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Ueli Aebi

Publications and source records attributed to Ueli Aebi.

At least 19 recordsLinked to original sources
PubMed2003

Cryo-electron tomography provides novel insights into nuclear pore architecture: implications for nucleocytoplasmic transport.

To go beyond the current structural consensus model of the nuclear pore complex (NPC), we performed cryo-electron tomography of fully native NPCs from Xenopus oocyte nuclear envelopes (NEs). The cytoplasmic face of the NPC revealed distinct anchoring sites for the cytoplasmic filaments, whereas the nuclear face was topped with a massive distal ring positioned above the central pore with indications of the anchoring sites for the nuclear basket filaments and putative intranuclear filaments. The rather "spongy" central framework of the NPC was perforated by an elaborate channel and void system, and at the membrane pore interface it exhibited distinct "handles" protruding into the lumen of the NE. The most variable structural moiety of the NPC was a rather tenuous central plug partially obstructing the central pore. Its mobile character was documented by time-lapse atomic force microscopy. Taken together, the new insights we gained into NPC structure support the notion that the NPC acts as a constrained diffusion pore for molecules and particles without retention signal and as an affinity gate for signal-bearing cargoes.

Active Transport, Cell Nucleus↗
PubMed2003

Full-length rat amylin forms fibrils following substitution of single residues from human amylin.

Pancreatic amyloid deposits, composed of the 37 amino acid residue peptide amylin, represent an integral part of type 2 diabetes mellitus pathology. Human amylin (hA) forms fibrils in vitro and is toxic to cultured pancreatic islet beta-cells. In contrast, rat amylin (rA) which differs from hA by only six amino acid residues in the central region of the peptide, residues 18-29, does not form fibrils and is not cytotoxic. To elucidate the role of individual residues in fibril formation, we have generated a series of full-length rA variants and examined their ability to form fibrils in vitro. Single-residue substitutions with amino acids from corresponding positions of the hA sequence, i.e. R18H, L23F, or V26I, were sufficient to render rA competent for fibril formation albeit at a small yield. Combining two or three of these substitutions generally increased the ability to produce fibrils. Variant rA fibril morphologies were examined by negative stain electron microscopy and found to be similar to those generated by hA itself. Bulk assays, i.e. involving thioflavin-T fluorescence and sedimentation, showed that the amount of fibril formation was relatively small for these rA variants when compared to hA under the same conditions. Fibril growth was demonstrated by time-lapse atomic force microscopy, and MALDI-TOF mass spectrometry was used to verify that fibrils consisted of full-length peptide. Our observations confirm previous reports that the three proline residues play a dominant negative role in fibril formation. However, their presence is not sufficient to completely abolish the ability of rA to form fibrils, as each of the other three implicated residues (i.e. R18, L23 and V26) also has a dominant modulating effect.

Amino Acid Sequence↗
PubMed2003

The single nuclear lamin of Caenorhabditis elegans forms in vitro stable intermediate filaments and paracrystals with a reduced axial periodicity.

The lamins of the tunicate Ciona intestinalis and the nematode Caenorhabditis elegans show unusual sequence features when compared to the more than 35 metazoan lamin sequences currently known. We therefore analyzed the in vitro assembly of these two lamins by electron microscopy using chicken lamin B2 as a control. While lamin dimers usually appear as a rod carrying two globules at one end, these globules are absent from Ciona lamin, which lacks the central 105-residue region of the tail domain. The deletion of 14 residues or two heptads from the coiled coil rod domain of the single C.elegans lamin results in a 1.5-nm shortening of the dimer rod. Similarly, the paracrystals assembled from the C.elegans lamin exhibit a 3.1-nm reduction of the true axial repeat compared to that of chicken lamin B2 paracrystals. We speculate that the banding pattern in the C.elegans lamin paracrystals arises from a relative stagger between dimers and/or a positioning of the globular tail domain relative to the central rod that is distinct from that observed in chicken lamin B2 paracrystals. Here we show that a nuclear lamin can assemble in vitro into 10-nm intermediate filaments (IFs). C.elegans lamin in low ionic strength Tris-buffers at a pH of 7.2-7.4 provides a stable population of lamin IFs. Some implications of this filament formation are discussed.

Amino Acid Sequence↗
PubMed2003

Molecular architecture of intermediate filaments.

Together with microtubules and actin microfilaments, approximately 11 nm wide intermediate filaments (IFs) constitute the integrated, dynamic filament network present in the cytoplasm of metazoan cells. This network is critically involved in division, motility and other cellular processes. While the structures of microtubules and microfilaments are known in atomic detail, IF architecture is presently much less understood. The elementary 'building block' of IFs is a highly elongated, rod-like dimer based on an alpha-helical coiled-coil structure. Assembly of cytoplasmic IF proteins, such as vimentin, begins with a lateral association of dimers into tetramers and gradually into the so-called unit-length filaments (ULFs). Subsequently ULFs start to anneal longitudinally, ultimately yielding mature IFs after a compaction step. For nuclear lamins, however, assembly starts with a head-to-tail association of dimers. Recently, X-ray crystallographic data were obtained for several fragments of the vimentin dimer. Based on the dimer structure, molecular models of the tetramer and the entire filament are now a possibility.

Amino Acid Sequence↗
PubMed2003

Functional complexity of intermediate filament cytoskeletons: from structure to assembly to gene ablation.

The cell biology of intermediate filament (IF) proteins and their filaments is complicated by the fact that the members of the gene family, which in humans amount to at least 65, are differentially expressed in very complex patterns during embryonic development. Thus, different tissues and cells express entirely different sets and amounts of IF proteins, the only exception being the nuclear B-type lamins, which are found in every cell. Moreover, in the course of evolution the individual members of this family have, within one species, diverged so much from each other with regard to sequence and thus molecular properties that it is hard to envision a unifying kind of function for them. The known epidermolytic diseases, caused by single point mutations in keratins, have been used as an argument for a role of IFs in mechanical "stress resistance," something one would not have easily ascribed to the beaded chain filaments, a special type of IF in the eye lens, or to nuclear lamins. Therefore, the power of plastic dish cell biology may be limited in revealing functional clues for these structural elements, and it may therefore be of interest to go to the extreme ends of the life sciences, i.e., from the molecular properties of individual molecules including their structure at the atomic level to targeted inactivation of their genes in living animals, mouse, and worm to define their role more precisely in metazoan cell physiology.

Animals↗
PubMed2003

The next ice age: cryo-electron tomography of intact cells.

Recent advances in electron tomography are beginning to reveal the internal structure of eukaryotic cells in their native states in three dimensions at molecular resolution. These observations represent the culmination of years of effort to develop protocols for automated data collection, image reconstruction and cryogenic preservation. Cryo-tomograms of Dictyostelium cells depict distinct populations of ribosomes, proteasomes and networks of actin filaments interconnected by branching or bundling, apparently controlled by strategically placed actin-associated proteins.

Animals↗
PubMed2003

Sac3 is an mRNA export factor that localizes to cytoplasmic fibrils of nuclear pore complex.

In eukaryotes, mRNAs are transcribed in the nucleus and exported to the cytoplasm for translation to occur. Messenger RNAs complexed with proteins referred to as ribonucleoparticles are recognized for nuclear export in part by association with Mex67, a key Saccharomyces cerevisiae mRNA export factor and homolog of human TAP/NXF1. Mex67, along with its cofactor Mtr2, is thought to promote ribonucleoparticle translocation by interacting directly with components of the nuclear pore complex (NPC). Herein, we show that the nuclear pore-associated protein Sac3 functions in mRNA export. Using a mutant allele of MTR2 as a starting point, we have identified a mutation in SAC3 in a screen for synthetic lethal interactors. Loss of function of SAC3 causes a strong nuclear accumulation of mRNA and synthetic lethality with a number of mRNA export mutants. Furthermore, Sac3 can be coimmunoprecipitated with Mex67, Mtr2, and other factors involved in mRNA export. Immunoelectron microscopy analysis shows that Sac3 localizes exclusively to cytoplasmic fibrils of the NPC. Finally, Mex67 accumulates at the nuclear rim when SAC3 is mutated, suggesting that Sac3 functions in Mex67 translocation through the NPC.

Biological Transport↗
PubMed2002

Exploring the 3D molecular architecture of Escherichia coli type 1 pili.

An integrated approach combining information gained by Fourier transformation, linear Markham superposition (real space) and mass-per-length measurement by scanning transmission electron microscopy was used to analyze the helical structure of the rod-like type 1 pili expressed by uropathogenic Escherichia coli strain W3110. The 3D reconstruction calculated from the experimental data showed the pili to be 6.9nm wide, right-handed helical tubes with a 19.31(+/-0.34)nm long helical repeat comprising 27 FimA monomers associated head-to-tail in eight turns of the genetic one-start helix. Adjacent turns of the genetic helix are connected via three binding sites making the pilus rod rather stiff. In situ immuno-electron microscopy experiments showed the minor subunit (FimH) mediating pilus adhesion to bladder epithelial cells to be the distal protein of the pilus tip, which had a spring-like appearance at higher magnification. The subunits FimG and FimF connect FimH to the FimA rod, the sequential orientation being FimA-FimF-FimG-FimH. The electron density map calculated at 18A resolution from an atomic model of the pilus rod (built using the pilin domain FimH together with the G1 strand of FimC as a template for FimA and applying the optimal helical parameters determined to the head-to-tail interaction model for pilus assembly) was practically identical with that of the actual 3D reconstruction.

Adhesins, Escherichia coli↗
PubMed2002

Actin's propensity for dynamic filament patterning.

Actin, through its various forms of assembly, provides the basic framework for cell motility, cell shape and intracellular organization in all eukaryotic cells. Many other cellular processes, for example endocytosis and cytokinesis, are also associated with dynamic changes of the actin cytoskeleton. Important prerequisites for actin's functional diversity are its intrinsic ability to rapidly assemble and disassemble filaments and its spatially and temporally well-controlled supramolecular organization. A large number of proteins that interact with actin, collectively referred to as actin-binding proteins (ABPs), carefully orchestrate different scenarios. Since its isolation in 1994 [Machesky, L.M. et al. (1994) J. Cell Biol. 127, 107-115], the Arp2/3 complex containing the actin-related proteins Arp2 and Arp3 has evolved to be one of the main players in the assembly and maintenance of many actin-based structures in the cell (for review see [Borths, E.L. and Welch, M.D. (2002) Structure 10, 131-135; May, R.C. (2001) Cell Mol. Life Sci. 58, 1607-1626; Pollard, T.D. et al. (2000) Rev. Biophys. Biomol. Struct. 29, 545-576; Welch, M.D. (1999) Trends Cell Biol. 11, 423-427]). In particular, when it comes to the assembly of the intricate branched actin network at the leading edge of lamellipodia, the Arp2/3 complex seems to have received all the attention in recent years. In parallel, but not so much in the spotlight, several reports showed that actin on its own can assume different conformations [Bubb, M.R. et al. (2002) J. Biol. Chem. 277, 20999-21006; Schoenenberger, C.-A. et al. (1999) Microsc. Res. Tech. 47, 38-50; Steinmetz, M.O. et al. (1998) J. Mol. Biol. 278, 793-811; Steinmetz, M.O. et al. (1997) J. Cell Biol. 138, 559-574; Millonig, R., Salvo, H. and Aebi, U. (1988) J. Cell Biol. 106, 785-796] through which it drives its supramolecular patterning, and which ultimately generate its functional diversity.

Actin Cytoskeleton↗
PubMed2002

Localization of uroplakin Ia, the urothelial receptor for bacterial adhesin FimH, on the six inner domains of the 16 nm urothelial plaque particle.

The binding of uropathogenic Escherichia coli to the urothelial surface is a critical initial event for establishing urinary tract infection, because it prevents the bacteria from being removed by micturition and it triggers bacterial invasion as well as host cell defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium and its urothelial receptor, uroplakin Ia (UPIa). To localize the UPIa receptor on the 16 nm particles that form two-dimensional crystals of asymmetric unit membrane (AUM) covering >90 % of the apical urothelial surface, we constructed a 15 A resolution 3-D model of the mouse 16 nm AUM particle by negative staining and electron crystallography. Similar to previous lower-resolution models of bovine and pig AUM particles, the mouse 16 nm AUM particle consists of six inner and six outer domains that are interconnected to form a twisted ribbon-like structure. Treatment of urothelial plaques with 0.02-0.1 % (v/v) Triton X-100 allowed the stain to penetrate into the membrane, revealing parts of the uroplakin transmembrane moiety with an overall diameter of 14 nm, which was much bigger than the 11 nm value determined earlier by quick-freeze deep-etch. Atomic force microscopy of native, unfixed mouse and bovine urothelial plaques confirmed the overall structure of the luminal 16 nm AUM particle that was raised by 6.5 nm above the luminal membrane surface and, in addition, revealed a circular, 0.5 nm high, cytoplasmic protrusion of approximately 14 nm diameter. Finally, a difference map calculated from the mouse urothelial plaque images collected in the presence and absence of recombinant bacterial FimH/FimC complex revealed the selective binding of FimH to the six inner domains of the 16 nm AUM particle. These results indicate that the 16 nm AUM particle is anchored by a approximately 14 nm diameter transmembrane stalk, and suggest that bacterial binding to UPIa that resides within the six inner domains of the 16 nm AUM particle may preferentially trigger transmembrane signaling involved in bacterial invasion and host cell defense.

Adhesins, Bacterial↗
PubMed2002

Conserved segments 1A and 2B of the intermediate filament dimer: their atomic structures and role in filament assembly.

Intermediate filaments (IFs) are key components of the cytoskeleton in higher eukaryotic cells. The elementary IF 'building block' is an elongated coiled-coil dimer consisting of four consecutive alpha-helical segments. The segments 1A and 2B include highly conserved sequences and are critically involved in IF assembly. Based on the crystal structures of three human vimentin fragments at 1.4-2.3 A resolution (PDB entries 1gk4, 1gk6 and 1gk7), we have established the molecular organization of these two segments. The fragment corresponding to segment 1A forms a single, amphipatic alpha-helix, which is compatible with a coiled-coil geometry. While this segment might yield a coiled coil within an isolated dimer, monomeric 1A helices are likely to play a role in specific dimer-dimer interactions during IF assembly. The 2B segment reveals a double-stranded coiled coil, which unwinds near residue Phe351 to accommodate a 'stutter'. A fragment containing the last seven heptads of 2B interferes heavily with IF assembly and also transforms mature vimentin filaments into a new kind of structure. These results provide the first insight into the architecture and functioning of IFs at the atomic level.

Amino Acid Sequence↗
PubMed2002

Self-assembly of human MxA GTPase into highly ordered dynamin-like oligomers.

Human MxA protein is a member of the interferon-induced Mx protein family and an important component of the innate host defense against RNA viruses. The Mx family belongs to a superfamily of large GTPases that also includes the dynamins and the interferon-regulated guanylate-binding proteins. A common feature of these large GTPases is their ability to form high molecular weight oligomers. Here we determined the capacity of MxA to self-assemble into homo-oligomers in vitro. We show that recombinant MxA protein assembles into long filamentous structures with a diameter of about 20 nm at physiological salt concentration as demonstrated by sedimentation assays and electron microscopy. In the presence of guanosine nucleotides the filaments rearranged into rings and more compact helical arrays. Our data indicate that binding and hydrolysis of GTP induce conformational changes in MxA that may be essential for viral target recognition and antiviral activity.

Aluminum Compounds↗
PubMed2002

Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins.

Now that it is likely that all yeast nucleoporins are known, one of the ultimate goals is the in vitro assembly of the entire nuclear pore complex from its approximately 30 individual components. Here, we report the reconstitution of seven proteins (Nup133p, Nup145p-C, Nup120p, Nup85p, Nup84p, Seh1p and Sec13p) into a heptameric 0.5 MDa nuclear pore subcomplex. We found that double plasmid transformation combined with bi-cistronic mRNA translation allow the expression and assembly of distinct subcomplexes of up to five nucleoporins in a single Escherichia coli cell. During the sequential reconstitution of the Nup84p complex, smaller assembly intermediates can be isolated, which exhibit modular structures determined by electron microscopy that finally make up the whole Y-shaped Nup84p complex. Importantly, a seventh subunit, Nup133p, was incorporated into the complex through its interaction with Nup84p, thereby elongating one arm of the Y-shaped assembly to an approximately 40 nm long stalk. Taken together, our data document that the Nup84p-Nup133p complex self-assembles in a modular concept from distinct smaller nucleoporin construction sets.

Escherichia coli↗
PubMed2002

Polymerisation of chemically cross-linked actin:thymosin beta(4) complex to filamentous actin: alteration in helical parameters and visualisation of thymosin beta(4) binding on F-actin.

The beta-thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin beta(4) with F-actin. Thymosin beta(4) at 200 microM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin beta(4) complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin beta(4) complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin beta(4) filaments was found to be 16.0(+/-0.8) kDa/nm and 18.0(+/-0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin beta(4) filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin beta(4). The deduced binding sites of thymosin beta(4) to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation.

Actins↗
PubMed2002

Felodipine inhibits nuclear translocation of p42/44 mitogen-activated protein kinase and human smooth muscle cell growth.

OBJECTIVE: Smooth muscle cell (SMC) proliferation contributes to vascular structural changes in cardiovascular disease. Ca(2+) antagonists exert antiproliferative effects and may also be clinically beneficial in the patients. However, the underlying mechanisms of action remain elusive. Activation of mitogen-activated protein kinases (MAPK), in particular p42/44mapk plays a central role in cell proliferation. We hypothesise that Ca(2+) antagonists inhibit cell proliferation by interfering with the p42/44mapk pathway in human SMC. METHODS: SMC were cultured from human aorta. Cell proliferation was analysed by [3H]thymidine incorporation. Activation of p42/44mapk and the nuclear target protein Elk-1 was analysed by phosphorylation and p42/44mapk nuclear translocation by confocal microscope. RESULTS: PDGF-BB (10 ng/ml) stimulated [3H]thymidine incorporation, phosphorylated p42/44mapk, caused nuclear translocation of the enzymes and phosphorylated the nuclear target protein Elk-1. Felodipine (10(-7) to 10(-5) mol/l) inhibited [3H]thymidine incorporation to PDGF-BB, had no effect on p42/44mapk phosphorylation. However, p42/44mapk nuclear translocation and Elk-1 activation stimulated by PDGF-BB were prevented by the Ca(2+) antagonist. CONCLUSION: Activation of p42/44mapk, subsequent nuclear translocation and activation of Elk-1 are essentially associated with human SMC proliferation. The Ca(2+) antagonist felodipine prevents p42/44mapk nuclear translocation (but not its activation) associated with inhibition of human SMC growth.

Becaplermin↗
PubMedDate not supplied

Sequence comparisons of intermediate filament chains: evidence of a unique functional/structural role for coiled-coil segment 1A and linker L1.

A comprehensive analysis of the sequences of all types of intermediate filament chains has been undertaken with a particular emphasis on those of segment 1A and linker L1. This has been done to assess whether structural characteristics can be recognized in the sequences that would be consistent with the role of each region in the recently proposed "swinging head" hypothesis. The analyses show that linker L1 is the most flexible rod domain region, that it is the most elongated structure (on a per residue basis), and that it is the most variable region as regards sequence and length. Segment 1A has one of the two most highly conserved regions of sequence in the rod domain (the other being at the end of segment 2B), with seven particular residues conserved across all chain types. It also contains one of the very few potential interchain ionic interactions that could be conserved across all chain types. However, the aggregation of chains in segment 1A is specified less precisely overall by interchain ionic interactions than are the other coiled-coil segments. The apolar residue contents in positions a and d of the heptad substructure are the highest of any coiled-coil segment in the intermediate filament family. Segment 1A also displays an amino acid composition atypical of not only coiled-coil segments 1B and 2B, but indeed of two-stranded coiled coils in general. Nonetheless, molecular modeling based on the crystal structure of the monomeric 1A fragment from human vimentin shows that coiled-coil formation is plausible. The most extensive regions of apolar/aromatic residues lie at the C-terminal end of segment 2B in the helix termination motif and in segment 1A in and close to the helix initiation motif. The predicted stability of the individual alpha-helices in segment 1A is greater than in those comprising segments 1B and 2B, though potential intrachain ionic interactions are either lacking or are minimal in number. Analysis of the 1A sequence and those regions immediately N- and C-terminal to it has shown that the capping residues are near optimal close to the previously predicted ends, thus adding to the likely stability of the alpha-helical structure. However, a second terminating sequence is predicted in 1A (about 10 residues back from the C-terminus). This allows the possibility of some unwinding of the alpha-helical structure of 1A immediately adjacent to linker L1 when the head domains no longer stabilize the coiled-coil structure. All of these data are consistent with the concept of a flexible hinge at L1 and with the ability of the two alpha-helical coiled-coil strands to separate under appropriate conditions and partly unwind at their C-terminal ends to allow the head domains a greater degree of mobility, thus facilitating function.

Amino Acid Motifs↗
PubMedDate not supplied

Characterization of early assembly intermediates of recombinant human keratins.

The intermediate filaments (IFs) form major structural elements of the cytoskeleton. In vitro analyses of these fibrous proteins reveal very different assembly properties for the nuclear and cytoplasmic IF proteins. However, keratins in particular, the largest and most heterogenous group of cytoplasmic IF proteins, have been difficult to analyze due to their rapid assembly dynamics under the near-physiological conditions used for other IF proteins. We show here that keratins, like other cytoplasmic IF proteins, go through a stage of assembling into full-width soluble complexes, i.e., "unit-length filaments" (ULFs). In contrast to other IF proteins, however, longitudinal annealing of keratin ULFs into long filaments quasi-coincides with their formation. In vitro assembly of IF proteins into filaments can be initiated by an increase of the ionic strength and/or lowering of the pH of the assembly buffer. We now document that 23-mer peptides from the head domains of various IF proteins can induce filament formation even under conditions of low salt and high pH. This suggests that the "heads" are involved in the formation and longitudinal association of the ULFs. Using a Tris-buffering protocol that causes formation of soluble oligomers at pH 9, the epidermal keratins K5/14 form less regular filaments and less efficiently than the simple epithelial keratins K8/18. In sodium phosphate buffers (pH 7.5), however, K5/14 were able to form long partially unraveled filaments which compacted into extended, regular filaments upon addition of 20 mM KCl. Applying the same assembly regimen to mutant K14 R125H demonstrated that mutations causing a severe disease phenotype and morphological filament abnormalities can form long, regular filaments with surprising efficiency in vitro.

Base Sequence↗