Cofilin-mediated actin filament network flexibility facilitates 2D to 3D actomyosin shape change.

The organization of actin filaments (F-actin) into crosslinked networks determines the transmission of mechanical stresses within the cytoskeleton and subsequent changes in cell and tissue shape. Principally mediated by proteins such as α-actinin, F-actin crosslinking increases both network connectivity and rigidity, thereby facilitating stress transmission at low crosslinking yet attenuating transmission at high crosslinker concentration. Here, we engineer a two-dimensional model of the actomyosin cytoskeleton, in which myosin-induced mechanical stresses are controlled by light. We alter the extent of F-actin crosslinking by the introduction of oligomerized cofilin. At pH 6.5, F-actin severing by cofilin is weak, but cofilin bundles and crosslinks filaments. Given its effect of lowering the F-actin bending stiffness, cofilin- crosslinked networks are significantly more flexible and softer in bending than networks crosslinked by α-actinin. Thus, upon local activation of myosin-induced contractile stress, the network bends out-of-plane in contrast to the in-plane compression as observed with networks crosslinked by α-actinin. Here, we demonstrate that local effects on filament mechanics by cofilin introduces novel large-scale network material properties that enable the sculpting of complex shapes in the cell cytoskeleton.

Intracellular tension sensor reveals mechanical anisotropy of the actin cytoskeleton.

The filamentous actin (F-actin) cytoskeleton is a composite material consisting of cortical actin and bundled F-actin stress fibers, which together mediate the mechanical behaviors of the cell, from cell division to cell migration. However, as mechanical forces are typically measured upon transmission to the extracellular matrix, the internal distribution of forces within the cytoskeleton is unknown. Likewise, how distinct F-actin architectures contribute to the generation and transmission of mechanical forces is unclear. Therefore, we have developed a molecular tension sensor that embeds into the F-actin cytoskeleton. Using this sensor, we measure tension within stress fibers and cortical actin, as the cell is subject to uniaxial stretch. We find that the mechanical response, as measured by FRET, depends on the direction of applied stretch relative to the cell's axis of alignment. When the cell is aligned parallel to the direction of the stretch, stress fibers and cortical actin both accumulate tension. By contrast, when aligned perpendicular to the direction of stretch, stress fibers relax tension while the cortex accumulates tension, indicating mechanical anisotropy within the cytoskeleton. We further show that myosin inhibition regulates this anisotropy. Thus, the mechanical anisotropy of the cell and the coordination between distinct F-actin architectures vary and depend upon applied load.

SPAK-dependent cotransporter activity mediates capillary adhesion and pressure during glioblastoma migration in confined spaces.

The invasive potential of glioblastoma cells is attributed to large changes in pressure and volume, driven by diverse elements, including the cytoskeleton and ion cotransporters.  However, how the cell actuates changes in pressure and volume in confinement, and how these changes contribute to invasive motion is unclear. Here, we inhibited SPAK activity, with known impacts on the cytoskeleton and cotransporter activity and explored its role on the migration of glioblastoma cells in confining microchannels to model invasive spread through brain tissue. First, we found that confinement altered cell shape, inducing a transition in morphology that resembled droplet interactions with a capillary vessel, from "wetting" (more adherent) at low confinement, to "nonwetting" (less adherent) at high confinement. This transition was marked by a change from negative to positive pressure by the cells to the confining walls, and an increase in migration speed. Second, we found that the SPAK pathway impacted the migration speed in different ways dependent upon the extent of wetting. For nonwetting cells, SPAK inhibition increased cell-surface tension and cotransporter activity. By contrast, for wetting cells, it also reduced myosin II and YAP phosphorylation. In both cases, membrane-to-cortex attachment is dramatically reduced. Thus, our results suggest that SPAK inhibition differentially coordinates cotransporter and cytoskeleton-induced forces, to impact glioblastoma migration depending on the extent of confinement.

Assessment of lumbar spinal disc injury in frontal crashes.

Frontal vehicle crashes have been a leading cause of spinal injuries in recent years. Reconstruction of frontal crashes using computational models and spinal load analysis helps us understand the patterns of injury and load propagation during frontal crashes. By reconstructing a real crash test and using a viscoelastic crash dummy model, spinal injury patterns were analyzed. The results indicated that a moderate crash with an impact speed of 56 km/h leads to injuries in L1-L2 and L5-S1 levels (L for lumbar and S for sacral vertebrae). The largest spinal loads and injuries were mainly observed immediately after the airbag deployment when the peak of the crash acceleration transpires. Also, the effects of impulse magnitude on the spinal loads and head injury criterion (HIC) showed that HIC is more sensitive than compressive forces to the magnitude of impulse. Moreover, the effects of disc preconditioning as a major factor in the risk of injury was evaluated. The results demonstrate that as the lumbar spine is subjected to a longer preloading, it will be more vulnerable to injury; preconditioning of the discs more adversely affected the risk of injury than a 10% increase in the crash impulse. Overall the results highlight the importance of spinal injury prevention in frontal crashes.

Effect of whole-body vibration and sitting configurations on lumbar spinal loads of vehicle occupants.

Whole-body vibration (WBV) has been identified as one of the serious risk factors leading to spinal disorders, particularly in professional drivers. Although the influential factors in this area have been investigated epidemiologically, finite element (FE) modeling can efficiently help us better understand the problem. In this study, a modified HYBRID III dummy FE model which was enhanced by detailed viscoelastic discs in the lumbar region was utilized to simulate the effect of WBV on lumbar spine loads. Spinal responses to the vertical sinusoidal vibrations of a generic seat were obtained and spinal injury risk factors were calculated. Effects of variation of excitation frequencies, three different seatback inclinations and four pre-defined occupant postures on the spinal loads were investigated as influential variables. Results showed that under sinusoidal loading with a frequency of 5 Hz and in a typical sitting configuration, disc forces remained in a safe range (<1700 N) for short term. Collagen Fibers strain (<0.3%) and intradiscal pressure (<1.15 MPa) also indicated that the spinal loads were in a safe range. Additionally, calculating the risk factor according to ISO 2631-5 (about R = 0.8) confirmed the low probability of an adverse health effect due to WBV in long term. Frequency-domain analysis showed the resonance frequency to be at f = 6.27 Hz. Although according to ISO/CD 2631-5 standard, the occupant experienced the highest risk of injury at f = 7 Hz, it was found that spinal compression load at f = 6 Hz was 7.7% higher than the compression load at f = 7 Hz. Seatback oriented at 75° exhibited the highest risk of injury, nevertheless, maximum von-Mises stress in disc annulus was observed at 70°. In the evaluation of occupant posture, lordotic and slouching postures were compared and the latter exhibited higher stress ranges resulting in higher injury risk factor. Results of the model demonstrated its aptness to predict the spinal disc injuries in response to various vibrational loading and boundary conditions.

Modeling and validation of a detailed FE viscoelastic lumbar spine model for vehicle occupant dummies.

The dummies currently used for predicting vehicle occupant response during frontal crashes or whole-body vibration provide insufficient information about spinal loads. Although they aptly approximate upper-body rotations in different loading scenarios, they overlook spinal loads, which are crucial to injury assessment. This paper aims to develop a modified dummy finite element (FE) model with a detailed viscoelastic lumbar spine. This model has been developed and validated against in-vitro and in-silico data under different loading conditions, and its predicted ranges of motion (RoM) and intradiscal pressure (IDP) maintain close correspondence with the in-vitro data. The dominant frequency of the model was f = 8.92 Hz, which was close to previous results. In the relaxation test, a force reduction of up to 21% was obtained, showing high agreement in force relaxation during the in-vitro test. The FE lumbar spine model was placed in the HYBRID III test dummy and aligned in a seated position based on available MRI data. Under two impulsive acceleration loadings in flexion and lateral directions with a peak acceleration of 60 m/s2, flexion responses of the modified and original dummies were close (RoMs of 29.1° and 29.6°, respectively), though not in lateral bending (RoMs of 34.1° and 15.6°, respectively), where the modified dummy was more flexible than the original. By reconstructing a real frontal crash, it was found that the modified dummy provided a 10% reduction in the Head Injury Criterion (HIC). Other than the more realistic behavior of this modified dummy, its capability of approximating lumbar loads and risk of lumbar spine injuries in vehicle crashes or whole-body vibration is of great importance.