A Study of Mechanosensing of an Osteoblast at Focal Adhesions Under Cyclic Strain Using Magnetic Micropillars

2019 ◽  
Author(s):  
Toshihiko Shiraishi ◽  
Kota Nagai
Keyword(s):  
Calcium Ion ◽  
Mechanical Vibration ◽  
Focal Adhesions ◽  
Cyclic Strain ◽  
Mechanical Stimuli ◽  
Osteoblast Cells ◽  
Intracellular Calcium Ion ◽  
A Cell ◽  
Sense And Respond ◽  
Biochemical Signals

Abstract It has been reported that cells sense and respond to mechanical stimuli. Mechanical vibration promotes the cell proliferation and the cell differentiation of osteoblast cells at 12.5 Hz and 50 Hz, respectively. It indicates that osteoblast cells have a mechansensing system for mechanical vibration. There may be some mechanosensors and we focus on cellular focal adhesions through which mechanical and biochemical signals may be transmitted from extracellular matrices into a cell. However, it is very difficult to directly apply mechanical stimuli to focal adhesions. We developed a magnetic micropillar substrate on which micron-sized pillars are deflected according to applied magnetic field strength and focal adhesions adhering to the top surface of the pillars are given mechanical stimuli. In this paper, we focus on intracellular calcium ion as a second messenger of cellular mechanosensing and investigate the mechanosensing mechanism of an osteoblast cell at focal adhesions under cyclic strain using a magnetic micropillar substrate. The experimental results indicate that the magnetic micropillars have enough performance to response to an electric current applied to a coil in an electromagnet and to apply the cyclic strain of less than 3% to a cell. In the cyclic strain of less than 3%, the calcium response of a cell was not observed.

Cell Response to Cyclic Strain at Focal Adhesions

2016 ◽  
Author(s):  
Toshihiko Shiraishi ◽  
Tomohiro Fukuno
Keyword(s):  
Focal Adhesion ◽  
Mechanical Vibration ◽  
Focal Adhesions ◽  
Traction Force ◽  
Cyclic Strain ◽  
Macromolecular Assemblies ◽  
Migration Direction ◽  
A Cell ◽  
Sense And Respond ◽  
The Relationship

Cells are known to sense and respond to mechanical stimulations. The fact shows that there are some cellular mechanosensors for mechanical stimulations. One of the candidates of the mechanosensors is focal adhesions which are large macromolecular assemblies via which mechanical force and regulatory signals may be transmitted between the extracellular matrix and an interacting cell. Although it is quite important to clarify the mechanism of sensing and responding to the mechanical vibration via focal adhesions, there was no micro device applying time-varied mechanical loading to a single focal adhesion of the order of a micrometer. In order to solve the challenging issue, we developed a magnetic micropillar substrate which is able to apply cyclic strain to focal adhesions of a cell. Using the substrate, we investigated how a single osteoblast-like cell changed the direction of migration on micropillars cyclically deflected at 5 Hz and revealed the relationship between the cell migration and the traction force. The experimental results indicate that a cell may sense the cyclic strain and reduce the traction force which is not enough to move the cell body forward leading to changing the migration direction toward the place without cyclic strain.

scholarly journals “An intrinsically disordered intracellular domain of PIEZO2 is required for force-from-filament activation of the channel”

2021 ◽  
Author(s):  
Clement Verkest ◽  
Irina Schaefer ◽  
Juri M. Jegelka ◽  
Timo A. Nees ◽  
Wang Na ◽  
...  
Keyword(s):  
Mechanical Stimuli ◽  
Force Transmission ◽  
Transmembrane Helices ◽  
Intrinsically Disordered ◽  
Membrane Stretch ◽  
Different Types ◽  
A Cell ◽  
Filament Activation ◽  
Transmission Pathways ◽  
Biochemical Signals

AbstractA central question in mechanobiology is how mechanical forces acting in or on a cell are transmitted to mechanically-gated PIEZO channels that convert these forces into biochemical signals. Here we show that PIEZO2 is sensitive to force-transmission via the membrane (force-from-lipids) as well as force transmission via the cytoskeleton (force-from-filament) and demonstrate that the latter requires the intracellular linker between the transmembrane helices nine and ten (IDR5). Moreover, we show that rendering PIEZO2 insensitive to force-from-filament by deleting IDR5 abolishes PIEZO2-mediated inhibition of neurite outgrowth, which relies on the detection of cellgenerated traction forces, while it only partially affects its sensitivity to cell indentation and does not at all alter its sensitivity to membrane stretch. Hence, we propose that PIEZO2 is a polymodal mechanosensor that detects different types of mechanical stimuli via different force transmission pathways, which highlights the importance of utilizing multiple complementary assays when investigating PIEZO channel function.

A Study of a Cell Mechanosensing System Under Mechanical Vibration Considering its Modes of Vibration and Calcium Ion Response

2017 ◽  
Author(s):  
Yuki Nakamura ◽  
Toshihiko Shiraishi
Keyword(s):  
Calcium Ion ◽  
Mechanical Vibration ◽  
Ion Concentration ◽  
Experimental Result ◽  
Osteoblastic Cells ◽  
Confocal Laser ◽  
Fluorescent Intensity ◽  
Modes Of Vibration ◽  
A Cell ◽  
Cell Mechanosensing

It was reported that osteoblastic cells respond to mechanical vibration and generate the bone mass with a peak at a specific frequency like a resonance curve [1]. There seems to be an analogy between its cell response and the resonance of a cell as a mechanical system. This paper describes a novel method to measure the cellular modes of vibration of a cell and its calcium ion response under mechanical vibration, and the evaluation of the obtained results to clarify the mechanism of the cell mechanosensing. Nuclei and calcium ion in osteoblastic cells were visualized with fluorescent labelling. Mechanical vibration was applied to cells in a dish in the horizontal direction under a confocal laser scanning microscope by an exciter. Since the fluorescent intensity was very weak due to high frame rate to capture moving cells under Mechanical vibration, we used a high-speed and high-sensitive camera adjusting various conditions such as exposure time. We realized the spatial resolution of approximately 2 μm in the captured micrographs even under mechanical vibration using the experimental setup. As a result, the modes of vibration of nuclei was not obtained in this resolution. We found that the intracellular calcium ion concentration began to increase in a few seconds after mechanical vibration was applied. This experimental result indicates that applying mechanical vibration to cells can produce calcium signals as a second messenger by causing the entry of the ion.

Response of an Osteoblast to Cyclic Strain at Its Focal Adhesions Using Magnetic Micropillars

2012 ◽  
Author(s):  
Toshihiko Shiraishi ◽  
Takuya Ohara ◽  
Shin Morishita ◽  
Ryohei Takeuchi
Keyword(s):  
Magnetic Field ◽  
External Magnetic Field ◽  
Focal Adhesion ◽  
Mechanical Stimulation ◽  
Magnetic Particles ◽  
Focal Adhesions ◽  
Cyclic Strain ◽  
Osteoblast Cell ◽  
A Cell ◽  
The Difference

This paper describes a micro device which applies cyclic strain to focal adhesions of a cell. In recent years, evidence has been growing that focal adhesions act as mechanosensors of cells which convert mechanical force into biomechanical signaling. However, there are no effective micro devices which can directly apply mechanical stimulation to each focal adhesion. Here we develop a micropillar substrate embedding micron-sized magnetic particles and enabling the micropillars to be deflected by external magnetic field. The combination of long and short micropillars produces the difference of deflection between them and enables the micropillars to apply strain to a cell. The long pillars were periodically deflected at the amplitude of approximately 1.4 μm whereas most of short pillars were not deflected. Using the magnetic micropillar substrate, we observed the deformation of an osteoblast cell at its focal adhesions. The findings indicate that the present micro device can be used for investigating mechanosensing systems of a cell.

A Method for Applying High Cyclic Strain to Focal Adhesions and Measuring the Cell Response

2013 ◽  
Author(s):  
Toshihiko Shiraishi ◽  
Takuya Ohara ◽  
Shin Morishita
Keyword(s):  
Magnetic Field ◽  
External Magnetic Field ◽  
Focal Adhesion ◽  
High Frequency ◽  
Magnetic Particles ◽  
Focal Adhesions ◽  
Cyclic Strain ◽  
Osteoblast Cell ◽  
A Cell ◽  
The Difference

This paper describes a method by which broadband cyclic strain can be applied to focal adhesions of a cell. In recent years, evidence has been growing that focal adhesions act as mechanosensors of cells which convert mechanical force into biomechanical signaling. However, there are no effective methods by which mechanical stimulation with high frequency can be directly applied to each focal adhesion. Here we develop a micropillar substrate embedding micron-sized magnetic particles and enabling the micropillars to be deflected by external magnetic field. The combination of long and short micropillars produces the difference of deflection between them and enables the micropillars to apply strain to a cell. We verified that the micropillars responded to external magnetic field up to at least 25 Hz without phase difference. Using the magnetic micropillar substrate, we observed the cytoskeletal deformation of an osteoblast cell. The findings indicate that the present micro device can be used for investigating mechanosensing systems of a cell.

scholarly journals Sticking around: Optimal cell adhesion patterning for energy minimization and substrate mechanosensing

2020 ◽  
Author(s):  
Josephine Solowiej-Wedderburn ◽  
Carina M. Dunlop
Keyword(s):  
Mechanical Properties ◽  
Focal Adhesion ◽  
Cell Model ◽  
Stress Component ◽  
Focal Adhesions ◽  
Cell Stiffness ◽  
Adhesion Sites ◽  
Cell Shapes ◽  
A Cell ◽  
Sense And Respond

AbstractCell mechanotransduction, in which cells sense and respond to the physical properties of their micro-environments, is proving fundamental to understanding cellular behaviours across biology. Tissue stiffness (Young’s modulus) is typically regarded as the key control parameter and bioengineered gels with defined mechanical properties have become an essential part of the toolkit for interrogating mechanotransduction. We here, however, show using a mechanical cell model that the effective substrate stiffness experienced by a cell depends not just on the engineered mechanical properties of the substrate but critically also on the particular arrangement of adhesions between cell and substrate. In particular, we find that cells with different adhesion patterns can experience two different gel stiffnesses as equivalent and will generate the same mean cell deformations. For small adhesive patches, which mimic experimentally observed focal adhesions, we demonstrate that the observed dynamics of adhesion growth and elongation can be explained by energy considerations. Significantly we show different focal adhesions dynamics for soft and stiff substrates with focal adhesion growth not preferred on soft substrates consistent with reported dynamics. Equally, fewer and larger adhesions are predicted to be preferred over more and smaller, an effect enhanced by random spot placing with the simulations predicting qualitatively realistic cell shapes in this case. The model is based on a continuum elasticity description of the cell and substrate system, with an active stress component capturing cellular contractility. This work demonstrates the necessity of considering the whole cell-substrate system, including the patterning of adhesion, when investigating cell stiffness sensing, with implications for mechanotransductive control in biophysics and tissue engineering.Author summaryCells are now known to sense the mechanical properties of their tissue micro-environments and use this as a signal to control a range of behaviours. Experimentally, such cell mechanotransduction is mostly investigated using carefully engineered gel substrates with defined stiffness. Here we show, using a model integrating active cellular contractility with continuum mechanics, that the way in which a cell senses its environment depends critically not just on the stiffness of the gel but also on the spatial patterning of adhesion sites. In this way, two gels of substantially different stiffnesses can be experienced by the cell as similar, if the adhesions are located differently. Exploiting this insight, we demonstrate that it is energetically favourable for small adhesions to grow and elongate on stiff substrates but that this is not the case on soft substrates. This is consistent with experimental observations that nascent adhesions only mature to stable focal adhesion (FA) sites on stiff substrates where they also grow and elongate. These focal adhesions (FAs) have been the focus of work on mechanotransduction. However, our paper demonstrates that there is a fundamental need to consider the combined cell and micro-environment system moving beyond a focus on individual FAs.

Study on a Cell Mechanosensing System by Measuring Structural Deformation and Biochemical Response

2015 ◽  
Author(s):  
Atsushi Horiguchi ◽  
Toshihiko Shiraishi
Keyword(s):  
Mechanical Stimulation ◽  
Calcium Ion ◽  
Quantitative Relationship ◽  
Ion Concentration ◽  
Structural Deformation ◽  
Biochemical Response ◽  
Intracellular Calcium Ion ◽  
A Cell ◽  
Calcium Ion Concentration ◽  
Cell Mechanosensing

Mechanical stimulation induces new bone formation in vivo and promotes the metabolic activity and the gene expression of osteoblasts in vitro. It was reported that biochemical signals of osteoblasts to sense mechanical stimulation are activated according to their actin cytoskeletal deformation. However, there have been not so many researches on the relationship between cytoskeletal deformation and biochemical response. Here we show an original method to investigate a cell mechanosensing system and the quantitative relationship between the deformation of cytoskeletal structure and the change of intracellular calcium ion concentration as biochemical response in a living cell stimulated by a micropipette. Gene transfection of green fluorescent protein to osteoblastic cells enabled visualization of actin in cells. When local deformation was applied to a single osteoblastic cell by a micropipette, the displacement distribution of cytoskeletal structure in the whole cell was automatically obtained from the two images of the cell before and after deformation by using Kanade-Lucas-Tomasi (KLT) method. Intracellular calcium ion response to mechanical stimulation was measured as the spatial and temporal changes of intensity of Fura Red loaded to a cell. As a result, we obtained the quantitative relationship between structural deformation and biochemical response of a cell and found that the change of calcium ion concentration increases with increasing the displacement of actin cytoskeleton. It indicates that the deformation of actin cytoskeleton is highly related to the cell mechanosensing system.

Effects of Cyclic Strain at Focal Adhesions on Migration of an Osteoblast

2015 ◽  
Author(s):  
Tomohiro Fukuno ◽  
Toshihiko Shiraishi
Keyword(s):  
Magnetic Particles ◽  
Protein Structures ◽  
Focal Adhesions ◽  
Cyclic Strain ◽  
Time Lapse ◽  
Mechanical Forces ◽  
Osteoblast Cell ◽  
Frequency Range ◽  
Cellular Mechanisms ◽  
Biochemical Signals

Cells respond to not only biochemical signals but also mechanical forces, which indicates that cells have some mechanosensors that convert mechanical forces into biochemical signals. According to recent reports, one of the candidates of the mechanosensors is focal adhesions that form multi-protein structures having mechanical links between intracellular cytoskeletons and extracellular matrices. Since the cellular mechanisms of sensing and responding to the mechanical stimulations at focal adhesions have not been clarified yet, we developed a micropillar substrate embedding micron-sized magnetic particles and enabling the micropillars to be cyclically deflected by a time-varied magnetic field. Using the magnetic micropillars, here we apply cyclic strain of some frequencies to a single osteoblast cell through focal adhesions and track cell migration with time-lapse observation to understand how the cell senses and responds to cyclic strain. Our data indicate that the cell may change the direction of migration to move away from the micropillar cyclically deflected in the frequency range from 0.1 to 50 Hz.

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