A biomimetic hydrogel culture system to facilitate cardiac reprogramming.

Direct cardiac reprogramming, in which fibroblasts are converted into induced cardiomyocytes (iCMs) with cardiogenic transcription factors, may be a promising approach for myocardial regeneration. Here, we present a protocol for cardiac reprogramming using a handmade hydrogel culture system. This system can recapitulate substrate stiffness comparable to that of the native myocardium.

Mechanical load regulates bone growth via periosteal Osteocrin.

Mechanical stimuli including loading after birth promote bone growth. However, little is known about how mechanical force triggers biochemical signals to regulate bone growth. Here, we identified a periosteal-osteoblast-derived secretory peptide, Osteocrin (OSTN), as a mechanotransducer involved in load-induced long bone growth.

Stiffness of the extracellular matrix regulates differentiation into beige adipocytes.

Beige/brite adipocytes, which express high levels of uncoupling protein 1 (UCP1) to generate heat using stored triglycerides, are induced under specific stimuli such as cold exposure in inguinal white adipose tissue (iWAT). Although extracellular microenvironments such as extracellular matrix (ECM) stiffness are known to regulate cell behaviors, including cell differentiation into adipocytes, the effect on iWAT cells is unknown. In this study, we show that rigid ECM promotes the cell spreading of iWAT-derived preadipocytes.

Local cyclical compression modulates macrophage function and alleviates immobilization-induced muscle atrophy.

Physical inactivity gives rise to numerous diseases and organismal dysfunctions, particularly those related to aging. Musculoskeletal disorders including muscle atrophy, which can result from a sedentary lifestyle, aggravate locomotive malfunction and evoke a vicious circle leading to severe functional disruptions of vital organs such as the brain and cardiovascular system. Although the significance of physical activity is evident, molecular mechanisms behind its beneficial effects are poorly understood.

Substrate Stiffness Influences Doxorubicin-Induced p53 Activation via ROCK2 Expression.

The physical properties of the extracellular matrix (ECM), such as stiffness, are involved in the determination of the characteristics of cancer cells, including chemotherapy sensitivity. Resistance to chemotherapy is often linked to dysfunction of tumor suppressor p53; however, it remains elusive whether the ECM microenvironment interferes with p53 activation in cancer cells. Here, we show that, in MCF-7 breast cancer cells, extracellular stiffness influences p53 activation induced by the antitumor drug doxorubicin.

MEKK1-dependent phosphorylation of calponin-3 tunes cell contractility.

MEKK1 (also known as MAP3K1), which plays a major role in MAPK signaling, has been implicated in mechanical processes in cells, such as migration. Here, we identify the actin-binding protein calponin-3 as a new MEKK1 substrate in the signaling that regulates actomyosin-based cellular contractility. MEKK1 colocalizes with calponin-3 at the actin cytoskeleton and phosphorylates it, leading to an increase in the cell-generated traction stress.

Activation of Yes-Associated Protein in Low-Grade Meningiomas Is Regulated by Merlin, Cell Density, and Extracellular Matrix Stiffness.

The NF2 gene product Merlin is a protein containing ezrin, radixin, and moesin domains; it is a member of the 4.1 protein superfamily associated with the membrane cytoskeleton and also interacts with cell surface molecules. The mammalian Hippo cascade, a downstream signaling cascade of merlin, inactivates the Yes-associated protein (YAP).

Coating extracellular matrix proteins on a (3-aminopropyl)triethoxysilane-treated glass substrate for improved cell culture.

We demonstrate that a (3-aminopropyl)triethoxysilane-treated glass surface is superior to an untreated glass surface for coating with extracellular matrix (ECM) proteins when used as a cell culture substrate to observe cell physiology and behavior. We found that MDCK cells cultured on untreated glass coated with ECM removed the coated ECM protein and secreted different ECM proteins. In contrast, the cells did not remove the coated ECM protein when seeded on (3-aminopropyl)triethoxysilane-treated (i.

p53-mediated activation of the mitochondrial protease HtrA2/Omi prevents cell invasion.

Oncogenic Ras induces cell transformation and promotes an invasive phenotype. The tumor suppressor p53 has a suppressive role in Ras-driven invasion. However, its mechanism remains poorly understood.

Design and construction of an equibiaxial cell stretching system that is improved for biochemical analysis.

We describe the design and validation of an equibiaxial stretching device in which cells are confined to regions of homogeneous strain. Using this device, we seek to overcome a significant limitation of existing equibiaxial stretching devices, in which strains are not homogeneous over the entire region of cell culture. We cast PDMS in a mold to produce a membrane with a cylindrical wall incorporated in the center, which was used to confine cell monolayers to the central membrane region subjected to homogeneous equibiaxial strain.

The role of the interaction of the vinculin proline-rich linker region with vinexin α in sensing the stiffness of the extracellular matrix.

Although extracellular matrix (ECM) stiffness is an important aspect of the extracellular microenvironment and is known to direct the lineage specification of stem cells and affect cancer progression, the molecular mechanisms that sense ECM stiffness have not yet been elucidated. In this study, we show that the proline-rich linker (PRL) region of vinculin and the PRL-region-binding protein vinexin are involved in sensing the stiffness of ECM substrates. A rigid substrate increases the level of cytoskeleton-associated vinculin, and the fraction of vinculin stably localizing at focal adhesions (FAs) is larger on rigid ECM than on soft ECM.

Dynamic chemotactic response of fibroblasts to local stimulation using EGF-immobilized microbeads.

Directional cellular migrations as a chemotactic response to spatially inhomogeneous growth factor stimulation play an important role in establishing physiological mechanisms and pathological events in cells. We developed epidermal growth factor (EGF)-immobilized microbeads by photoreaction and evaluated its local stimulatory effects on the dynamic chemotactic motility of fibroblasts. The local stimulation resulted in global activation of ERK 1/2 and directionality of cellular migration.

Substrate stiffness regulates temporary NF-κB activation via actomyosin contractions.

Physical properties of the extracellular matrix (ECM) can control cellular phenotypes via mechanotransduction, which is the process of translation of mechanical stresses into biochemical signals. While current research is clarifying the relationship between mechanotransduction and cytoskeleton or adhesion complexes, the contribution of transcription factors to mechanotransduction is not well understood. The results of this study revealed that the transcription factor NF-κB, a major regulator for immunoreaction and cancer progression, is responsive to substrate stiffness.

Leachate tests with sewage sludge contaminated by radioactive cesium.

The sewer systems of eastern Japan have transported radioactive fallout from the Fukushima Dai-ichi nuclear power plant accident to wastewater treatment plants, where the radioisotopes have accumulated. To better understand the potential problems associated with the disposal of contaminated sewage sludge in landfills, leachate tests were conducted with radioactive incinerator ash, cement solidification incinerator ash, and dewatered sludge cake. Radioactivity was undetectable in the eluate from incinerator ash and dewatered sludge cake, but about 30% of the radioactivity initially in cement solidification incinerator ash appeared in the eluate during the leaching experiments.

Cellular response to substrate rigidity is governed by either stress or strain.

Cells sense the rigidity of their substrate; however, little is known about the physical variables that determine their response to this rigidity. Here, we report traction stress measurements carried out using fibroblasts on polyacrylamide gels with Young's moduli ranging from 6 to 110 kPa. We prepared the substrates by employing a modified method that involves N-acryloyl-6-aminocaproic acid (ACA).

Src, p130Cas, and Mechanotransduction in Cancer Cells.

Anchorage-independent growth is the most significant hallmark of cell transformation, which has an intimate relevance to cancer. Anchorage or adhesion physically links cells to the extracellular matrix and allows the transmission of external mechanical cues to intracellular signaling machineries. Transformation involves acquiring the ability to proliferate without requiring mechanically initiated signal transduction, known as mechanotransduction.

[Space flight/bedrest immobilization and bone. Mechanical stress : a double-edged sword to osteoarthritis].

Osteoarthritis (OA) is a disease caused by the degeneration and destruction of joint cartilage followed by peri-articular or subchondral bone formation and concomitant degeneration of other components of joints including synovium. Several animal OA models that adopt surgically induced joint instability have been developed. Analysis of those OA models indicates that increased mechanical stress exacerbates OA.

Dual inhibition of Src and GSK3 maintains mouse embryonic stem cells, whose differentiation is mechanically regulated by Src signaling.

Recent studies reveal that the mechanical environment influences the behavior and function of various types of cells, including stem cells. However, signaling pathways involved in the mechanical regulation of stem cell properties remain largely unknown. Using polyacrylamide gels with varying Young's moduli as substrates, we demonstrate that mouse embryonic stem cells (mESCs) are induced to differentiate on substrates with defined elasticity, involving the Src-ShcA-MAP kinase pathway.

Local mechanical stimulation of Mardin-Darby canine kidney cell sheets on temperature-responsive hydrogel.

Collective motion of cell sheets plays a role not only in development and repair, but also in devastating diseases such as cancer. However, unlike single-cell motility, collective motion of cell sheets involves complex cell-cell communication during migration; therefore, its mechanism is largely unknown. To elucidate propagation of signaling transduced by cell-cell interaction, we designed a hydrogel substrate that can cause local mechanical stretching of cell sheets.

Direct detection of cellular adaptation to local cyclic stretching at the single cell level by atomic force microscopy.

The cellular response to external mechanical forces has important effects on numerous biological phenomena. The sequences of molecular events that underlie the observed changes in cellular properties have yet to be elucidated in detail. Here we have detected the responses of a cultured cell against locally applied cyclic stretching and compressive forces, after creating an artificial focal adhesion under a glass bead attached to the cantilever of an atomic force microscope.

Direct manipulation of intracellular stress fibres using a hook-shaped AFM probe.

Atomic force microscopy (AFM) is a highly successful technique for imaging nanometre-sized samples and measuring pico- to nano-newton forces acting between atoms and molecules. When it comes to the manipulation of larger samples with forces of tens and hundreds of nano-newtons, however, the present chemistry-based modification protocols for functionalizing AFM cantilevers to achieve the formation of covalent/non-covalent linkages between the AFM probe and the sample surface do not produce strong enough bonds. For the purpose of measuring the fracture strength and other mechanical properties of stress fibres (SFs) in living as well as semi-intact fibroblast cells, we fabricated an AFM probe with a hooking function by focused ion beam technology and used the AFM probe hook to capture, pull and eventually sever a chosen SF labelled with green or red fluorescent protein.

Regulation of cellular morphology using temperature-responsive hydrogel for integrin-mediated mechanical force stimulation.

A new culture substrate was developed for cells to be equibiaxially stretched using fibronectin (Fn)-immobilized temperature-responsive hydrogel. The cells cultured on the gel substrate were equibiaxially stretched with swelling of the gel, which was accompanied by slight changes of temperature. During gel swelling, changes of cell shape were clearly observed by optical microscopy because of high transparency of the gel.

Embryonic undifferentiated cells show scattering activity on a surface coated with immobilized E-cadherin.

Rearrangement of cell-cell adhesion is a critical event in embryonic development and tissue formation. We investigated the regulatory function of E-cadherin, a key adhesion protein, in the developmental process by using E-cadherin/IgG Fc fusion protein as an adhesion matrix in cell culture. F9 embryonal carcinoma cells usually form colonies when cultured on gelatin or fibronectin matrices.

A simple combined floating and anchored collagen gel for enhancing mechanical strength of culture system.

In this study, a simple combined method consisting of floating and anchored collagen gel in a ligament or tendon equivalent culture system was used to produce the oriented fibrils in fibroblast-populated collagen matrices (FPCMs) during the remodeling and contraction of the collagen gel. Orientation of the collagen fibrils along single axis occurred over the whole area of the floating section and most of the fibroblasts were elongated and aligned along the oriented collagen fibrils, whereas no significant orientation of fibrils was observed in normally contracted FPCMs by the floating method. Higher elasticity and enhanced mechanical strength were obtained using our simple method compared with normally contracted floating FPCMs.