Megan T. Valentine

Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro

Eg5, a member of the kinesin superfamily of microtubule-based motors, is essential for bipolar spindle assembly and maintenance during mitosis, yet little is known about the mechanisms by which it accomplishes these tasks. Here, we used an automated optical trapping apparatus in conjunction with a novel motility assay that employed chemically modified surfaces to probe the mechanochemistry of Eg5. Individual dimers, formed by a recombinant human construct Eg5–513–5His, stepped processively along microtubules in 8-nm increments, with short run lengths averaging approximately eight steps. By varying the applied load (with a force clamp) and the ATP concentration, we found that the velocity of Eg5 was slower and less sensitive to external load than that of conventional kinesin, possibly reflecting the distinct demands of spindle assembly as compared with vesicle transport. The Eg5–513–5His velocity data were described by a minimal, three-state model where a force-dependent transition follows nucleotide binding.

Measuring the mechanical stress induced by an expanding multicellular tumor system: a case study

Rapid volumetric growth and extensive invasion into brain parenchyma are hallmarks of malignant neuroepithelial tumors in vivo. Little is known, however, about the mechanical impact of the growing brain tumor on its microenvironment. To better understand the environmental mechanical response, we used multiparticle tracking methods to probe the environment of a dynamically expanding, multicellular brain tumor spheroid that grew for 6 days in a three-dimensional Matrigel-based in vitro assay containing 1.0-microm latex beads. These beads act as reference markers for the gel, allowing us to image the spatial displacement of the tumor environment using high-resolution time-lapse video microscopy. The results show that the volumetrically expanding tumor spheroid pushes the gel outward and that this tumor-generated pressure propagates to a distance greater than the initial radius of the tumor spheroid. Intriguingly, beads near the tips of invasive cells are displaced inward, toward the advancing invasive cells. Furthermore, this localized cell traction correlates with a marked increase in total invasion area over the observation period. This case study presents evidence that an expanding microscopic tumor system exerts both significant mechanical pressure and significant traction on its microenvironment.

Forces on a colloidal particle in a polymer solution: a study using optical tweezers

We report a study of the dynamical behaviour of a polystyrene latex sphere in a telechelic poly(ethylene oxide) solution using optical tweezers. With this new technique, we use a position-sensing detector and a lock-in amplifier to measure the displacement magnitude and phase responses of one latex sphere driven sinusoidally by optical tweezers. For a single particle in solution, the equation of motion of the particle is simply that of the forced oscillation problem with damping from viscous drag and the restoring force from the elasticity of the solution medium and that of the optical tweezers. Because the system is overdamped, it is not feasible to probe the high-frequency regime. Thus we cannot measure the viscosity and elastic moduli separately from frequency-dependent measurements alone. At low polymer concentration, measurements of the viscosity have been achieved. We compared the measured viscosity with that obtained with other measurements. Key issues for further development of this technique, such as measuring the elastic modulus, are briefly discussed.

Precision steering of an optical trap by electro-optic deflection

We designed, constructed, and tested a single-beam optical trapping instrument employing twin electro-optic deflectors (EODs) to steer the trap in the specimen plane. Compared with traditional instruments based on acousto-optic deflectors (AODs), EOD-based traps offer a significant improvement in light throughput and a reduction in deflection-angle (pointing) errors. These attributes impart improved force and position resolution, making EOD-based traps a promising alternative for high-precision nanomechanical measurements of biomaterials.

Eg5 steps it up

Understanding how molecular motors generate force and move microtubules in mitosis is essential to understanding the physical mechanism of cell division. Recent measurements have shown that one mitotic kinesin superfamily member, Eg5, is mechanically processive and capable of crosslinking and sliding microtubules in vitro. In this review, we highlight recent work that explores how Eg5 functions under load, with an emphasis on the nanomechanical properties of single enzymes.

Microscope-based static light-scattering instrument

We describe a new design for a microscope-based static light-scattering instrument that provides simultaneous high-resolution images and static light-scattering data. By correlating real space images with scattering patterns, we can interpret measurements from heterogeneous samples, which we illustrate by using biological tissue.

Microscopic origin of light scattering in tissue.

A newly designed instrument, the static light-scattering (SLS) microscope, which combines light microscopy with SLS, enables us to characterize local light-scattering patterns of thin tissue sections. Each measurement is performed with an illumination beam of 70-microm diameter. On these length scales, tissue is not homogeneous. Both structural ordering and small heterogeneities contribute to the scattering signal. Raw SLS data consist of a two-dimensional intensity distribution map I(theta, phi), showing the dependence of the scattered intensity I on the scattering angle theta and the azimuthal angle phi. In contrast to the majority of experiments and to simulations that consider only the scattering angle, we additionally perform an analysis of the azimuthal dependence I(phi). We estimate different contributions to the azimuthal scattering variation and show that a significant fraction of the azimuthal amplitude is the result of tissue structure. As a demonstration of the importance of the structure-dependent part of the azimuthal signal, we show that this function of the scattered light alone can be used to classify tissue types with surprisingly high specificity and sensitivity.

Microrheology of highly crosslinked microtubule networks is dominated by force-induced crosslinker unbinding

We determine the time- and force-dependent viscoelastic responses of reconstituted networks of microtubules that have been strongly crosslinked by biotin-streptavidin bonds. To measure the microscale viscoelasticity of such networks, we use a magnetic tweezers device to apply localized forces. At short time scales, the networks respond nonlinearly to applied force, with stiffening at small forces, followed by a reduction in the stiffening response at high forces, which we attribute to the force-induced unbinding of crosslinks. At long time scales, force-induced bond unbinding leads to local network rearrangement and significant bead creep. Interestingly, the network retains its elastic modulus even under conditions of significant plastic flow, suggesting that crosslinker breakage is balanced by the formation of new bonds. To better understand this effect, we developed a finite element model of such a stiff filament network with labile crosslinkers obeying force-dependent Bell model unbinding dynamics. The coexistence of dissipation, due to bond breakage, and the elastic recovery of the network is possible because each filament has many crosslinkers. Recovery can occur as long as a sufficient number of the original crosslinkers are preserved under the loading period. When these remaining original crosslinkers are broken, plastic flow results.

Toughening elastomers using mussel-inspired iron-catechol complexes

Combining stiffness and stretchiness There is usually a trade-off between making a material stretchy, so that it can absorb energy on deformation, and making a material stiff, so that it does not extend very much when stretched. Mussels have long been an inspiration for developing adhesives that work when wet. Filippidi et al. produced an extensible polymeric material containing catechol groups whose mechanical properties were augmented when dry through the addition of iron ions (see the Perspective by Winey). The iron ions lead to sacrificial metal coordination bonds, creating a reversible load-bearing network that does not trade extensibility for stiffness. Science, this issue p. 502; see also p. 449 Mussel-inspired iron complexes improve dry elastomer stiffness, strength, and toughness via enhanced energy dissipation. Materials often exhibit a trade-off between stiffness and extensibility; for example, strengthening elastomers by increasing their cross-link density leads to embrittlement and decreased toughness. Inspired by cuticles of marine mussel byssi, we circumvent this inherent trade-off by incorporating sacrificial, reversible iron-catechol cross-links into a dry, loosely cross-linked epoxy network. The iron-containing network exhibits two to three orders of magnitude increases in stiffness, tensile strength, and tensile toughness compared to its iron-free precursor while gaining recoverable hysteretic energy dissipation and maintaining its original extensibility. Compared to previous realizations of this chemistry in hydrogels, the dry nature of the network enables larger property enhancement owing to the cooperative effects of both the increased cross-link density given by the reversible iron-catecholate complexes and the chain-restricting ionomeric nanodomains that they form.

Force and Premature Binding of ADP Can Regulate the Processivity of Individual Eg5 Dimers

Using a high-resolution optical trapping instrument, we directly observed the processive motions of individual Eg5 dimers over a range of external loads and ATP, ADP, and phosphate concentrations. To constrain possible models for dissociation from the microtubule, we measured Eg5 run lengths and also compared the duration of the last step of a processive run to all previous step durations. We found that the application of large longitudinal forces in either hindering or assisting directions could induce Eg5-microtubule dissociation. At a constant moderate force, maintained with a force clamp, the premature binding of ADP strongly promoted microtubule release by Eg5, whereas the addition of ATP or phosphate had little effect on dissociation. These results imply that run length is determined not only by the load, but also by the concentration and type of nucleotides present, and therefore that the biochemical cycles of the two motor domains of the Eg5 dimer are coordinated to promote processive stepping.