Biophysical journal最新文献

The apparent bending moduli (K_C) of bilayers composed of binary mixtures of lipids with different spontaneous curvatures have been obtained using x-ray diffuse scattering (XDS). The mixtures that were studied are POPC/POPE, POPC/POPA, POPC/POPS, and DLPC/DiPhyPC. The data are qualitatively consistent with what is expected from the theory of diffusional softening for lipids with different spontaneous curvatures. However, the derived spontaneous curvature differences are larger than those obtained from the hexagonal_II (H_II) phase and from a recent giant unilamellar vesicle (GUV) study. We propose that the interactions between lipids, which we have added to the theory, also play an important role in the values of K_C obtained at the short length scale of XDS. Inclusion of a mean field term in the analysis brings the calculated difference in spontaneous curvatures ΔC₀ of the two lipids closer to the values from the H_II and GUV methods. The use of XDS opens a new experimental window on diffusional softening and the interactions between lipids in mixtures.

Single-molecule manipulation techniques are used to elucidate mechanisms in biological systems. Optical tweezers are powerful tools because of their ease of use in combination with optical microscopy and appropriate torque range. However, the use of optical tweezers to generate rotational motion is difficult owing to the complexity of applying constant torque to a moving molecule. The magnitude of the torque applied with optical tweezers depends on the positional relationship with the trapping particle and requires positional feedback. In this study, we found that the adaptation of optical vortices (OVs) generated by phase modulation of optical tweezers enabled quantitative mechanical manipulations. Moreover, optical tweezers with an OV could be applied to measure the torque generated by a molecular motor. We used an OV to apply torque via a precise handle consisting of a DNA origami rod to a rotating molecular motor, F₁-ATPase. Using the constant torque generated by the OV, we applied torques to F₁-ATPase and succeeded in stalling and reversing its rotation. This technique is useful for applying constant torque to biomolecules.

Cells sense substrate mechanical properties through the integrin-talin-F-actin linkage. Talin's N-terminal head domain binds β-integrin, whereas its C-terminal domain connects to F-actin directly via two actin binding sites (ABS) and indirectly through cryptic vinculin binding sites (VBS) within rod domain bundles. Force-induced unfolding of these alpha helical bundles exposes VBS, recruiting vinculin to strengthen the talin-actin bond. This system is sensitive to the loading rate and is influenced by rates of F-actin movement and substrate stiffness. Though the components of this pathway are well studied, how talin, vinculin and actin synergize to mechanically buffer loads and mediate cellular stiffness sensing remains incompletely understood. We developed a multiscale stochastic finite element model to simulate talin unfolding during interactions with retrograde actin flows, and analyzed the contributions of ABS2, ABS3, and VBS to talin mechanosensitivity. Vinculin attachments strengthened the force-bearing capacity in talin, stabilized the actin-talin contact, and regulated binding site activity at R3. The lifetime of dynamic bond formed between talin, and actin decreased with increase in actin flow velocity. Higher substrate stiffness enhanced the lifetime at low actin flow velocity but negatively impacted it at higher velocities. ABS3 primarily mediated force transfer from actin to talin at rapid actin flows, while vinculin and ABS2 reinforced the F-actin bond under slower flows. Stiffer substrates enhanced force transmission through VBS. Our results show that stretch rate modulated force feedback between the unfolding of talin rod domains and VBS attachments, drive sensitivity of talin to substrate stiffness.

In 1952, Alan Turing showed that analog reaction-diffusion equations were extremely powerful models of biological development and of distributed cellular automata. Analog circuits have been shown to accelerate the simulation of chemical reactions by many orders of magnitude, including in stochastic (noisy) cytomorphic chips which are useful for drug-cocktail formulation and in systems medicine. However, the simulation of the partial differential equations of diffusion is expensive to architect in analog systems. Here, we show how to simulate diffusion as though it were a chemical reaction such that reaction-reaction analog systems can simulate reaction-diffusion systems. As an example, we show that the BMP-SOX9-WNT reaction-diffusion system can be simulated in analog cytomorphic integrated circuits that only have reaction circuits but no explicit diffusion circuits. Experimental data from reaction-reaction circuits show excellent agreement with MATLAB and COPASI simulations of biological models. Even cytomorphic chips with relatively sparse sampling appear to demonstrate decaying and unstable growing waves. Our work is the first step towards large-scale simulations of spatiotemporal reaction-diffusion equations.

Microtubules (MTs) are a major component of the eukaryotic cytoskeleton. MT architecture is highly regulated by microtubule-associated proteins (MAPs) such as Tau as well as a number of MT-targeted chemotherapeutic agents such as paclitaxel (PTX). In this study, we examined the ability of each of the six different alternatively spliced isoforms of human wild-type (WT) Tau (4R2N, 4R1N, 4R0N, 3R2N, 3R1N and 3R0N) and PTX to bind to MTs as well as their effects upon MT structure. MTs were assembled in the physiologically relevant experimental regime of mixing WT Tau protein with unpolymerized tubulin and then treating the resulting MTs with PTX (i.e. Tau-coassembled MTs). The extent of Tau and PTX binding to MTs were assayed by co-sedimentation/western blotting and high-performance liquid chromatography, respectively. Radial size of MTs was determined by synchrotron small-angle X-ray scattering (SAXS). We observed that 4R Tau and PTX compete for binding to MTs while 3R Tau and PTX exhibit only limited competition. These observations suggest that both 4R and 3R Tau bind initially to the well-studied binding sites on the outer surface of MTs, followed by binding to the less-well understood binding site within the MT lumen in an isoform-specific manner. These binding events also lead to distinct effects on MT radial structure compared with MTs formed by PTX and then treated with Tau (i.e. PTX-stabilized MTs). Specifically, the inner radius of MTs first increased and then markedly decreased with increasing Tau concentrations. In addition to providing fundamental insights in the basic biochemistry of MTs, our results have implications the onset and progression of chemotherapy-induced peripheral neuropathy (CIPN), a consequence of many MT-targeted anti-cancer therapeutics including PTX. The differential use of the luminal Tau binding site in 4R versus 3R further raises the possibility of differential Tau isoform action in fetal versus adult nervous systems.