulk (30) Berg, O. G.; von Hippel, P. H. Annu. ReV. Biophys. Biophys. Chem. 1985, 14, 131–158.
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Figure 4. AFM images of tubulin polymerization on a mica surface after 300 s of incubation in polymerization buffer containing 80 nM tubulin. The scale bars are (a) 500 and (b) 200 nm.
concentration that is about 2 orders of magnitude lower than Cc (80 nM compared to 10 µM), protofilaments of tubulin (Figure 4a) are rapidly produced on the mica surface with shape ranging from straight to ring structures (Figure 4b), following the same process as for FtsZ polymerization. In conclusion, we demonstrated that the mica surface fosters the formation of stable protein filaments, and this may provide a novel opportunity to study cytoskeletal protein dynamics and their interactions with partners at the single-molecule level as well as the surface autoassembly of nanostructures. The possibility that other surfaces may drive the assembly of cytoskeletal proteins
is realistic provided that these proteins are attracted to the surface and that the attraction is weak enough to allow their diffusion on the surface. Similarly, other biological polymers may be the subject of the surface-driven assembly. Acknowledgment. This work was supported by funds from the Institut National de la Sante´ et de la Recherche Me´dicale and l’Association pour la Recherche contre le Cancer. We gratefully acknowledge the Genopole EVRY for constant support of the laboratory. LA8035743
