Nuclear Pore Complex Structure and Dynamics

Nuclear Pore Complex Structure and Dynamics Revealed by Cryoelectron Tomography Martin Beck, et al. Science 306, 1387 (2004); DOI: 10.1126/science.1104808 The following resources related to this article are available online at www.sciencemag.org (this information is current as of February 1, 2008 ):

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Nuclear Pore Complex Structure and Dynamics Revealed by Cryoelectron Tomography Martin Beck, Friedrich Fo¨rster, Mary Ecke, Ju¨rgen M. Plitzko, Frauke Melchior,* Gu¨nther Gerisch, Wolfgang Baumeister,. Ohad Medalia. Nuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. To analyze their structure in a close-to-life state, we studied transport-active, intact nuclei from Dictyostelium discoideum by means of cryoelectron tomography. Subvolumes of the tomograms containing individual NPCs were extracted in silico and subjected to three-dimensional classification and averaging, whereby distinct structural states were observed. The central plug/ transporter (CP/T) was variable in volume and could occupy different positions along the nucleocytoplasmic axis, which supports the notion that it essentially represents cargo in transit. Changes in the position of the CP/T were accompanied by structural rearrangements in the NPC scaffold. Nuclear pore complexes (NPCs) mediate the exchange of macromolecules between the nucleus and the cytoplasm. These large assemblies (È120 megadaltons in metazoa) are constructed from about 30 different proteins, the nucleoporins (1). Functional and structural characterization of NPCs is challenging, due mainly to their sheer size. Structures of isolated NPCs have been resolved to 12 nm by means of cryo-EM (2), albeit with a nonisotropic resolution (see below). The structural analysis of active NPCs has obvious advantages over studies with isolated and detergentextracted NPCs: The procedures that are traditionally used for the purification of NPCs are susceptible to loss of transport machinery components and cargo. In principle, cryoelectron tomography (cryo-ET) allows one to analyze the three-dimensional (3D) architecture of organelles or even whole cells embedded in vitreous ice, i.e., in a close-to-life state (3, 4). Therefore, we have applied cryo-ET to whole Dictyostelium discoideum nuclei, which are relatively small (È2 mm) and can be isolated by a gentle procedure. In a projection image of such a nucleus after vitrification, an intact nuclear envelope is evident (Fig. 1A). These nuclei were fully competent for active nuclear import (Fig. 1, B and C; fig. S1), in a manner similar to permeabilized cells (5, 6). We acquired 16 tilt series of frozenhydrated Dictyostelium nuclei and reconstructed the respective volumes. The framed area of the nucleus shown in Fig. 1A was used for recording a tomogram. Three differMax Planck Institute of Biochemistry, D-82152 Martinsried, Germany. *Present address: Department of Biochemistry, University of Go¨ttingen, D-37073 Go¨ttingen, Germany. .To whom correspondence should be addressed. E-mail: [email protected] (W.B.) and [email protected] (O.M.)

ent x-y slices (along the z axis) through this tomogram are shown (Fig. 1D). Both the outer nuclear membrane and a patch of

connected endoplasmic reticulum are decorated with globular complexes that resemble 80S ribosomes in size and shape (È27 nm). In slices that are approximately perpendicular to the nuclear envelope, but more obviously in grazing slices, individual NPCs are clearly discernible. After surface rendering of the tomograms (Fig. 1E), clear pictures of individual NPCs were obtained. The canonical features such as cytoplasmic filaments, the three rings (cytoplasmic, lumenal spoke, and nuclear rings, respectively), parts of the basket, and the central plug/transporter (CP/T) were visible without any postprocessing. We extracted 267 NPC-containing subvolumes from our tomograms. Given that our NPCs are transport-competent, one would expect them to be arrested in a variety of different transport states. Averaging without prior classification would therefore be expected to emphasize nonvariable features, whereas variable features would be deemphasized or even eliminated. Averaging procedures resulted in an isotropically sampled 3D density map of the NPC with a resolution of 8 to 9 nm. The cytoplasmic face (Fig. 2A)

Fig. 1. Cryo-ET of transport-competent nuclei. (A) Transmission electron micrograph of a vitrified Dictyostelium nucleus. The image was recorded after acquisition of a complete tilt series; the frame marks the area representative for the reconstruction shown in (D). (B) Phase-contrast image and (C) the corresponding fluorescence image showing uptake of the transport substrate (FITC-BSANLS) into isolated, enriched nuclei. (D) Three-dimensional reconstruction of an intact nucleus. Three sequential x-y slices of 10 nm thickness along the z axis through a typical tomogram are indicated. Different orientations of NPCs are shown: top-views (left) and side-views (right, arrows). Ribosomes connected to the outer nuclear membrane are visible, as is a patch of rough ER (right, arrowheads). (E) Surfacerendered representation of a segment of nuclear envelope (NPCs in blue, membranes in yellow). The dimensions of the rendered volume are 1680 nm 984 nm 558 nm. The number of NPCs was È45/mm2.

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calculated. The analysis of the occupied volumes (Fig. 3A) supports the notion that the CP/T makes up, at least in part, cargo complexes arrested during translocation (2). The distribution of the centers of gravity along the nucleocytoplasmic axis indicates

that two preferred positions of mass within the central channel exist, which are likely to correspond to different NPC states (Fig. 3B). It provides a means for an objective classification of individual NPCs. In order to visualize differences in the two NPC states,


shows eight cytoplasmic filaments (È35 nm) arranged around the central channel. They protrude from the cytoplasmic ring and point toward the center of the structure. In spite of their flexible nature, these filaments show a distinct shape with a pointed kink. The CP/T comprises two overlapping, sphere-shaped densities. The smaller one (È20 nm) is inplane with the cytoplasmic filaments; the larger one (È40 nm) is located farther down within the central channel (Fig. 2A). The most conspicuous feature on the nuclear face (Fig. 2B) is the nuclear basket. The nuclear filaments that connect the distal ring to the nuclear ring do not appear to be entirely straight but rather appear bent in the proximity of the nuclear ring (Fig. 2C, nuclear basket). The cytoplasmic as well as the nuclear filaments appear to be more delicate than in previous work that used metal coating, a technique that gives more prominence to filiform structures (7). The dimensions of the main features are revealed in a cutaway view without the CP/T (Fig. 2C). If one considers the corresponding dimensions that are known from different organisms, the diameter of the NPC from Dictyostelium is more similar to that of Metazoa than to yeasts (7, 8), whereas the stack of rings is less elongated in the direction of the nucleocytoplasmic axis when compared with structures obtained previously by cryo-EM (2, 9, 10). The nuclear envelopes or detergent-extracted NPCs that were investigated in these studies assume a preferred orientation on the EM-grid. Consequently, the resulting structures were not isotropically sampled and lack information (the Bmissing cone[) that leads to an artificial elongation along the z axis (11). In contrast, the NPCs of an intact nucleus assume free spatial orientations that represent all Eularian angles (fig. S3B). Gold-labeling experiments have shown that most of the components of the NPC (at least 18 different nucleoporins) are localized symmetrically in relation to an imaginary central plane through the lumenal spoke ring (12). In fact, when one looks at a slice oriented parallel to the nucleocytoplasmic axis, the upper and lower part of the lumenal spoke ring appear similar, whereas the CP/T appears rather asymmetrical (fig. S2B, right). The CP/T located within the central channel has been described previously (10, 13), but its role is still a matter of speculation. It is not found in every NPC (13), possibly because it is lost during the isolation procedure. Here, we found the CP/T in almost all NPCs that were examined, but its size, shape, and position varied substantially (fig. S2). Consequently, the average is rather featureless. We performed a quantitative analysis of this substructure in individual NPCs. The CP/Ts were extracted in silico and the occupied volumes, as well as the positions of the centers of gravity, were

Fig. 2. Structure of the Dictyostelium NPC. (A). Cytoplasmic face of the NPC in stereo view. The cytoplasmic filaments are arranged around the central channel; they are kinked and point toward the CP/T. (B) Nuclear face of the NPC in stereo view. The distal ring of the basket is connected to the nuclear ring by the nuclear filaments. (C) Cutaway view of the NPC with the CP/T removed. The dimensions of the main features are indicated. All views are surface-rendered (nuclear basket in brown).

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we divided our stack of particles into two classes: one designated a Bcytoplasmic filament class[ (CF class), and the second, a Blumenal spoke ring class[ (LR class). Probability clouds for both classes have been calculated from the positions of the centers of gravity (Fig. 3C). The variance for the CF class is significantly smaller than for the LR class, which suggests that the position of the mass surrounded by the lumenal spoke ring is more diffuse. The two stacks of NPCs assigned to the CF class or the LR class were averaged separately. Slices through the structures of CF- and LR classes are shown in Fig. 3, D and E. The slices oriented along the nucleocytoplasmic axis (left) show the different positions of the CP/T. The cytoplasmic filaments are well defined in the CF

class and have an apparent length of È35 nm. An elongated density connects them to the CP/T (arrowhead). As revealed by the surface-rendered view shown in Fig. 3F (left), they are kinked, with the kink pointing toward the CP/T, whereas their tips point to adjacent filaments. The cytoplasmic face of the LR class shows only the base of the cytoplasmic filaments (Fig. 3G, left). The densities of the cytoplasmic and lumenal spoke ring are similar for both classes (Fig. 3, D and E, as CR, LR). The eight spokes of the lumenal spoke ring point toward the central channel in the LR class, but they appear bent in the CF class. Moreover, structural changes of the nuclear ring, which is connected to the nuclear basket, can be seen (Fig. 3, D and E, NR). Substantial differences

Fig. 3. Position of the CP/T correlates with structural changes of the NPC. (A) The distribution of the occupied volume in individual CP/Ts is significantly broader than the fitted Gaussian function, which would indicate that the CP/T in different NPCs has essentially the same components. The observed mass is thus likely to represent an average of different structures, rather than a constitutive substructure within the central channel of the NPC. (B) The distribution of the centers of gravity of individual CP/Ts along the nucleocytoplasmic axis shows two distinct peaks (LR class shaded in red). It matches a double-Gaussian function, which is indicative of two preferred positions of mass along the same axis. The quality of the fits in (A) and (B) was evaluated with a chi-square test. The disjunction criterion was chosen at the minimum between the two peaks and is also indicated in the average viewed in fig. S2B (right). (C) Probability clouds of the centers of gravity corresponding to both classes. The position of the centers of gravity is shown superimposed on a centered slice through the structure (CF class in blue, LR class in red). The center of gravity is better defined in the CF class. (D) Slices through the structure of the CF class. Centered slices along the nucleocytoplasmic axis (left) and slices in-plane with the cytoplasmic filaments (CF), cytoplasmic ring (CR), lumenal spoke ring (LR), and nuclear ring (NR), corresponding to 7 nm in thickness. The cytoplasmic filaments are connected to the CP/T by an elongated density (arrowhead). (E) Same as (D) but for the LR class. (F) Surface-rendered views of the CF class. Cytoplasmic face view (left) and nuclear face view (right). (G) Same as (F) but for the LR class (nuclear basket in brown).

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in the structure of the nuclear basket are revealed in the surface-rendered views in Fig. 3, F and G (right). In the CF class, the distal ring has an opening in the middle and a smaller diameter; however, in the LR class it is more massive, indicative of the presence of additional mass bound to NPCs of this class. The major differences between CF and LR classes are shown in Fig. 4. The schematic illustration shows the cytoplasmic filaments connected to the CP/T in the CF class (Fig. 4A), as indicated by the thin connection evident in the slice in Fig. 3D (arrowhead). We suggest that in this class, some of the cytoplasmic filaments are engaged in interactions with cargo that is represented by the CP/T and have a preferred orientation. As a result, the structure of the CF class represents the shape of a filament that interacts with cargo. Since it is not likely that all of the cytoplasmic filaments interact at the same time, their density is slightly Bdiluted[ when they are averaged with imposed 8-fold symmetry. In the LR class, the cargo is not situated in the plane with the cytoplasmic filaments, which faded almost entirely during averaging. This suggests that the filaments

Fig. 4. Comparison of the two structural states of the NPC. (A) Schematic illustration of the structural changes of the cytoplasmic filaments. Surface-rendered views of the structure superimposed with the CP/Ts at a lower threshold (CF class in red, LR class in blue). In the CF class, the cytoplasmic filaments are in a defined orientation and interact with the CP/T; the latter is situated within the same plane. In the LR class, the CP/T is located in the plane with the lumenal spoke ring, and the disengaged cytoplasmic filaments are variable in shape and fade out in the average. Therefore, we added four with arbitrary shapes for the sake of completeness. (B) Contour-line-view of slices along the nucleocytoplasmic axis. The position of the narrowest constriction in the central channel is indicated for both classes (arrows).

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REPORTS predominant rate-limiting steps in both processes is not yet possible, the application of cryo-ET to transport-competent, intact nuclei holds great potential for a structural dissection of the key steps involved. The use of defined cargo and the trapping of distinct transport intermediates should ultimately enable us to arrive at a detailed mechanistic understanding of the nuclear pore complex. References and Notes 1. M. Suntharalingam, S. R. Wente, Dev. Cell 4, 775 (2003). 2. D. Stoffler et al., J. Mol. Biol. 328, 119 (2003). 3. W. Baumeister, Curr. Opin. Struct. Biol. 12, 679 (2002). 4. O. Medalia et al., Science 298, 1209 (2002). 5. F. Melchior, B. Paschal, J. Evans, L. Gerace, J. Cell Biol. 123, 1649 (1993). 6. Because the NPCs of spread Xenopus nuclear envelopes have a preferred orientation (17), an isotropically resolved structure cannot be obtained from such samples (although transport active), owing to the missing cone problem (11). 7. M. W. Goldberg, T. D. Allen, J. Mol. Biol. 257, 848 (1996). 8. E. Kiseleva et al., J. Struct. Biol. 145, 272 (2004).

Anabaena Sensory Rhodopsin: A Photochromic Color Sensor at 2.0 A˚ Lutz Vogeley,1 Oleg A. Sineshchekov,3,5 Vishwa D. Trivedi,3 Jun Sasaki,3 John L. Spudich,3,4* Hartmut Luecke1,2* Microbial sensory rhodopsins are a family of membrane-embedded photoreceptors in prokaryotic and eukaryotic organisms. Structures of archaeal rhodopsins, which function as light-driven ion pumps or photosensors, have been reported. We present the structure of a eubacterial rhodopsin, which differs from those of previously characterized archaeal rhodopsins in its chromophore and cytoplasmic-side portions. Anabaena sensory rhodopsin exhibits light-induced interconversion between stable 13-cis and all-trans states of the retinylidene protein. The ratio of its cis and trans chromophore forms depends on the wavelength of illumination, thus providing a mechanism for a single protein to signal the color of light, for example, to regulate color-sensitive processes such as chromatic adaptation in photosynthesis. Its cytoplasmic half channel, highly hydrophobic in the archaeal rhodopsins, contains numerous hydrophilic residues networked by water molecules, providing a connection from the photoactive site to the cytoplasmic surface believed to interact with the receptor’s soluble 14-kilodalton transducer. Over the past 4 years, microbial genomics has revealed a large family of photoactive, seven-transmembrane-helix retinylidene proteins called microbial rhodopsins in phylogenetically diverse species, including haloarchaea, proteobacteria, cyanobacteria, fungi, and algae (1–4). The first members of this family were discovered in halophilic archaea: the light-driven ion pumps bacteriorhodopsin and halorhodopsin and the phototaxis receptors sensory rhodopsins I and II. These four related haloarchaeal pigments are among the best-characterized membrane proteins in

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terms of structure and function, and nearly all of our knowledge of the properties of microbial rhodopsins, such as isomeric configuration and conformation of their chromophore, photochemical reactions, light-induced conformational changes in the protein, and function, derives from the study of these four, including atomic resolution structures that have been obtained for three of them (5–9). Studies of non-haloarchaeal rhodopsins, of which 9800 are known to exist (10, 11), are needed to examine the diversity of properties of this widespread family (12). Anabaena

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9. Q. Yang, M. P. Rout, C. W. Akey, Mol. Cell 1, 223 (1998). 10. C. W. Akey, M. Radermacher, J. Cell Biol. 122, 1 (1993). 11. K. Grunewald, O. Medalia, A. Gross, A. C. Steven, W. Baumeister, Biophys. Chem. 100, 577 (2003). 12. M. P. Rout et al., J. Cell Biol. 148, 635 (2000). 13. E. Kiseleva, M. W. Goldberg, T. D. Allen, C. W. Akey, J. Cell Sci. 111, 223 (1998). 14. N. Pante, U. Aebi, Science 273, 1729 (1996). 15. B. Fahrenkrog, U. Aebi, Nat. Rev. Mol. Cell Biol. 4, 757 (2003). 16. W. Yang, J. Gelles, S. M. Musser, Proc. Natl. Acad. Sci. U.S.A. 101, 12887 (2004). 17. J. P. Siebrasse, R. Peters, EMBO Rep. 3, 887 (2002). 18. We thank R. Hegerl for help with the image processing and A. Leis, V. Lucic, and P. Zwickl for critical reading of the manuscript.

Supporting Online Material www.sciencemag.org/cgi/content/full/1104808/DC1 Materials and Methods Figs. S1 to S3 References and Notes

3 September 2004; accepted 24 September 2004 Published online 28 October 2004; 10.1126/science.1104808 Include this information when citing this paper.

sensory rhodopsin, a recently discovered sensory representative outside of archaea (2), is well suited for exploration. It is the only bacterial sensory rhodopsin so far expressed in a photoactive form. Unlike the haloarchaeal sensory rhodopsins, which transmit signals to other integral membrane proteins, its function appears to involve modulation of a soluble cytoplasmic transducer, analogous to animal visual pigments (2). In this study, we report the structure of the retinal-complexed protein at 2.0 ) resolution, obtained by X-ray diffraction of crystals grown in a cubic lipid phase (table S1). The overall membrane-embedded seven-helical structure is similar to those of the archaeal rhodopsins. However, distinct differences in the photoactive site prompted analysis of the isomeric configuration of the retinal and the photochemical reactions of the pigment. Despite intense white-light illumination Elight adaptation (13)^ of the crystals before cryocooling and X-ray data collection, which results in a fully all-trans retinal configuration in bacteriorhodopsin, maps of the retinal and Schiff base region of Anabaena sensory rhodopsin show electron density incompati1

Department of Molecular Biology and Biochemistry, Department of Physiology and Biophysics and Department of Informatics and Computer Sciences, University of California, Irvine, CA 92697, USA. 3 Center for Membrane Biology, Department of Biochemistry and Molecular Biology, 4Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, TX 77030, USA. 5 Biology Department, Moscow State University, Moscow, Russia. 2

*To whom correspondence should be addressed. E-mail: [email protected] (H.L.) or john.l.spudich@ uth.tmc.edu (J.L.S.)

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that do not interact with the CP/T at this stage are variable in shape, while binding to cargo restricts their freedom of movement (14). The contour line view reveals that the narrowest constriction of the central channel is situated at the cytoplasmic side of the lumenal spoke ring in the CF class, even though it is at the nuclear side of the same ring in the LR class (Fig. 4B). These observations indicate that major rearrangements in the spokes might play a critical role in the translocation of cargo. Both classes represent major structural states of the NPC. Since the CP/T is better defined in the CF class, this state might represent the slow incorporation or release of cargo complexes into or from the FXFGframework residing in the central channel (15), which involves interaction with the cytoplasmic filaments. The more diffuse CP/ T of the LR class indicates that cargo complexes can be found in various positions once they have entered the channel Ein agreement with (16)^. Although an assignment of the classes to import, export or to