Self-assembly of polysaccharides gives rise to distinct mechanical signatures in marine gels.

Marine-gel biopolymers were recently visualized at the molecular level using atomic force microscopy (AFM) to reveal fine fibril-forming networks with low to high degrees of cross-linking. In this work, we use force spectroscopy to quantify the intra- and intermolecular forces within the marine-gel network. Combining force measurements, AFM imaging, and the known chemical composition of marine gels allows us to identify the microscopic origins of distinct mechanical responses. At the single-fibril level, we uncover force-extension curves that resemble those of individual polysaccharide fibrils. They exhibit entropic elasticity followed by extensions associated with chair-to-boat transitions specific to the type of polysaccharide at high forces. Surprisingly, a low degree of cross-linking leads to sawtooth patterns that we attribute to the unraveling of polysaccharide entanglements. At a high degree of cross-linking, we observe force plateaus that arise from unzipping, as well as unwinding, of helical bundles. Finally, the complex 3D network structure gives rise to force staircases of increasing height that correspond to the hierarchical peeling of fibrils away from the junction zones. In addition, we show that these diverse mechanical responses also arise in reconstituted polysaccharide gels, which highlights their dominant role in the mechanical architecture of marine gels.

Mechanical manipulation of Alzheimer's amyloid beta1-42 fibrils.

The 39- to 42-residue-long amyloid beta-peptide (Abeta-peptide) forms filamentous structures in the neuritic plaques found in the neuropil of Alzheimer's disease patients. The assembly and deposition of Abeta-fibrils is one of the most important factors in the pathogenesis of this neurodegenerative disease. Although the structural analysis of amyloid fibrils is difficult, single-molecule methods may provide unique insights into their characteristics. In the present work, we explored the nanomechanical properties of amyloid fibrils formed from the full-length, most neurotoxic Abeta1-42 peptide, by manipulating individual fibrils with an atomic force microscope. We show that Abeta-subunit sheets can be mechanically unzipped from the fibril surface with constant forces in a reversible transition. The fundamental unzipping force (approximately 23 pN) was significantly lower than that observed earlier for fibrils formed from the Abeta1-40 peptide (approximately 33 pN), suggesting that the presence of the two extra residues (Ile and Ala) at the peptide's C-terminus result in a mechanical destabilization of the fibril. Deviations from the constant force transition may arise as a result of geometrical constraints within the fibril caused by its left-handed helical structure. The nanomechanical fingerprint of the Abeta1-42 is further influenced by the structural dynamics of intrafibrillar interactions.

Nanomechanical properties of desmin intermediate filaments.

Desmin intermediate filaments play important role in the mechanical integrity and elasticity of muscle cells. The mechanisms of how desmin contributes to cellular mechanics are little understood. Here, we explored the nanomechanics of desmin by manipulating individual filaments with atomic force microscopy. In complex, hierarchical force responses we identified recurring features which likely correspond to distinct properties and structural transitions related to desmin's extensibility and elasticity. The most frequently observed feature is an initial unbinding transition that corresponds to the removal of approximately 45-nm-long coiled-coil dimers from the filament surface with 20-60 pN forces in usually two discrete steps. In tethers longer than 60 nm we most often observed force plateaus studded with bumps spaced approximately 16 nm apart, which are likely caused by a combination of protofilament unzipping, dimer-dimer sliding and coiled-coil-domain unfolding events. At high stresses and strains non-linear, entropic elasticity was dominant, and sometimes repetitive sawtooth force transitions were seen which might arise because of slippage within the desmin protofilament. A model is proposed in which mechanical yielding is caused by coiled-coil domain unfolding and dimer-dimer sliding/slippage, and strain hardening by the entropic elasticity of partially unfolded protofilaments.

Effect of lysine-28 side-chain acetylation on the nanomechanical behavior of alzheimer amyloid beta25-35 fibrils.

Amyloid fibrils are self-associating filamentous structures formed from the 39- to 42-residue-long amyloid beta peptide (Abeta peptide). The deposition of Abeta fibrils is one of the most important factors in the pathogenesis of Alzheimer's disease. Abeta25-35 is a fibril-forming peptide that is thought to represent the biologically active, toxic form of the full-length Abeta peptide. We have recently shown that beta sheets can be mechanically unzipped from the fibril surface with constant forces in a reversible transition, and the unzipping forces differ in fibrils composed of different peptides. In the present work, we explored the effect of epsilon-amino acetylation of the Lys28 residue on the magnitude of the unzipping force of Abeta25-35 fibrils. Although the gross structure of the Lys28-acetylated (Abeta25-35_K28Ac) and wild-type Abeta25-35 (Abeta25-35wt) fibrils were similar, as revealed by atomic force microscopy, the fundamental unzipping forces were significantly lower for Abeta25-35_K28Ac (20 +/- 4 pN SD) than for Abeta25-35wt (42 +/- 9 pN SD). Simulations based on a simple two-state model suggest that the decreased unzipping forces, caused most likely by steric constraints, are likely due to a destabilized zippered state of the fibril.

Hierarchical extensibility in the PEVK domain of skeletal-muscle titin.

Titin is the main determinant of passive muscle force. Physiological extension of titin derives largely from its PEVK (Pro-Glu-Val-Lys) domain, which has a different length in different muscle types. Here we characterized the elasticity of the full-length, human soleus PEVK domain by mechanically manipulating its contiguous, recombinant subdomain segments: an N-terminal (PEVKI), a middle (PEVKII), and a C-terminal (PEVKIII) one third. Measurement of the apparent persistence lengths revealed a hierarchical arrangement according to local flexibility: the N-terminal PEVKI is the most rigid and the C-terminal PEVKIII is the most flexible segment within the domain. Immunoelectron microscopy supported the hierarchical extensibility within the PEVK domain. The effective persistence lengths decreased as a function of ionic strength, as predicted by the Odijk-Skolnick-Fixman model of polyelectrolyte chains. The ionic strength dependence of persistence length was similar in all segments, indicating that the residual differences in the elasticity of the segments derive from nonelectrostatic mechanisms.

Molecular basis of passive stress relaxation in human soleus fibers: assessment of the role of immunoglobulin-like domain unfolding.

Titin (also known as connectin) is the main determinant of physiological levels of passive muscle force. This force is generated by the extensible I-band region of the molecule, which is constructed of the PEVK domain and tandem-immunoglobulin segments comprising serially linked immunoglobulin (Ig)-like domains. It is unresolved whether under physiological conditions Ig domains remain folded and act as "spacers" that set the sarcomere length at which the PEVK extends or whether they contribute to titin's extensibility by unfolding. Here we focused on whether Ig unfolding plays a prominent role in stress relaxation (decay of force at constant length after stretch) using mechanical and immunolabeling studies on relaxed human soleus muscle fibers and Monte Carlo simulations. Simulation experiments using Ig-domain unfolding parameters obtained in earlier single-molecule atomic force microscopy experiments recover the phenomenology of stress relaxation and predict large-scale unfolding in titin during an extended period (> approximately 20 min) of relaxation. By contrast, immunolabeling experiments failed to demonstrate large-scale unfolding. Thus, under physiological conditions in relaxed human soleus fibers, Ig domains are more stable than predicted by atomic force microscopy experiments. Ig-domain unfolding did not become more pronounced after gelsolin treatment, suggesting that the thin filament is unlikely to significantly contribute to the mechanical stability of the domains. We conclude that in human soleus fibers, Ig unfolding cannot solely explain stress relaxation.

Global configuration of single titin molecules observed through chain-associated rhodamine dimers.

The global configuration of individual, surface-adsorbed molecules of the giant muscle protein titin, labeled with rhodamine conjugates, was followed with confocal microscopy. Fluorescence-emission intensity was reduced because of self-quenching caused by the close spacing between rhodamine dye molecules that formed dimers. In the presence of chemical denaturants, fluorescence intensity increased, reversibly, up to 5-fold in a fast reaction; the kinetics were followed at the single-molecule level. We show that dimers formed in a concentrated rhodamine solution dissociate when exposed to chemical denaturants. Furthermore, titin denaturation, followed by means of tryptophan fluorescence, is dominated by a slow reaction. Therefore, the rapid fluorescence change of the single molecules reflects the direct action of the denaturants on rhodamine dimers rather than the unfolding/refolding of the protein. Upon acidic denaturation, which we have shown not to dissociate rhodamine dimers, fluorescence intensity change was minimal, suggesting that dimers persist because the unfolded molecule has contracted into a small volume. The highly contractile nature of the acid-unfolded protein molecule derives from a significant increase in chain flexibility. We discuss the potential implications this finding could have for the passive mechanical behavior of striated muscle.

Titin-actin interaction in mouse myocardium: passive tension modulation and its regulation by calcium/S100A1.

Passive tension in striated muscles derives primarily from the extension of the giant protein titin. However, several studies have suggested that, in cardiac muscle, interactions between titin and actin might also contribute to passive tension. We expressed recombinant fragments representing the subdomains of the extensible region of cardiac N2B titin (tandem-Ig segments, the N2B splice element, and the PEVK domain), and assayed them for binding to F-actin. The PEVK fragment bound F-actin, but no binding was detected for the other fragments. Comparison with a skeletal muscle PEVK fragment revealed that only the cardiac PEVK binds actin at physiological ionic strengths. The significance of PEVK-actin interaction was investigated using in vitro motility and single-myocyte mechanics. As F-actin slid relative to titin in the motility assay, a dynamic interaction between the PEVK domain and F-actin retarded filament sliding. Myocyte results suggest that a similar interaction makes a significant contribution to the passive tension. We also investigated the effect of calcium on PEVK-actin interaction. Although calcium alone had no effect, S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, inhibited PEVK-actin interaction in a calcium-dependent manner. Gel overlay analysis revealed that S100A1 bound the PEVK region in vitro in a calcium-dependent manner, and S100A1 binding was observed at several sites along titin's extensible region in situ, including the PEVK domain. In vitro motility results indicate that S100A1-PEVK interaction reduces the force that arises as F-actin slides relative to the PEVK domain, and we speculate that S100A1 may provide a mechanism to free the thin filament from titin and reduce titin-based tension before active contraction.

Mechanical fatigue in repetitively stretched single molecules of titin.

Relaxed striated muscle cells exhibit mechanical fatigue when exposed to repeated stretch and release cycles. To understand the molecular basis of such mechanical fatigue, single molecules of the giant filamentous protein titin, which is the main determinant of sarcomeric elasticity, were repetitively stretched and released while their force response was characterized with optical tweezers. During repeated stretch-release cycles titin becomes mechanically worn out in a process we call molecular fatigue. The process is characterized by a progressive shift of the stretch-force curve toward increasing end-to-end lengths, indicating that repeated mechanical cycles increase titin's effective contour length. Molecular fatigue occurs only in a restricted force range (0-25 pN) during the initial part of the stretch half-cycle, whereas the rest of the force response is repeated from one mechanical cycle to the other. Protein-folding models fail to explain molecular fatigue on the basis of an incomplete refolding of titin's globular domains. Rather, the process apparently derives from the formation of labile nonspecific bonds cross-linking various sites along a pre-unfolded titin segment. Because titin's molecular fatigue occurs in a physiologically relevant force range, the process may play an important role in dynamically adjusting muscle's response to the recent history of mechanical perturbations.

Mechanical manipulation of single titin molecules with laser tweezers.

Titin (also known as connectin) is a giant filamentous polypeptide of multi-domain construction spanning between the Z- and M-lines of the vertebrate muscle sarcomere. The molecule is significant in maintaining sarcomeric structural integrity and generating passive muscle force via its elastic properties. Here we summarize our efforts to characterize titin's elastic properties by manipulating single molecules with force-measuring laser tweezers. The titin molecules can be described as an entropic spring in which domain unfolding occurs at high forces during stretch and refolding at low forces during release. Statistical analysis of a large number (> 500) of stretch-release experiments and comparison of experimental data with the predictions of the wormlike chain theory permit the estimation of unfolded titin's mean persistence length as 16.86 A (+/- 0.11 SD). The slow rates of unfolding and refolding compared with the rates of stretch and release, respectively, result in a state of non-equilibrium and the display of force hysteresis. Folding kinetics as the source of non-equilibrium is directly demonstrated here by the abolishment of force hysteresis in the presence of chemical denaturant. Experimental observations were well simulated by superimposing a simple domain folding kinetics model on the wormlike chain behavior of titin and considering the characteristics of the compliant laser trap. The original video presentation of this paper may be viewed on the web at http:¿www.pote.hu/mm/prezentacio/mkpres/++ +mkpres.htm.

Complete unfolding of the titin molecule under external force.

Titin (also known as connectin) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere. Several earlier studies have implicated titin as playing a fundamental role in maintaining sarcomeric structural integrity and generating the passive force of muscle. The elastic properties of titin were characterized in recent single-molecule mechanical works that described the molecule as an entropic spring in which partial unfolding may take place at high forces during stretch and refolding at low forces during release. In the present work titin molecules were stretched using a laser tweezer with forces above 400 pN. The high external forces resulted in complete mechanical unfolding of the molecule, characterized by the disappearance of force hysteresis at high forces. Titin refolded following complete denaturation, as the hysteresis at low forces reappeared in subsequent stretch-release cycles. The broad force range throughout which unfolding occurred indicates that the various globular domains in titin require different unfolding forces due to differences in the activation energies for their unfolding.

Delayed dissociation of in vitro moving actin filaments from heavy meromyosin induced by low concentrations of Triton X-100.

The in vitro motility of fluorescent actin filaments over heavy meromyosin (HMM) was studied in the presence of the nonionic detergent Triton X-100. Below 0.004% Triton X-100 concentration, motility was not affected. Above 0.007%, motility was not observed because actin filaments were dissociated from HMM. In the Triton X-100 concentration range of 0.004-0.007%, the sliding actin filaments dissociated from HMM with a delay. The dissociation delay time decreased with increasing Triton X-100 concentration, increasing ATP (adenosine-5'-triphosphate) concentration, and increasing temperature. The delayed acto-HMM dissociation was absent when weak-binding kinetic intermediates of the myosin ATPase cycle (M.ATP and M.ADP-Pi) were used. The presence of sliding movement was necessary to evoke the delayed acto-HMM dissociation. The acto-HMM dissociation delay was independent of actin filament length. For a given Triton X-100 concentration, the dissociation delay time was found to be inversely proportional to sliding velocity, indicating that actin filaments travel a more or less constant distance prior to dissociation from HMM. The actin-activated HMM ATPase activity was not inhibited by Triton X-100; rather, it was slightly enhanced. The results imply the presence of a motility-associated conformational change in acto-HMM.

Folding-unfolding transitions in single titin molecules characterized with laser tweezers.

Titin, a giant filamentous polypeptide, is believed to play a fundamental role in maintaining sarcomeric structural integrity and developing what is known as passive force in muscle. Measurements of the force required to stretch a single molecule revealed that titin behaves as a highly nonlinear entropic spring. The molecule unfolds in a high-force transition beginning at 20 to 30 piconewtons and refolds in a low-force transition at approximately 2.5 piconewtons. A fraction of the molecule (5 to 40 percent) remains permanently unfolded, behaving as a wormlike chain with a persistence length (a measure of the chain's bending rigidity) of 20 angstroms. Force hysteresis arises from a difference between the unfolding and refolding kinetics of the molecule relative to the stretch and release rates in the experiments, respectively. Scaling the molecular data up to sarcomeric dimensions reproduced many features of the passive force versus extension curve of muscle fibers.

Actin-filament motion in the in vitro motility assay has a periodic component.

The interaction between actin and myosin can be studied in the in vitro motility assay, where fluorescently labelled actin filaments are observed to move over a lawn of myosin heads. To examine details of this movement, we measured systematically the velocities of the front end, rear end, and centroid of the actin filament as the filament translated over the assay surface. We found that these velocities exhibited an unexpectedly periodic component, alternating regularly between high and low values, superimposed on the steady velocity component. The period of the oscillatory component was approximately 380 ms. When translation was stopped by an increase in osmolarity, the filaments wiggled with a periodicity similar to the translating filament, implying that wiggling and translation may be related. Rigor filaments showed no periodicity. From the frequency content of the auto- and cross-correlation functions derived from the velocities of the front end, rear end, and centroid of the actin filament, we infer a deterministic, possibly wave-like process travelling along the actin filament. Potential molecular mechanisms underlying this phenomenon are considered.

Rescue of in vitro actin motility halted at high ionic strength by reduction of ATP to submicromolar levels.

The combined effects of ATP concentration and ionic strength were studied in an actomyosin in vitro motility assay using skeletal and cardiac myosin. The velocity of actin filaments increased up to a critical ionic strength, at which filament sliding stopped. At or above the critical ionic strength, filaments did not slide, but wiggled while focally attached to the surface. At these high ionic strengths, when the ATP concentration (originally 1 mM) was progressively reduced (down to submicromolar levels) by rigor-solution washes, the stationary, wiggling actin filaments promptly started to slide. The effect was reversible; upon adding ATP again, the sliding movement stopped, and wiggling began. The ATP washout-induced motility at high ionic strength may be explained by an electrostatic mechanism which determines the affinity of myosin to actin. The critical ionic strength was different for skeletal and cardiac myosin. For skeletal it was 77 mM, while for cardiac it was only 57 mM. Cardiac myosin's lower critical ionic strength implies a lower affinity to actin.

Elastic properties of single titin molecules made visible through fluorescent F-actin binding.

Titin (also known as connection) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere. Several indirect observations have implicated titin as playing a fundamental role in the generation of passive force of muscle, driven by titin's elastic properties. A direct observation of the mechanical properties of titin, however, has not been demonstrated. Here we have used the recently shown strong actin-binding property of titin to indirectly visualize and manipulate single molecules of titin. Titin molecules were immobilized on a microscope coverslip by attaching them to anti-titin antibody. The titin molecules were detected by attaching fluorescent actin filaments to them. The titin molecules were subsequently stretched by manipulating the free end of the attached actin filaments with a glass microneedle. Titin is shown here to possess a high degree of torsional and longitudinal flexibility. The molecule can be repetitively stretched at least fourfold, followed by recoil. Titin's unloaded elastic recoil proceeded in two stages: an initial rapid process (15 ms time constant) was followed by a slower one (400 ms time constant). The force necessary to fully extend titin--estimated by observing the breakage of the titin-bound actin filaments--may reach above approximately 100 pN (longitudinal tensile strength of actin). Attachment of fluorescent actin filaments to titin provides a useful tool to further probe titin's molecular properties.

Calcium-dependent inhibition of in vitro thin-filament motility by native titin.

Titin ( also known as connectin) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere and plays a fundamental role in the generation of passive tension. Titin has been shown to bind strongly to myosin, making it tightly associated to the thick filament in the sarcomere. Recent observations have suggested the possibility that titin also interacts with actin, implying further functions of titin in muscle contraction. We show -- using in vitro motility and binding assays -- that native titin interacts with both filamentous actin and reconstituted thin filaments. The interaction results in the inhibition of the filaments' in vitro motility. Furthermore, the titin-thin filament interaction occurs in a calcium-dependent manner: increased calcium results in enhanced binding of thin filaments to titin and greater suppression of in vitro motility.

Analysis of the bound nucleotide in the acto-heavy meromyosin in vitro motility assay.

Muscle contraction and various forms of cell motility are driven by the interaction of actin and myosin with the simultaneous binding and hydrolysis of ATP. The process can be reconstituted in in vitro motility assays, where actin filaments slide over myosin in an ATP-dependent fashion. We have recently shown that in vitro actin motility persists at unexpectedly low, nanomolar free ATP concentrations if the actomyosin is pretreated with millimolar levels of the nucleotide (10). In these experiments, however, the amount of bound ATP--which could potentially support motility--was not exactly known. In the present work, the amount of nucleotide bound in the in vitro motility assay is directly measured by using radiolabeled ATP analogs in a novel capillary binding assay. The results indicate that although a low quantity of nucleotide remains bound, it is stable and does not seem to be available to support motility.

ATP induces dissociation of the 90 kDa heat shock protein (hsp90) from F-actin: interference with the binding of heavy meromyosin.

The 90 kDa heat shock protein (hsp90) is a major cytoplasmic molecular chaperone associating with various other proteins such as steroid receptors, protein kinases and filamentous actin. hsp90 has also been shown to bind ATP, which causes a conformational change of the protein. The physiological role and significance of ATP binding by hsp90, however, has remained unclear. Here we show through direct, microscopic observations, that ATP induces the dissociation of actin filaments from immobilized molecules of hsp90 as well as the dissociation of F-actin from heavy meromyosin in the presence of hsp90.