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.

Keywords

Cell-substrate adhesion, Focal adhesions, Paxillin, Cellular contractility, Micropatterning, Mechanobiology

Paper information

Shinji DEGUCHI, Akira C. SAITO, Tsubasa S. MATSUI, Wenjing HUANG, Masaaki SATO, “The opposite mechano-response of paxillin phosphorylation between subcellular and whole-cell levels is explained by a minimal model of cell-substrate adhesions”, Journal of Biomechanical Science and Engineering, Vol.12, No.2 (2017), p.16-00670. doi:10.1299/jbse.16-00670. Final Version Released on June 09, 2017, Advance Publication Released on May 02, 2017.

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.

Keywords

Allometry, Scaling law, Animal vocalization, Phonation, Falsetto voice, Self-excited oscillation, Internal flow, Flow-structure interaction, Strouhal number, Dimensionless number

Paper information

Shinji DEGUCHI, “A possible common physical principle that underlies animal vocalization: theoretical considerations with an unsteady airflow-structure interaction model”, Journal of Biomechanical Science and Engineering, Vol.11, No.4 (2016), p.16-00414. doi:10.1299/jbse.16-00414. Final Version Released on December 16, 2016, Advance Publication Released on November 24, 2016.

Actin stress fibers (SFs) play a key role in regulation of cell adhesion, but the biochemical and biophysical properties intrinsic to SFs remain unclear. Here we extracted SFs from rat embryonic smooth muscle cells by deroofing, and evaluated the effects of varying ionic strength and temperature on their intactness. Wash buffers with ionic strength ranging from 90 to 490 mM were prepared, and the extracted SFs were incubated in a buffer with a particular ionic strength for 10 min or 24 h. Light and electron microscopy revealed that the extracted SFs comprised tightly packed straight bundles at low ionic strengths that became looser and exhibited a ragged pattern at high ionic strengths. The expression of α-actinin associated with the extracted SFs decreased with the increase in ionic strength. Unexpectedly, non-muscle myosin II and smooth muscle myosin in the extracted SFs displayed comparable expression levels over the different ionic strengths. ATP-induced contractility was better preserved at low ionic strengths, including the physiological ionic strength of 170 mM. The rate of ATP-induced enzymatic activity increased with increase in temperature, but the difference was not statistically significant. These results demonstrate that low ionic strength produces extracted SFs that are more intact with regard to structure and function.

Keywords

Stress Fibers, Smooth Muscle Cells, Ionic Strength, Contractility

Paper information

Shinji DEGUCHI, Tsubasa S. MATSUI, Daiki KOMATSU and Masaaki SATO, “Contraction of Stress Fibers Extracted from Smooth Muscle Cells: Effects of Varying Ionic Strength”, Journal of Biomechanical Science and Engineering, Vol. 7, No. 4 (2012), pp.388-398 . doi:10.1299/jbse.7.388

Oscillation of the vocal folds makes a sound source of the human voiced sound. Understanding of the oscillation mechanism, which is a complex flow-structure interaction problem in the airway, is crucial for considering clinical diagnosis of voice disorders. However, details of the oscillation mechanism are still unclear partly because, from a fluid mechanical viewpoint, the effect of oscillation of the vocal fold wall during the phonation on airflow behaviors remains elusive. In the present study, flow characteristics in a sinusoidally-oscillating constriction mimicking the vocal fold were investigated by numerical and experimental approaches. The numerical analyses focused in particular on the effect of constriction oscillation on flow separation demonstrated that the flow separation point moves continuously and periodically in a frequency-dependent manner. In the experimental study, an apparatus was newly designed, with a view to detect the oscillation-induced movement of the flow separation point, to enable detailed measurement of pressure distribution along the constriction with an interval of 2 mm that is synchronized with measurement of constriction displacement. Although movement of the separation point as seen in the numerical analyses was not detected by this limiting resolution of the apparatus, we obtained pressure-width relations that is partly contrary to the numerical results but is presumably dependent on the inlet boundary condition. These findings indicate that appropriate evaluations of separation point and inlet boundary conditions are key factors to characterize the flow in oscillating constriction, which is crucial for better understanding of the vocal fold mechanics.

Keywords

Vocal Fold, Flow-Structure Interaction, Flow Separation, Airway, Phonation

Paper information

Toru HYAKUTAKE, Shinji DEGUCHI, Akiya SHIOTA, Yasunobu NISHIOKA, Shinichiro YANASE and Seiichi WASHIO, “Effect of Constriction Oscillation on Flow for Potential Application to Vocal Fold Mechanics: Numerical Analysis and Experiment”, Journal of Biomechanical Science and Engineering, Vol. 1, No. 2 (2006), pp.290-303 . doi:10.1299/jbse.1.290

The vocal folds in the larynx experience a self-excited oscillation with a wavelike motion during speech owing to interaction with respiratory airflow. The mechanism of the onset of the oscillation remains elusive partly because of compound effects of laryngeal muscles, although its better understanding has clinical significance in determining the ease with which phonation can be achieved. Approaches to the mechanism using a mechanical vocal fold model are useful because it allows investigating the roles of interested parameters in isolation. Here, we designed a mechanical vocal fold model made of a pair of rubber sheets. A key feature of the experimental setup is that it enables observations of high-speed deformation of the oscillating vocal fold model, together with pressure evaluations while changing separately isolated parameters associated with the laryngeal muscle functions. The observations of the oscillation onset demonstrated a gradually developed wavelike oscillation that spreads out over the rubber sheets. The magnitude of the motion is restricted by either increase in rubber restoring force or reduction in flow path width, each of the effects mimics the actual laryngeal muscle functions and reduces, in the experimental results, the threshold upstream pressure that induces the onset of the self-excitation. Thus, the present study highlights close association between degrees of oscillation, flow-tissue interaction, and threshold pressure required for the onset.

Keywords

Self-excited Oscillation, Flow-Structure Interaction, High-Speed Video Camera, Pressure Measurement, Vocal Fold, Airway, Phonation

Paper information

Shinji DEGUCHI, Yusuke MIYAKE, Yoshihiko TAMURA and Seiichi WASHIO, “Wavelike Motion of a Mechanical Vocal Fold Model at the Onset of Self-Excited Oscillation”, Journal of Biomechanical Science and Engineering, Vol. 1, No. 1 (2006), pp.246-255 . doi:10.1299/jbse.1.246