Hans I.Chen Harn

Mechanosensitive store‐operated calcium entry regulates the formation of cell polarity

Ca2+‐mediated formation of cell polarity is essential for directional migration which plays an important role in physiological and pathological processes in organisms. To examine the critical role of store‐operated Ca2+ entry, which is the major form of extracellular Ca2+ influx in non‐excitable cells, in the formation of cell polarity, we employed human bone osteosarcoma U2OS cells, which exhibit distinct morphological polarity during directional migration. Our analyses showed that Ca2+ was concentrated at the rear end of cells and that extracellular Ca2+ influx was important for cell polarization. Inhibition of store‐operated Ca2+ entry using specific inhibitors disrupted the formation of cell polarity in a dose‐dependent manner. Moreover, the channelosomal components caveolin‐1, TRPC1, and Orai1 were concentrated at the rear end of polarized cells. Knockdown of TRPC1 or a TRPC inhibitor, but not knockdown of Orai1, reduced cell polarization. Furthermore, disruption of lipid rafts or overexpression of caveolin‐1 contributed to the downregulation of cell polarity. On the other hand, we also found that cell polarity, store‐operated Ca2+ entry activity, and cell stiffness were markedly decreased by low substrate rigidity, which may be caused by the disorganization of actin filaments and microtubules that occurs while regulating the activity of the mechanosensitive TRPC1 channel. J. Cell. Physiol. 230: 2086–2097, 2015.

Using surfaces to modulate the morphology and structure of attached cells – a case of cancer cells on chitosan membranes

This paper describes the development of physically and/or chemically modified chitosan membranes to probe cellular behaviors and molecular-level structural responses of NIH-3T3 fibroblasts (normal cells) and Ha-ras-transformed cells (abnormal cells) adhered onto these modified membranes. To prepare chitosan membranes with nanometrically scaled physical features, we have demonstrated an inexpensive and easy-to-handle method that could be easily integrated with IC-based manufacturing processes with mass production potential. These physically or chemically modified chitosan membranes were examined via scanning electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy, and water contact angle measurement, in order to gain a better understanding of chitosan membrane surface characteristics including surface morphology, stiffness, functional groups, and surface hydrophobicity/hydrophilicity. NIH-3T3 fibroblasts and their Ha-ras-transformed progeny were cultured on these modified chitosan membranes. After 12, 24 and 48 h of culture, these cells were investigated to decipher cellular behaviors. We found that NIH-3T3 fibroblasts and their Ha-ras-transformed progeny exhibited distinct structurally based responses attributable to chitosan membrane surface chemical or physical properties that we demonstrate as possibly applicable, for drug screening applications. Secondarily, but crucially to this study, we developed a chitosan-based micropatterning procedure that allowed us to re-arrange mammalian cells (i.e., HeLa cells in this study, for cancer drug screening) at the desired locations (with a single-cell array format). This procedure was based on cell affinity to different surface topographies of chitosan membranes that we prepared. This cell-based patterning approach has the potential for use in a wide range of applications including use as a promising platform for drug discovery, cytotoxicity studies, functional genomics, and investigations of cellular microenvironment. We believe that this study would provide further understanding of naturally derived biomaterials, lay the foundation for broadening the applications of chitosan, and facilitate the development of new biomedical devices (i.e., artificial stents, implantable artificial tissues, and sustainable implantable biosensors) with unique cell–material interface properties and characteristics, such as in vitro cell culture and diagnostic platforms.

Mechanical coupling of cytoskeletal elasticity and force generation is crucial for understanding the migrating nature of keloid fibroblasts

One of the key features of keloid is its fibroblasts migrating beyond the original wound border. During migration, cells not only undergo molecular changes but also mechanical modulation. This process is led by actin filaments serving as the backbone of intra‐cellular force and transduces external mechanical signal via focal adhesion complex into the cell. Here, we focus on determining the mechanical changes of actin filaments and the spatial distribution of forces in response to changing chemical stimulations and during cell migration. Atomic force microscopy and micropost array detector are used to determine and compare the magnitude and distribution of filament elasticity and force generation in fibroblasts and keloid fibroblasts. We found both filament elasticity and force generation show spatial distribution in a polarized and migrating cell. Such spatial distribution is disrupted when mechano‐signalling is perturbed by focal adhesion kinase inhibitor and in keloid fibroblasts. The demonstration of keloid pathology at the nanoscale highlights the coupling of cytoskeletal function with physical characters at the subcellular level and provides new research directions for migration‐related disease such as keloid.

The Tension Biology of Wound Healing

Following skin wounding, the healing outcome can be: regeneration, repair with normal scar tissue, repair with hypertrophic scar tissue or the formation of keloids. The role of chemical factors in wound healing has been extensively explored, and while there is evidence suggesting the role of mechanical forces, its influence is much less well defined. Here, we provide a brief review on the recent progress of the role of mechanical force in skin wound healing by comparing laboratory mice, African spiny mice, fetal wound healing and adult scar keloid formation. A comparison across different species may provide insight into key regulators. Interestingly, some findings suggest tension can induce an immune response, and this provides a new link between mechanical and chemical forces. Clinically, manipulating skin tension has been demonstrated to be effective for scar prevention and treatment, but not for tissue regeneration. Utilising this knowledge, specialists may modulate regulatory factors and develop therapeutic strategies to reduce scar formation and promote regeneration.

Caveolin-1 Controls Hyperresponsiveness to Mechanical Stimuli and Fibrogenesis-Associated RUNX2 Activation in Keloid Fibroblasts

Keloids are pathological scars characterized by excessive extracellular matrix production that are prone to form in body sites with increased skin tension. CAV1, the principal coat protein of caveolae, has been associated with the regulation of cell mechanics, including cell softening and loss of stiffness sensing ability in NIH3T3 fibroblasts. Although CAV1 is present in low amounts in keloid fibroblasts (KFs), the causal association between CAV1 down-regulation and its aberrant responses to mechanical stimuli remain unclear. In this study, atomic force microscopy showed that KFs were softer than normal fibroblasts with a loss of stiffness sensing. The decrease of CAV1 contributed to the hyperactivation of fibrogenesis-associated RUNX2, a transcription factor germane to osteogenesis/chondrogenesis, and increased migratory ability in KFs. Treatment of KFs with trichostatin A, which increased the acetylation level of histone H3, increased CAV1 and decreased RUNX2 and fibronectin. Trichostatin A treatment also resulted in cell stiffening and decreased migratory ability in KFs. Collectively, these results suggest a role for CAV1 down-regulation in linking the aberrant responsiveness to mechanical stimulation and extracellular matrix accumulation with the progression of keloids, findings that may lead to new developments in the prevention and treatment of keloid scarring.

Revealing the three dimensional architecture of focal adhesion components to explain Ca2 +-mediated turnover of focal adhesions

BACKGROUND Focal adhesions (FAs) are large, dynamic protein complexes located close to the plasma membrane, which serve as the mechanical linkages and a biochemical signaling hub of cells. The coordinated and dynamic regulation of focal adhesion is required for cell migration. Degradation, or turnover, of FAs is a major event at the trailing edge of a migratory cell, and is mediated by Ca2+/calpain-dependent proteolysis and disassembly. Here, we investigated how Ca2+ influx induces cascades of FA turnover in living cells. METHODS Images obtained with a total internal reflection fluorescence microscope (TIRFM) showed that Ca2+ ions induce different processes in the FA molecules focal adhesion kinase (FAK), paxillin, vinculin, and talin. Three mutated calpain-resistant FA molecules, FAK-V744G, paxillin-S95G, and talin-L432G, were used to clarify the role of each FA molecule in FA turnover. RESULTS Vinculin was resistant to degradation and was not significantly affected by the presence of mutated calpain-resistant FA molecules. In contrast, talin was more sensitive to calpain-mediated turnover than the other molecules. Three-dimensional (3D) fluorescence imaging and immunoblotting demonstrated that outer FA molecules were more sensitive to calpain-mediated proteolysis than internal FA molecules. Furthermore, cell contraction is not involved in degradation of FA. CONCLUSIONS These results suggest that Ca2+-mediated degradation of FAs was mediated by both proteolysis and disassembly. The 3D architecture of FAs is related to the different dynamics of FA molecule degradation during Ca2+-mediated FA turnover. GENERAL SIGNIFICANCE This study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca2+.

Spatial distribution of filament elasticity determines the migratory behaviors of a cell

ABSTRACT Any cellular response leading to morphological changes is highly tuned to balance the force generated from structural reorganization, provided by actin cytoskeleton. Actin filaments serve as the backbone of intracellular force, and transduce external mechanical signal via focal adhesion complex into the cell. During migration, cells not only undergo molecular changes but also rapid mechanical modulation. Here we focus on determining, the role of spatial distribution of mechanical changes of actin filaments in epithelial, mesenchymal, fibrotic and cancer cells with non-migration, directional migration, and non-directional migration behaviors using the atomic force microscopy. We found 1) non-migratory cells only generated one type of filament elasticity, 2) cells generating spatially distributed two types of filament elasticity showed directional migration, and 3) pathologic cells that autonomously generated two types of filament elasticity without spatial distribution were actively migrating non-directionally. The demonstration of spatial regulation of filament elasticity of different cell types at the nano-scale highlights the coupling of cytoskeletal function with physical characters at the sub-cellular level, and provides new research directions for migration related disease.

Mechanical forces in skin disorders

Mechanical forces are known to regulate homeostasis of the skin and play a role in the pathogenesis of skin diseases. The epidermis consists of keratinocytes that are tightly adhered to each other by cell junctions. Defects in keratins or desmosomal/hemidesmosomal proteins lead to the attenuation of mechanical strength and formation of intraepidermal blisters in the case of epidermolysis bullosa simplex. The dermis is rich in extracellular matrix, especially collagen, and provides the majority of tensile force in the skin. Keloid and hypertrophic scar, which is the result of over-production of collagen by fibroblasts during the wound healing, are associated with extrinsic tensile forces and changes of intrinsic mechanical properties of the cell. Increasing evidences shows that stiffness of the skin environment determines the regenerative ability during wound healing process. Mechanotransduction pathways are also involved in the morphogenesis and cyclic growth of hair follicles. The development of androgenetic alopecia is correlated to tensile forces generated by the fibrous tissue underlying the scalp. Acral melanoma predominantly occurs in the weight-bearing area of the foot suggesting the role of mechanical stress. Increased dermal stiffness from fibrosis might be the cause of recessive dystrophic epidermolysis bullosa associated squamous cell carcinoma. Strategies to change the mechanical forces or modify the mechanotransduction signals may lead to a new way to treat skin diseases and promote skin regeneration.