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D Stoffler

Publications and source records attributed to D Stoffler.

14 recordsLinked to original sources
PubMed2000

Polymerization, three-dimensional structure and mechanical properties of Ddictyostelium versus rabbit muscle actin filaments.

To assess more systematically functional differences among non-muscle and muscle actins and the effect of specific mutations on their function, we compared actin from Dictyostelium discoideum (D-actin) with actin from rabbit skeletal muscle (R-actin) with respect to the formation of filaments, their three-dimensional structure and mechanical properties. With Mg(2+) occupying the single high-affinity divalent cation-binding site, the course of polymerization is very similar for the two types of actin. In contrast, when Ca(2+ )is bound, D-actin exhibits a significantly longer lag phase at the onset of polymerization than R-actin. Crossover spacing and helical screw angle of negatively stained filaments are similar for D and R-F-actin filaments, irrespective of the tightly bound divalent cation. However, three-dimensional helical reconstructions reveal that the intersubunit contacts along the two long-pitch helical strands of D-(Ca)F-actin filaments are more tenuous compared to those in R-(Ca)F-actin filaments. D-(Mg)F-actin filaments on the other hand exhibit more massive contacts between the two long-pitch helical strands than R-(Mg)F-actin filaments. Moreover, in contrast to the structure of R-F-actin filaments which is not significantly modulated by the divalent cation, the intersubunit contacts both along and between the two long-pitch helical strands are weaker in D-(Ca)F-actin compared to D-(Mg)F-actin filaments. Consistent with these structural differences, D-(Ca)F-actin filaments were significantly more flexible than D-(Mg)F-actin. Taken together, this work documents that despite being highly conserved, muscle and non-muscle actins exhibit subtle differences in terms of their polymerization behavior, and the three-dimensional structure and mechanical properties of their F-actin filaments which, in turn, may account for their functional diversity.

Actin Cytoskeleton↗
PubMed2000

Polymerization and structure of nucleotide-free actin filaments.

Two factors have limited studies of the properties of nucleotide-free actin (NFA). First, actin lacking bound nucleotide denatures rapidly without stabilizing agents such as sucrose; and second, without denaturants such as urea, it is difficult to remove all of the bound nucleotide. We used apyrase, EDTA and Dowex-1 to prepare actin that is stable in sucrose and approximately 99 % free of bound nucleotide. In high concentrations of sucrose where NFA is stable, it polymerizes more favorably with a lag phase shorter than ATP-actin and a critical concentration close to zero. NFA filaments are stable, but depolymerize at low sucrose concentrations due to denaturation of subunits when they dissociate from filament ends. By electron microscopy of negatively stained specimens, NFA forms long filaments with a persistence length 1.5 times greater than ADP-actin filaments. Three-dimensional helical reconstructions of NFA and ADP-actin filaments at 2.5 nm resolution reveal similar intersubunit contacts along the two long-pitch helical strands but statistically significant less mass density between the two strands of NFA filaments. When compared with ADP-actin filaments, the major difference peak of NFA filaments is near, but does not coincide with, the vacated nucleotide binding site. The empty nucleotide binding site in these NFA filaments is not accessible to free nucleotide in the solution. The affinity of NFA filaments for rhodamine phalloidin is lower than that of native actin filaments, due to a lower association rate. This work confirms that bound nucleotide is not essential for actin polymerization, so the main functions of the nucleotide are to stabilize monomers, modulate the mechanical and dynamic properties of filaments through ATP hydrolysis and phosphate release, and to provide an internal timer for the age of the filament.

Actins↗
PubMed2000

Monitoring biomolecular interactions by time-lapse atomic force microscopy.

The atomic force microscope (AFM) is a unique imaging tool that enables the tracking of single macromolecule events in response to physiological effectors and pharmacological stimuli. Direct correlation can therefore be made between structural and functional states of individual biomolecules in an aqueous environment. This review explores how time-lapse AFM has been used to learn more about normal and disease-associated biological processes. Three specific examples have been chosen to illustrate the capabilities of this technique. In the cell, actin polymerizes into filaments, depolymerizes, and undergoes interactions with numerous effector molecules (i.e., severing, capping, depolymerizing, bundling, and cross-linking proteins) in response to many different stimuli. Such events are critical for the function and maintenance of the molecular machinery of muscle contraction and the dynamic organization of the cytoskeleton. One goal is to use time-lapse AFM to examine and manipulate some of these events in vitro, in order to learn more about how these processes occur in the cell. Aberrant protein polymerization into amyloid fibrils occurs in a multitude of diseases, including Alzheimer's and type 2 diabetes. Local amyloid deposits may cause organ dysfunction and cell death; hence, it is of interest to learn how to interfere with fibril formation. One application of time-lapse AFM in this area has been the direct visualization of amyloid fibril growth in vitro. This experimental approach holds promise for the future testing of potential therapeutic drugs, for example, by directly visualizing at which level of fibril assembly (i.e., nucleation, elongation, branching, or lateral association of protofibrils) a given active compound will interfere. Nuclear pore complexes (NPCs) are large supramolecular assemblies embedded in the nuclear envelope. Transport of ions, small molecules, proteins, RNAs, and RNP particles in and out of the nucleus occurs via NPCs. Time-lapse AFM has been used to structurally visualize the response of individual NPC particles to various chemical and physical effectors known to interfere with nucleocytoplasmic transport. Taken together, such time-lapse AFM studies could provide novel insights into the molecular mechanisms of fundamental biological processes under both normal and pathological conditions at the single molecule level.

Actins↗
PubMed1999

Structure, assembly, and dynamics of actin filaments in situ and in vitro.

Actin, though highly conserved, exhibits a myriad of diverse functions, most of which ultimately depend on its intrinsic ability to rapidly assemble and disassemble filamentous structures. Many organisms synthesize multiple actin isoforms even within the same cell. Tissue-specific expression patterns and tight developmental regulation as well as a high conservation across species emphasize the functional importance of isoforms. The detailed knowledge of the structure, assembly, and dynamic behavior of actin provides important pieces in solving the puzzle of how the different isoforms can be so versatile despite their extremely high sequence identity.

Actins↗
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

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

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↗
PubMed1998

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.

Actins↗
PubMed1997

Actin: from cell biology to atomic detail.

Over the past 2 decades our knowledge about actin filaments has evolved from a rigid "pearls on a string" model to that of a complex, highly dynamic protein polymer which can now be analyzed at atomic detail. To achieve this, exploring actin's oligomerization, polymerization, polymorphism, and dynamic behavior has been crucial to understanding in detail how this abundant and ubiquitous protein can fulfill its various functions within living cells. In this review, a correlative view of a number of distinct aspects of actin is presented, and the functional implications of recent structural, biochemical, and mechanical data are critically evaluated. Rational analysis of these various experimental data is achieved using an integrated structural approach which combines intermediate-resolution electron microscopy-based 3-D reconstructions of entire actin filaments with atomic resolution X-ray data of monomeric and polymeric actin.

Actin Cytoskeleton↗
PubMed1995

Molecular mimicry in halothane hepatitis: biochemical and structural characterization of lipoylated autoantigens.

Exposure of human individuals to halothane causes, in about 20% of all cases, a mild transient form of hepatotoxicity. A small subset of exposed individuals, however, develops a potentially severe and life-threatening form of hepatic damage, coined halothane hepatitis. Halothane hepatitis is thought to have an immunological basis. Sera of afflicted individuals contain a wide variety of autoantibodies against hepatic proteins, in both trifluoroacetylated form (CF3CO-proteins) and, at least in part, in native form. CF3CO-proteins are elicited in the course of oxidative biotransformation of halothane, and include the trifluoroacetylated forms of protein disulfide isomerase, microsomal carboxylesterase, calreticulin, ERp72, GRP 78, and ERp99. Current evidence suggests that CF3CO-proteins arise in all halothane-exposed individuals; however, the vast majority of individuals appear to immunochemically tolerate CF3CO-proteins. The lack of immunological responsiveness of these individuals towards CF3CO-proteins might be due to tolerance, induced through the occurrence of structures in the repertoire of self-determinants, which immunochemically and structurally mimic CF3CO-proteins very closely. In fact, lipoic acid, the prosthetic group of the constitutively expressed E2 subunits of the family of mammalian 2-oxoacid dehydrogenase complexes and of protein X, was shown by immunochemical and molecular modelling analysis to be a perfect structural mimic of N6-trifluoroacetyl-L-lysine (CF3 CO-Lys), the major haptenic group of CF3CO-proteins. As a consequence of molecular mimicry, autoantibodies in patients' sera not only recognize CF3CO-proteins, but also the E2 subunit proteins of the 2-oxoacid dehydrogenase complexes and protein X, as autoantigens associated with halothane hepatitis. Furthermore, a fraction of patients with halothane hepatitis exhibit irregularities in the hepatic expression levels of these native, not trifluoroacetylated autoantigens. Collectively, these data suggest that molecular mimicry of CF3CO-Lys by lipoic acid, or the impairment thereof, might play a role in the susceptibility of individuals for the development of halothane hepatitis.

Autoantigens↗