No abstract

Proteolytic degradation of fibrin, the major structural component in blood clots, is critical both during normal wound healing and in the treatment of ischemic stroke and myocardial infarction. Fibrin-containing clots experience substantial strain due to platelet contraction, fluid shear, and mechanical stress at the wound site. However, little is understood about how mechanical forces may influence fibrin dissolution. We used video microscopy to image strained fibrin clots as they were degraded by plasmin, a major fibrinolytic enzyme. Applied strain causes up to 10-fold reduction in the rate of fibrin degradation. Analysis of our data supports a quantitative model in which the decrease in fibrin proteolysis rates with strain stems from slower transport of plasmin into the clot. We performed fluorescence recovery after photobleaching (FRAP) measurements to further probe the effect of strain on diffusive transport. We find that diffusivity perpendicular to the strain axis decreases exponentially with increasing strain, while diffusivity along the strain axis remains unchanged. Our results suggest that the properties of the fibrin network have evolved to protect mechanically loaded fibrin from degradation, consistent with its function in wound healing. The pronounced effect of strain upon diffusivity within fibrin networks offers a means of tuning the transport of proteins and other soluble factors within fibrin-based biomaterials, potentially addressing a key challenge in engineering complex tissues in vitro. The mechanical and functional properties of DNA arise from its double helical structure. It is now widely accepted that the torsional properties of DNA and DNA supercoiling play an important role in the kinetics of many DNAbinding proteins, but the mechanism underlying this relationship remains unclear. To address this gap in our understanding, we need an instrument that can accurately measure and control torsional stress applied to DNA. We have developed a high-bandwidth electromagnetic trapping system that can generate a uniform magnetic field in the sample region and apply constant torque above 10 2 pN$nm on the samples under study. The octupole magnetic trap is integrated into a microscope-based particle tracking system and can rotate superparamagnetic particles with three degrees of rotational freedom. The large signal bandwidth of the current in the coils can reach above 3kHz at 800uH inductive load and the heat generated by the current is dissipated by an active PID-controlled cooling system to prevent heating biological samples. The magnetic trap is being designed to independently control force and torque, allowing us to confine superparamagnetic particles in a trap with low torsional stiffness that is suitable for torque application and measurement at biologically relevant scales. To directly measure the torsional strain in DNA, we are planning to use superparamagnetic beads coated with metal on one hemisphere. Our magnetic torque tweezers are intended to quantitatively measure the changes of torsion...

We are using tethered particle motion (TPM) microscopy to observe protein-mediated DNA looping in the lactose repressor system in DNA constructs with varying AT / CG content. We use these data to determine the persistence length of the DNA as a function of its sequence content and compare the data to direct micromechanical measurements with constant-force axial optical tweezers. The data from the TPM experiments show a much smaller sequence effect on the persistence length than the optical tweezers experiments.

different locations along the genome and changes during the cell cycle, little is known about how localized changes in DNA supercoiling, under enzymegenerated DNA tension, perturb critical protein-DNA interactions. Here we investigated how DNA supercoiling affects stability of the lambda repressormediated DNA loop that acts as a genetic switch between lysogeny (quiescence) and lysis (virulence). We performed single molecule magnetic tweezers measurements to record lambda repressor-mediated DNA loop formation and breakdown and to measure the stability of the loop as a function of negative supercoiling, loop size and DNA tension at physiological repressor concentration. The level of negative supercoiling required for loop formation increases with loop size and that, in general, negative supercoiling stabilizes the loop. Since genomic supercoiling depends on the energy level of the cell which is tightly associated with its health status, we propose that the switch to lysis is favored by the destabilization of the lambda repressor-mediated loop that follows loss of DNA supercoiling in suffering cells. By using a dual laser optical tweezers with a fast force feedback, we were able to record the exponential elongation following force steps imposed on D-phage ds-DNA molecule and on two~3000 bp segments of the molecule, with either high (59%) or low (46%) CG content. The rate of elongation (r) following a 2pN step imposed on the whole molecule in the region of the overstretching transition varies with the force in a U shaped way, while its corresponding elongation varies in a reverse-U shaped way (Bianco et al., Biophys. J. 101, 866-874, 2011). The minimum of the r -force relation (rmin) and the corresponding maximum elongation (DLe) do not change significantly with temperature in the range 25-10 C (mean values 4.9 5 0.2 s-1 and 3.9 5 0.2 mm respectively) and are shifted progressively to higher forces at lower temperature. The load-and temperature-dependence of the elongation rate supports the two state (B-S) nature of the transition, yielding a cooperativity of 22 bp and revealing the absence of an enthalpic contribution to the transition free energy barrier. At room temperature the AT rich segment shows large hysteresis on relaxation, implying a predominance of melting on overstretching, while the CG rich segment does not show hysteresis. At temperature below 10 C hysteresis completely disappears for both segments. rmin and DLe are 3.9 51.4 s-1 and 0.3 5 0.1 nm for the CG and 4.3 5 1.3 s-1 and 0.3 5 0.1 nm for the AT. Fitting the data with the two state reaction model shows a cooperativity of~15 and 32 for the AT and CG respectively, which correlates with the average distance between groups of more than four consecutive A or T bases.