Tsubasa S. Matsui

Non-muscle myosin II induces disassembly of actin stress fibres independently of myosin light chain dephosphorylation

Dynamic remodelling of actin stress fibres (SFs) allows non-muscle cells to adapt to applied forces such as uniaxial cell shortening. However, the mechanism underlying rapid and selective disassembly of SFs oriented in the direction of shortening remains to be elucidated. Here, we investigated how myosin crossbridge cycling induced by MgATP is associated with SF disassembly. Moderate concentrations of MgATP, or [MgATP], induced SF contraction. Meanwhile, at [MgATP] slightly higher than the physiological level, periodic actin patterns emerged along the length of SFs and dispersed within seconds. The actin fragments were diverse in length, but comparable to those in characteristic sarcomeric units of SFs. These results suggest that MgATP-bound non-muscle myosin II dissociates from the individual actin filaments that constitute the sarcomeric units, resulting in unbundling-induced disassembly rather than end-to-end actin depolymerization. This rapid SF disassembly occurred independent of dephosphorylation of myosin light chain. In terms of effects on actin–myosin interactions, a rise in [MgATP] is functionally equivalent to a temporal decrease in the total number of actin–myosin crossbridges. Actin–myosin crossbridges are known to be reduced by an assisting load on myosin. Thus, the present study suggests that reducing the number of actin–myosin crossbridges promotes rapid and orientation-dependent disassembly of SFs after cell shortening.

Viscoelastic and optical properties of four different PDMS polymers

Polydimethylsiloxane (PDMS) is the most commonly used silicone elastomer with a wide range of applications including microfluidics and microcontact printing. Various types of PDMS are currently available, and their bulk material properties have been extensively investigated. However, because the properties are rarely compared in a single study, it is often unclear whether the large disparity of the reported data is attributable to the difference in methodology or to their intrinsic characteristics. Here we report on viscoelastic properties and optical properties of four different PDMS polymers, i.e. Sylgard-184, CY52-276, SIM-360, and KE-1606. Our results show that all the PDMSs are highly elastic rather than viscoelastic at the standard base/curing agent ratios, and their quantified elastic modulus, refractive index, and optical cleanness are similar but distinct in magnitude.

Contact guidance of smooth muscle cells is associated with tension-mediated adhesion maturation.

Contact guidance is a cellular phenomenon observed during wound healing and developmental patterning, in which adherent cells align in the same direction due to physical cues. Despite numerous studies, the molecular mechanism underlying the consistent cell orientation is poorly understood. Here we fabricated microgrooves with a pitch of submicrons to study contact guidance of smooth muscle cells. We show that both integrin-based cell-substrate adhesions and cellular tension are necessary to achieve contact guidance along microgrooves. We further show through analyses on paxillin that cell-substrate adhesions are more prone to become mature when they run along microgrooves than align at an angle to the direction of microgrooves. Because cellular tension promotes the maturation of cell-substrate adhesions, we propose that the adhesions aligning across microgrooves are not physically efficient for bearing cellular tension compared to those aligning along microgrooves. Thus, the proposed model describes a mechanism of contact guidance that cells would finally align preferentially along microgrooves because cellular tensions are more easily borne within the direction, and the direction of resulting mature adhesions determines the direction of the whole cells.

The position and size of individual focal adhesions are determined by intracellular stress-dependent positive regulation †

It remains unclear how the subcellular positions and sizes of individual focal adhesions (FAs) are determined in stationary cells. The elucidation of spatial regulation mechanisms is important for accurate understanding of the cellular response to mechanical stress. Through a theoretical analysis on previously reported cell behavior, the present study demonstrates a close correlation between the appearances of mechanosensitive elements and intracellular stress reflecting traction stress that the cell exerts on the substrate. The magnitude and distribution of stress were predicted in this analysis by mimicking intrinsic actomyosin contraction independent of extracellular stimuli. Positions of FAs and actin stress fibers corresponded to the local maximum and minimum stress points, respectively, and thus were determined by the global configuration of cell adhesions. Furthermore, their subcellular sizes were in agreement with the predicted stress magnitudes that were dependent on the local mechanical environment. These results suggest that a positive regulation (i.e., force and cell adhesion enhance each other) functions in the organization of individual FAs in nonmigrating cells.

Actin stress fibers are at a tipping point between conventional shortening and rapid disassembly at physiological levels of MgATP

Stress fibers (SFs) composed of nonmuscle actin and myosin II play critical roles in various cellular functions such as structural remodeling in response to changes in cell stress or strain. Previous studies report that SFs rapidly disassemble upon loss of tension caused by reduced myosin activity or sudden cell shortening, but the mechanism is unclear. Here, we showed that Rho-kinase inhibition with Y-27632 led to detachment of intact actin filaments from the SFs rather than depolymerization. Loss of tension may allow SFs to shorten via MgATP-driven cross-bridge cycling, thus we investigated the effects of MgATP concentration on SF shortening and stability. We performed the experiments using extracted SFs to allow control over MgATP concentration. SF contraction and disassembly rates each increased with increasing MgATP concentration. SFs transitioned from conventional SF shortening to rapid disassembly as MgATP concentration increases from 2 to 5mM, which is within the physiological range of intracellular MgATP concentrations. Thus, we submit that SFs in intact cells are inherently on the verge of disassembly, which is likely due to the small number of actomyosin cross-bridges in SFs compared to those found in relatively stable myofibrils. Given that recent studies have revealed that loss of resistive force against myosin II could lower the fraction of the MgATPase cycle time that the myosin head is attached to actin (i.e., the duty ratio), binding of cytoplasmic levels of MgATP to myosin II may be sufficient to cause the disassembly of unloaded SFs. The present study thus describes a putative mechanism for rapid SF disassembly caused by decreased myosin activity or sudden cell shortening.

A versatile micro-mechanical tester for actin stress fibers isolated from cells

Conventional atomic force microscopy is one of the major techniques to evaluate mechanical properties of cells and subcellular components. The use of a cantilever probe for sample manipulation within the vertical plane often makes absolute positioning of the probe, subject to thermal drift, difficult. In addition, the vertical test is unable to observe changes in the sample structure responsible for mechanical behavior detected by the probe. In the present study, an alternative mechanical tester was developed that incorporated a pair of micro-needles to manipulate a sample in a project plane, allowing acquisition of the accurate probe position and entire sample image. Using a vision-based feedback control, a micro-needle driven by a piezo actuator is moved to give user-defined displacements or forces to sample. To show its usefulness and versatility, three types of viscoelastic measurements on actin stress fibers isolated from smooth muscle cells were demonstrated: strain rate-controlled tensile tests, relaxation tests and creep tests. Fluorescence imaging of the stress fibers using Qdots over the course of the measurements, obtained through multiple image detectors, was also carried out. The technique described here is useful for examining the quantitative relationship between mechanical behavior and related structural changes of biomaterials.

Piezoelectric actuator-based cell microstretch device with real-time imaging capability

Cellular response to physical stretch has been extensively studied as a regulator of various physiological functions. For live cell microscopy combined with stretch experiments, cells are typically seeded on an extensible elastomer sheet. In this case, the position of the cells of interest tends to shift out of the field of view upon stretch, making real-time imaging of identical cells difficult. To circumvent this situation, here we describe a robust methodology in which these cell shifts are minimized. Cells are plated in a custom-designed stretch chamber with an elastomer sheet of a small cell culture area. The cell-supporting chamber is stretched on an inverted microscope by using a piezoelectric actuator that provides small, but precisely controlled displacements. Even under this small displacement within the filed of view, our device allows the cells to undergo physiologically relevant levels of stretch. Identical cells can thus be continuously observed during stretching, thereby potentially enablin...

Microcontact peeling as a new method for cell micropatterning.

Micropatterning is becoming a powerful tool for studying morphogenetic and differentiation processes of cells. Here we describe a new micropatterning technique, which we refer to as microcontact peeling. Polydimethylsiloxane (PDMS) substrates were treated with oxygen plasma, and the resulting hydrophilic layer of the surface was locally peeled off through direct contact with a peeling stamp made of aluminum, copper, or silicon. A hydrophobic layer of PDMS could be selectively exposed only at the places of the physical contact as revealed by water contact angle measurements and angle-resolved X-ray photoelectron spectroscopy, which thus enabled successful micropatterning of cells with micro-featured peeling stamps. This new micropatterning technique needs no procedure for directly adsorbing proteins to bare PDMS in contrast to conventional techniques using a microcontact printing stamp. Given the several unique characteristics, the present technique based on the peel-off of inorganic materials may become a useful option for performing cell micropatterning.

Development of motorized plasma lithography for cell patterning

The micropatterning of cells, which restricts the adhesive regions on the substrate and thus controls cell geometry, is used to study mechanobiology-related cell functions. Plasma lithography is a means of providing such patterns and uses a spatially-selective plasma treatment. Conventional plasma lithography employs a positionally-fixed mask with which the geometry of the patterns is determined and thus is not suited for producing on-demand geometries of patterns. To overcome this, we have manufactured a new device with a motorized mask mounted in a vacuum chamber of a plasma generator, which we designate motorized plasma lithography. Our pilot tests indicate that various pattern geometries can be obtained with the control of a shielding mask during plasma treatment. Our approach can thus omit the laborious process of preparing photolithographically microfabricated masks required for the conventional plasma lithography.

Aligning cells in arbitrary directions on a membrane sheet using locally formed microwrinkles

Sheets of cells can be used for tissue regenerative medicine. Cell alignment within the sheet is now a key factor in the next generation of this technology. Anisotropic cell sheets without random cell orientations have been conventionally produced with photolithographically, microfabricated substrates using special facilities and equipment. Here we demonstrate a more accessible approach to the fabrication of anisotropic substrates. We locally deformed part of an elastic membrane and simultaneously oxidized the surface to create microwrinkles as well as to enable adhesion to the extracellular matrix. The approach with the local loading made it possible to orient cells in controlled directions within a single membrane sheet depending on the strains determined by the controllable deformation. This technique potentially enables a versatile design of microwrinkles for target-compatible cell alignments.