Cell-free reconstitution reveals centriole cartwheel assembly mechanisms.

How cellular organelles assemble is a fundamental question in biology. The centriole organelle organizes around a nine-fold symmetrical cartwheel structure typically ∼100 nm high comprising a stack of rings that each accommodates nine homodimers of SAS-6 proteins. Whether nine-fold symmetrical ring-like assemblies of SAS-6 proteins harbour more peripheral cartwheel elements is unclear. Furthermore, the mechanisms governing ring stacking are not known. Here we develop a cell-free reconstitution system for core cartwheel assembly. Using cryo-electron tomography, we uncover that the Chlamydomonas reinhardtii proteins CrSAS-6 and Bld10p together drive assembly of the core cartwheel. Moreover, we discover that CrSAS-6 possesses autonomous properties that ensure self-organized ring stacking. Mathematical fitting of reconstituted cartwheel height distribution suggests a mechanism whereby preferential addition of pairs of SAS-6 rings governs cartwheel growth. In conclusion, we have developed a cell-free reconstitution system that reveals fundamental assembly principles at the root of centriole biogenesis.

Unravelling the structure of the lamellipodium.

Summary Pushing at the cell front is the business of lamellipodia and understanding how lamellipodia function requires knowledge of their structural organization. Analysis of extracted, critical-point-dried cells by electron microscopy has led to a current dogma that the lamellipodium pushes as a branched array of actin filaments, with a branching angle of 70 degrees , defined by the Arp2/3 complex. Comparison of different preparative methods indicates that the critical-point-drying-replica technique introduces distortions into actin networks, such that crossing filaments may appear branched. After negative staining and from preliminary studies by cryo-electron tomography, no clear evidence could be found for actin filament branching in lamellipodia. From recent observations of a sub-class of actin speckles in lamellipodia that exhibit a dynamic behaviour similar to speckles in the lamella region behind, it has been proposed that the lamellipodium surfs on top of the lamella. Negative stain electron microscopy and cryo-electron microscopy of fixed cells, which reveal the entire complement of filaments in lamellipodia show, however, that there is no separate, second array of filaments beneath the lamellipodium network. From present data, we conclude that the lamellipodium is a distinct protrusive entity composed of a network of primarily unbranched actin filaments. Cryo-electron tomography of snap-frozen intact cells will be required to finally clarify the three-dimensional arrangement of actin filaments in lamellipodia in vivo.

Complex formation with kinesin motor domains affects the structure of microtubules.

Microtubules are highly dynamic components of the cytoskeleton. They are important for cell movement and they are involved in a variety of transport processes together with motor proteins, such as kinesin. The exact mechanism of these transport processes is not known and so far the focus has been on structural changes within the motor domains, but not within the underlying microtubule structure. Here we investigated the interaction between kinesin and tubulin and our experimental data show that microtubules themselves are changing structure during that process. We studied unstained, vitrified samples of microtubules composed of 15 protofilaments using cryo electron microscopy and helical image analysis. 3D maps of plain microtubules and microtubules decorated with kinesin have been reconstructed to approximately 17A resolution. The alphabeta-tubulin dimer could be identified and, according to our data, alpha- and beta-tubulin adopt different conformations in plain microtubules. Significant differences were detected between maps of plain microtubules and microtubule-kinesin complexes. Most pronounced is the continuous axial inter-dimer contact in the microtubule-kinesin complex, suggesting stabilized protofilaments along the microtubule axis. It seems, that mainly structural changes within alpha-tubulin are responsible for this observation. Lateral effects are less pronounced. Following our data, we believe, that microtubules play an active role in intracellular transport processes through modulations of their core structure.

Modulation of antigenicity related to changes in antibody flexibility upon lyophilization.

Lyophilization is frequently used to increase the shelf-life of biopharmaceuticals containing antibodies. A case in which an anti-idiotypic antibody, MMA 383, substantially lost its in vivo immunogenic properties although the protein was not degraded, is investigated. The scanning transmission electron microscope allowed the MMA 383 Fab and Fc moieties to be resolved. By averaging the single antibodies, the angle between the Fab moieties can be calculated. Non-lyophilized antibodies displayed a wider range of shapes than their reconstituted, lyophilized counterparts. Accordingly, the angle between the two Fab fragments varied more, indicating greater flexibility. The tryptophan steady-state fluorescence intensity, steady-state fluorescence anisotropy and fluorescence lifetime, were smaller for the lyophilized antibodies. These were also more resistant towards thermal denaturation/aggregation. Circular dichroism spectra detected temperature-dependent differences between the two antibody types in the 236 nm region. The subtle but reproducible structural changes induced by lyophilization may be related to the loss of in vivo immunogenic properties.

A new look at the microtubule binding patterns of dimeric kinesins.

The interactions of monomeric and dimeric kinesin and ncd constructs with microtubules have been investigated using cryo-electron microscopy (cryo-EM) and several biochemical methods. There is a good consensus on the structure of dimeric ncd when bound to a tubulin dimer showing one head attached directly to tubulin, and the second head tethered to the first. However, the 3D maps of dimeric kinesin motor domains are still quite controversial and leave room for different interpretations. Here we reinvestigated the microtubule binding patterns of dimeric kinesins by cryo-EM and digital 3D reconstruction under different nucleotide conditions and different motor:tubulin ratios, and determined the molecular mass of motor-tubulin complexes by STEM. Both methods revealed complementary results. We found that the ratio of bound kinesin motor-heads to alphabeta-tubulin dimers was never reaching above 1.5 irrespective of the initial mixing ratios. It appears that each kinesin dimer occupies two microtubule-binding sites, provided that there is a free one nearby. Thus the appearances of different image reconstructions can be explained by non-specific excess binding of motor heads. Consequently, the use of different apparent density distributions for docking the X-ray structures onto the microtubule surface leads to different and mutually exclusive models. We propose that in conditions of stoichiometric binding the two heads of a kinesin dimer separate and bind to different tubulin subunits. This is in contrast to ncd where the two heads remain tightly attached on the microtubule surface. Using dimeric kinesin molecules crosslinked in their neck domain we also found that they stabilize protofilaments axially, but not laterally, which is a strong indication that the two heads of the dimers bind along one protofilament, rather than laterally bridging two protofilaments. A molecular walking model based on these results summarizes our conclusions and illustrates the implications of symmetry for such models.

Op18/stathmin caps a kinked protofilament-like tubulin tetramer.

Oncoprotein 18/stathmin (Op18), a regulator of microtubule dynamics, was recombinantly expressed and its structure and function analysed. We report that Op18 by itself can fold into a flexible and extended alpha-helix, which is in equilibrium with a less ordered structure. In complex with tubulin, however, all except the last seven C-terminal residues of Op18 are tightly bound to tubulin. Digital image analysis of Op18:tubulin electron micrographs revealed that the complex consists of two longitudinally aligned alpha/beta-tubulin heterodimers. The appearance of the complex was that of a kinked protofilament-like structure with a flat and a ribbed side. Deletion mapping of Op18 further demonstrated that (i) the function of the N-terminal part of the molecule is to 'cap' tubulin subunits to ensure the specificity of the complex and (ii) the complete C-terminal alpha-helical domain of Op18 is necessary and sufficient for stable Op18:tubulin complex formation. Together, our results suggest that besides sequestering tubulin, the structural features of Op18 enable the protein specifically to recognize microtubule ends to trigger catastrophes.

Calcium-mediated structural changes of native nuclear pore complexes monitored by time-lapse atomic force microscopy.

Nuclear pore complexes (NPCs) are large macromolecular assemblies embedded in the double membrane nuclear envelope. They are the major gateways mediating transport of ions, small molecules, proteins, RNAs, and ribonucleoprotein particles in and out of the nucleus in interphase cells. Understanding structural changes at the level of individual pores will be a prerequisite to eventually correlate the molecular architecture of the NPC with its distinct functional states during nucleocytoplasmic transport. Toward this goal, we have employed time-lapse atomic force microscopy of native NPCs kept in buffer, and recorded calcium-mediated structural changes such as the opening (i.e. +Ca2+) and closing (i.e. -Ca2+) of individual nuclear baskets. Most likely, this structural change of the nuclear basket involves its distal ring which may act as an iris-like diaphragm. In order to directly correlate distinct structural features with corresponding functional states and dynamic aspects, we also addressed the question of whether the "central plug" or "transporter" actually represents a calcium-sensitive component of the NPC involved in mediating nucleocytoplasmic transport. Our data indicate that in the absence of ATP, cytoplasmic plugging/unplugging of the NPC is insensitive to calcium.

Reconstitution, characterisation and mass analysis of the pentacylindrical allophycocyanin core complex from the cyanobacterium Anabaena sp. PCC 7120.

The phycobilisome (PBS) of Anabaena sp. PCC 7120 was allowed to dissociate into its constituents and the resulting allophycocyanin (AP) fraction was purified. Its reconstitution yielded a complex which according to negative stain electron microscopy and spectral analysis was identical to the native pentacylindrical PBS core domain. Each cylinder of the central tricylindric unit was comprised of four AP (alphabeta)3 disks. Mass analysis using the scanning transmission electron microscope (STEM) showed the presence of 16 AP trimers in the intact reconstitute, which had a total mass of 1966(+/-66) kDa. Composition analysis indicated an AP trimer distribution of (AP-II):(AP-LCM):(AP-B):(AP-I)=6:2:2:6, i.e. an addition of two AP-I and two AP-II complexes compared to a tricylindrical PBS core domain. Therefore, we suggest that each supplementary half-core cylinder found in pentacylindrical AP core domains is comprised of one AP-I and one AP-II trimer, in agreement with the current model. The structural significance of the 127 kDa core membrane linker polypeptide was further investigated by subjecting the AP core reconstitute to mild chymotryptic degradation. After isolation, the digested complex exhibited a tricylindrical appearance while STEM mass analysis confirmed the presence of only 12 AP complexes. Polypeptide analysis by SDS-PAGE and Edman degradation related the half-cylinder loss to cleavage of the Rep4 domain of the core membrane linker polypeptide. On the basis of these data, a general model for the assembly of the three hemidiscoidal PBS types known to date is discussed.

Evaluating atomic models of F-actin with an undecagold-tagged phalloidin derivative.

We have prepared an undecagold-tagged phalloidin derivative to determine this mushroom toxin's binding site and orientation within the F-actin filament by scanning transmission electron microscopy (STEM) and 3-D helical reconstruction. Remarkably, when stoichiometrically bound to F-actin, the undecagold moiety of the derivative could be directly visualized by STEM along the two half-staggered long-pitch helical strands of single filaments. Most importantly, the structural data obtained when combined with various biochemical constraints enabled us to critically evaluate two distinct atomic models of the F-actin filament (i.e. the Holmes-Lorenz versus the Schutt-Lindberg model). Taken together, our data are in excellent agreement with the Holmes-Lorenz model.

A correlative analysis of actin filament assembly, structure, and dynamics.

The effect of the type of metal ion (i.e., Ca2+, Mg2+, or none) bound to the high-affinity divalent cation binding site (HAS) of actin on filament assembly, structure, and dynamics was investigated in the absence and presence of the mushroom toxin phalloidin. In agreement with earlier reports, we found the polymerization reaction of G-actin into F-actin filaments to be tightly controlled by the type of divalent cation residing in its HAS. Moreover, novel polymerization data are presented indicating that LD, a dimer unproductive by itself, does incorporate into growing F-actin filaments. This observation suggests that during actin filament formation, in addition to the obligatory nucleation- condensation pathway involving UD, a productive filament dimer, a facultative, LD-based pathway is implicated whose abundance strongly depends on the exact polymerization conditions chosen. The "ragged" and "branched" filaments observed during the early stages of assembly represent a hallmark of LD incorporation and might be key to producing an actin meshwork capable of rapidly assembling and disassembling in highly motile cells. Hence, LD incorporation into growing actin filaments might provide an additional level of regulation of actin cytoskeleton dynamics. Regarding the structure and mechanical properties of the F-actin filament at steady state, no significant correlation with the divalent cation residing in its HAS was found. However, compared to native filaments, phalloidin-stabilized filaments were stiffer and yielded subtle but significant structural changes. Together, our data indicate that whereas the G-actin conformation is tightly controlled by the divalent cation in its HAS, the F-actin conformation appears more robust than this variation. Hence, we conclude that the structure and dynamics of the Mg-F-actin moiety within the thin filament are not significantly modulated by the cyclic Ca2+ release as it occurs in muscle contraction to regulate the actomyosin interaction via troponin.

Polymorphic fibrillar assembly of human amylin.

Human amylin forms fibrillar amyloid between pancreatic islet cells in patients with non-insulin-dependent (type 2) diabetes mellitus. Fibrillar assemblies also form in vitro in aqueous solutions of synthetic human amylin. We now report on the structural polymorphism of these fibrils. The thinnest fibril, referred to as the protofibril, has an apparent width of 5 nm but is only rarely observed by itself. These protofibrils spontaneously assemble into higher order fibrillar structures with distinct morphologies. Prominent among these is an 8-nm fibril with a distinct 25-nm axial crossover repeat which is formed by left-handed coiling of two 5-nm protofibrils. Coiling of more than two 5-nm protofibrils results in cable-like structures of variable width depending on the number of protofibrils involved. Lateral (side-by-side) assembly of 5-nm protofibrils is also observed and produces ribbons which may contain two, three, four, or more protofibrils and occasionally large single-layered sheets. The mass-per-length (MPL) of the 5-nm protofibril is 10 kDa/nm. This has been established in two ways: first, the 8-nm fibril, which is formed by coiling two 5-nm protofibrils around each other, has an MPL of 20 kDa/nm. Second, higher order fibrils differ by increments of 10 kDa/nm. Hence, about 2.6 human amylin molecules (3904 Da) are packed in 1 nm of protofibril length. Similarities exist between amylin fibrils and those formed from other amyloid proteins, suggesting that the in vitro assembly of synthetic protein may serve as a useful model system in advancing our understanding of amyloid formation in disease.

Structure and assembly properties of the intermediate filament protein vimentin: the role of its head, rod and tail domains.

We have investigated the functional role of the non-helical end domains of vimentin on its assembly properties using truncated Xenopus and human recombinant proteins. Removal of the amino-terminal "head" domain yielded a molecule that did not assemble into 10 nm filaments but remained in a soluble oligomeric particle form with a sedimentation coefficient considerably smaller than that of wild-type vimentin (Vim(wt)). In contrast, removal of the carboxy-terminal "tail" domain had no obvious effect on the sedimentation characteristics. In particular, sedimentation equilibrium analysis under low ionic strength conditions yielded oligomeric particle species of Mr 135,000 to 360,000, indistinguishable from those obtained with Vim(wt). When induced to form filaments from this state by rapid dilution into filament forming buffer, Vim(wt) and Vim(deltaT) protein generated similar viscosity profiles. However, as determined by scanning transmission electron microscopy, under these conditions Vim(deltaT) formed filaments of heterogeneous diameter, corresponding to various distinct mass-per-length (MPL) values: whereas Vim(wt) yielded MPL values peaking between 40 and 45 kDa/nm, Vim(deltaT) filaments produced histograms which could be fitted by three Gaussian curves peaking between 37 and 131 kDa/nm. In contrast, when dialyzed against, instead of being rapidly diluted into, filament forming buffer, Vim(deltaT) gave histograms with one major peak at about 54 kDa/nm. The MPL heterogeneity observed for Vim(deltaT) was already evident at the earliest stages of assembly. For example, ten seconds after initiation, "unit-length" filament segments (58 to 63 nm) were formed with both wt and deltaT proteins, but the diameters were considerably larger for Vim(deltaT) compared to Vim(wt) (20(+/- 3) nm versus 16(+/- 3)nm), indicating a distinct role of the carboxy-terminal tail domain in the width control during unit-length filament formation. Despite this difference both Vim(deltaT) and Vim(wt) filaments appeared to grow stepwise in a modular fashion from such unit-length filament segments. This suggests that assembly occurred by a principally similar mechanism involving the end-on-fusion or annealing of unit-length filaments.

Functional significance of symmetrical versus asymmetrical GroEL-GroES chaperonin complexes.

The Escherichia coli chaperonin GroEL and its regulator GroES are thought to mediate adenosine triphosphate-dependent protein folding as an asymmetrical complex, with substrate protein bound within the GroEL cylinder. In contrast, a symmetrical complex formed between one GroEL and two GroES oligomers, with substrate protein binding to the outer surface of GroEL, was recently proposed to be the functional chaperonin unit. Electron microscopic and biochemical analyses have now shown that unphysiologically high magnesium concentrations and increased pH are required to assemble symmetrical complexes, the formation of which precludes the association of unfolded polypeptide. Thus, the functional significance of GroEL:(GroES)2 particles remains to be demonstrated.

Towards atomic interpretation of F-actin filament three-dimensional reconstructions.

We have recorded dark field images of negatively stained F-actin filaments polymerized with 2 mM MgCl2 and 50 mM KCl with a scanning transmission electron microscope and computed 3-D reconstructions using a helical parameter search to optimize simultaneously the helical repeat length, the radial position of the filament axis, and the helical selection rule. The resulting optimized averaged filament 3-D reconstruction at 2.5 nm resolution is remarkably similar to an atomic model of the F-actin filament. By comparison, several structural features of the reconstruction can be interpreted at the level of distinct secondary structure elements, and predictions made by the atomic model could be verified: for instance, the density connecting the two long-pitch helical strands in our reconstruction co-localizes with an extended beta-hairpin, the "hydrophobic loop" (i.e. residues 262 to 274), which according to the atomic model establishes the major intersubunit contact between the two long-pitch helical strands. The most pronounced structural variations among individual filament 3-D reconstructions were observed in (1) the details of the intersubunit contact pattern between the two long-pitch helical strands, and (2) the exact size and shape of subdomain 2 of the F-actin molecule, which appears rather flexible and easily deformed. In addition, we found that all phenotypes of F-actin filament 3-D reconstructions that arise from small deviations from the optimal helical parameters or from lowering the nominal resolution exhibited stronger intersubunit contacts between than along the two long-pitch helical strands, a structural feature that has been emphasized for a number of F-actin filament 3-D reconstructions in the past. Since this is clearly at variance with the relative strength of the intersubunit contacts as predicted by the atomic model, it may represent an artifactual structural feature arising from low-resolution data or suboptimal helical data processing, and should therefore be interpreted with caution in terms of indicating chemical, mechanical or conformational states of the F-actin filament.

Topology of the morphological domains of the chaperonin GroEL visualized by immuno-electron microscopy.

Electron microscopy of the tetradecameric double-ring complex of GroEL reveals a four-layered structure, indicating that the 58 kDa subunits are composed of two major morphological domains. We have used immuno-electron microscopy to assign these domains to the corresponding segments of the GroEL sequence. Upon chemical modification of GroEL with N-ethylmaleimide, protease treatment in the presence of ATP or ADP generates GroEL fragments of 15 kDa (N15; residues 1-141) and 40 kDa (C40; residues 153-531). As visualized by scanning transmission electron microscopy, affinity-purified antibodies directed against C40 recognize the outer layers, whereas antibodies against N15 interact with the equatorial portions of the GroEL double-ring. Thus, the two major domains of the subunits in the chaperonin complex are arranged in the order C40-N15:N15-C40. The single-ring chaperonin co-factor GroES interacts with the C40 domain while the ATP-binding site of GroEL is probably close to the junction between N15 and C40.

Structure of selenocysteine synthase from Escherichia coli and location of tRNA in the seryl-tRNA(sec)-enzyme complex.

Selenocysteine synthase of Escherichia coli catalyses the biosynthesis of selenocysteine in the form of the aminoacyl-tRNA complex, the reaction intermediate being aminoacrylyl-tRNA(sec) covalently bound to the prosthetic group of the enzyme. Selenocysteine synthase and the specific aminoacrylyl-tRNA(sec)-enzyme complex as well as the isolated seryl-tRNA(sec) were investigated in the electron microscope and analysed by means of image processing to a resolution of 2 nm in projection. The stoichiometric composition of the selenocysteine synthase molecule was elucidated by scanning transmission electron microscopic mass determination. The enzyme has a fivefold symmetric structure and consists of 10 monomers arranged in two rings. The tRNA is bound near the margin of the dimeric subunits. Principal component analysis of the tRNA-enzyme complexes revealed that the selenocysteine synthase appears to bind only one seryl-tRNA(sec) per dimer, which is consistent with the result of biochemical binding studies.