Micron 56 (2014) 37–43
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X-ray solution structure of the native neuronal porosome-synaptic vesicle complex: Implication in neurotransmitter release Ladislau C. Kovari a,1 , Joseph S. Brunzelle b,1 , Kenneth T. Lewis c,d , Won Jin Cho c , Jin-Sook Lee c , Douglas J. Taatjes e , Bhanu P. Jena c,∗ a
Wayne State University School of Medicine, Department of Biochemistry and Molecular Biology, Detroit, MI, USA Life Sciences Collaborative Access Team, Synchrotron Research Center, Northwestern University, Argonne, IL 60439, USA c Department of Physiology, Wayne State University School of Medicine, Detroit, MI, USA d Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI, USA e Department of Pathology, Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, VT 05405, USA b
a r t i c l e
i n f o
Article history: Received 19 September 2013 Received in revised form 29 September 2013 Accepted 2 October 2013 Keywords: Neuronal porosome complex X-ray solution scattering Photon correlation spectroscopy Electron microscopy
a b s t r a c t Nanoportals at the cell plasma membrane called porosomes, mediate secretion from cells. In neurons porosomes are 15 nm cup-shaped lipoprotein structure composed of nearly 40 proteins. The size and complexity of the porosome has precluded determination of its atomic structure. Here we report at nanometer resolution the native 3D structure of the neuronal porosome-synaptic vesicle complex within isolated nerve terminals using small-angle X-ray solution scattering. In addition to furthering our understanding of the porosome structure, results from the study suggests the molecular mechanism involved in neurotransmitter release at the nerve terminal. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction The chemistry of life processes is governed at the molecular level. Hence a major challenge is the determination of atomic structure of cellular organelles and macromolecules required for understand their function in cells. Determination of the atomic structure of cellular organelles and macromolecules is required for understand their cellular function. It is especially challenging, when such organelles involve membrane proteins. Cup-shaped lipoprotein structures called porosomes (Schneider et al., 1997; Cho et al., 2002a, 2004, 2008; Jena et al., 2003; Jena, 2009; Jeremic et al., 2003; Lee et al., 2012; Anderson et al., 2004; Anderson and Scanes, 2012) are the universal secretory nanoportals at the cell plasma membrane. Membrane-bound secretory vesicles transiently dock and fuse at the base of the porosome to deliver intravesicular contents outside the cell. Fusion of membrane-bound secretory vesicles at the porosome base is mediated by calcium and a specialized set of three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors called SNAREs (Jeremic et al., 2004a; Malhotra et al., 1988; Trimble et al., 1988; Oyler et al., 1989). In neurons,
∗ Corresponding author. Tel.: +1 313 577 1532. E-mail address: [email protected] (B.P. Jena). 1 These authors contributed equally to the study. 0968-4328/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.micron.2013.10.002
target membrane proteins SNAP-25 and syntaxin called t-SNAREs present at the base of neuronal porosomes, and a synaptic vesicleassociated membrane protein (VAMP) or v-SNARE, are part of the conserved protein complex involved in membrane fusion and neurotransmission. Neuronal porosomes measure approximately 15 nm in diameter, and are present at the presynaptic membrane of nerve terminals called synaptosomes. Nearly 40 proteins (Cho et al., 2004; Lee et al., 2012) including SNARE and lipids that include cholesterol (Cho et al., 2007), compose the neuronal porosome complex. The overall morphology (Figs. 1 and 2) (Cho et al., 2004, 2008) and dynamics (Cho et al., 2010) of the neuronal porosome complex which has previously been studied and reported using both atomic force microscopy (AFM) and electron microscopy (EM), is presented (Figs. 1 and 2) for clarity and to introduce the reader to the subject. Additionally, the neuronal porosome has been functionally reconstituted into artificial lipid membrane (Cho et al., 2004), and the 3D contour map of its protein backbone at nanometer scale established (Cho et al., 2008). A set of eight protein units lining the neuronal porosome cup is present (Cho et al., 2004, 2008), each connected via spoke-like elements to a central plug, suggested to be involved in the rapid opening and closing of the structure to the outside during neurotransmission. AFM micrographs of the presynaptic membrane of isolated synaptosome preparations demonstrates the presence of the neuronal porosome plug at various conformations (Cho et al., 2010). The central plug
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Fig. 1. Neuronal porosomes at the presynaptic membrane of nerve terminals determined in previous (Cho et al., 2004; Jena et al., 2003; Lee et al., 2012; Jeremic et al., 2004a) studies. (a) Atomic force micrograph (AFM) amplitude image Bar = 100 nm (black arrowhead points to presynaptic membrane), and (b) electron micrograph (EM) of isolated synaptosome Bar = 100 nm. (c) High resolution AFM micrograph of isolated neuronal porosome from synaptosomes followed by reconstitution into a lipid membrane. Note the near 10 nm neuronal porosome complex with a central plug (red arrowhead) and eight peripheral densities. (d) EM micrograph of the cup-shaped neuronal porosome complex with its central plug (red arrowhead) and a docked synaptic vesicle at its base. (e) Nagative staining EM of an isolated neuronal porosome complex. Note the nano scale structure and assembly of proteins within the complex, and a schematic drawing (f) of the cup-shaped neuronal porosome (P) associated with a docked synaptic vesicle (SV). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
when fully retracted is in the closed conformation, when partially retracted, it is in its semi-open state, and when completely inserted into the porosome cup, in its open conformation (Cho et al., 2010). The presence of such a mechanism in the neuronal porosome complex makes sense since it would allow the rapid open and close states of the organelle during neurotransmission at the nerve terminal. This is opposed to a slow secretory cell such as the exocrine pancreas (Schneider et al., 1997) or the neuroendocrine growth hormone secreting cell (Cho et al., 2002a) where no such plug is present at the porosome opening; instead the porosome opening dilates during secretion and returns to its resting size following completion of the process (Schneider et al., 1997; Cho et al., 2002a). The size and complexity of the membrane-associated porosome has precluded determination of its atomic structure. For example,
solution NMR has not been possible primarily due to the large molecular size of the porosome complex, which is beyond the operating limits of current NMR’s. Similarly, X-ray crystallography is impractical, due in part to the solubility problems of this membrane-associated structure composed of neatly 40 proteins. Although these limitations have been partially overcome by the use of AFM and EM in furthering our understanding of the fine structure and nano-arrangement of proteins within the native porosome complex (Cho et al., 2008), its 3D structure in association with a docked secretory vesicle in intact synaptosome had not been observed. In recent years, with major advances in instrumentation and computational power, small angle X-ray scattering (SAXS) has become a powerful method to study biological material at nanometer to sub-nanometer resolution in solution (Round
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2.2. Photon correlation spectroscopy (PCS) on isolated synaptosome preparation
Fig. 2. AFM micrograph obtained from an earlier study of the cytosolic compartment at the presynaptic membrane of a synaptosome (Cho et al., 2004). Note SV (blue arrowheads) docked at the base of porosomes (P, red arrowheads). The mushroom-shaped head represents the SV and the stalk represents the P complex. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
et al., 2008; Hura et al., 2009; Forster et al., 2008; Mertens and Svergun, 2010). In SAXS, a focused X-ray beam from a synchrotron is scattered by the sample, and the scattering data is collected and recorded in real-time. The scattering patterns generated from a supramolecular structure in suspension are presented as radially averaged single dimensional curves, from which important parameters such as the overall three-dimensional shape and size of structures are obtained. In the current study, the native structure and interaction between SV and the porosome complex was studied using SAXS. Results from the study confirm earlier findings (Cho et al., 2004, 2007, 2008, 2010; Lee et al., 2012; Okuneva et al., 2012; Drescher et al., 2011; Siksou et al., 2007), demonstrating the neuronal porosome complex to be cup-shaped structures measuring approximately 15 nm in diameter where on average 35 nm in diameter synaptic vesicles transiently dock and fuse to release neurotransmitters. These results further provide an understanding of the possible mechanisms involved in neurotransmitter release at the nerve terminal. 2. Materials and methods
Synaptosome size was determined using photon correlation spectroscopy (PCS). PCS is a well-known technique for the measurement of m to nm size particles and macromolecules. PCS measurements were performed in a Zetasizer Nano ZS, (Malvern Instruments, UK). In a typical experiment, the size distribution of isolated synaptosomes was determined using built-in software provided by Malvern Instruments. Prior to determination of the synaptosome hydrodynamic radius, calibration of the instrument was performed using latex spheres of known size. In PCS, subtle fluctuations in the sample scattering intensity are correlated across microsecond time scales. The correlation function was calculated, from which the diffusion coefficient was determined. Using the Stokes–Einstein equation, hydrodynamic radius can be acquired from the diffusion coefficient (Higashijima et al., 1988). The intensity size distribution, which is obtained as a plot of the relative intensity of light scattered by particles in various size classes, is then calculated from a correlation function using built-in software. The particle scattering intensity is proportional to the molecular weight squared. Volume distribution can be derived from the intensity distribution using Mie theory (Vitale et al., 1993; Weingarten et al., 1990). The transforms of the PCS intensity distribution to volume distributions can be obtained using the provided software by Malvern Instruments.
2.3. Electron microscopy of isolated synaptosomes Electron microscopy was performed on isolated synaptosomes as described in a previously published procedure (Wang et al., 2012). Briefly, isolated synaptosomes were fixed in 2% glutaraldehyde/2% paraformaldehyde (GA-PFA) in ice-cold PBS for 24 h, washed, embedded in 2% SeaPrep agarose, followed by postfixation for 1 h at 4 ◦ C using 1% OsO4 in 0.1 M cacodylate buffer. Finally, the samples were dehydrated in a graded series of ethanol, through propylene oxide, and infiltrated and embedded in Spurr’s resin. Ultrathin sections were cut with a diamond knife, retrieved onto 200 mesh nickel thin-bar grids, and contrasted with alcoholic uranyl acetate and lead citrate. Grids were viewed with a JEOL 1400 transmission electron microscope (JEOL USA, Inc., Peabody, MA) operating at 60 or 80 kV, and digital images were acquired with an AMT-XR611 11 megapixel CCD camera (Advanced Microscopy Techniques, Danvers, MA).
2.1. Synaptosome preparation 2.4. Small-angle X-ray solution scattering Synaptosomes were prepared from rat brains, with minor modifications of established published methods (Cho et al., 2004, 2007, 2010; Lee et al., 2012). For each experiment, Sprague-Dawley rats weighing 100–150 g were euthanized by CO2 inhalation, with all animal procedures preapproved by the Institution Animal Care & Use Committee (IACUC). Whole brains were isolated and placed in ice-cold buffered sucrose solution (5 mM Hepes, pH 7.4, 0.32 M sucrose) supplemented with protease inhibitor cocktail (SigmaAldrich, St. Louis, MO). The brain tissue was homogenized using 12 strokes in a Teflon-glass homogenizer, and the total homogenate was centrifuged for 3 min at 2500 × g. The resulting supernatant fraction was further centrifuged for 15 min at 14,500 × g, to obtain a pellet. The resultant pellet was re-suspended in buffered sucrose solution, and loaded onto a 3–10–23% Percoll gradient. Following centrifugation at 28,000 × g for 6 min, only the top half layer of the enriched synaptosome fraction at the 10–23% Percoll gradient interface was collected, and re-suspended in PBS pH 7.4 containing 0.2% paraformaldehyde (PFA).
Isolated synaptosomes suspended in PBS-PFA at a concentration of 2.5 mg/ml were subjected to small-angle X-ray solution scattering experiments. All small angle X-ray scattering data were collected on the SAXS/WAXS setup located at the 5-ID-D beamline of the DND-CAT synchrotron research center, Advanced Photon Source, Argonne National Laboratory (Argonne, IL). The SAXS data were recorded on a Mar-USA 165 mm CCD and covered the momentum transfer range on two-dimensional CCD detectors. Four exposures of 10 s were acquired for each sample at a sample-todetector distance of 2.6 m and over a range of momentum transfer 0.007 < q < 0.24 A˚ −1 , Q = 4 × sin /. The WAXS data were recorded on a custom Roper CCD and covered a range of momentum transfer 0.20 < q < 1.0 A˚ −1 . Data and a buffer blank were collected for seven different concentrations (2.5–0.04 mg/ml) of each sample to check for concentration-dependent scattering effects, such as aggregation or interparticle interference. No measurable radiation damage was observed under these conditions.
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Fig. 3. Synaptosomes isolated for the present study. (a) Photon correlation spectroscopy (PCS) of isolated synaptosome suspension demonstrating nearly 93% of the suspended particles to measure 955–1219 nm, typical of synaptosome size. (b and c) EM micrographs of isolated synaptosomes demonstrating the presence of docked SV at the presynaptic membrane. Note the sparse presence of free or non-docked SVs within the isolated synaptosomes. (d) Image enhancement of the boxed area in (c) is shown. The figure depicts a cup-shaped porosome complex (P) at the presynaptic membrane (PSM) with a docked SV that has sheared off during sectioning for EM. Note the cap or mushroom shape of the docked SV at the cup-shaped neuronal porosome complex.
3. Results and discussion To determine the 3D structure of the neuronal porosome with docked synaptic vesicles in the native state at nanometer resolution, we isolate synaptosomes containing primarily docked synaptic vesicles at the presynaptic membrane for SAXS studies. In this way, majority of the structures in the synaptosome suspension would represent porosomes-associated synaptic vesicle complexes. Using a minor modification of a published procedure, synaptosomes containing primarily docked synaptic vesicles were isolated and assessed using photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) (Fig. 3). As outlined in the materials and methods section, briefly, the brain tissue was homogenized using 12 strokes in a Teflon-glass homogenizer, and the total homogenate was centrifuged for 3 min at 2500 × g. The resulting supernatant fraction was further centrifuged for 15 min at 14,500 × g, to obtain a pellet. The resultant pellet was re-suspended in buffered sucrose solution, and loaded onto a 3–10–23% Percoll gradient. Following centrifugation at 28,000 × g for 6 min, only the top half layer of the enriched synaptosome fraction at the 10–23% Percoll gradient interface was collected. The top layer of the synaptosome fraction was lighter and enriched in synaptosomes containing just docked synaptic vesicles. Most of the undocked synaptic vesicles were lost from the preparation, making it ideal for the SAXS study. Isolated synaptosomes are known to measure approximately 0.8–1.5 m in diameter (Lee et al., 2010; Chen et al., 2011). PCS measurements of isolated synaptosome preparation from the top layer of the synaptosome fraction demonstrate that >92% of the particles measure between 0.955–1.219 m (Fig. 3A), suggesting them to represent isolated synaptosomes. Further examination of the isolated synaptosome preparations using TEM (Fig. 3B–D), confirmed them to be synaptosomes containing primarily docked synaptic vesicles. Therefore, in the synaptosome suspension used for SAXS, the structure that is frequently encountered are docked SVs at the porosome complex, most likely in repetitive lattice conformation. Hence the synaptosome
preparation was ideal for study of the interactions between SV and the neuronal porosome complex in the native state using SAXS. When the isolated synaptosome suspensions were subjected to small-angle X-ray solution scattering, and the scattering data was collected on the SAXS/WAXS setup, an averaged 3-D image of SV-porosome complex at nm resolution in the native state was obtained (Fig. 4). Scattering data were radially averaged to produce one-dimensional profiles of scattering intensity vs. q (Koch et al., 2003). The innermost portions of the scattering curves were used for fitting to the equation I(q) = I(0) exp(−42 RG 2 q2 /3), where I(0) is the forward scattering intensity at q = 0 and RG is the radius of gyration (Guinier and Fournet, 1955). Values for RG were extracted from the Guinier plot log {I(q)} vs. q2 or the pair distribution function P(r) using the GNOM package (Svergun, 1992). A final P(r) plot was generated with GNOM v4.6, including Q values from 0.0075 to 0.8794. The output was the basis for ab initio shape reconstruction using DAMMIN v5.3 running in slow mode, to generate a set of 15 models (28) (Svergun, 1999). The 15 bead models were aligned and then averaged using DAMAVER (Volkov and Svergun, 2003), and the resulting DAMMIN averaged model was then converted to a volume envelope by using the UCSF Chimera package (UCSF Chimera – a visualization system for exploratory research and analysis (Pettersen et al., 2004). The SAXS 3-D structure obtained (Fig. 4) clearly demonstrates a similar pattern of porosome-SV interaction as previously observed (Cho et al., 2004) in the 3-D AFM micrograph presented in Fig. 2 and EM micrograph in Fig. 1 (Fig. 5A and B). Since, no structural information from earlier AFM and EM studies (Cho et al., 2004, 2008; Lee et al., 2012), nor the known composition and molecular weight of the neuronal porosome complex were used to obtain the 3-D image of SV-porosome complex from SAXS results, the SAXS study was blind in arriving at the ‘averaged’ 3-D structure of the SV-porosome complex in its native state at the presynaptic membrane in synaptosomes. In the 3-D SAXS image, a 35 nm SV is seen docked at a 15 nm cup-shaped porosome complex having a central plug element that as noted earlier, has previously been implicated in the regulation of
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Fig. 4. Solution small-angle X-ray scattering structure of synaptic vesicle docked at the porosome complex within native synaptosomes suspended in buffer. (a) Experimental 1-D scattering curve. Inset shows the Guinier fit with a calculated RG of 91.56 ± 0.65 A˚ with limits of 0.667–1.316 (Rodic, I and Rambo, R., SCÅTTER, Software for SAXS Analysis). ˚ and (c) “Averaged” SAXS 3-D structure (center) of synaptic vesicle (b) Corresponding P(r) pairwise distance distribution plot; calculated RG of 91.91 ± 0.21 A˚ and Dmax 301 A, docked at the cup-shaped neuronal porosome complex at the presynaptic membrane. The image to the left of (c) [c1] is the top-view of the synaptic vesicle, and both the center image [c2] and the image to the right [c3] are opposite side views of the averaged 3-D structure of the synaptic vesicle docked to a porosome complex. Note the prominent central plug of the porosome connected to the periphery of the structure via a robust arm.
the open and closed conformation states of the neuronal porosome complex (Cho et al., 2010). In these earlier ultra-high resolution AFM studies (Cho et al., 2010) of the outer presynaptic membrane of isolated synaptosome preparations, the central plug at the porosome opening was found either retracted into the porosome cup or exhibit various degrees of projection out of the structure (Cho et al., 2010), suggesting that the central plug is capable of vertical motion, and in doing so may regulate the opening and closing of the porosome complex. In agreement with this hypothesis, the detailed structure of the SV-porosome complex obtained in the current SAXS study (Figs. 4C and 5C) provides further structural information to speculate on the possible molecular mechanism involved in neurotransmitter release via the neuronal porosome complex at the nerve terminal. First, it appears from the SAXS structure that the SV needs to dock extremely tightly with the cupshaped porosome complex, that would result in the SV to form a cap-like or mushroom-head appearance (Figs. 3 and 5). Second, following stimulation of neurotransmission, the arm of the central plug that appears to be connected to the lip of the porosome opening (Fig. 4C2 and C3), could be pushed inward, resulting in a small bump that would be close in size to the diameter of the foot of the central plug which from the SAXS image (Figs. 4 and 5) appears to be approximately 3–4 nm. Consequently, the membrane at the porosome base constituting this 3–4 nm in diameter bump must be under enormous tension and simultaneously have established tight apposition and contact with the outer leaflet of the SV membrane via SNARE proteins. The consequent establishment of t-/v-SNARE complex in a ring or rosette pattern (Cho et al., 2002b, 2005a, 2009, 2011; Jeremic et al., 2006) at this contact site, and in the presence
of Ca2+ ions (Jeremic et al., 2004a; Mohrmann et al., 2010; Jeremic et al., 2004b; Cho et al., 2005b; Potoff et al., 2008) would result in the fusion of the opposing lipid membranes. The resultant fusion pore would establishment of continuity between the SV lumen and the porosome for the release of neurotransmitters at the nerve terminal. Subsequently, the movement of the porosome plug toward the porosome opening to the outside would release tension off the membrane at the porosome base, resulting in a rapid closure or resealing of the fusion pore that had been transiently established (Fig. 5). Since actin, myosin, and several structural proteins constitute the neuronal porosome proteome (Lee et al., 2012), the motile central plug element is likely composed of these motor proteins requiring ATP for movement. In summary, results from the current SAXS study provides at nanometer resolution, a detailed 3-D structure of the interactions between the SV and the porosome complex in its native state at the presynaptic membrane. This new information has allowed the formulation of a hypothesis for the molecular mechanism involved in neurotransmitter release at the nerve terminal. Furthermore, results form the present study confirm earlier findings that the neuronal porosome complex is a cup-shaped supramolecular lipoprotein structure measuring approximately 15 nm in diameter where 30–50 nm in diameter SVs dock and transiently fuse to release neurotransmitters. The ultrahigh resolution 3-D structure of the porosome-synaptic vesicle complex elucidated from the present study, and the consequent formulation of the hypothesis for the molecular mechanism of neurotransmitter release, is in agreement with earlier studies demonstrating that “single synaptic vesicles fuse transiently and successively without loss of identity”
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Fig. 5. Docked synaptic vesicles at porosome complexes in the presynaptic membrane of the nerve terminal. (a) AFM micrograph obtained in fluid of a synaptic vesicle (SV) docked at the cup-shaped porosome complex (P) at the cytosolic compartment of the presynaptic membrane.3 Note the 35 nm SV docked to a 15 nm porosome complex. (b) An EM micrograph of a 35 nm SV docked to a 15 nm P at the presynaptic membrane (Cho et al., 2004). Note the central plug of the porosome complex in the electron micrograph. (c) The averaged SAXS 3-D structure of synaptic vesicle (purple) docked at the cup-shaped neuronal porosome complex (pink) at the presynaptic membrane in isolated synaptosomes is presented. Note that AFM, EM, and SAXS, all demonstrating similarity in the docking and interaction of synaptic vesicles at the neuronal porosome complex. (d) Schematic drawing of a possible mechanism of porosome (P) involvement in synaptic vesicle (SV) docking at the presynaptic membrane (PSM) in neurotransmitter release [1–5]. Note the arm (A) of the central plug (CP) that enables the vertical movement of the plug [1]. As the foot of the CP is pushed inwards following stimulation of neurotransmission, it results in a small bump (b) that would be close in size to the diameter of the foot of the CP, which is approximately 3–4 nm in diameter. Consequently, the membrane at the porosome base constituting this 3–4 nm in diameter bump is under enormous tension and simultaneously establishes tight apposition and contact with the outer leaflet of the SV membrane via SNARE proteins, resulting in the establishment of t-/v-SNARE complex in a ring or rosette pattern [3], resulting in fusion pore (FP) formation for neurotransmitter release. Subsequently, the lifting of the porosome plug back to its original resting position results in release of the tension off the membrane at the porosome base, and the rapid FP closure that had been transiently established [4]. The spent SV then undocks from the porosome, refilled with neurotransmitters via the neurotransmitter transporters (NTT) present at the SV membrane [5], and be ready for the next round of docking, fusion, and transmitter release. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
(Aravanis et al., 2003). Future high-resolution SAXS, combined with cryo-EM measurements of the porosome and the porosome-SV complex, in the presence and absence of ATP, will provide further insights into the molecular underpinnings of porosome function and neurotransmitter release at the nerve terminal. Conflicts of interest The authors declare no competing financial interests or conflicts. Acknowledgements Supported by grants from NIH NS-39918 and Wayne State University Research Enhancement Award (BPJ). We are grateful to Steven Weigand for technical assistance and help with data collection at DND-CAT. The DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT, sector 5) Synchrotron Research Center at the Advanced Photon Source (APS, Argonne, IL) is supported by E. I. DuPont de Nemours & Co., The Dow Chemical Company, the
National Science Foundation, and the State of Illinois. Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. Contributions: BPJ designed the research and wrote the paper. JSB and LCK performed the SAXS and associated calculation and analysis of the data. KTL isolated synaptosomes, performed PCS on them, and helped in preparation of the micrographs for the manuscript. DJT performed the EM studies on the isolated synaptosomes. WJC and JSL prepared synaptosomes, helped in drawing the cartoon, and in the preparation of the micrographs. BPJ, DJT, JSB and LCK critically analyzed results and proof read the manuscript. References Anderson, L.L., Jeftinija, S., Scanes, C.G., 2004. Growth hormone secretion: molecular and cellular mechanisms and in vivo approaches. Exp. Biol. Med. 229, 291–302. Anderson, L.L., Scanes, C.G., 2012. Nanobiology and physiology of growth hormone secretion. Exp. Biol. Med. 237, 126–142.
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Micron journal homepage: www.elsevier.com/locate/micron
X-ray solution structure of the native neuronal porosome-synaptic vesicle complex: Implication in neurotransmitter release Ladislau C. Kovari a,1 , Joseph S. Brunzelle b,1 , Kenneth T. Lewis c,d , Won Jin Cho c , Jin-Sook Lee c , Douglas J. Taatjes e , Bhanu P. Jena c,∗ a
Wayne State University School of Medicine, Department of Biochemistry and Molecular Biology, Detroit, MI, USA Life Sciences Collaborative Access Team, Synchrotron Research Center, Northwestern University, Argonne, IL 60439, USA c Department of Physiology, Wayne State University School of Medicine, Detroit, MI, USA d Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI, USA e Department of Pathology, Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, VT 05405, USA b
a r t i c l e
i n f o
Article history: Received 19 September 2013 Received in revised form 29 September 2013 Accepted 2 October 2013 Keywords: Neuronal porosome complex X-ray solution scattering Photon correlation spectroscopy Electron microscopy
a b s t r a c t Nanoportals at the cell plasma membrane called porosomes, mediate secretion from cells. In neurons porosomes are 15 nm cup-shaped lipoprotein structure composed of nearly 40 proteins. The size and complexity of the porosome has precluded determination of its atomic structure. Here we report at nanometer resolution the native 3D structure of the neuronal porosome-synaptic vesicle complex within isolated nerve terminals using small-angle X-ray solution scattering. In addition to furthering our understanding of the porosome structure, results from the study suggests the molecular mechanism involved in neurotransmitter release at the nerve terminal. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction The chemistry of life processes is governed at the molecular level. Hence a major challenge is the determination of atomic structure of cellular organelles and macromolecules required for understand their function in cells. Determination of the atomic structure of cellular organelles and macromolecules is required for understand their cellular function. It is especially challenging, when such organelles involve membrane proteins. Cup-shaped lipoprotein structures called porosomes (Schneider et al., 1997; Cho et al., 2002a, 2004, 2008; Jena et al., 2003; Jena, 2009; Jeremic et al., 2003; Lee et al., 2012; Anderson et al., 2004; Anderson and Scanes, 2012) are the universal secretory nanoportals at the cell plasma membrane. Membrane-bound secretory vesicles transiently dock and fuse at the base of the porosome to deliver intravesicular contents outside the cell. Fusion of membrane-bound secretory vesicles at the porosome base is mediated by calcium and a specialized set of three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors called SNAREs (Jeremic et al., 2004a; Malhotra et al., 1988; Trimble et al., 1988; Oyler et al., 1989). In neurons,
∗ Corresponding author. Tel.: +1 313 577 1532. E-mail address: [email protected] (B.P. Jena). 1 These authors contributed equally to the study. 0968-4328/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.micron.2013.10.002
target membrane proteins SNAP-25 and syntaxin called t-SNAREs present at the base of neuronal porosomes, and a synaptic vesicleassociated membrane protein (VAMP) or v-SNARE, are part of the conserved protein complex involved in membrane fusion and neurotransmission. Neuronal porosomes measure approximately 15 nm in diameter, and are present at the presynaptic membrane of nerve terminals called synaptosomes. Nearly 40 proteins (Cho et al., 2004; Lee et al., 2012) including SNARE and lipids that include cholesterol (Cho et al., 2007), compose the neuronal porosome complex. The overall morphology (Figs. 1 and 2) (Cho et al., 2004, 2008) and dynamics (Cho et al., 2010) of the neuronal porosome complex which has previously been studied and reported using both atomic force microscopy (AFM) and electron microscopy (EM), is presented (Figs. 1 and 2) for clarity and to introduce the reader to the subject. Additionally, the neuronal porosome has been functionally reconstituted into artificial lipid membrane (Cho et al., 2004), and the 3D contour map of its protein backbone at nanometer scale established (Cho et al., 2008). A set of eight protein units lining the neuronal porosome cup is present (Cho et al., 2004, 2008), each connected via spoke-like elements to a central plug, suggested to be involved in the rapid opening and closing of the structure to the outside during neurotransmission. AFM micrographs of the presynaptic membrane of isolated synaptosome preparations demonstrates the presence of the neuronal porosome plug at various conformations (Cho et al., 2010). The central plug
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Fig. 1. Neuronal porosomes at the presynaptic membrane of nerve terminals determined in previous (Cho et al., 2004; Jena et al., 2003; Lee et al., 2012; Jeremic et al., 2004a) studies. (a) Atomic force micrograph (AFM) amplitude image Bar = 100 nm (black arrowhead points to presynaptic membrane), and (b) electron micrograph (EM) of isolated synaptosome Bar = 100 nm. (c) High resolution AFM micrograph of isolated neuronal porosome from synaptosomes followed by reconstitution into a lipid membrane. Note the near 10 nm neuronal porosome complex with a central plug (red arrowhead) and eight peripheral densities. (d) EM micrograph of the cup-shaped neuronal porosome complex with its central plug (red arrowhead) and a docked synaptic vesicle at its base. (e) Nagative staining EM of an isolated neuronal porosome complex. Note the nano scale structure and assembly of proteins within the complex, and a schematic drawing (f) of the cup-shaped neuronal porosome (P) associated with a docked synaptic vesicle (SV). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
when fully retracted is in the closed conformation, when partially retracted, it is in its semi-open state, and when completely inserted into the porosome cup, in its open conformation (Cho et al., 2010). The presence of such a mechanism in the neuronal porosome complex makes sense since it would allow the rapid open and close states of the organelle during neurotransmission at the nerve terminal. This is opposed to a slow secretory cell such as the exocrine pancreas (Schneider et al., 1997) or the neuroendocrine growth hormone secreting cell (Cho et al., 2002a) where no such plug is present at the porosome opening; instead the porosome opening dilates during secretion and returns to its resting size following completion of the process (Schneider et al., 1997; Cho et al., 2002a). The size and complexity of the membrane-associated porosome has precluded determination of its atomic structure. For example,
solution NMR has not been possible primarily due to the large molecular size of the porosome complex, which is beyond the operating limits of current NMR’s. Similarly, X-ray crystallography is impractical, due in part to the solubility problems of this membrane-associated structure composed of neatly 40 proteins. Although these limitations have been partially overcome by the use of AFM and EM in furthering our understanding of the fine structure and nano-arrangement of proteins within the native porosome complex (Cho et al., 2008), its 3D structure in association with a docked secretory vesicle in intact synaptosome had not been observed. In recent years, with major advances in instrumentation and computational power, small angle X-ray scattering (SAXS) has become a powerful method to study biological material at nanometer to sub-nanometer resolution in solution (Round
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2.2. Photon correlation spectroscopy (PCS) on isolated synaptosome preparation
Fig. 2. AFM micrograph obtained from an earlier study of the cytosolic compartment at the presynaptic membrane of a synaptosome (Cho et al., 2004). Note SV (blue arrowheads) docked at the base of porosomes (P, red arrowheads). The mushroom-shaped head represents the SV and the stalk represents the P complex. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
et al., 2008; Hura et al., 2009; Forster et al., 2008; Mertens and Svergun, 2010). In SAXS, a focused X-ray beam from a synchrotron is scattered by the sample, and the scattering data is collected and recorded in real-time. The scattering patterns generated from a supramolecular structure in suspension are presented as radially averaged single dimensional curves, from which important parameters such as the overall three-dimensional shape and size of structures are obtained. In the current study, the native structure and interaction between SV and the porosome complex was studied using SAXS. Results from the study confirm earlier findings (Cho et al., 2004, 2007, 2008, 2010; Lee et al., 2012; Okuneva et al., 2012; Drescher et al., 2011; Siksou et al., 2007), demonstrating the neuronal porosome complex to be cup-shaped structures measuring approximately 15 nm in diameter where on average 35 nm in diameter synaptic vesicles transiently dock and fuse to release neurotransmitters. These results further provide an understanding of the possible mechanisms involved in neurotransmitter release at the nerve terminal. 2. Materials and methods
Synaptosome size was determined using photon correlation spectroscopy (PCS). PCS is a well-known technique for the measurement of m to nm size particles and macromolecules. PCS measurements were performed in a Zetasizer Nano ZS, (Malvern Instruments, UK). In a typical experiment, the size distribution of isolated synaptosomes was determined using built-in software provided by Malvern Instruments. Prior to determination of the synaptosome hydrodynamic radius, calibration of the instrument was performed using latex spheres of known size. In PCS, subtle fluctuations in the sample scattering intensity are correlated across microsecond time scales. The correlation function was calculated, from which the diffusion coefficient was determined. Using the Stokes–Einstein equation, hydrodynamic radius can be acquired from the diffusion coefficient (Higashijima et al., 1988). The intensity size distribution, which is obtained as a plot of the relative intensity of light scattered by particles in various size classes, is then calculated from a correlation function using built-in software. The particle scattering intensity is proportional to the molecular weight squared. Volume distribution can be derived from the intensity distribution using Mie theory (Vitale et al., 1993; Weingarten et al., 1990). The transforms of the PCS intensity distribution to volume distributions can be obtained using the provided software by Malvern Instruments.
2.3. Electron microscopy of isolated synaptosomes Electron microscopy was performed on isolated synaptosomes as described in a previously published procedure (Wang et al., 2012). Briefly, isolated synaptosomes were fixed in 2% glutaraldehyde/2% paraformaldehyde (GA-PFA) in ice-cold PBS for 24 h, washed, embedded in 2% SeaPrep agarose, followed by postfixation for 1 h at 4 ◦ C using 1% OsO4 in 0.1 M cacodylate buffer. Finally, the samples were dehydrated in a graded series of ethanol, through propylene oxide, and infiltrated and embedded in Spurr’s resin. Ultrathin sections were cut with a diamond knife, retrieved onto 200 mesh nickel thin-bar grids, and contrasted with alcoholic uranyl acetate and lead citrate. Grids were viewed with a JEOL 1400 transmission electron microscope (JEOL USA, Inc., Peabody, MA) operating at 60 or 80 kV, and digital images were acquired with an AMT-XR611 11 megapixel CCD camera (Advanced Microscopy Techniques, Danvers, MA).
2.1. Synaptosome preparation 2.4. Small-angle X-ray solution scattering Synaptosomes were prepared from rat brains, with minor modifications of established published methods (Cho et al., 2004, 2007, 2010; Lee et al., 2012). For each experiment, Sprague-Dawley rats weighing 100–150 g were euthanized by CO2 inhalation, with all animal procedures preapproved by the Institution Animal Care & Use Committee (IACUC). Whole brains were isolated and placed in ice-cold buffered sucrose solution (5 mM Hepes, pH 7.4, 0.32 M sucrose) supplemented with protease inhibitor cocktail (SigmaAldrich, St. Louis, MO). The brain tissue was homogenized using 12 strokes in a Teflon-glass homogenizer, and the total homogenate was centrifuged for 3 min at 2500 × g. The resulting supernatant fraction was further centrifuged for 15 min at 14,500 × g, to obtain a pellet. The resultant pellet was re-suspended in buffered sucrose solution, and loaded onto a 3–10–23% Percoll gradient. Following centrifugation at 28,000 × g for 6 min, only the top half layer of the enriched synaptosome fraction at the 10–23% Percoll gradient interface was collected, and re-suspended in PBS pH 7.4 containing 0.2% paraformaldehyde (PFA).
Isolated synaptosomes suspended in PBS-PFA at a concentration of 2.5 mg/ml were subjected to small-angle X-ray solution scattering experiments. All small angle X-ray scattering data were collected on the SAXS/WAXS setup located at the 5-ID-D beamline of the DND-CAT synchrotron research center, Advanced Photon Source, Argonne National Laboratory (Argonne, IL). The SAXS data were recorded on a Mar-USA 165 mm CCD and covered the momentum transfer range on two-dimensional CCD detectors. Four exposures of 10 s were acquired for each sample at a sample-todetector distance of 2.6 m and over a range of momentum transfer 0.007 < q < 0.24 A˚ −1 , Q = 4 × sin /. The WAXS data were recorded on a custom Roper CCD and covered a range of momentum transfer 0.20 < q < 1.0 A˚ −1 . Data and a buffer blank were collected for seven different concentrations (2.5–0.04 mg/ml) of each sample to check for concentration-dependent scattering effects, such as aggregation or interparticle interference. No measurable radiation damage was observed under these conditions.
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Fig. 3. Synaptosomes isolated for the present study. (a) Photon correlation spectroscopy (PCS) of isolated synaptosome suspension demonstrating nearly 93% of the suspended particles to measure 955–1219 nm, typical of synaptosome size. (b and c) EM micrographs of isolated synaptosomes demonstrating the presence of docked SV at the presynaptic membrane. Note the sparse presence of free or non-docked SVs within the isolated synaptosomes. (d) Image enhancement of the boxed area in (c) is shown. The figure depicts a cup-shaped porosome complex (P) at the presynaptic membrane (PSM) with a docked SV that has sheared off during sectioning for EM. Note the cap or mushroom shape of the docked SV at the cup-shaped neuronal porosome complex.
3. Results and discussion To determine the 3D structure of the neuronal porosome with docked synaptic vesicles in the native state at nanometer resolution, we isolate synaptosomes containing primarily docked synaptic vesicles at the presynaptic membrane for SAXS studies. In this way, majority of the structures in the synaptosome suspension would represent porosomes-associated synaptic vesicle complexes. Using a minor modification of a published procedure, synaptosomes containing primarily docked synaptic vesicles were isolated and assessed using photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) (Fig. 3). As outlined in the materials and methods section, briefly, the brain tissue was homogenized using 12 strokes in a Teflon-glass homogenizer, and the total homogenate was centrifuged for 3 min at 2500 × g. The resulting supernatant fraction was further centrifuged for 15 min at 14,500 × g, to obtain a pellet. The resultant pellet was re-suspended in buffered sucrose solution, and loaded onto a 3–10–23% Percoll gradient. Following centrifugation at 28,000 × g for 6 min, only the top half layer of the enriched synaptosome fraction at the 10–23% Percoll gradient interface was collected. The top layer of the synaptosome fraction was lighter and enriched in synaptosomes containing just docked synaptic vesicles. Most of the undocked synaptic vesicles were lost from the preparation, making it ideal for the SAXS study. Isolated synaptosomes are known to measure approximately 0.8–1.5 m in diameter (Lee et al., 2010; Chen et al., 2011). PCS measurements of isolated synaptosome preparation from the top layer of the synaptosome fraction demonstrate that >92% of the particles measure between 0.955–1.219 m (Fig. 3A), suggesting them to represent isolated synaptosomes. Further examination of the isolated synaptosome preparations using TEM (Fig. 3B–D), confirmed them to be synaptosomes containing primarily docked synaptic vesicles. Therefore, in the synaptosome suspension used for SAXS, the structure that is frequently encountered are docked SVs at the porosome complex, most likely in repetitive lattice conformation. Hence the synaptosome
preparation was ideal for study of the interactions between SV and the neuronal porosome complex in the native state using SAXS. When the isolated synaptosome suspensions were subjected to small-angle X-ray solution scattering, and the scattering data was collected on the SAXS/WAXS setup, an averaged 3-D image of SV-porosome complex at nm resolution in the native state was obtained (Fig. 4). Scattering data were radially averaged to produce one-dimensional profiles of scattering intensity vs. q (Koch et al., 2003). The innermost portions of the scattering curves were used for fitting to the equation I(q) = I(0) exp(−42 RG 2 q2 /3), where I(0) is the forward scattering intensity at q = 0 and RG is the radius of gyration (Guinier and Fournet, 1955). Values for RG were extracted from the Guinier plot log {I(q)} vs. q2 or the pair distribution function P(r) using the GNOM package (Svergun, 1992). A final P(r) plot was generated with GNOM v4.6, including Q values from 0.0075 to 0.8794. The output was the basis for ab initio shape reconstruction using DAMMIN v5.3 running in slow mode, to generate a set of 15 models (28) (Svergun, 1999). The 15 bead models were aligned and then averaged using DAMAVER (Volkov and Svergun, 2003), and the resulting DAMMIN averaged model was then converted to a volume envelope by using the UCSF Chimera package (UCSF Chimera – a visualization system for exploratory research and analysis (Pettersen et al., 2004). The SAXS 3-D structure obtained (Fig. 4) clearly demonstrates a similar pattern of porosome-SV interaction as previously observed (Cho et al., 2004) in the 3-D AFM micrograph presented in Fig. 2 and EM micrograph in Fig. 1 (Fig. 5A and B). Since, no structural information from earlier AFM and EM studies (Cho et al., 2004, 2008; Lee et al., 2012), nor the known composition and molecular weight of the neuronal porosome complex were used to obtain the 3-D image of SV-porosome complex from SAXS results, the SAXS study was blind in arriving at the ‘averaged’ 3-D structure of the SV-porosome complex in its native state at the presynaptic membrane in synaptosomes. In the 3-D SAXS image, a 35 nm SV is seen docked at a 15 nm cup-shaped porosome complex having a central plug element that as noted earlier, has previously been implicated in the regulation of
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Fig. 4. Solution small-angle X-ray scattering structure of synaptic vesicle docked at the porosome complex within native synaptosomes suspended in buffer. (a) Experimental 1-D scattering curve. Inset shows the Guinier fit with a calculated RG of 91.56 ± 0.65 A˚ with limits of 0.667–1.316 (Rodic, I and Rambo, R., SCÅTTER, Software for SAXS Analysis). ˚ and (c) “Averaged” SAXS 3-D structure (center) of synaptic vesicle (b) Corresponding P(r) pairwise distance distribution plot; calculated RG of 91.91 ± 0.21 A˚ and Dmax 301 A, docked at the cup-shaped neuronal porosome complex at the presynaptic membrane. The image to the left of (c) [c1] is the top-view of the synaptic vesicle, and both the center image [c2] and the image to the right [c3] are opposite side views of the averaged 3-D structure of the synaptic vesicle docked to a porosome complex. Note the prominent central plug of the porosome connected to the periphery of the structure via a robust arm.
the open and closed conformation states of the neuronal porosome complex (Cho et al., 2010). In these earlier ultra-high resolution AFM studies (Cho et al., 2010) of the outer presynaptic membrane of isolated synaptosome preparations, the central plug at the porosome opening was found either retracted into the porosome cup or exhibit various degrees of projection out of the structure (Cho et al., 2010), suggesting that the central plug is capable of vertical motion, and in doing so may regulate the opening and closing of the porosome complex. In agreement with this hypothesis, the detailed structure of the SV-porosome complex obtained in the current SAXS study (Figs. 4C and 5C) provides further structural information to speculate on the possible molecular mechanism involved in neurotransmitter release via the neuronal porosome complex at the nerve terminal. First, it appears from the SAXS structure that the SV needs to dock extremely tightly with the cupshaped porosome complex, that would result in the SV to form a cap-like or mushroom-head appearance (Figs. 3 and 5). Second, following stimulation of neurotransmission, the arm of the central plug that appears to be connected to the lip of the porosome opening (Fig. 4C2 and C3), could be pushed inward, resulting in a small bump that would be close in size to the diameter of the foot of the central plug which from the SAXS image (Figs. 4 and 5) appears to be approximately 3–4 nm. Consequently, the membrane at the porosome base constituting this 3–4 nm in diameter bump must be under enormous tension and simultaneously have established tight apposition and contact with the outer leaflet of the SV membrane via SNARE proteins. The consequent establishment of t-/v-SNARE complex in a ring or rosette pattern (Cho et al., 2002b, 2005a, 2009, 2011; Jeremic et al., 2006) at this contact site, and in the presence
of Ca2+ ions (Jeremic et al., 2004a; Mohrmann et al., 2010; Jeremic et al., 2004b; Cho et al., 2005b; Potoff et al., 2008) would result in the fusion of the opposing lipid membranes. The resultant fusion pore would establishment of continuity between the SV lumen and the porosome for the release of neurotransmitters at the nerve terminal. Subsequently, the movement of the porosome plug toward the porosome opening to the outside would release tension off the membrane at the porosome base, resulting in a rapid closure or resealing of the fusion pore that had been transiently established (Fig. 5). Since actin, myosin, and several structural proteins constitute the neuronal porosome proteome (Lee et al., 2012), the motile central plug element is likely composed of these motor proteins requiring ATP for movement. In summary, results from the current SAXS study provides at nanometer resolution, a detailed 3-D structure of the interactions between the SV and the porosome complex in its native state at the presynaptic membrane. This new information has allowed the formulation of a hypothesis for the molecular mechanism involved in neurotransmitter release at the nerve terminal. Furthermore, results form the present study confirm earlier findings that the neuronal porosome complex is a cup-shaped supramolecular lipoprotein structure measuring approximately 15 nm in diameter where 30–50 nm in diameter SVs dock and transiently fuse to release neurotransmitters. The ultrahigh resolution 3-D structure of the porosome-synaptic vesicle complex elucidated from the present study, and the consequent formulation of the hypothesis for the molecular mechanism of neurotransmitter release, is in agreement with earlier studies demonstrating that “single synaptic vesicles fuse transiently and successively without loss of identity”
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L.C. Kovari et al. / Micron 56 (2014) 37–43
Fig. 5. Docked synaptic vesicles at porosome complexes in the presynaptic membrane of the nerve terminal. (a) AFM micrograph obtained in fluid of a synaptic vesicle (SV) docked at the cup-shaped porosome complex (P) at the cytosolic compartment of the presynaptic membrane.3 Note the 35 nm SV docked to a 15 nm porosome complex. (b) An EM micrograph of a 35 nm SV docked to a 15 nm P at the presynaptic membrane (Cho et al., 2004). Note the central plug of the porosome complex in the electron micrograph. (c) The averaged SAXS 3-D structure of synaptic vesicle (purple) docked at the cup-shaped neuronal porosome complex (pink) at the presynaptic membrane in isolated synaptosomes is presented. Note that AFM, EM, and SAXS, all demonstrating similarity in the docking and interaction of synaptic vesicles at the neuronal porosome complex. (d) Schematic drawing of a possible mechanism of porosome (P) involvement in synaptic vesicle (SV) docking at the presynaptic membrane (PSM) in neurotransmitter release [1–5]. Note the arm (A) of the central plug (CP) that enables the vertical movement of the plug [1]. As the foot of the CP is pushed inwards following stimulation of neurotransmission, it results in a small bump (b) that would be close in size to the diameter of the foot of the CP, which is approximately 3–4 nm in diameter. Consequently, the membrane at the porosome base constituting this 3–4 nm in diameter bump is under enormous tension and simultaneously establishes tight apposition and contact with the outer leaflet of the SV membrane via SNARE proteins, resulting in the establishment of t-/v-SNARE complex in a ring or rosette pattern [3], resulting in fusion pore (FP) formation for neurotransmitter release. Subsequently, the lifting of the porosome plug back to its original resting position results in release of the tension off the membrane at the porosome base, and the rapid FP closure that had been transiently established [4]. The spent SV then undocks from the porosome, refilled with neurotransmitters via the neurotransmitter transporters (NTT) present at the SV membrane [5], and be ready for the next round of docking, fusion, and transmitter release. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
(Aravanis et al., 2003). Future high-resolution SAXS, combined with cryo-EM measurements of the porosome and the porosome-SV complex, in the presence and absence of ATP, will provide further insights into the molecular underpinnings of porosome function and neurotransmitter release at the nerve terminal. Conflicts of interest The authors declare no competing financial interests or conflicts. Acknowledgements Supported by grants from NIH NS-39918 and Wayne State University Research Enhancement Award (BPJ). We are grateful to Steven Weigand for technical assistance and help with data collection at DND-CAT. The DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT, sector 5) Synchrotron Research Center at the Advanced Photon Source (APS, Argonne, IL) is supported by E. I. DuPont de Nemours & Co., The Dow Chemical Company, the
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