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U Aebi

Publications and source records attributed to U Aebi.

At least 55 records · Page 3Linked to original sources
PubMed1999

Domain analysis of cortexillin I: actin-bundling, PIP(2)-binding and the rescue of cytokinesis.

Cortexillins are actin-bundling proteins that form a parallel two-stranded coiled-coil rod. Actin-binding domains of the alpha-actinin/spectrin type are located N-terminal to the rod and unique sequence elements are found in the C-terminal region. Domain analysis in vitro revealed that the N-terminal domains are not responsible for the strong actin-filament bundling activity of cortexillin I. The strongest activity resides in the C-terminal region. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) suppresses this bundling activity by binding to a C-terminal nonapeptide sequence. These data define a new PIP(2)-regulated actin-bundling site. In vivo the PIP(2)-binding motif enhances localization of a C-terminal cortexillin I fragment to the cell cortex and improves the rescue of cytokinesis. This motif is not required, however, for translocation to the cleavage furrow. A model is presented proposing that cortexillin translocation is based on a mitotic cycle of polar actin polymerization and midzone depolymerization.

Actins↗
PubMed1999

Purification and analysis of authentic CLIP-170 and recombinant fragments.

We have purified authentic CLIP-170 (cytoplasmic linker protein of 170 kDa) and fragments comprising functional domains of the protein to characterize the structural basis of the function of CLIP-170. Analysis of authentic CLIP-170 and the recombinant fragments by electron microscopy after glycerol spraying/low angle rotary metal shadowing reveals CLIP-170 as a thin, 135-nm-long molecule with two kinks in its central rod domain, which are approximately equally spaced from the two ends of the protein. The central domain consisting of heptad repeats, which is alpha-helical in nature and forms a 2-stranded coiled-coil, mediates dimerization of CLIP-170. The rod domain harbors two kinks, each spaced approximately 37 nm from the corresponding end of the molecule, thus providing mechanical flexibility to the highly elongated molecule. The N-terminal domain of CLIP-170 binds to microtubules in vitro with a stoichiometry of one dimeric head domain per four tubulin heterodimers. Authentic CLIP-170 binds to microtubules with lower stoichiometry, indicating that the rod and tail domains affect microtubule binding of CLIP-170. These results document that CLIP-170 is a highly elongated polar molecule with the microtubule-binding domain and the organelle-interacting domains at opposite ends of the homodimer, thus providing a structural basis for the function of CLIP-170 as a microtubule-organelle linker protein.

Binding Sites↗
PubMed1999

Intermediate filament assembly: temperature sensitivity and polymorphism.

Intermediate filament (IF) proteins are encoded by a large multigene family and form polymers with a uniform diameter of approximately 10 nm. However, although the cytoplasmic representatives all confirm to a unit-type structural principle leading to the formation of extended coiled coils, it is becoming increasingly clear that subunit arrangements and physical properties vary among the different filaments. Thus, the intricate tissue-specific expression pattern of individual IF proteins (especially, their co-expression with other members of the IF protein family or with IF-associated proteins to form obligatory heteropolymers) points to distinct functions acquired during evolution relevant to cellular homeostasis in various tissues.

Amino Acid Sequence↗
PubMed1999

Isolation, electron microscopic imaging, and 3-D visualization of native cardiac thin myofilaments.

An increasing number of cardiac diseases are currently pinpointed to reside at the level of the thin myofilaments (e.g., cardiomyopathies, reperfusion injury). Hence the aim of our study was to develop a new method for the isolation of mammalian thin myofilaments suitable for subsequent high-resolution electron microscopic imaging. Native cardiac thin myofilaments were extracted from glycerinated porcine myocardial tissue in the presence of protease inhibitors. Separation of thick and thin myofilaments was achieved by addition of ATP and several centrifugation steps. Negative staining and subsequent conventional and scanning transmission electron microscopy (STEM) of thin myofilaments permitted visualization of molecular details; unlike conventional preparations of thin myofilaments, our method reveals the F-actin moiety and allows direct recognition of thin myofilament-associated porcine cardiac troponin complexes. They appear as "bulges" at regular intervals of approximately 36 nm along the actin filaments. Protein analysis using SDS-polyacrylamide gel electrophoresis revealed that only approximately 20% troponin I was lost during the isolation procedure. In a further step, 3-D helical reconstructions were calculated using STEM dark-field images. These 3-D reconstructions will allow further characterization of molecular details, and they will be useful for directly visualizing molecular alterations related to diseased cardiac thin myofilaments (e.g., reperfusion injury, alterations of Ca2+-mediated tropomyosin switch).

Actin Cytoskeleton↗
PubMed1999

Ectopic overexpression of Drosophila lamin C is stage-specific lethal.

To gain insight into the function of the developmentally regulated A-type lamins we transformed Drosophila melanogaster with a construct containing the hsp70 promoter followed by the Drosophila lamin C (an analog of vertebrate A-type lamins) cDNA. Lamin C was expressed ectopically after heat shock of embryos and localized to the nucleus. No phenotypic change was observed after lamin C expression in embryos that normally do not contain lamin C. However, ectopic expression of lamin C during most larval (but not pupal) stages stalled growth, inhibited ecdysteroid signaling (in particular during the larval-prepupal transition), resulted in development of melanotic tumors, and finally caused death. During pupation in control animals, when massive apoptosis of larval tissues takes place, lamin C is proteolyzed into a fragment with a size similar to that predicted by caspase cleavage. The ectopically expressed lamin C is identically cleaved, resulting in a large increase of the steady-state level of the lamin C fragment. A null mutation of the dcp-1 gene, one of the two known Drosophila caspase genes, also results in development of melanotic tumors and larval death, suggesting that the ectopically expressed lamin C inhibits apoptosis through competitive inhibition of caspase activity.

Age Factors↗
PubMed1999

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.

Animals↗
PubMed1999

Characterization of distinct early assembly units of different intermediate filament proteins.

We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from the four cytoplasmic sequence homology classes, and compared these values with those of the corresponding mature filaments. After two seconds under standard assembly conditions (i.e. 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 37 degrees C), vimentin, desmin and the neurofilament triplet protein NF-L aggregated into similar types of "unit-length filaments" (ULFs), whereas cytokeratins (CKs) 8/18 already yielded long IFs at this time point, so the ionic strength had to be reduced. The number of molecules per filament cross-section, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 at two seconds compared to 16 and 21 at one hour. NF-L exhibited corresponding values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, with major peak values of 32 and 45 at two seconds and 30, 37 and 44 after one hour. Desmin formed filaments of distinctly higher mass with 47 molecules per cross-section, at two seconds and after one hour of assembly. This indicates that individual types of IF proteins generate filaments with distinctly different numbers of molecules per cross-section. Also, the observed significant reduction of apparent filament diameter of ULFs compared to the corresponding mature IFs is the result of a "conservative" radial compaction-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subunits per cross-section. Moreover, the MPL composition of filaments is strikingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significantly increased number of molecules per cross-section (56 and 84) compared to assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature "pathological" ULFs form that are arrested in this state, indicating that the system is forced into strong but unproductive interactions between subunits. Similar "dead-end" structures were obtained with vimentins mutated to introduce principal alterations in subdomains presumed to be of general structural importance, indicating that these sequence changes led to new modes of intermolecular interactions.

Amino Acid Sequence↗
PubMed1999

Three-dimensional analysis of the 16 nm urothelial plaque particle: luminal surface exposure, preferential head-to-head interaction, and hinge formation.

The luminal surface of mouse urothelium in contact with the urine is almost entirely covered with plaques consisting of uroplakin-containing particles that form p6 hexagonal crystals with a center-to-center distance of 16 nm. A combination of quick-freeze/deep-etch images and our previous negative staining data indicate that the head domain of the uroplakin particle, which is exposed without an extensive glycocalyx shield, interacts closely with the head domains of the neighboring particles, while the membrane-embedded tail domains are farther apart; and that urothelial particles and plaques are not rigid structures as they can change their configuration in response to mechanical perturbations. Based on these data, we have constructed three-dimensional models depicting the structural organization of urothelial particles and plaques. Our models suggest that the head-to-head interaction may play a key role in determining the shape and size of the urothelial plaques. These models can explain many properties of urothelial plaques including their unique shape, detergent-insolubility, and morphological changes during vesicle maturation.

Animals↗
PubMed1999

Watching amyloid fibrils grow by time-lapse atomic force microscopy.

Late-onset diabetes is typically associated with amyloid deposits of fibrillar amylin in the pancreatic islets. Aqueous synthetic human amylin spontaneously forms polymorphic fibrils in vitro, and this system was used to examine the dynamics of fibril assembly. By time-lapse atomic force microscopy (AFM), the growth of individual amylin fibrils on a mica surface was observed over several hours. Prominent was the assembly of a protofibril with an elongation rate in these experiments of 1.1(+/-0.5) nm/minute. The assembly of higher order polymorphic fibrils was also observed. Growth of the protofibrils was bidirectional, i.e. it occurred by elongation at both ends. This ability of AFM to continuously monitor growth, directionality, and changes in morphology for individual fibrils, provides a significant advantage over spectroscopy-based bulk methods which average the growth of many fibrils and typically require 100 to 1000-fold more protein. The time-lapse AFM procedure used for human amylin here is thus likely to be applicable to fibril formation from other amyloid proteins and peptides.

Aluminum Silicates↗
PubMed1999

Interactions between coiled-coil proteins: Drosophila lamin Dm0 binds to the bicaudal-D protein.

In a yeast two-hybrid screen we identified an interaction between Drosophila lamin Dm0, a structural nuclear protein, and BICD, a protein involved in oocyte development. The interaction can be reconstituted in vitro and takes place between segments of both proteins predicted to form coiled coils. The affinity for lamin Dm0 of the minimal binding site on BICD is modulated in a complex fashion by other BICD segments. A point mutation, F684I, that causes the dominant, bicaudal, Bic-D phenotype inhibits lamin binding in the context of the minimal lamin-binding site, but not in a larger BICD fragment. The minimal lamin-binding site of BICD binds to a few other coiled-coil proteins, but binding to these proteins is not influenced by the F684I point mutation, suggesting that the interaction with lamin may play a role in Bic-D function. Our structural studies demonstrated that BICD is 60-70% alpha-helical, is a dimer, and consists of two parts: a thin rod-shaped part of about 32 nm, and a thicker rod-shaped part of about 26 nm. Likely, the thinner rod-shaped part of full-length BICD consists of the N-terminal half of the protein, and the lamin-binding site is located within the thicker rod-shaped part.

Animals↗
PubMed1999

The nuclear pore complex: from molecular architecture to functional dynamics.

Toward dissecting the molecular composition and architecture of the nuclear pore complex (NPC), over the past 18 months novel nucleoporins and NPC subcomplexes were identified and characterized. The three-dimensional structure of isolated yeast NPCs was determined by electron cryomicroscopy. New specimen preparation and labeling protocols localized a number of nucleoporins and NPC subcomplexes within the three-dimensional architecture of the yeast NPC. Structural changes of native NPCs mediated by physiological effectors such as calcium or ATP were monitored by time-lapse atomic force microscopy, thus revealing a first glimpse of the NPC's functional dynamics.

Animals↗
PubMed1999

Determination of the inelastic mean free path of electrons in vitrified ice layers for on-line thickness measurements by zero-loss imaging.

The inelastic mean free path of 120 keV electrons in vitrified ice layers has been determined in an energy-filtering TEM. From the ratio of the unfiltered and zero-loss-filtered image intensities recorded with a slow-scan CCD camera, the relative sample thickness t/Lambda can be calculated. For calibration, the geometric ice thickness was measured by imaging a tilted view of a cylindrical hole which had been burnt into the ice layer. The total inelastic mean free path was found to be 161 nm, and the partial inelastic mean free path for an acceptance angle of 4.2 mrad was 232 nm. These results were built into a standard protocol for use in cryo-electron microscopy allowing on-line measurements of local ice-layer thicknesses by zero-loss-filtered/unfiltered imaging.

Journal Article↗
PubMed1999

Differential epitope tagging of actin in transformed Drosophila produces distinct effects on myofibril assembly and function of the indirect flight muscle.

We have tested the impact of tags on the structure and function of indirect flight muscle (IFM)-specific Act88F actin by transforming mutant Drosophila melanogaster, which do not express endogenous actin in their IFMs, with tagged Act88F constructs. Epitope tagging is often the method of choice to monitor the fate of a protein when a specific antibody is not available. Studies addressing the functional significance of the closely related actin isoforms rely almost exclusively on tagged exogenous actin, because only few antibodies exist that can discriminate between isoforms. Thereby it is widely presumed that the tag does not significantly interfere with protein function. However, in most studies the tagged actin is expressed in a background of endogenous actin and, as a rule, represents only a minor fraction of the total actin. The Act88F gene encodes the only Drosophila actin isoform exclusively expressed in the highly ordered IFM. Null mutations in this gene do not affect viability, but phenotypic effects in transformants can be directly attributed to the transgene. Transgenic flies that express Act88F with either a 6x histidine tag or an 11-residue peptide derived from vesicular stomatitis virus G protein at the C terminus were flightless. Overall, the ultrastructure of the IFM resembled that of the Act88F null mutant, and only low amounts of C-terminally tagged actins were found. In contrast, expression of N-terminally tagged Act88F at amounts comparable with that of wild-type flies yielded fairly normal-looking myofibrils and partially reconstituted flight ability in the transformants. Our findings suggest that the N terminus of actin is less sensitive to modifications than the C terminus, because it can be tagged and still polymerize into functional thin filaments.

Actins↗
PubMed1999

Substitution of flight muscle-specific actin by human (beta)-cytoplasmic actin in the indirect flight muscle of Drosophila.

The human (beta)-cytoplasmic actin differs by only 15 amino acids from Act88F actin which is the only actin expressed in the indirect flight muscle (IFM) of Drosophila melanogaster. To test the structural and functional significance of this difference, we ectopically expressed (beta)-cytoplasmic actin in the IFM of Drosophila that lack endogenous Act88F. When expression of the heterologous actin was regulated by approximately 1.5 kb of the 5' promoter region of the Act88F gene, little (beta)-cytoplasmic actin accumulated in the IFM of the flightless transformants. Including Act88F-specific 5' and 3' untranslated regions (UTRs) yielded transformants that expressed wild-type amounts of (beta)-cytoplasmic actin. Despite the assembly of (beta)-cytoplasmic actin containing thin filaments to which endogenous myosin crossbridges attached, sarcomere organization was deficient, leaving the transformants flightless. Rather than affecting primarily actin-myosin interactions, our findings suggest that the (beta)-cytoplasmic actin isoform is not competent to interact with other actin-binding proteins in the IFM that are involved in the organization of functional myofibrils.

Actins↗
PubMed1998

An autonomous folding unit mediates the assembly of two-stranded coiled coils.

Subunit oligomerization of many proteins is mediated by coiled-coil domains. Although the basic features contributing to the thermodynamic stability of coiled coils are well understood, the mechanistic details of their assembly have not yet been dissected. Here we report a 13-residue sequence pattern that occurs with limited sequence variations in many two-stranded coiled coils and that is absolutely required for the assembly of the Dictyostelium discoideum actin-bundling protein cortexillin I and the yeast transcriptional activator GCN4. The functional relationship between coiled-coil "trigger" sequences was manifested by replacing the intrinsic trigger motif of GCN4 with the related sequence from cortexillin I. We demonstrate that these trigger sequences represent autonomous helical folding units that, in contrast to arbitrarily chosen heptad repeats, can mediate coiled-coil formation. Aside from being of general interest for protein folding, trigger motifs should be of particular importance in the protein de novo design.

Amino Acid Sequence↗
PubMed1998

Molecular architecture of the yeast nuclear pore complex: localization of Nsp1p subcomplexes.

The nuclear pore complex (NPC), a supramolecular assembly of approximately 100 different proteins (nucleoporins), mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus. Extensive structural studies have revealed the three- dimensional (3D) architecture of Xenopus NPCs, and eight of the approximately 12 cloned and characterized vertebrate nucleoporins have been localized within the NPC. Thanks to the power of yeast genetics, 30 yeast nucleoporins have recently been cloned and characterized at the molecular level. However, the localization of these nucleoporins within the 3D structure of the NPC has remain elusive, mainly due to limitations of preparing yeast cells for electron microscopy (EM). We have developed a new protocol for preparing yeast cells for EM that yielded structurally well-preserved yeast NPCs. A direct comparison of yeast and Xenopus NPCs revealed that the NPC structure is evolutionarily conserved, although yeast NPCs are 15% smaller in their linear dimensions. With this preparation protocol and yeast strains expressing nucleoporins tagged with protein A, we have localized Nsp1p and its interacting partners Nup49p, Nup57p, Nup82p, and Nic96p by immuno-EM. Accordingly, Nsp1p resides in three distinct subcomplexes which are located at the entry and exit of the central gated channel and at the terminal ring of the nuclear basket.

Animals↗
PubMed1998

Assembly and architecture of invertebrate cytoplasmic intermediate filaments reconcile features of vertebrate cytoplasmic and nuclear lamin-type intermediate filaments.

The two major intermediate filament (IF) proteins from the esophagus epithelium of the snail Helix pomatia and the two major IF proteins from muscle tissue of the nematode Ascaris suum were investigated under a variety of assembly conditions. The lowest-order complexes from each of the four protostomic invertebrate (p-INV) IF proteins are parallel, unstaggered dimers involving two-stranded alpha-helical coiled coil formation of their approximately 350 amino acid residue central rod domain (i.e. long-rod). In the electron microscope these are readily recognized by their distinct approximately 56 nm long rod with two globular domains (i.e. representing the non-helical carboxy-terminal tail domain of the p-INV IF proteins) attached at one end, closely resembling vertebrate lamin dimers. The next-higher-order oligomers are tetramers, which are easily recognized by their two pairs of globular tail domains attached at either end of a approximately 72 nm long central rod portion. According to their size and shape, these tetramers are built from two dimers associated laterally in an antiparallel, approximately half-staggered fashion via the amino-terminal halves of their rod domains. This is similar to the NN-type tetramers found as the most abundant oligomer species in all types of vertebrate cytoplasmic IF proteins, which contain a approximately 310 amino acid residue central rod domain (i.e. short-rod). As a first step toward filament formation, the p-INV IF tetramers anneal longitudinally into protofilaments by antiparallel CC-type association of the carboxy-terminal halves of their dimer rods. The next step involves radial growth, occurring initially through lateral association of two four-chain protofilaments into octameric subfibrils, which then further associate into mature, full-width filaments. Head-to-tail polymers of dimers and paracrystalline fibers commonly observed with vertebrate lamins were only rarely seen with p-INV IF proteins. The globular domains residing at the carboxy-terminal end of p-INV IF dimers were studding the surface of the filaments at regular, approximately 24.5 nm intervals, thereby giving them a "beaded" appearance with an axial periodicity of about 24.5 nm, which is approximately 3 nm longer than the corresponding approximately 21.5 nm repeat pattern exhibited by short-rod vertebrate IFs.

Animals↗