Glycosphingolipid GM2 Induces Invasiveness in Irradiation-tolerant Lung Cancer Cells.

Glycans, including glycosphingolipids, are broadly expressed in plasma membranes and play important roles in cell-cell interactions. Recently, it has been revealed that glycans participate in the regulation of malignant phenotypes of cancer cells, e.g. growth and invasion. However, their roles in irradiation-tolerant cancer cells have not yet been elucidated. In this study, we show that specific glycosphingolipids are highly expressed in invasive, irradiation-tolerant lung cancer cells. Particularly, the glycosphingolipid GM2 contributes to the development of an invasive phenotype in these lung cancer cells. Our results suggest that glycosphingolipids, including GM2, are implicated in the regulation of invasiveness in irradiation-tolerant lung cancer cells and may therefore serve as potential therapeutic targets for lung cancers following radiotherapy.Key words: glycosphingolipids, GM2, invasion, lung cancer cells, radiotherapy.

Publisher Correction: Cytoplasmic localization of GRHL3 upon epidermal differentiation triggers cell shape change for epithelial morphogenesis.

The original version of this Article contained an error in the labelling of Fig. 4. In panel i, the sixth column was incorrectly labelled as NSC23766 negative, and should have been NSC23766 positive. This has now been corrected in both the PDF and HTML versions of the Article.

Cytoplasmic localization of GRHL3 upon epidermal differentiation triggers cell shape change for epithelial morphogenesis.

Epithelial cell shape change is a pivotal driving force for morphogenesis of complex three-dimensional architecture. However, molecular mechanisms triggering shape changes of epithelial cells in the course of growth and differentiation have not been entirely elucidated. Grhl3 plays a crucial role as a downstream transcription factor of Wnt/ß-catenin in epidermal differentiation. Here, we show Grhl3 induced large, mature epidermal cells, enriched with actomyosin networks, from embryoid bodies in vitro. Such epidermal cells were apparently formed by the simultaneous activation of canonical and non-canonical Wnt signaling pathways. A nuclear transcription factor, GRHL3 is localized in the cytoplasm and cell membrane during epidermal differentiation. Subsequently, such extranuclear GRHL3 is essential for the membrane-associated expression of VANGL2 and CELSR1. Cytoplasmic GRHL3, thereby, allows epidermal cells to acquire mechanical properties for changes in epithelial cell shape. Thus, we propose that cytoplasmic localization of GRHL3 upon epidermal differentiation directly triggers epithelial morphogenesis.

Distinct Motion of GFP-Tagged Histone Expressing Cells Under AC Electrokinetics in Electrode-Multilayered Microfluidic Device.

The distinct motion of GFP-tagged histone expressing cells (Histone-GFP type cells) has been investigated under ac electrokinetics in an electrode-multilayered microfluidic device as compared with Wild type cells and GFP type cells in terms of different intracellular components. The Histone-GFP type cells were modified by the transfection of green fluorescent protein-fused histone from the human lung fibroblast cell line. The velocity of the Histone-GFP type cells obtained by particle tracking velocimetry technique is faster than Wild type cells by 24.9% and GFP type cells by 57.1%. This phenomenon is caused by the more amount of proteins in the intracellular of single Histone-GFP type cell than that of the Wild type and GFP type cells. The more amount of proteins in the Histone-GFP type cells corresponds to a lower electric permittivity ϵc of the cells, which generates a lower dielectrophoretic force exerting on the cells. The velocity of Histone-GFP type cells is well agreed with Eulerian-Lagrangian two-phase flow simulation by 4.2% mean error, which proves that the fluid motion driven by thermal buoyancy and electrothermal force dominates the direction of cells motion, while the distinct motion of Histone-GFP type cells is caused by dielectrophoretic force. The fluid motion does not generate a distinct drag motion for Histone-GFP type cells because the Histone-GFP type cells have the same size to the Wild type and GFP type cells. These results clarified the mechanism of cells motion in terms of intracellular components, which helps to improve the cell manipulation efficiency with electrokinetics.

Human myosin VIIa is a very slow processive motor protein on various cellular actin structures.

Human myosin VIIa (MYO7A) is an actin-linked motor protein associated with human Usher syndrome (USH) type 1B, which causes human congenital hearing and visual loss. Although it has been thought that the role of human myosin VIIa is critical for USH1 protein tethering with actin and transportation along actin bundles in inner-ear hair cells, myosin VIIa's motor function remains unclear. Here, we studied the motor function of the tail-truncated human myosin VIIa dimer (HM7AΔTail/LZ) at the single-molecule level. We found that the HM7AΔTail/LZ moves processively on single actin filaments with a step size of 35 nm. Dwell-time distribution analysis indicated an average waiting time of 3.4 s, yielding ∼0.3 s-1 for the mechanical turnover rate; hence, the velocity of HM7AΔTail/LZ was extremely slow, at 11 nm·s-1 We also examined HM7AΔTail/LZ movement on various actin structures in demembranated cells. HM7AΔTail/LZ showed unidirectional movement on actin structures at cell edges, such as lamellipodia and filopodia. However, HM7AΔTail/LZ frequently missed steps on actin tracks and exhibited bidirectional movement at stress fibers, which was not observed with tail-truncated myosin Va. These results suggest that the movement of the human myosin VIIa motor protein is more efficient on lamellipodial and filopodial actin tracks than on stress fibers, which are composed of actin filaments with different polarity, and that the actin structures influence the characteristics of cargo transportation by human myosin VIIa. In conclusion, myosin VIIa movement appears to be suitable for translocating USH1 proteins on stereocilia actin bundles in inner-ear hair cells.

High-Resolution Vertical Observation of Intracellular Structure Using Magnetically Responsive Microplates.

A vertical confocal observation system capable of high-resolution observation of intracellular structure is demonstrated. The system consists of magnet-active microplates to rotate, incline, and translate single adherent cells in the applied magnetic field. Appended to conventional confocal microscopes, this system enables high-resolution cross-sectional imaging with single-molecule sensitivity in single scanning.

Role of ATF5 in the invasive potential of diverse human cancer cell lines.

Activating transcription factor 5 (ATF5) is a member of the ATF/cAMP response element-binding protein family. Our research group recently revealed that ATF5 expression increases the invasiveness of human lung carcinoma cells. However, the effects of ATF5 on the invasive potential of other cancer cells lines remain unclear. Therefore, in this study, we investigated the role of ATF5 in the invasive activity of diverse human cancer cell lines. Invasiveness was assessed using Matrigel invasion assays. ATF5 knockdown resulted in decreased invasiveness in seven of eight cancer cell lines tested. These results suggest that ATF5 promotes invasiveness in several cancer cell lines. Furthermore, the roles of ATF5 in the invasiveness were evaluated in three-dimensional (3D) culture conditions. In 3D collagen gel, HT-1080 and MDA-MB-231 cells exhibited high invasiveness, with spindle morphology and high invasion speed. In both cell lines, knockdown of ATF5 resulted in rounded morphology and decreased invasion speed. Next, we showed that ATF5 induced integrin-α2 and integrin-ß1 expression and that the depletion of integrin-α2 or integrin-ß1 resulted in round morphology and decreased invasion speed. Our results suggest that ATF5 promotes invasion by inducing the expression of integrin-α2 and integrin-ß1 in several human cancer cell lines.

Data set for comparison of cellular dynamics between human AAVS1 locus-modified and wild-type cells.

This data article describes cellular dynamics, such as migration speed and mobility of the cytoskeletal protein, of wild-type human fibroblast cells and cells with a modified adeno-associated virus integration site 1 (AAVS1) locus on human chromosome 19. Insertion of exogenous gene into the AAVS1 locus has been conducted in recent biological researches. Previously, our data showed that the AAVS1-modification changes cellular contractile force (Mizutani et al., 2015 [1]). To assess if this AAVS1-modification affects cell migration, we compared cellular migration speed and turnover of cytoskeletal protein in human fibroblasts and fibroblasts with a green fluorescent protein gene knocked-in at the AAVS1 locus in this data article. Cell nuclei were stained and changes in their position attributable to cell migration were analyzed. Fluorescence recovery was observed after photobleaching for the fluorescent protein-tagged myosin regulatory light chain. Data here are related to the research article "Transgene Integration into the Human AAVS1 Locus Enhances Myosin II-Dependent Contractile Force by Reducing Expression of Myosin Binding Subunit 85" [1].

An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions.

Western blotting is a widely used method for detection and quantification of specific proteins extracted from mammalian cells. In the conventional method of protein extraction, we found that collagen-containing gels interfered with detection of the p65 protein (one of the subunits in the NF-κB family of proteins) in human lung adenocarcinoma A549 cells cultured on a collagen gel containing serum. In contrast, the collagen gels did not affect detection of the GAPDH protein. Then, we established an improved method for preparation of protein extracts (using trichloroacetic acid fixation and collagenase treatment) from the cells cultured on the collagen gel. Using the improved method, we were able to detect p65 proteins without loss in A549 cells cultured on a collagen gel under serum-free conditions, but we could not detect the proteins if serum was present in cell culture. Thus, using western blotting and serum-free culture conditions, we succeeded in comparing the p65 expression between the cells grown in a plastic dish and cells grown on a collagen gel.

Development of the evaluation system for barrier functions of engineered epithelial lumens.

We have investigated the effects of a diameter of engineered epithelial lumen on cellar architectures and a barrier function. For this investigation, we have developed a system to evaluate the barrier function of engineered epithelial lumens. To test the utility of our system, we constructed the engineered epithelial lumens by culturing Madin-Darby Canine Kidney cells (MDCK) on the gold wires with different diameters ranging from 50 µm-200 µm. Confocal laser scanning microscopy revealed that long actin stress fibers and a low focal adhesion density were observed at the gold wire diameter of 200 µm, whereas the mesh-like morphology consisted of short actin stress fibers and high focal adhesion densities were found at the gold wire diameters of 50 µm and 100 µm. The expression pattern of ZO-1 that localizes at the tight junction was independent on the gold wire diameter. The electrical impedance measurement indicates that the barrier function for the samples constructed at the gold wire diameter of 200 µm was significantly higher than those at the gold wire diameters of 50 µm and 100 µm. The difference in the barrier functions of epithelial lumens might be attributed to the changes in cellular architectures with increasing the curvature of gold wire.

Heterogeneous filament network formation by myosin light chain isoforms effects on contractile energy output of single cardiomyocytes derived from human induced pluripotent stem cells.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are expected to play an important role in heart therapies, in which hiPSC-CMs should generate sufficient contractile force to pump blood. However, recent studies have shown that the contractility of myocardial mimics composed of hiPSC-CMs is lower than that of adult human myocardium. To examine the mechanism by which contractile force output of hiPSC-CMs is weakened, we measured the contractile force of single hiPSC-CMs and observed the fibrous distribution of myosin II regulatory light chain (MRLC) of cardiac (contributes to beating) and non-cardiac (does not contribute to beating) isoforms. Single hiPSC-CMs were cultured on an extracellular matrix gel, and the contractile force and strain energy exerted on the gel were measured. Strain energy was not uniform between cells and ranged from 0.2 to 5.8 pJ. The combination of contractile force measurement and immunofluorescent microscopy for MRLC isoforms showed that cells with higher strain energy expressed the weakened non-cardiac myosin II fibers compared to those of cells with lower strain energy. Observation of cardiac and non-cardiac MRLC showed that the MRLC isoforms formed heterogeneous filament networks. These results suggest that strain energy output from single hiPSC-CMs depends both cardiac and non-cardiac myosin fibers, which prevent deformation of the cell body.

Three-dimensional morphogenesis of MDCK cells induced by cellular contractile forces on a viscous substrate.

Substrate physical properties are essential for many physiological events such as embryonic development and 3D tissue formation. Physical properties of the extracellular matrix such as viscoelasticity and geometrical constraints are understood as factors that affect cell behaviour. In this study, we focused on the relationship between epithelial cell 3D morphogenesis and the substrate viscosity. We observed that Madin-Darby Canine Kidney (MDCK) cells formed 3D structures on a viscous substrate (Matrigel). The structures appear as a tulip hat. We then changed the substrate viscosity by genipin (GP) treatment. GP is a cross-linker of amino groups. Cells cultured on GP-treated-matrigel changed their 3D morphology in a substrate viscosity-dependent manner. Furthermore, to elucidate the spatial distribution of the cellular contractile force, localization of mono-phosphorylated and di-phosphorylated myosin regulatory light chain (P-MRLCs) was visualized by immunofluorescence. P-MRLCs localized along the periphery of epithelial sheets. Treatment with Y-27632, a Rho-kinase inhibitor, blocked the P-MRLCs localization at the edge of epithelial sheets and halted 3D morphogenesis. Our results indicate that the substrate viscosity, the substrate deformation, and the cellular contractile forces induced by P-MRLCs play crucial roles in 3D morphogenesis.

Transgene integration into the human AAVS1 locus enhances myosin II-dependent contractile force by reducing expression of myosin binding subunit 85.

The adeno-associated virus site 1 (AAVS1) locus in the human genome is a strong candidate for gene therapy by insertion of an exogenous gene into the locus. The AAVS1 locus includes the coding region for myosin binding subunit 85 (MBS85). Although the function of MBS85 is not well understood, myosin II-dependent contractile force may be affected by altered expression of MBS85. The effect of altered expression of MBS85 on cellular contractile force should be examined prior to the application of gene therapy. In this study, we show that transgene integration into AAVS1 and consequent reduction of MBS85 expression changes myosin II-dependent cellular contractile force. We established a human fibroblast cell line with exogenous DNA knocked-in to AAVS1 (KI cells) using the CRISPR/Cas9 genome editing system. Western blotting analysis showed that KI cells had significantly reduced MBS85 expression. KI cells also showed greater cellular contractile force than control cells. The increased contractile force was associated with phosphorylation of the myosin II regulatory light chain (MRLC). Transfection of KI cells with an MBS85 expression plasmid restored cellular contractile force and phosphorylation of MRLC to the levels in control cells. These data suggest that transgene integration into the human AAVS1 locus induces an increase in cellular contractile force and thus should be considered as a gene therapy to effect changes in cellular contractile force.

Filamin B Enhances the Invasiveness of Cancer Cells into 3D Collagen Matrices.

Numerous types of cancer cells migrate into extracellular tissues. This phenomenon is termed invasion, and is associated with poor prognosis in cancer patients. In this study, we demonstrated that filamin B (FLNb), an actin-binding protein, is highly expressed in cancer cell lines that exhibit high invasiveness, with a spindle morphology, into 3D collagen matrices. In addition, we determined that knockdown of FLNb in invasive cancer cells converts cell morphology from spindle-shaped, which is associated with high invasiveness, to round-shaped with low invasiveness. Furthermore, di-phosphorylation of myosin regulatory light chain (MRLC) and phosphorylation of focal adhesion kinase (FAK) are inhibited in FLNb-knockdown cancer cells. These results suggest that FLNb enhances invasion of cancer cells through phosphorylation of MRLC and FAK. Therefore, FLNb may be a new therapeutic target for invasive cancers.

Compressive stress induces dephosphorylation of the myosin regulatory light chain via RhoA phosphorylation by the adenylyl cyclase/protein kinase A signaling pathway.

Mechanical stress that arises due to deformation of the extracellular matrix (ECM) either stretches or compresses cells. The cellular response to stretching has been actively studied. For example, stretching induces phosphorylation of the myosin regulatory light chain (MRLC) via the RhoA/RhoA-associated protein kinase (ROCK) pathway, resulting in increased cellular tension. In contrast, the effects of compressive stress on cellular functions are not fully resolved. The mechanisms for sensing and differentially responding to stretching and compressive stress are not known. To address these questions, we investigated whether phosphorylation levels of MRLC were affected by compressive stress. Contrary to the response in stretching cells, MRLC was dephosphorylated 5 min after cells were subjected to compressive stress. Compressive loading induced activation of myosin phosphatase mediated via the dephosphorylation of myosin phosphatase targeting subunit 1 (Thr853). Because myosin phosphatase targeting subunit 1 (Thr853) is phosphorylated only by ROCK, compressive loading may have induced inactivation of ROCK. However, GTP-bound RhoA (active form) increased in response to compressive stress. The compression-induced activation of RhoA and inactivation of its effector ROCK are contradictory. This inconsistency was due to phosphorylation of RhoA (Ser188) that reduced affinity of RhoA to ROCK. Treatment with the inhibitor of protein kinase A that phosphorylates RhoA (Ser188) induced suppression of compression-stimulated MRLC dephosphorylation. Incidentally, stretching induced phosphorylation of MRLC, but did not affect phosphorylation levels of RhoA (Ser188). Together, our results suggest that RhoA phosphorylation is an important process for MRLC dephosphorylation by compressive loading, and for distinguishing between stretching and compressing cells.

Leader cells regulate collective cell migration via Rac activation in the downstream signaling of integrin ß1 and PI3K.

Collective cell migration plays a crucial role in several biological processes, such as embryonic development, wound healing, and cancer metastasis. Here, we focused on collectively migrating Madin-Darby Canine Kidney (MDCK) epithelial cells that follow a leader cell on a collagen gel to clarify the mechanism of collective cell migration. First, we removed a leader cell from the migrating collective with a micromanipulator. This then caused disruption of the cohesive migration of cells that followed in movement, called "follower" cells, which showed the importance of leader cells. Next, we observed localization of active Rac, integrin ß1, and PI3K. These molecules were clearly localized in the leading edge of leader cells, but not in follower cells. Live cell imaging using active Rac and active PI3K indicators was performed to elucidate the relationship between Rac, integrin ß1, and PI3K. Finally, we demonstrated that the inhibition of these molecules resulted in the disruption of collective migration. Our findings not only demonstrated the significance of a leader cell in collective cell migration, but also showed that Rac, integrin ß1, and PI3K are upregulated in leader cells and drive collective cell migration.

Application of Multichannel Collagen Gels in Construction of Epithelial Lumen-like Engineered Tissues.

Introduction of epithelial lumen-like structures such as blood and lymphatic vessels, as well as renal tubules, is a prerequisite for successful construction and function of artificially engineered giant tissues. Here, we demonstrate a methodology for construction of various epithelial lumen-like structures by using multichannel collagen gels (MCCGs). MCCGs were prepared and used as template scaffolds for constructing epithelial lumen structures in a controlled fashion. The effect of NaCl concentration on the multichannel structure of MCCGs was investigated by using confocal laser scanning microscopy along with fluorescent staining. The channel diameter increased with increasing NaCl concentrations in the collagen solution and the phosphate buffer solution. In contrast, the channel number decreased with increasing NaCl concentrations. Engineered tissues with various lumen-like structures were constructed by seeding and culturing Madin-Darby canine kidney cells on MCCGs. The diameter of the lumen and the number of lumens per unit area were controllable by regulating the multichannel structure of cylindrical MCCG. We believe that our methodology for the construction of engineered tissues possessing epithelial lumen-like structures will prove helpful in regeneration of giant tissues with various hierarchical structures.

Partially photodegradable hybrid hydrogels with elasticity tunable by light irradiation.

This paper reports a simple technique to synthesize elasticity tunable hybrid hydrogels using photocleavable (N-hydroxysuccinimide terminated photocleavable tetra-arm poly(ethylene glycol); NHS-PC-4armPEG) and non-photocleavable (N-hydroxysuccinimide terminated tetra-arm poly(ethylene glycol); NHS-4armPEG) activated-ester type crosslinkers. Partially photodegradable hybrid hydrogels were synthesized by reacting the crosslinker mixture with amino-terminated tetra-arm poly(ethylene glycol) (amino-4armPEG). The photocleavable crosslinks are cleaved by irradiating light while the non-photocleavable crosslinks remain intact, resulting in decreased elasticity. We demonstrate that hydrogel elasticity can be controlled by adjusting the ratio of photocleavable NHS-PC-4armPEG and non-photocleavable NHS-4armPEG, and by varying the light exposure energy. We also show how micropatterned elasticity can be obtained in the hydrogels by irradiating with micropatterned light. These techniques could provide a novel platform to tailor the elasticity of hydrogels with microscale precision for biological studies in the near future.

Epithelial sheet folding induces lumen formation by Madin-Darby canine kidney cells in a collagen gel.

Lumen formation is important for morphogenesis; however, an unanswered question is whether it involves the collective migration of epithelial cells. Here, using a collagen gel overlay culture method, we show that Madin-Darby canine kidney cells migrated collectively and formed a luminal structure in a collagen gel. Immediately after the collagen gel overlay, an epithelial sheet folded from the periphery, migrated inwardly, and formed a luminal structure. The inhibition of integrin-ß1 or Rac1 activity decreased the migration rate of the peripheral cells after the sheets folded. Moreover, lumen formation was perturbed by disruption of apical-basolateral polarity induced by transforming growth factor-ß1. These results indicate that cell migration and cell polarity play an important role in folding. To further explore epithelial sheet folding, we developed a computer-simulated mechanical model based on the rigidity of the extracellular matrix. It indicated a soft substrate is required for the folding movement.

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.e., silanized) glass coated with ECM. Furthermore, the morphology and motility of cells grown on silanized glass differed from those grown on non-treated glass, even when both types of glass were initially coated with laminin. We also found that cells on silanized glass coated with laminin had higher motility than those on silanized glass coated with fibronectin. Based on our results, we suggest that silanized glass is a more suitable cell culture substrate than conventional non-treated glass when coated by ECM for observations of ECM effects on cell physiology.