Architecture of the Xenopus nuclear pore complex revealed by three- dimensional cryo-electron microscopy

The nuclear pore complex spans the nuclear envelope and functions as a macromolecular transporter in the ATP-dependent process of nucleocytoplasmic transport. In this report, we present three dimensional (3D) structures for both membrane-associated and detergent- extracted Xenopus NPCs, imaged in frozen buffers by cryo-electron microscopy. A comparison of the differing configurations present in the 3D maps suggests that the spokes may possess an intrinsic conformational flexibility. When combined with recent data from a 3D map of negatively stained NPCs (Hinshaw, J. E., B. O. Carragher, and R. A. Milligan. 1992. Cell. 69:1133-1141), these observations suggest a minimal domain model for the spoke-ring complex which may account for the observed plasticity of this assembly. Moreover, lumenal domains in adjacent spokes are interconnected by radial arm dimers, forming a lumenal ring that may be responsible for anchoring the NPC within the nuclear envelope pore. Importantly, the NPC transporter is visualized as a centrally tapered cylinder that spans the entire width of the NPC, in a direction normal to the nuclear envelope. The central positioning, tripartite structure, and hollow nature of the transporter suggests that it may form a macromolecular transport channel, with a globular gating domain at each end. Finally, the packing of the transporter within the spokes creates a set of eight internal channels that may be responsible, in part, for the diffusion of ions and small molecules across the nuclear envelope.     

Localization of calmodulin and dynein light chain LC8 in flagellar radial spokes

Genetic and in vitro analyses have revealed that radial spokes play a crucial role in regulation of ciliary and flagellar motility, including control of waveform. However, the mechanisms of regulation are not understood. Here, we developed a novel procedure to isolate intact radial spokes as a step toward understanding the mechanism by which these complexes regulate dynein activity. The isolated radial spokes sediment as 20S complexes that are the size and shape of radial spokes. Extracted radial spokes rescue radial spoke structure when reconstituted with isolated axonemes derived from the radial spoke mutant pf14. Isolated radial spokes are composed of the 17 previously defined spoke proteins as well as at least five additional proteins including calmodulin and the ubiquitous dynein light chain LC8. Analyses of flagellar mutants and chemical cross-linking studies demonstrated calmodulin and LC8 form a complex located in the radial spoke stalk. We postulate that calmodulin, located in the radial spoke stalk, plays a role in calcium control of flagellar bending.     

Global Motions of the Nuclear Pore Complex: Insights from Elastic Network Models

The nuclear pore complex (NPC) is the gate to the nucleus. Recent determination of the configuration of proteins in the yeast NPC at ∼5 nm resolution permits us to study the NPC global dynamics using coarse-grained structural models. We investigate these large-scale motions by using an extended elastic network model (ENM) formalism applied to several coarse-grained representations of the NPC. Two types of collective motions (global modes) are predicted by the ENMs to be intrinsically favored by the NPC architecture: global bending and extension/contraction from circular to elliptical shapes. These motions are shown to be robust against tested variations in the representation of the NPC, and are largely captured by a simple model of a toroid with axially varying mass density. We demonstrate that spoke multiplicity significantly affects the accessible number of symmetric low-energy modes of motion; the NPC-like toroidal structures composed of 8 spokes have access to highly cooperative symmetric motions that are inaccessible to toroids composed of 7 or 9 spokes. The analysis reveals modes of motion that may facilitate macromolecular transport through the NPC, consistent with previous experimental observations.     

On the universality of nuclear pore complex structure

Summary Substructural details of the nuclear pore complex were studied in diverse plant and animal cells with both section technique and negative staining of isolated nuclear envelope pieces. The structures observed after the different techniques, including a variety of fixation procedures, are compared and their significance is discussed. It is shown that, down to the 15–20 Å level, the architecture of the nuclear pore complex is universal among such diverse cell types as from, e. g., onion root tips, bean leaves, mammalian liver parenchyma, HeLa cell cultures, and amphibian germ material. The fundamental substructures of the pore complex such as (1) the annular granules, (2) the fibrils attached to the annuli, (3) the central granules, (4) the fibrils in the pore interior including those which make up the inner ring and/or those which connect the central granule to the pore margin, are recognized in all cell types studied. The dynamic variability of the central granule morphology is emphasized and observations are presented which suggest that the view of such centrally located material as representing ribonucleoproteins in a transitory state of nucleocytoplasmic migration can be extended to generality. General concepts of the nuclear pore complex structure are presented as alternative model views revealing either a more compact, predominantly granular, or a more fibrillar aspect.The author gratefully acknowledges the frequent discussions and cooperation with his team-colleagues Drs. H. Falk (in the work on leaf material) and U. Scheer (in working with amphibian oocytes) as well as the skillful technical assistance of Miss Marianne Winter and Miss Sigrid Krien. The work was supported by the Deutsche Forschungsgemeinschaft.     

Interactions and structure of the nuclear pore complex revealed by cryo- electron microscopy

Nuclear pore complexes (NPCs) play a central role in mediating nucleocytoplasmic transport and exchange processes in eukaryotic cells. The arrangement and interactions of NPCs within amphibian nuclear envelopes have been studied using cryo-electron microscopy of unfixed and frozen hydrated specimens. The nuclear lamina in Necturus forms an orthogonal network with crossover distances which vary between 1,600 and 4,000 A and which may be related to the basic filament repeat of lamins. Furthermore, the NPCs are attached randomly within the confines of the lamin network, presumably by their nucleoplasmic rings. Image analysis of edge-on and en face projections of detergent-extracted NPCs has been combined with data on the coaxial thin rings to provide a quantitative evaluation of the triple ring model of NPC architecture proposed previously (Unwin, P. N. T., and R. Milligan. 1982. J. Cell Biol. 93:63-75). Additional details of the complex have been visualized including an intimate association of the inner spoke domains as an inner spoke ring, extensive domains within the spokes and coaxial thin rings, and interestingly, a central channel-like feature. Membrane-associated NPCs and detergent-extracted NPCs both possess peripherally located radial arms resulting in an effective diameter of approximately 1,450-1,500 A. In projection, the radial arms possess approximate mirror symmetry suggesting that they originate from both sides of the assembly. Moreover, membrane-associated NPCs are asymmetric at most radii and right-handed as viewed from the cytoplasm; detergent-extracted NPCs appear to be symmetric and have approximately 822 symmetry. Taken together, the data suggests that the framework of membrane-associated NPCs is perturbed from a symmetrical configuration, either during isolation of nuclei or by interactions with the lamina and the nuclear envelope in vivo. However, detergent extraction of nuclei appears to result in a more symmetrical alignment of components in apposing halves of the assembly.     

Appearance of nuclear pore complexes after Bernhard's staining procedure

Summary Bernhard's electron microscopical differential staining procedure for nucleoproteins (1969) was employed with plant (onion root tip) and animal (HeLa) material for a detailed study of the nuclear pore complex structures. All non-membraneous components such as the annular and the inner pore material including the central granule and the pore-associated fibrils are strongly stained in contrast to the adjacent chromatin. No difference in staining behaviour was observed between the different components. In general, however, the pore complex structures showed slightly less contrast than the cytoplasmic ribosomes and the nucleolar particles with which they appear to be connected in fibrillar continuity. Although this preferential staining speaks against a considerable deoxyribonucleoprotein content of the structures in question, it cannot serve to differentiate between ribonucleoproteins and pure protein structures.The work was supported in part by the Deutsche Forschungsgemeinschaft.The authors are indebted to Miss Sigrid Krien for careful technical assistance as well as to Dr. Hans Kleinig for help with the HeLa material.     

Dimeric novel HSP40 is incorporated into the radial spoke complex during the assembly process in flagella

The radial spoke is a stable structural complex in the 9 + 2 axoneme for the control of flagellar motility. However, the spokes in Chlamydomonas mutant pf24 are heterogeneous and unstable, whereas several spoke proteins are reduced differentially. To elucidate the defective mechanism, we clone RSP16, a prominent spoke protein diminished in pf24 axonemes. Unexpectedly, RSP16 is a novel HSP40 member of the DnaJ superfamily that assists chaperones in various protein-folding-related processes. Importantly, RSP16 is uniquely excluded from the 12S spoke precursor complex that is packaged in the cell body and transported toward the flagellar tip to be converted into mature 20S axonemal spokes. Rather, RSP16, transported separately, joins the precursor complex in flagella. Furthermore, RSP16 molecules in vitro and in flagella form homodimers, a characteristic required for the cochaperone activity of HSP40. We postulate that the spoke HSP40 operates as a cochaperone to assist chaperone machinery at the flagellar tip to actively convert the smaller spoke precursor and itself into the mature stable complex; failure of the interaction between the spoke HSP40 and its target polypeptide results in heterogeneous unstable radial spokes in pf24.     

Substructure of inner dynein arms, radial spokes, and the central pair/projection complex of cilia and flagella

The substructure of the components of the axoneme interior--the inner dynein arms, the radial spokes, and the central pair/projection complex--was analyzed for Chlamydomonas. Tetrahymena, Strongelocentrotus, and Mnemiopsis using the quick-freeze, deep-etch technique. The inner arms are shown to resemble the outer arms in overall molecular organization, but they are disposed differently on the microtubule and have two distinct morphologies--dyads with two heads and triads with three. The dyads associate with spokes S3 and S2; the triads associate with S1. The spokes form a three-start right-handed helix with a 288-nm rise; the central pair makes a shallow left-handed twist. The spoke heads are shown to be made up of four major subunits; two bind to the spoke shaft and two bind to a pair of central-sheath projections.     

Two anti-radial spoke monoclonal antibodies inhibit Chlamydomonas axonemal motility by different mechanisms

In the 9 + 2 axoneme, radial spokes are structural components attached to the A-tubules of the nine outer doublet microtubules. They protrude toward the central pair microtubule complex with which they have transient but regular interactions for the normal flagellar motility to occur. Flagella of Chlamydomonas mutants deficient in entire radial spokes or spoke heads are paralyzed. In this study the importance of two radial spoke proteins in the flagellar movement is exemplified by the potent inhibitory action of two monoclonal antibodies on the axonemal motility of demembranated-reactivated Chlamydomonas models. We show that one of these proteins is localized on the stalk of the radial spokes, whereas the other is a component of the head of the same structure and most likely correspond to radial spoke protein 2 and 1, respectively. Fine motility analysis by videomicrography further indicates that these two anti-radial spoke protein antibodies at low concentration affect motility of demembranated-reactivated Chlamydomonas by changing the flagellar waveform without modifying axonemal beat frequency. They also modify wave amplitude differently during motility inhibition. This brings more direct evidence for the involvement of both radial spoke stalk and head in the fine tuning of the waveform during flagellar motility.     

Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella

Radial spokes (RSs) play an essential role in the regulation of axonemal dynein activity and thus of ciliary and flagellar motility. However, few details are known about the complexes involved. Using cryo-electron tomography and subtomogram averaging, we visualized the three-dimensional structure of the radial spokes in Chlamydomonas flagella in unprecedented detail. Unlike many other species, Chlamydomonas has only two spokes per axonemal repeat, RS1 and RS2. Our data revealed previously uncharacterized features, including two-pronged spoke bases that facilitate docking to the doublet microtubules, and that inner dyneins connect directly to the spokes. Structures of wild type and the headless spoke mutant pf17 were compared to define the morphology and boundaries of the head, including a direct RS1-to-RS2 interaction. Although the overall structures of the spokes are very similar, we also observed some differences, corroborating recent findings about heterogeneity in the docking of RS1 and RS2. In place of a third radial spoke we found an uncharacterized, shorter electron density named "radial spoke 3 stand-in," which structurally bears no resemblance to RS1 and RS2 and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes are heterogeneous in structure and may play functionally distinct roles in axoneme regulation.     

THE STRUCTURAL BASIS OF CILIARY BEND FORMATION : Radial Spoke Positional Changes Accompanying Microtubule Sliding

The sliding microtubule model of ciliary motility predicts that cumulative local displacement (Δl) of doublet microtubules relative to one another occurs only in bent regions of the axoneme. We have now tested this prediction by using the radial spokes which join the A subfiber of each doublet to the central sheath as markers of microtubule alignment to measure sliding displacements directly. Gill cilia from the mussel Elliptio complanatus have radial spokes lying in groups of three which repeat at 860 Å along the A subfiber. The spokes are aligned with the two rows of projections along each of the central microtubules that form the central sheath. The projections repeat at 143 Å and form a vernier with the radial spokes in the precise ratio of 6 projection repeats to 1 spoke group repeat. In straight regions of the axoneme, either proximal or distal to a bend, the relative position of spoke groups between any two doublets remains constant for the length of that region. However, in bent regions, the position of spoke groups changes systematically so that Δl (doublet 1 vs. 5) can be seen to accumulate at a maximum of 122 Å per successive 860-Å spoke repeat. Local contraction of microtubules is absent. In straight regions of the axoneme, the radial spokes lie in either of two basic configurations: (a) the parallel configuration where spokes 1–3 of each group are normal (90°) to subfiber A, and (b) the tilted spoke 3 configuration where spoke 3 forms an angle (θ) of 9–20°. Since considerable sliding of doublets relative to the central sheath (~650 Å) has usually occurred in these regions, the spokes must be considered, functionally, as detached from the sheath projections. In bent regions of the axoneme, two additional spoke configurations occur where all three spokes of each group are tilted to a maximum of ± 33° from normal. Since the spoke angles do not lie on radii through the center of bend curvature, and Δl accumulates in the bend, the spokes must be considered as attached to the sheath when bending occurs. The observed radial spoke configurations strongly imply that there is a precise cycle of spoke detachment-reattachment to the central sheath which we conclude forms the main part of the mechanism converting active interdoublet sliding into local bending.     

The ultrastructure of the nuclear envelope of amphibian oocytes

Summary In order to investigate the chemical composition of the nuclear pore complexes isolated nuclei from matureXenopus laevis oocytes were manually fractioned into nucleoplasmic aggregates and the nuclear envelopes. The whole isolation procedure takes no more than 60–90 sec, and the pore complexes of the isolated envelopes are well preserved as demonstrated by electron microscopy. Minor nucleoplasmic and cytoplasmic contaminations associated with the isolated nuclear envelopes were determined with electron microscopic morphometry and were found to be quantitatively negligible as far as their mass and nucleic acid content is concerned. The RNA content of the fractions was determined by direct phosphorus analysis after differential alkaline hydrolysis. Approximately 9% of the total nuclear RNA of the matureXenopus egg was found to be attached to the nuclear envelope. The nonmembranous elements of one pore complex contain 0.41×10^–16 g RNA. This value agrees well with the content estimated from morphometric data. The RNA package density in the pore complexes (270×10^–15 g/³) is compared with the nucleolar, nucleoplasmic and cytoplasmic RNA concentration and is discussed in context with the importance of the pore complexes for the nucleo-cytoplasmic transport of RNA-containing macromolecules.Additionally, the results of the chemical analyses as well as of the³H-actinomycin D autoradiography and of the nucleoprotein staining method of Bernhard (1969) speak against the occurence of considerable amounts of DNA in the nuclear pore complex structures.The author thanks Miss Ulrika Lempert, Miss Marianne Winter, and Miss Sigrid Krien for skilful technical help as well as Dr. W. W. Franke for many helpful discussions. The work has been supported by a Deutsche Forschungsgemeinschaft grant given to Dr. W. W. Franke (SFB Molgrudent, 46).     

The CSC proteins FAP61 and FAP251 build the basal substructures of radial spoke 3 in cilia

Dynein motors and regulatory complexes repeat every 96 nm along the length of motile cilia. Each repeat contains three radial spokes, RS1, RS2, and RS3, which transduct signals between the central microtubules and dynein arms. Each radial spoke has a distinct structure, but little is known about the mechanisms of assembly and function of the individual radial spokes. In Chlamydomonas, calmodulin and spoke-associated complex (CSC) is composed of FAP61, FAP91, and FAP251 and has been linked to the base of RS2 and RS3. We show that in Tetrahymena, loss of either FAP61 or FAP251 reduces cell swimming and affects the ciliary waveform and that RS3 is either missing or incomplete, whereas RS1 and RS2 are unaffected. Specifically, FAP251-null cilia lack an arch-like density at the RS3 base, whereas FAP61-null cilia lack an adjacent portion of the RS3 stem region. This suggests that the CSC proteins are crucial for stable and functional assembly of RS3 and that RS3 and the CSC are important for ciliary motility.     

Nuclear surface complex as observed with the high resolution scanning electron microscope. Visualization of the membrane surfaces of the neclear envelope and the nuclear cortex from Xenopus laevis oocytes

The nuclear envelope and associated structures from Xenopus laevis oocytes (stage VI) have been examined with the high resolution scanning electron microscope (SEM). The features of the inner and outer surfaces of the nuclear surface complex were revealed by manual isolation , whereas the membranes facing the perinuclear space (the space between the inner and outer nuclear membranes) were observed by fracturing the nuclear envelope in this plane and splaying the corresponding regions apart. Pore complexes were observed on all four membrane surfaces of this double-membraned structure. The densely packed pore complexes (55/micron2) are often clustered into triplets with shared walls (outer diameter = 90 nm; inner diameter = 25 nm; wall thickness = aproximately 30 nm), and project aproximately 20 nm above each membrane except where they are flush with the innermost surface. The pore complex appears to be an aggregate of four 30-nm subunits. The nuclear cortex, a fibrous layer (300 nm thickness) associated with the inner surface of the nuclear envelope, has been revealed by rapid fixation. This cortical layer is interrupted by funnel-shaped intranuclear channels (120-640 nm diam) which narrow towards the pore complexes. Chains of particles, arranged in spirals, are inserted into these intranuclear channels. The fibers associated with the innermost face of the nuclear envelope can be extraced with 0.6 MKI to reveal the pore complexes. A model of the nuclear surface complex, compiled from the visualization of all the membrane faces and the nuclear cortex, demonstrates relations between the intranuclear channels (3.2/micron2) and the numerous pore complexes, and the possibility of their role in nucleocytoplasmic interactions.     

Asymmetric arrangement of auxiliary subunits of skeletal muscle voltage-gated l-type Ca(2+) channel

Highly purified L-type Ca(2+) channel complexes containing all five subunits (alpha(1), alpha(2), beta, gamma, and delta) and complexes of alpha(1)-beta subunits were obtained from skeletal muscle triad membranes by three-step purification and by 1% Triton X-100 treatment, respectively. Their structures and the subunit arrangements were analyzed by electron microscopy. Projection images of negatively stained Ca(2+) channels and alpha(1)-beta complexes were aligned, classified and averaged. The alpha(1)-beta complex showed a hollow trapezoid shape of 12 nm height. In top view, four asymmetric domains surrounded a central depression predicted to form the channel pore. The complete Ca(2+) channel complex exhibited the cylindrical shape of 20 nm in height binding a spherical domain on one edge. Further image analysis of higher complexes of the Ca(2+) channel using a monoclonal antibody against the beta subunit showed that the alpha(1)-beta complex forms the non-decorated side of the cylinder, which can traverse the membrane from outside the cell to the cytoplasm. Based on these results, we propose that the Ca(2+) channel exhibits an asymmetric arrangement of auxiliary subunits.     

Architecture and design of the nuclear pore complex.

A three-dimensional analysis of the nuclear pore complex reveals the underlying, highly symmetric framework of this supramolecular assembly, how it is anchored in the nuclear membrane, and how it is built from many distinct, interconnected subunits. The arrangement of the subunits within the membrane pore creates a large central channel, through which active nucleocytoplasmic transport is known to occur, and eight smaller peripheral channels that are probable routes for passive diffusion of ions and small molecules.     

Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.     

Bend propagation drives central pair rotation in Chlamydomonas reinhardtii flagella

Regulation of motile 9+2 cilia and flagella depends on interactions between radial spokes and a central pair apparatus. Although the central pair rotates during bend propagation in flagella of many organisms and rotation correlates with a twisted central pair structure, propulsive forces for central pair rotation and twist are unknown. Here we compared central pair conformation in straight, quiescent flagella to that in actively beating flagella using wild-type Chlamydomonas reinhardtii and mutants that lack radial spoke heads. Twists occur in quiescent flagella in both the presence and absence of spoke heads, indicating that spoke--central pair interactions are not needed to generate torque for twisting. Central pair orientation in propagating bends was also similar in wild type and spoke head mutant strains, thus orientation is a passive response to bend formation. These results indicate that bend propagation drives central pair rotation and suggest that dynein regulation by central pair--radial spoke interactions involves passive central pair reorientation to changes in bend plane.     

The CSC is required for complete radial spoke assembly and wild-type ciliary motility

The ubiquitous calcium binding protein, calmodulin (CaM), plays a major role in regulating the motility of all eukaryotic cilia and flagella. We previously identified a CaM and Spoke associated Complex (CSC) and provided evidence that this complex mediates regulatory signals between the radial spokes and dynein arms. We have now used an artificial microRNA (amiRNA) approach to reduce expression of two CSC subunits in Chlamydomonas. For all amiRNA mutants, the entire CSC is lacking or severely reduced in flagella. Structural studies of mutant axonemes revealed that assembly of radial spoke 2 is defective. Furthermore, analysis of both flagellar beating and microtubule sliding in vitro demonstrates that the CSC plays a critical role in modulating dynein activity. Our results not only indicate that the CSC is required for spoke assembly and wild-type motility, but also provide evidence for heterogeneity among the radial spokes.