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.

Keywords

FRET, Tension sensor, Cytoskeleton, Actin filament, α-actinin, MC3T3-E1, Adhesion

Paper information

Junfeng WANG, Masahiro ITO, Wenhao ZHONG, Shukei SUGITA, Tatsuo MICHIUE, Takashi TSUBOI, Tetsuya KITAGUCHI, Takeo MATSUMOTO, “Observations of intracellular tension dynamics of MC3T3-E1 cells during substrate adhesion using a FRET-based actinin tension sensor”, Journal of Biomechanical Science and Engineering, Vol.11, No.4 (2016), p.16-00504. doi:10.1299/jbse.16-00504. Final Version Released on December 16, 2016, Advance Publication Released on October 24, 2016.

In order to determine how cells change their morphology during adhesion process to a substrate, we focused on the actin cytoskeleton and investigated its morphological change along with that of the whole cell during adhesion process. An osteoblastic cell line MC3T3-E1 was used as the test model. We plated cells whose cell cycle had been synchronized by serum starvation on fibronectin-coated glass plate and cultured them for 10 min to 24 h. We then stained their F-actin and nucleus and observed them with a fluorescent microscope for cell area and shape index and 2D parameters for actin morphology, and with a laser scanning microscope for 3D morphology of actin and nucleus. In the beginning of adhesion, the trypsinized cells were round and their nuclei were surrounded uniformly by thick layer of actin. The actin layer in the upper side became actin aggregate (AA) and lower side dense peripheral band (DPB) in 30 min. The upper AA then became smaller and finally to actin filaments (AFs) spanning the cell top. The DPB expanded and finally became AFs on cell bottom by 1 h. The nucleus becomes flattened possibly due to compression by the cell membrane caused by the expansion of the DPB in the early stage of adhesion. In the later stage of adhesion, the number of AFs continuously increased and nucleus became flattened more and more until 12 h. This may be caused by the increase in the top AFs that may compress the nucleus. Cells become more elongated in response to further alignment of AFs until 12 h. These results indicate that change in AFs during adhesion process is complicated not only temporally but also spatially.

Keywords

Cytoskeleton, Actin aggregate, Actin filament, Morphology, MC3T3-E1, Adhesion

Paper information

Junfeng WANG, Shukei SUGITA, Kazuaki NAGAYAMA, Takeo MATSUMOTO, “Dynamics of actin filaments of MC3T3-E1 cells during adhesion process to substrate”, Journal of Biomechanical Science and Engineering, Vol.11, No.2 (2016), p.15-00637. doi:10.1299/jbse.15-00637. Final Version Released on June 24, 2016, Advance Publication Released on January 04, 2016.

Estimation of wall strength of the aortic aneurysms is necessary for the prediction of their rupture risk. We previously found a significant correlation between their tensile strength σ<i>_MAX</i> and a yielding parameter τ_σ, which is the stress when tangent elastic modulus reaches at 63% of the plateau level. This may indicate that the wall strength is estimated from their pressure-diameter relationship. Here we show a possible mechanism of the correlation between τ_σ and σ<i>_MAX</i> by focusing on alignment of collagen fibers. Thin (150-µm) slices of porcine thoracic aortas were uniaxially stretched in circumferential direction until failure under a microscope, and a retardance, a phase shift when polarized light passes through a birefringent material, was measured as the degree of collagen fiber alignment. Strength σ<i>_MAX</i> correlated significantly with τ_σ as obtained previously. The retardance increased with the increase in the stress and reached a plateau at the stress σ<i>_Ret-plateau</i>, indicating that σ<i>_Ret-plateau</i> is the stress at which most of the intramural collagen fibers have aligned. The stress σ<i>_Ret-plateau</i> correlated significantly with τ<i>_σ</i> and both parameters has similar values. This may indicate that the aortic wall yields when all of collagen fibers become straight. Smaller σ<i>_Ret-plateau</i> means that most collagen fibers are stretched and loaded at smaller stress, resulting in failure at smaller stress. This seems to be a reason for the significant correlation between τ_σ and σ<i>_MAX</i>.

Keywords

Collagen Fibers, Failure Stress, Porcine Thoracic Aorta, Stiffness, Birefringence

Paper information

Shukei SUGITA and Takeo MATSUMOTO, “Yielding Phenomena of Aortic Wall and Intramural Collagen Fiber Alignment: Possible Link to Rupture Mechanism of Aortic Aneurysms”, Journal of Biomechanical Science and Engineering, Vol. 8, No. 2 (2013), pp. 104-113. doi:10.1299/jbse.8.104

Control of the gliding directions of kinesin-driven microtubules (MTs) in vitro has good feasibility for the development of nano-scale transport systems. A requirement for the control of transporters in these systems includes detecting the positions of gliding MTs; however, no studies have reported on the monitoring of the positions of gliding MTs. Here, we suggest an algorithm to detect tip coordinates of gliding MTs by binarization, skeletonization, and filtration of fluorescent images of MTs. The algorithm was first applied to artificially drawn segments with given lengths (10-80 pixels), widths (1-10 pixels), and curvature radii (20-120 pixels) to verify the effect of the sizes of MTs on accuracy of tip coordinates extracted by the algorithm, and error was estimated by referring to the true coordinates. The estimated errors were as small as 2 pixels in the width and were not affected by the length and the curvature radius, indicating that our algorithm is useful to extract the tips of MTs. The algorithm was subsequently applied to images of gliding MTs. Since distances from the trajectories of the MTs to the centers of gravity of the MTs (3.7 ± 2.1 pixels) were significantly larger than those to the tips (1.9 ± 0.5 pixels), the use of the tips as representative points of gliding MTs was verified. A detection method using tips of MTs, as suggested in this study, may be a useful technique for monitoring each MT in nanoscale transport systems.

Keywords

Image Analysis, Tip Detection, Microtubule, Kinesin, Nano-Scale Transport System

Paper information

Shukei SUGITA, Naoya SAKAMOTO, Toshiro OHASHI and Masaaki SATO, “Novel Image Analysis for Trajectory of Microtubules Gliding on Kinesins with Tip Detection”, Journal of Biomechanical Science and Engineering, Vol. 4, No. 3 (2009), pp.404-414 . doi:10.1299/jbse.4.404

Kinesins, biomolecular motors moving along microtubules (MTs) in cells, can potentially be utilized as nano-scale transport systems with an inverted gliding assay, in which the MTs glide on a kinesin-coated surface. Although the key requirements include controls of gliding direction and velocity of MTs, the details of motility properties of MTs have not been well known. In this study, we quantitatively measured angular and gliding velocities, particularly focusing on the effects of MT length and kinesin density. The gliding assay of MTs of up to 20 μm in length was performed on a substrate coated with the kinesin density of 7.5, 38, and 75 μg/ml that resulted in the kinesin spacing of 7.8, 4.2, and 3.1 μm, respectively. The angular velocity for MTs shorter than kinesin spacings significantly decreased with increasing their length, and that for MTs longer than kinesin spacings was not affected by their length. Moreover, the angular velocity was substantially higher at lower kinesin density. These results suggest that both the number of associated kinesins with MTs and the kinesin spacings may contribute to the gliding direction. In contrast, the gliding velocity was independent of the MT length, ranging from 0.3 to 0.5 μm/s with decreasing the kinesin density. This may potentially imply the existence of an underlying mechanism with respect to the number of kinesins per the unit length of MTs. Towards development of high throughput nano-scale transport systems, long MTs and low kinesin densities would be effective for high directionality and high velocity, respectively.

Keywords

Nanoscale Transport System, Motor Protein, Microtubule, Angular Velocity, Gliding Velocity

Paper information

Shukei SUGITA, Naoya SAKAMOTO, Toshiro OHASHI and Masaaki SATO, “Characterization of Motility Properties of Kinesin-Driven Microtubules Towards Nano-Scale Transporter: Focusing on Length of Microtubules and Kinesin Density”, Journal of Biomechanical Science and Engineering, Vol. 3, No. 4 (2008), pp.510-519 . doi:10.1299/jbse.3.510