Interaction Forces between F-Actin and Titin PEVK Domain Measured with Optical Tweezers

AbstractTitin is a giant protein that determines the elasticity of striated muscle and is thought to play important roles in numerous regulatory processes. Previous studies have shown that titin's PEVK domain interacts with F-actin, thereby creating viscous forces of unknown magnitude that may modulate muscle contraction. Here we measured, with optical tweezers, the forces necessary to dissociate F-actin from individual molecules of recombinant PEVK fragments rich either in polyE or PPAK motifs. Rupture forces at a stretch rate of 250 nm/s displayed a wide, nonnormal distribution with a peak at ∼8 pN in the case of both fragments. Dynamic force spectroscopy experiments revealed low spontaneous off-rates that were increased even by low forces. The loading-rate dependence of rupture force was biphasic for polyE in contrast with the monophasic response observed for PPAK. Analysis of the molecular lengths at which rupture occurred indicated that there are numerous actin-binding regions along the PEVK fragments’ contour, suggesting that the PEVK domain is a promiscuous actin-binding partner. The complexity of PEVK-actin interaction points to an adaptable viscoelastic mechanism that safeguards sarcomeric structural integrity in the relaxed state and modulates thixotropic behavior during contraction

Spatially and Temporally Synchronized Atomic Force and Total Internal Reflection Fluorescence Microscopy for Imaging and Manipulating Cells and Biomolecules

AbstractThe atomic force microscope is a high-resolution scanning-probe instrument which has become an important tool for cellular and molecular biophysics in recent years but lacks the time resolution and functional specificities offered by fluorescence microscopic techniques. To exploit the advantages of both methods, here we developed a spatially and temporally synchronized total internal reflection fluorescence and atomic force microscope system. The instrument, which we hereby call STIRF-AFM, is a stage-scanning device in which the mechanical and optical axes are coaligned to achieve spatial synchrony. At each point of the scan the sample topography (atomic force microscope) and fluorescence (photon count or intensity) information are simultaneously recorded. The tool was tested and validated on various cellular (monolayer cells in which actin filaments and intermediate filaments were fluorescently labeled) and biomolecular (actin filaments and titin molecules) systems. We demonstrate that with the technique, correlated sample topography and fluorescence images can be recorded, soft biomolecular systems can be mechanically manipulated in a targeted fashion, and the fluorescence of mechanically stretched titin can be followed with high temporal resolution

Cross-Species Mechanical Fingerprinting of Cardiac Myosin Binding Protein-C

AbstractCardiac myosin binding protein-C (cMyBP-C) is a member of the immunoglobulin (Ig) superfamily of proteins and consists of 8 Ig- and 3 fibronectin III (FNIII)-like domains along with a unique regulatory sequence referred to as the MyBP-C motif or M-domain. We previously used atomic force microscopy to investigate the mechanical properties of murine cMyBP-C expressed using a baculovirus/insect cell expression system. Here, we investigate whether the mechanical properties of cMyBP-C are conserved across species by using atomic force microscopy to manipulate recombinant human cMyBP-C and native cMyBP-C purified from bovine heart. Force versus extension data obtained in velocity-clamp experiments showed that the mechanical response of the human recombinant protein was remarkably similar to that of the bovine native cMyBP-C. Ig/Fn-like domain unfolding events occurred in a hierarchical fashion across a threefold range of forces starting at relatively low forces of ∼50 pN and ending with the unfolding of the highest stability domains at ∼180 pN. Force-extension traces were also frequently marked by the appearance of anomalous force drops suggestive of additional mechanical complexity such as structural coupling among domains. Both recombinant and native cMyBP-C exhibited a prominent segment ∼100 nm-long that could be stretched by forces <50 pN before the unfolding of Ig- and FN-like domains. Combined with our previous observations of mouse cMyBP-C, these results establish that although the response of cMyBP-C to mechanical load displays a complex pattern, it is highly conserved across species

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

AbstractTitin (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