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Visualization of inositol 1,4,5-trisphosphate receptor by atomic force microscopy
Neuroscience Letters 391 (2006) 102–107
Visualization of inositol 1,4,5-trisphosphate receptor by atomic force microscopy Wakako Suhara a , Mime Kobayashi b , Hiroshi Sagara c , Kozo Hamada d , Touichiro Goto b , Ichiro Fujimoto a , Keiichi Torimitsu a,b,e,∗ , Katsuhiko Mikoshiba a,d,f,g a
The Division of Neural Signal Information NTT-IMSUT, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan b Materials Science Laboratory, NTT Basic Research Laboratories, NTT Corporation, Atsugi, Kanagawa 243-0198, Japan c Fine Morphology Laboratory, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan d Calcium Oscillation Project, International Cooperative Research Project (ICORP), Japan Science and Technology Agency (JST), Minato-ku, Tokyo 108-0071, Japan e Solution Oriented Research for Science and Technology (SORST), Japan Science and Technology Agency (JST), Toyonaka, Osaka 560-0082, Japan f The Division of Molecular Neurobiology, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan g Laboratory for Developmental Neurobiology, Brain Research Institute, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan Received 23 March 2005; received in revised form 12 August 2005; accepted 21 August 2005
Abstract Inositol 1,4,5-trisphosphate (IP3 ) receptor (IP3 R) acts as a ligand-gated channel that mediates neuronal signals by releasing Ca2+ from the endoplasmic reticulum. The three-dimensional (3D) structure of tetrameric IP3 R has been demonstrated by using electron microscopy (EM) with static specimens; however, the dynamic aspects of the IP3 R structure have never been visualized in a native environment. Here we attempt to measure the surface topography of IP3 R in solution using atomic force microscopy (AFM). AFM revealed large protrusions extending ∼4.3 nm above a flat membrane prepared from Spodoptera frugiperda (Sf9) cells overexpressing mouse type 1 IP3 R (Sf9-IP3 R1). The average diameter of the large protrusions was ∼32 nm. A specific antibody against a cytosolic epitope close to the IP3 -binding site enabled us to gold-label the Sf9-IP3 R1 membrane as confirmed by EM. AFM images of the gold-labeled membrane revealed 7.7-nm high protrusions with a diameter of ∼30 nm, which should be IP3 R1-antibody complexes. Authentic IP3 R1 immuno-purified from mouse cerebella had approximately the same dimensions as those of the IP3 R-like protrusions on the membrane. Altogether, these results suggest that the large protrusions on the Sf9-IP3 R1 membrane correspond to the cytosolic domain of IP3 R1. Our study provides the first 3D representation of individual IP3 R1 particles in an aqueous solution. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Atomic force microscopy; Inositol 1,4,5-trisphosphate receptor; Calcium signaling; Molecular imaging; Protein structure
Inositol 1,4,5-trisphosphate receptor (IP3 R) is an intricately regulated channel that releases Ca2+ from the endoplasmic reticulum (ER) in response to the binding of 1,4,5-trisphosphate (IP3 ), which is a second messenger generated by various stimuli, such as neurotransmitters, neuromodulators, and hormones [4]. Three isoforms, namely IP3 R1-3, have different binding properties for IP3 and various regulatory molecules, and they play distinct roles in the temporal organization of Ca2+ signaling in living cells [8]. IP3 R1, a predominant type in the cerebellar ER and spine apparatus, has been shown to be essential for higher
∗
Corresponding author. Tel.: +81 46 240 3360; fax: +81 46 270 2362. E-mail address: [email protected] (K. Torimitsu).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.08.066
functions including long-term depression and neural development [10,14,18]. Recent protein-protein interaction research has revealed multiple binding-partners for IP3 R1 (reviewed in [21]), e.g. scaffolding proteins such as Homer protein, which tethers IP3 R1 to glutamate receptors [27], ankyrin, 4.1N [30], Ca2+ binding proteins such as calmodulin, CaBP, plasma-membrane channels such as the TRP channel, and ER lumenal proteins such as ERp44, which regulate IP3 R1 activity depending on the pH and redox state within ER [9]. These divergent regulations imply the dynamic behavior of IP3 R1-containing complex and/or of IP3 R1 itself, which would provide a mechanistic basis for IP3 R1-related functions in the central nervous system. IP3 R is structurally divided into three regions: a large cytoplasmic region with an IP3 -binding site close to the N-terminus,
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a transmembrane region near the C-terminus, and a short Cterminal tail [4]. The structures of the IP3 -binding site and the N-terminal suppresser domain have been obtained with a high resolution by using X-ray crystallography [1,2]. However, information about the overall structure of tetrameric IP3 R has only been acquired by electron microscopy (EM) because of its inherently large size (>1.2 MDa). In previous EM studies, IP3 R1 protein purified from rodent or bovine cerebella was shown to have a four-fold symmetry with a top width of 15–25 nm [3,5,6,12,22,25]. Although the appearances of reported IP3 R1 models are inconsistent, they can be classified into two types: square-shaped and windmill-like. Since the windmill type, which has four wings about 30 nm in diameter, is relatively abundant in the presence of Ca2+ , it has been suggested that dynamic changes occur within the IP3 R1 structure [5,6]. However, these EM data were obtained from static IP3 R1 in a vacuum; therefore there is still a need for a real-time visualization of the conformational states to be demonstrated in aqueous solution. Atomic force microscopy (AFM) is an established technique for evaluating the nanostructures of metal or silicon surfaces. Recent improvements in AFM have made it feasible to apply the method to soft biological samples such as proteins and DNA under a thin layer of solution [7]. A biomolecule in solution is generally expected to represent a native structure as a functional entity; AFM therefore offers the significant advantage of allowing the dynamic visualization of solution structures, unlike EM where the samples must be static for observation. AFM in the contact mode simply scans the sample surface with a probe. This approach has been used to image the surface topography of highly packed two-dimensional crystals of membrane proteins such as bacteriorhodopsin [16], aquaporin [23], and connexin 26 gap junctions [15] at subnanometer resolution. However, this technique is only useful for physically stable crystals of membrane proteins. AFM in the “tapping” or “dynamic force” mode, where a probe is excited with resonance oscillation during surface scanning to reduce the lateral force applied to the sample, has made it possible to visualize the conformational states of non-crystalline proteins such as insulin receptor [26], GroEL [28], and 20S proteasome [20] in a native environment. The crystallization of membrane protein is generally so difficult that AFM in the dynamic force mode should provide a practical way to understand the three-dimensional (3D) structure of a membrane protein in aqueous solution. In an attempt to visualize membrane-embedded IP3 R1 in solution, we prepared recombinant mouse IP3 R1 protein, which was overexpressed in insect Spodoptera frugiperda (Sf9) cells (Sf9-IP3 R1) using a baculovirus system. After infecting the cells, we confirmed the proper expression and the IP3 binding and Ca2+ release activities of the protein [11]. The cells overexpressing the protein were homogenized in lysis buffer (250 mM sucrose, 5 mM HEPES-pH 7.2, 1 mM EDTA, 0.5 mM EGTA, 1 mM 2-mercaptoethanol, protease inhibitor cocktail tablet (Roche)) and centrifuged at 2500 × g for 5 min. The supernatant was ultracentrifuged in a TLA100.3 rotor (Beckman) at 100,000 × g for 30 min to obtain a crude microsome fraction. The pellet was suspended with 51% sucrose in gradient
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buffer (10 mM Tris–HCl, pH 7.4). To form a discontinuous gradient, 3 ml each of 38, 31, and 23% sucrose buffer were successively layered on top of the sample and subjected to centrifugation in a SW41 rotor (Beckman) at 100,000 × g for 3 h at 4 ◦ C. We collected the 38%/51% interface fraction based on the result of a Western blot with anti-IP3 R1 antibody (18A10). The fraction was diluted in gradient buffer and centrifuged in a TLA100.3 rotor at 100,000 × g for 1 h at 4 ◦ C and the pellet was re-suspended in 46% sucrose buffer. The second discontinuous gradient consisting of 3 ml of 72% sucrose buffer at the bottom, 6 ml of the sample in the middle, and 3 ml of 27% sucrose buffer at the top was centrifuged in a SW41 rotor at 50,000 × g for 20 h at 4 ◦ C. The 27%/46% interface was collected and separated by 12% SDS-polyacrylamide gel electrophoresis. The presence of IP3 R1 was confirmed by Western blotting using conventional procedures (Fig. 1A). Briefly, the proteins were transferred to a PVDF membrane (Millipore), blocked, and incubated with anti-IP3 R monoclonal antibody (18A10) followed by secondary, alkaline phosphatase-conjugated antibody. The proteins were visualized by using the BCIP/NBT system (Promega). AFM experiments were performed using a SPI3800 or SPI4000/SPA300-HV (SII NanoTechnology Inc.) with a 20 m scanner and a liquid chamber. The sample was scanned in imaging buffer (10 mM Tris–HCl, pH 7.4, 100 mM KCl, 0.5 mM EGTA) using the dynamic force mode (DFM). Oxide sharpened Si3 N4 probes were used that had a resonance frequency of 34 kHz and a nominal spring constant of 0.08 N/m (Olympus Corporation). The resonant frequency of the tip was tuned to ∼12 kHz with an amplitude of around 0.5 V. Images were obtained at a scan rate of 0.5–1.0 Hz. Membranes prepared from Sf9-IP3 R1 cells were diluted in adsorption buffer (10 mM Tris, pH 7.4, 150 mM KCl, 0.5 mM EGTA), dropped onto freshly cleaved mica, and incubated for 30 min. The membrane vesicles formed a flat membrane surface with protrusions on the mem-
Fig. 1. Expression of recombinant IP3 R1 in Sf9 cells. (A) IP3 R1 protein was detected by Western blotting using 18A10 antibody. After two rounds of ultracentrifugation, the samples were separated by SDS-polyacrylamide gel electrophoresis (12% gel) and subjected to Western blotting. The arrow indicates IP3 R1 detected in Sf9-IP3 R1 cells (lane +) but not in control Sf9 cells (lane −). (B and C) AFM images of membranes from native Sf9 cells (B) and Sf9-IP3 R1 cells (C). The sample was adsorbed on mica and imaged in solution. Except for the flipped edges of the membrane (white arrows), few protrusions could be found on the membrane of native Sf9 cells in contrast to that of Sf9-IP3 R1 cells (red arrows).
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Fig. 2. AFM topographs of the Sf9-IP3 R overexpressed membrane. (A) Scanning image of a 1500 nm × 1500 nm area. A section analysis along the line in the image is shown at the bottom. The center of the membrane surrounded by the dotted square was scanned again and the image shown in (B) was obtained. (B) A higher resolution image of (A) showing a 600 nm × 600 nm area. A section analysis along the line in the image is shown at the bottom. The large protrusions had an average diameter of 31.5 ± 4.9 nm (n = 89) and an average height of 4.3 ± 0.8 nm (n = 89). (C) 3D reconstructed image of (B).
branes of IP3 R1 overexpressed Sf9 cells (Fig. 1). A quantitative analysis indicated that the membranes of Sf9-IP3 R1 cells had six times as many protrusions as native Sf9 membranes. A few protrusions could be found on a membrane prepared from a negative control native Sf9. We assumed that these protrusions were endogenous IP3 R [19] and/or other proteins. We found clustered (Fig. 2) or unequally dispersed protrusions (Fig. 3A) in the membranes. The two-dimensional crystalline array of IP3 R that was reported previously [13] could not be found in the Sf9-IP3 R1 membrane. At best, we observed densely gathered protrusions in the center of the membrane as shown in Fig. 2. Large structures near the edge of the membrane are likely to be the flipped edges of the membrane. The height of the membrane from the mica surface was 4.0 ± 0.6 nm (n = 22), which corresponds to the thickness of a lipid bilayer (Fig. 2A, section analysis). The center of the IP3 R1 overexpressed membrane was scanned at high resolution, and the image and its 3D reconstructed view are shown in Fig. 2B and C, respectively. The average height of the large protrusions was 4.3 ± 0.8 nm from the membrane surface and they were 31.5 ± 4.9 nm in diameter (n = 89). We found that these large protrusions did not move laterally in the membrane during AFM imaging, suggesting that the extra-membranous region is immobilized by being directly and/or indirectly attached to the mica surface. To characterize the protrusions on the Sf9-IP3 R1 membrane, we carried out immuno-labeling with a gold-conjugated antibody [24]. A Sf9-IP3 R1 membrane adsorbed on mica was incubated with a rat monoclonal antibody (4C11) against the cytoplasmic region close to IP3 -binding site. After 2 h incubation at room temperature, the membrane was washed three times with adsorption buffer. Next, 10 nm gold particles conjugated with anti-rat secondary antibody (Sigma) diluted ten-fold in adsorption buffer were added and incubated for 2 h at room temperature. The membrane was washed three times with imaging buffer, and then AFM images were obtained (Fig. 3B). We selected protrusions with diameters of between 25 and 40 nm,
which is the size estimated from previous EM studies, and membranes that had at least two protrusions per m2 . Several small-unidentified protrusions were not included in this analysis (white arrows with an asterisk in Fig. 3A–C). The average height of the protrusions was increased from 4.3 ± 0.8 nm (n = 89) to 7.7 ± 1.6 nm (n = 49) by the treatment (Fig. 3A and B, section analyses). The fact that the protrusions increase in height with antibody labeling strongly supports the idea that the protruding structure is cytosolic domain of IP3 R1. When we used a rat antibody against non-related protein (anti-CD3; R&D systems), the height remained the same (4.3 ± 0.7 nm, n = 46, Fig. 3C). The histograms in Fig. 3D and E summarize the distribution of the protrusion height. It was clear that the height increase was specific to 4C11 antibody treatment (Fig. 3E). To confirm the immuno-reactivity of the Sf9-IP3 R1 membrane, we undertook EM observation. Sf9-IP3 R1 membranes were adsorbed on Formvar (polyvinyl formal) coated nickel grids by incubating them for 5 min at room temperature. They were successively incubated with 4C11 antibody and 10 nm gold-conjugated secondary antibody for 1h each. After extensive washing, immuno-labeled membranes were fixed with 2% glutaraldehyde. They were then washed with distilled water and negatively stained with 2% phosphotungstic acid solution (pH 7.0). The dried grids were examined on a JEM1200EX transmission electron microscope (TEM) operated at an acceleration voltage of 80 kV. In a representative view, dispersed gold particles were found on the 4C11 treated membrane at a rate of approximately two dots per 100 nm × 100 nm (Fig. 3F). Without 4C11 treatment, no gold particles were observed (data not shown). These results clearly confirmed that the Sf9-IP3 R1 membranes contained a high density of immuno-reactive IP3 R1 particles. To obtain a better comparison of our AFM images with those of previous EM studies, we used immuno-purified IP3 R1 protein. The IP3 R1 protein was highly purified from mouse brain in the presence of 1% CHAPS detergent [5,6], which is capable of purifying functional IP3 R1 tetramers from the membrane [17].
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Fig. 3. Protrusions on the membrane are characterized by immuno-labeling. AFM images of a membrane prepared from Sf9-IP3 R1 on mica without treatment (A), after treatment with an IP3 R1 specific antibody, 4C11 (B) or after treatment with an unrelated antibody (C) followed by a secondary antibody conjugated with gold particles. Section analyses along the lines show that the height of the protrusions was increased by 4C11 treatment. The blue, green, and red arrows indicate examples of protrusions whose heights were measured for the analyses presented in (D) and (E). The color corresponds to those presented in the histograms in (D) and (E). White arrows with an asterisk indicate examples of small unidentified protrusions that were not included in the analyses. The large structures near the edge of the membrane are likely to be flipped edges of the membrane, and these examples are indicated by black arrows with an asterisk. (D and E) Histograms show the protrusion height distributions. The vertical axis indicates the number of protrusions and the horizontal axis indicates the height of the protrusions rounded to nm. Results are shown for membranes that were untreated (D), treated with unrelated antibody (E: green bars) or 4C11 (E: red bars). (F) TEM image of a membrane prepared from Sf9-IP3 R1 on a nickel-grid treated with 4C11 followed by a secondary gold-conjugated antibody. Black dots that constitute 10 nm gold particles dispersed on the membrane can be clearly seen.
Si probes with a resonance frequency of 27 kHz and a nominal spring constant of 1.9 N/m (SII NanoTechnology Inc.) were used at a resonant frequency of ∼9 kHz. Mica surface was treated with 10mM MgCl2 to immobilize the IP3 R1 in a relatively preferred direction by utilizing the electrostatic interaction with IP3 R1, and IP3 R1 in a buffer (50 mM Tris, pH 7.4, 150 mM KCl, 1% CHAPS, 0.5 mM EGTA) was applied. Individual IP3 R1 particles on mica were successfully observed in imaging buffer without detergent, and representative images are shown in Fig. 4. We found small particles with widths of about 10–15 nm that might be debris, and large particles whose apparent size and the height (Fig. 4) closely resemble those of the large protrusion on the Sf9IP3 R1 membrane. The rectangular structure shown in Fig. 4A with a width of about 25 nm closely resembles the model proposed in previous EM studies [5,6,22,25]. This evidence strongly supports our notion that the large protrusion on the membrane could be IP3 R1 itself.
The height of the detergent-solubilized and membraneembedded IP3 R1s measured by AFM (4–8.3 nm) is clearly less than the value of ∼17 nm estimated from the side view image of negatively stained IP3 R1 [6] or other reported 3D reconstructions [3,5,6,22,25] obtained by EM. If the AFM estimation is accurate, it provides new information about the IP3 R1 structure in aqueous solution. However, we must consider the technical issues posed by AFM, namely the possibility that the force applied with the AFM probe and/or by the adsorption force of the mica surface could have resulted in IP3 R1 compression under these conditions. In this context, the height of the purified IP3 R (4–6 nm) was somewhat less than the calculated value of ∼8.3 nm, which is the sum of the 4.3-nm protruding extra-membrane domain and the 4-nm thick membrane. We can assume that the membrane-embedded IP3 R1 could be relatively resistant to the compressive force perpendicular to the membrane surface. The IP3 R1 compression might result in apparently
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of structural changes within IP3 R1 and other membrane proteins in a natural environment. Acknowledgements We thank M. Iwai (University of Tokyo) for the Sf9 expression system, and T. Michikawa (University of Tokyo) and Dr. K. Furukawa and other members of the Materials Science Laboratory at NTT Basic Research Laboratories for helpful discussions. References
Fig. 4. AFM topographs of IP3 R1 immuno-purified from mouse cerebella. AFM topographs of single IP3 R1 adsorbed on mica (A–C). Plausible 3D models and section analyses along the dotted line in the main images are shown in the lower left insets and at the bottom, respectively. The upper right inset in (A) is a 3D reconstructed image.
larger sizes than in the reported EM models because of particle flattening. However, we cannot exclude the possibility that the particle diameter was over-estimated due to the triangular geometry of the probe [29]. This is the first time that AFM has been used to reveal the surface topography of an Sf9-IP3 R1 membrane in aqueous solution. However, we were unable to obtain convincing results with sufficient resolution to observe the detailed structure revealed by previous EM studies [3,5,6,22,25]. Recent biochemical studies have revealed that IP3 R1 is unique in that it has more than 25 kinds of binding partner. One reason for the apparently low resolution might be the associations of several binding partners with IP3 R1, which could modify the overall shape and size of protrusions containing IP3 R1. Another possibility is the vibration of the protein as a result of the membrane’s fluidity because AFM cannot scan the surface topography precisely when objects vibrate faster than the scanning speed. It took more than two minutes to obtain the overall AFM topography shown in Fig. 2. In addition, the resolution mainly depends on the forces interacting between the cantilever and the sample. Therefore, the inherent flexibility due to windmill/square transition [5,6] and the softness attributed to the multi-porous structure of IP3 R1 [25] might have made it difficult to observe their detailed structures in this study. Further technical improvements that address the issue of spatiotemporal resolution will enable AFM to become a powerful technique for directly visualizing the 3D structure of IP3 R1 itself, as well as the dynamic association/dissociation of binding partners during supramolecular formation in real time. In conclusion, we succeeded in imaging the surface topography of an IP3 R1 overexpressed membrane in solution by using AFM in the dynamic force mode. We observed many large protrusions on the membrane that we consider to be IP3 R1 by immuno-gold labeling and TEM. Using immuno-purified IP3 R1, we obtained approximately the same dimensions as those of an IP3 R1-like protrusion in an Sf9-IP3 R1 membrane. To the best of our knowledge, this is the first successful imaging of individual IP3 R1 particles in physiological solutions. We believe that our study is an important step towards the dynamic visualization
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Visualization of inositol 1,4,5-trisphosphate receptor by atomic force microscopy Wakako Suhara a , Mime Kobayashi b , Hiroshi Sagara c , Kozo Hamada d , Touichiro Goto b , Ichiro Fujimoto a , Keiichi Torimitsu a,b,e,∗ , Katsuhiko Mikoshiba a,d,f,g a
The Division of Neural Signal Information NTT-IMSUT, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan b Materials Science Laboratory, NTT Basic Research Laboratories, NTT Corporation, Atsugi, Kanagawa 243-0198, Japan c Fine Morphology Laboratory, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan d Calcium Oscillation Project, International Cooperative Research Project (ICORP), Japan Science and Technology Agency (JST), Minato-ku, Tokyo 108-0071, Japan e Solution Oriented Research for Science and Technology (SORST), Japan Science and Technology Agency (JST), Toyonaka, Osaka 560-0082, Japan f The Division of Molecular Neurobiology, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan g Laboratory for Developmental Neurobiology, Brain Research Institute, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan Received 23 March 2005; received in revised form 12 August 2005; accepted 21 August 2005
Abstract Inositol 1,4,5-trisphosphate (IP3 ) receptor (IP3 R) acts as a ligand-gated channel that mediates neuronal signals by releasing Ca2+ from the endoplasmic reticulum. The three-dimensional (3D) structure of tetrameric IP3 R has been demonstrated by using electron microscopy (EM) with static specimens; however, the dynamic aspects of the IP3 R structure have never been visualized in a native environment. Here we attempt to measure the surface topography of IP3 R in solution using atomic force microscopy (AFM). AFM revealed large protrusions extending ∼4.3 nm above a flat membrane prepared from Spodoptera frugiperda (Sf9) cells overexpressing mouse type 1 IP3 R (Sf9-IP3 R1). The average diameter of the large protrusions was ∼32 nm. A specific antibody against a cytosolic epitope close to the IP3 -binding site enabled us to gold-label the Sf9-IP3 R1 membrane as confirmed by EM. AFM images of the gold-labeled membrane revealed 7.7-nm high protrusions with a diameter of ∼30 nm, which should be IP3 R1-antibody complexes. Authentic IP3 R1 immuno-purified from mouse cerebella had approximately the same dimensions as those of the IP3 R-like protrusions on the membrane. Altogether, these results suggest that the large protrusions on the Sf9-IP3 R1 membrane correspond to the cytosolic domain of IP3 R1. Our study provides the first 3D representation of individual IP3 R1 particles in an aqueous solution. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Atomic force microscopy; Inositol 1,4,5-trisphosphate receptor; Calcium signaling; Molecular imaging; Protein structure
Inositol 1,4,5-trisphosphate receptor (IP3 R) is an intricately regulated channel that releases Ca2+ from the endoplasmic reticulum (ER) in response to the binding of 1,4,5-trisphosphate (IP3 ), which is a second messenger generated by various stimuli, such as neurotransmitters, neuromodulators, and hormones [4]. Three isoforms, namely IP3 R1-3, have different binding properties for IP3 and various regulatory molecules, and they play distinct roles in the temporal organization of Ca2+ signaling in living cells [8]. IP3 R1, a predominant type in the cerebellar ER and spine apparatus, has been shown to be essential for higher
∗
Corresponding author. Tel.: +81 46 240 3360; fax: +81 46 270 2362. E-mail address: [email protected] (K. Torimitsu).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.08.066
functions including long-term depression and neural development [10,14,18]. Recent protein-protein interaction research has revealed multiple binding-partners for IP3 R1 (reviewed in [21]), e.g. scaffolding proteins such as Homer protein, which tethers IP3 R1 to glutamate receptors [27], ankyrin, 4.1N [30], Ca2+ binding proteins such as calmodulin, CaBP, plasma-membrane channels such as the TRP channel, and ER lumenal proteins such as ERp44, which regulate IP3 R1 activity depending on the pH and redox state within ER [9]. These divergent regulations imply the dynamic behavior of IP3 R1-containing complex and/or of IP3 R1 itself, which would provide a mechanistic basis for IP3 R1-related functions in the central nervous system. IP3 R is structurally divided into three regions: a large cytoplasmic region with an IP3 -binding site close to the N-terminus,
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a transmembrane region near the C-terminus, and a short Cterminal tail [4]. The structures of the IP3 -binding site and the N-terminal suppresser domain have been obtained with a high resolution by using X-ray crystallography [1,2]. However, information about the overall structure of tetrameric IP3 R has only been acquired by electron microscopy (EM) because of its inherently large size (>1.2 MDa). In previous EM studies, IP3 R1 protein purified from rodent or bovine cerebella was shown to have a four-fold symmetry with a top width of 15–25 nm [3,5,6,12,22,25]. Although the appearances of reported IP3 R1 models are inconsistent, they can be classified into two types: square-shaped and windmill-like. Since the windmill type, which has four wings about 30 nm in diameter, is relatively abundant in the presence of Ca2+ , it has been suggested that dynamic changes occur within the IP3 R1 structure [5,6]. However, these EM data were obtained from static IP3 R1 in a vacuum; therefore there is still a need for a real-time visualization of the conformational states to be demonstrated in aqueous solution. Atomic force microscopy (AFM) is an established technique for evaluating the nanostructures of metal or silicon surfaces. Recent improvements in AFM have made it feasible to apply the method to soft biological samples such as proteins and DNA under a thin layer of solution [7]. A biomolecule in solution is generally expected to represent a native structure as a functional entity; AFM therefore offers the significant advantage of allowing the dynamic visualization of solution structures, unlike EM where the samples must be static for observation. AFM in the contact mode simply scans the sample surface with a probe. This approach has been used to image the surface topography of highly packed two-dimensional crystals of membrane proteins such as bacteriorhodopsin [16], aquaporin [23], and connexin 26 gap junctions [15] at subnanometer resolution. However, this technique is only useful for physically stable crystals of membrane proteins. AFM in the “tapping” or “dynamic force” mode, where a probe is excited with resonance oscillation during surface scanning to reduce the lateral force applied to the sample, has made it possible to visualize the conformational states of non-crystalline proteins such as insulin receptor [26], GroEL [28], and 20S proteasome [20] in a native environment. The crystallization of membrane protein is generally so difficult that AFM in the dynamic force mode should provide a practical way to understand the three-dimensional (3D) structure of a membrane protein in aqueous solution. In an attempt to visualize membrane-embedded IP3 R1 in solution, we prepared recombinant mouse IP3 R1 protein, which was overexpressed in insect Spodoptera frugiperda (Sf9) cells (Sf9-IP3 R1) using a baculovirus system. After infecting the cells, we confirmed the proper expression and the IP3 binding and Ca2+ release activities of the protein [11]. The cells overexpressing the protein were homogenized in lysis buffer (250 mM sucrose, 5 mM HEPES-pH 7.2, 1 mM EDTA, 0.5 mM EGTA, 1 mM 2-mercaptoethanol, protease inhibitor cocktail tablet (Roche)) and centrifuged at 2500 × g for 5 min. The supernatant was ultracentrifuged in a TLA100.3 rotor (Beckman) at 100,000 × g for 30 min to obtain a crude microsome fraction. The pellet was suspended with 51% sucrose in gradient
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buffer (10 mM Tris–HCl, pH 7.4). To form a discontinuous gradient, 3 ml each of 38, 31, and 23% sucrose buffer were successively layered on top of the sample and subjected to centrifugation in a SW41 rotor (Beckman) at 100,000 × g for 3 h at 4 ◦ C. We collected the 38%/51% interface fraction based on the result of a Western blot with anti-IP3 R1 antibody (18A10). The fraction was diluted in gradient buffer and centrifuged in a TLA100.3 rotor at 100,000 × g for 1 h at 4 ◦ C and the pellet was re-suspended in 46% sucrose buffer. The second discontinuous gradient consisting of 3 ml of 72% sucrose buffer at the bottom, 6 ml of the sample in the middle, and 3 ml of 27% sucrose buffer at the top was centrifuged in a SW41 rotor at 50,000 × g for 20 h at 4 ◦ C. The 27%/46% interface was collected and separated by 12% SDS-polyacrylamide gel electrophoresis. The presence of IP3 R1 was confirmed by Western blotting using conventional procedures (Fig. 1A). Briefly, the proteins were transferred to a PVDF membrane (Millipore), blocked, and incubated with anti-IP3 R monoclonal antibody (18A10) followed by secondary, alkaline phosphatase-conjugated antibody. The proteins were visualized by using the BCIP/NBT system (Promega). AFM experiments were performed using a SPI3800 or SPI4000/SPA300-HV (SII NanoTechnology Inc.) with a 20 m scanner and a liquid chamber. The sample was scanned in imaging buffer (10 mM Tris–HCl, pH 7.4, 100 mM KCl, 0.5 mM EGTA) using the dynamic force mode (DFM). Oxide sharpened Si3 N4 probes were used that had a resonance frequency of 34 kHz and a nominal spring constant of 0.08 N/m (Olympus Corporation). The resonant frequency of the tip was tuned to ∼12 kHz with an amplitude of around 0.5 V. Images were obtained at a scan rate of 0.5–1.0 Hz. Membranes prepared from Sf9-IP3 R1 cells were diluted in adsorption buffer (10 mM Tris, pH 7.4, 150 mM KCl, 0.5 mM EGTA), dropped onto freshly cleaved mica, and incubated for 30 min. The membrane vesicles formed a flat membrane surface with protrusions on the mem-
Fig. 1. Expression of recombinant IP3 R1 in Sf9 cells. (A) IP3 R1 protein was detected by Western blotting using 18A10 antibody. After two rounds of ultracentrifugation, the samples were separated by SDS-polyacrylamide gel electrophoresis (12% gel) and subjected to Western blotting. The arrow indicates IP3 R1 detected in Sf9-IP3 R1 cells (lane +) but not in control Sf9 cells (lane −). (B and C) AFM images of membranes from native Sf9 cells (B) and Sf9-IP3 R1 cells (C). The sample was adsorbed on mica and imaged in solution. Except for the flipped edges of the membrane (white arrows), few protrusions could be found on the membrane of native Sf9 cells in contrast to that of Sf9-IP3 R1 cells (red arrows).
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Fig. 2. AFM topographs of the Sf9-IP3 R overexpressed membrane. (A) Scanning image of a 1500 nm × 1500 nm area. A section analysis along the line in the image is shown at the bottom. The center of the membrane surrounded by the dotted square was scanned again and the image shown in (B) was obtained. (B) A higher resolution image of (A) showing a 600 nm × 600 nm area. A section analysis along the line in the image is shown at the bottom. The large protrusions had an average diameter of 31.5 ± 4.9 nm (n = 89) and an average height of 4.3 ± 0.8 nm (n = 89). (C) 3D reconstructed image of (B).
branes of IP3 R1 overexpressed Sf9 cells (Fig. 1). A quantitative analysis indicated that the membranes of Sf9-IP3 R1 cells had six times as many protrusions as native Sf9 membranes. A few protrusions could be found on a membrane prepared from a negative control native Sf9. We assumed that these protrusions were endogenous IP3 R [19] and/or other proteins. We found clustered (Fig. 2) or unequally dispersed protrusions (Fig. 3A) in the membranes. The two-dimensional crystalline array of IP3 R that was reported previously [13] could not be found in the Sf9-IP3 R1 membrane. At best, we observed densely gathered protrusions in the center of the membrane as shown in Fig. 2. Large structures near the edge of the membrane are likely to be the flipped edges of the membrane. The height of the membrane from the mica surface was 4.0 ± 0.6 nm (n = 22), which corresponds to the thickness of a lipid bilayer (Fig. 2A, section analysis). The center of the IP3 R1 overexpressed membrane was scanned at high resolution, and the image and its 3D reconstructed view are shown in Fig. 2B and C, respectively. The average height of the large protrusions was 4.3 ± 0.8 nm from the membrane surface and they were 31.5 ± 4.9 nm in diameter (n = 89). We found that these large protrusions did not move laterally in the membrane during AFM imaging, suggesting that the extra-membranous region is immobilized by being directly and/or indirectly attached to the mica surface. To characterize the protrusions on the Sf9-IP3 R1 membrane, we carried out immuno-labeling with a gold-conjugated antibody [24]. A Sf9-IP3 R1 membrane adsorbed on mica was incubated with a rat monoclonal antibody (4C11) against the cytoplasmic region close to IP3 -binding site. After 2 h incubation at room temperature, the membrane was washed three times with adsorption buffer. Next, 10 nm gold particles conjugated with anti-rat secondary antibody (Sigma) diluted ten-fold in adsorption buffer were added and incubated for 2 h at room temperature. The membrane was washed three times with imaging buffer, and then AFM images were obtained (Fig. 3B). We selected protrusions with diameters of between 25 and 40 nm,
which is the size estimated from previous EM studies, and membranes that had at least two protrusions per m2 . Several small-unidentified protrusions were not included in this analysis (white arrows with an asterisk in Fig. 3A–C). The average height of the protrusions was increased from 4.3 ± 0.8 nm (n = 89) to 7.7 ± 1.6 nm (n = 49) by the treatment (Fig. 3A and B, section analyses). The fact that the protrusions increase in height with antibody labeling strongly supports the idea that the protruding structure is cytosolic domain of IP3 R1. When we used a rat antibody against non-related protein (anti-CD3; R&D systems), the height remained the same (4.3 ± 0.7 nm, n = 46, Fig. 3C). The histograms in Fig. 3D and E summarize the distribution of the protrusion height. It was clear that the height increase was specific to 4C11 antibody treatment (Fig. 3E). To confirm the immuno-reactivity of the Sf9-IP3 R1 membrane, we undertook EM observation. Sf9-IP3 R1 membranes were adsorbed on Formvar (polyvinyl formal) coated nickel grids by incubating them for 5 min at room temperature. They were successively incubated with 4C11 antibody and 10 nm gold-conjugated secondary antibody for 1h each. After extensive washing, immuno-labeled membranes were fixed with 2% glutaraldehyde. They were then washed with distilled water and negatively stained with 2% phosphotungstic acid solution (pH 7.0). The dried grids were examined on a JEM1200EX transmission electron microscope (TEM) operated at an acceleration voltage of 80 kV. In a representative view, dispersed gold particles were found on the 4C11 treated membrane at a rate of approximately two dots per 100 nm × 100 nm (Fig. 3F). Without 4C11 treatment, no gold particles were observed (data not shown). These results clearly confirmed that the Sf9-IP3 R1 membranes contained a high density of immuno-reactive IP3 R1 particles. To obtain a better comparison of our AFM images with those of previous EM studies, we used immuno-purified IP3 R1 protein. The IP3 R1 protein was highly purified from mouse brain in the presence of 1% CHAPS detergent [5,6], which is capable of purifying functional IP3 R1 tetramers from the membrane [17].
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Fig. 3. Protrusions on the membrane are characterized by immuno-labeling. AFM images of a membrane prepared from Sf9-IP3 R1 on mica without treatment (A), after treatment with an IP3 R1 specific antibody, 4C11 (B) or after treatment with an unrelated antibody (C) followed by a secondary antibody conjugated with gold particles. Section analyses along the lines show that the height of the protrusions was increased by 4C11 treatment. The blue, green, and red arrows indicate examples of protrusions whose heights were measured for the analyses presented in (D) and (E). The color corresponds to those presented in the histograms in (D) and (E). White arrows with an asterisk indicate examples of small unidentified protrusions that were not included in the analyses. The large structures near the edge of the membrane are likely to be flipped edges of the membrane, and these examples are indicated by black arrows with an asterisk. (D and E) Histograms show the protrusion height distributions. The vertical axis indicates the number of protrusions and the horizontal axis indicates the height of the protrusions rounded to nm. Results are shown for membranes that were untreated (D), treated with unrelated antibody (E: green bars) or 4C11 (E: red bars). (F) TEM image of a membrane prepared from Sf9-IP3 R1 on a nickel-grid treated with 4C11 followed by a secondary gold-conjugated antibody. Black dots that constitute 10 nm gold particles dispersed on the membrane can be clearly seen.
Si probes with a resonance frequency of 27 kHz and a nominal spring constant of 1.9 N/m (SII NanoTechnology Inc.) were used at a resonant frequency of ∼9 kHz. Mica surface was treated with 10mM MgCl2 to immobilize the IP3 R1 in a relatively preferred direction by utilizing the electrostatic interaction with IP3 R1, and IP3 R1 in a buffer (50 mM Tris, pH 7.4, 150 mM KCl, 1% CHAPS, 0.5 mM EGTA) was applied. Individual IP3 R1 particles on mica were successfully observed in imaging buffer without detergent, and representative images are shown in Fig. 4. We found small particles with widths of about 10–15 nm that might be debris, and large particles whose apparent size and the height (Fig. 4) closely resemble those of the large protrusion on the Sf9IP3 R1 membrane. The rectangular structure shown in Fig. 4A with a width of about 25 nm closely resembles the model proposed in previous EM studies [5,6,22,25]. This evidence strongly supports our notion that the large protrusion on the membrane could be IP3 R1 itself.
The height of the detergent-solubilized and membraneembedded IP3 R1s measured by AFM (4–8.3 nm) is clearly less than the value of ∼17 nm estimated from the side view image of negatively stained IP3 R1 [6] or other reported 3D reconstructions [3,5,6,22,25] obtained by EM. If the AFM estimation is accurate, it provides new information about the IP3 R1 structure in aqueous solution. However, we must consider the technical issues posed by AFM, namely the possibility that the force applied with the AFM probe and/or by the adsorption force of the mica surface could have resulted in IP3 R1 compression under these conditions. In this context, the height of the purified IP3 R (4–6 nm) was somewhat less than the calculated value of ∼8.3 nm, which is the sum of the 4.3-nm protruding extra-membrane domain and the 4-nm thick membrane. We can assume that the membrane-embedded IP3 R1 could be relatively resistant to the compressive force perpendicular to the membrane surface. The IP3 R1 compression might result in apparently
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of structural changes within IP3 R1 and other membrane proteins in a natural environment. Acknowledgements We thank M. Iwai (University of Tokyo) for the Sf9 expression system, and T. Michikawa (University of Tokyo) and Dr. K. Furukawa and other members of the Materials Science Laboratory at NTT Basic Research Laboratories for helpful discussions. References
Fig. 4. AFM topographs of IP3 R1 immuno-purified from mouse cerebella. AFM topographs of single IP3 R1 adsorbed on mica (A–C). Plausible 3D models and section analyses along the dotted line in the main images are shown in the lower left insets and at the bottom, respectively. The upper right inset in (A) is a 3D reconstructed image.
larger sizes than in the reported EM models because of particle flattening. However, we cannot exclude the possibility that the particle diameter was over-estimated due to the triangular geometry of the probe [29]. This is the first time that AFM has been used to reveal the surface topography of an Sf9-IP3 R1 membrane in aqueous solution. However, we were unable to obtain convincing results with sufficient resolution to observe the detailed structure revealed by previous EM studies [3,5,6,22,25]. Recent biochemical studies have revealed that IP3 R1 is unique in that it has more than 25 kinds of binding partner. One reason for the apparently low resolution might be the associations of several binding partners with IP3 R1, which could modify the overall shape and size of protrusions containing IP3 R1. Another possibility is the vibration of the protein as a result of the membrane’s fluidity because AFM cannot scan the surface topography precisely when objects vibrate faster than the scanning speed. It took more than two minutes to obtain the overall AFM topography shown in Fig. 2. In addition, the resolution mainly depends on the forces interacting between the cantilever and the sample. Therefore, the inherent flexibility due to windmill/square transition [5,6] and the softness attributed to the multi-porous structure of IP3 R1 [25] might have made it difficult to observe their detailed structures in this study. Further technical improvements that address the issue of spatiotemporal resolution will enable AFM to become a powerful technique for directly visualizing the 3D structure of IP3 R1 itself, as well as the dynamic association/dissociation of binding partners during supramolecular formation in real time. In conclusion, we succeeded in imaging the surface topography of an IP3 R1 overexpressed membrane in solution by using AFM in the dynamic force mode. We observed many large protrusions on the membrane that we consider to be IP3 R1 by immuno-gold labeling and TEM. Using immuno-purified IP3 R1, we obtained approximately the same dimensions as those of an IP3 R1-like protrusion in an Sf9-IP3 R1 membrane. To the best of our knowledge, this is the first successful imaging of individual IP3 R1 particles in physiological solutions. We believe that our study is an important step towards the dynamic visualization
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