Cell-substrate adhesions are a mechanosensitive protein complex that regulates various cellular functions. Molecular mechanisms underlying the physical force-dependent regulation remain elusive partly because endogenous forces are distributed in a spatially heterogeneous manner within cells, thus complicating the interpretation on the effect of forces. Here we use a micropatterning technique to focus spatially distributed intracellular contractile forces onto a particular subcellular area, with which mechanical and pharmacological effects are separately analyzed. Single human osteosarcoma U2OS cells were plated within square micropatterns, and phosphorylation of an adhesion-associated adaptor protein paxillin was analyzed. Paxillin, visualized with immunostaining, was highly accumulated in the proximity of the corners of the square micropatterns where cellular forces are concentrated, but the huge paxillin-labeled adhesions were less phosphorylated compared to those present elsewhere as a small patch. Pharmacological inhibition of the endogenous forces resulted in disassembly of the huge dephoshorylated paxillin clusters; in contrast, the small, highly phosphorylated paxillin patches persisted. Similar negative regulation of paxillin phosphorylation is also induced upon loading of exogenous forces. Unexpectedly, on the other hand, immunoblot of cell lysates showed a tendency of a reduction in paxillin phosphorylation upon the same pharmacological inhibition of the endogenous forces. Thus, the response of paxillin phosphorylation to mechanical forces was the opposite between the immunostaining and immunoblot data; i.e., the phosphorylation is reduced and enhanced at subcellular level and whole-cell level, respectively, in response to loading of mechanical forces. To reconcile the contradictory results, we submit a simple model that is consistent with not only the present but also previous reports on the regulation of paxillin. The model implies that similar opposite response can generally emerge if the protein activation is negatively regulated at a local place while the activation trigger alters the assembly of the protein to the local place.

Cell migration is an important process both in physiological and pathological conditions. Migrating cells in vivo respond to various extracellular environmental factors and change their migratory behavior. Thus, it is important to take into account extracellular environmental factors in studies on cell migration. This study specifically focused on fibroblast migration in a three-dimensional microenvironment. We fabricated a polydimethylsiloxane cell culture substrate with intersecting grooves as a model to mimic a feature of the complex porous microenvironment experienced by fibroblasts in vivo. The sizes of the grooves allowed fibroblasts to penetrate into the grooves. The effects of branched grooved structures on cell migration, and on cellular organization of the actin filaments and phosphorylated myosin light chain, were analyzed. Fibroblasts migrated along intersecting lattice grooves that were 5 μm wide, 13 μm deep, and spaced 10 μm apart. Analysis of the cellular distribution of actin filaments and phosphorylated myosin light chain demonstrated two effects of the intersecting grooved structure on actin cytoskeletal organization in the fibroblast. One was the enhancement of filopodia protrusions into the branched groove at the junction, and the other was the formation of stress fibers to cross the opening of the junction. These results suggest that the filopodia protrusions are guided by the groove and are followed by the cytoplasmic protrusion, then the rear of the cell retracts due to stress fiber contraction, leading to fibroblast guided migration in the branched intersecting groove.

Mechanisms of development, growth, and rupture of a cerebral aneurysm have been studied by computational fluid dynamics (CFD), calculating hemodynamic parameters, such as wall shear stress, oscillatory shear index (OSI), and relative residence time (RRT). It is well known that the upstream velocity profile and upstream vessel shape influence the computational results. However, few studies have dealt with cases involving a bifurcation upstream of a cerebral aneurysm. Furthermore, the fluid-dynamic effects of multiple structural elements of upstream vessel shape, such as a bifurcation and a bend, on the results of CFD remain unknown. The purpose of this study was to elucidate the fluid-dynamic effects of multiple structural elements of upstream vessel shape such as bifurcation and bend on blood flow and hemodynamic parameters inside an aneurysm. Computations were performed for blood flow in a cerebral aneurysm with upstream sequential elements of a bifurcation, a bend, and a straight section, using four models with different upstream boundaries, i.e., before the bifurcation, after the bifurcation before the bend, after the bend, and after the straight section just before the cerebral aneurysm. The results were compared to elucidate the effect of each upstream structural element on the blood flow and hemodynamic parameters in the cerebral aneurysm. Differences in OSI and RRT resulting from changes in the velocity distribution were observed locally in the aneurysm between the models. It was also found that the effect of the bifurcation on the velocity distribution was greater than that of the bend.

Adherent cells generate traction forces, which are generated by and transmitted along the actin cytoskeleton to the underlying external substrate via focal adhesions. These cell generated forces are fundamental for maintaining cell shape and driving cell migration as well as triggering signaling pathways to promote processes such as differentiation and proliferation. Here we investigate how mechanical stretch affects traction forces. Following moderate mechanical stretching and release, traction forces are increased and then return to basal levels. However, when cells are stretched with relatively large strains, >10%, traction forces fail to return to basal levels and are attenuated. In this study, we investigate whether cells experiencing attenuated traction forces, following a large strain, can still respond to subsequent mechanical stretching. Traction forces were measured in cells following a consecutive 12% increase in mechanical stretch. We show that, even after traction force attenuation occurs, cells are able to respond to consecutive mechanical stretching by increasing traction force. Visualization of stress fibers, with GFP-actin, indicates that the attenuation of traction force is accompanied by a decrease in stress fiber integrity during mechanical stretching. The ability of cells to increase traction force during a second round of stretching, following attenuation, maybe generated by intact stress fibers that escape damage.

We previously described an analytical model regarding how human falsetto voice is produced upon interaction between the respiratory airflow and vocal fold motion. This theory highlights the role of an unsteady flow effect―or specifically, convective acceleration of wall motion-induced flow―in inducing a Hopf bifurcation or aerodynamic flutter of vocal folds, reminiscent of falsetto voice production. Importantly, the mucosal wave motion and glottal closure of the vocal folds―typically observed in human modal voice production but absent in falsetto vocalization of high voice pitch―are dispensable in this analytical model. Thus, given its rigorous applicability to high-pitched vocalization, our model may function as a universal physical mechanism underlying the vocalization of not only humans but also other diverse vertebrate animals that share a basic anatomical design. Here we show that the relationship between the vocal frequency and animal body size and mass, derived from the present model, captures the actual features reported elsewhere, thus suggesting that the allometric scaling of animal vocalization is explained theoretically. Moreover, the critical biomechanical conditions that induce the vocalization are rewritten to highlight an intriguing consequence from our model that the voice pitch can be controlled simply and extensively with the mechanical tension in vocal membranes. Furthermore, several dimensionless numbers that characterize the aerodynamic flutter are introduced to shed light on the physical essence of the possible universal mechanism underlying vertebrate animal vocalization: whether the animal prefers to falsetto or modal vocalization is determined by its communication frequency.

Tension in actin filaments plays crucial roles in multiple cellular functions, although little is known about tension dynamics of cells during adhesion to substrates. In this study, we visualized intracellular tension in actin filaments using a newly developed Förster resonance energy transfer (FRET)-based tension sensor (Actinin-sstFRET-GR). Tension dynamics were monitored during adhesion in MC3T3-E1 mouse osteoblastic cells after introduction of the sensor. Whole cell areas continued to increase from 10 to 180 min after plating and tension was monitored in a typical section close to the bottom, where the mean fluorescence was the largest. Tension on the bottom side was negatively correlated with cell area from 10 to 110 min. Thereafter, this correlation was positive until 180 min. We then analyzed central-peripheral differences in tension close to the bottom. In these experiments, tension in the cell periphery increased during expansion of the area and decreased during contraction. As a result, fluctuations of tension in this area were much larger than those in the central area of the cell. Finally, we analyzed upper-lower differences in tension development during initial adhesion and showed continuous decreases in the lower side of the cell from 10 to 110 min after plating, and decreases in the upper side until 70 min, followed by increases. In this study, we successfully visualized dynamic changes in intracellular tension at the sub-cellular level and found that development of tension during initial adhesion processes is time-dependent and has central-peripheral and upper-lower differences. The present FRET tension sensor may become a powerful tool in studies of cell biomechanics.