Kazushige Kawabata

Elasticity mapping of living fibroblasts by AFM and immunofluorescence observation of the cytoskeleton

Using the force mapping mode of atomic force microscopy (AFM), we measured spatial distribution of elastic moduli of living mouse fibroblasts (NIH3T3) in a physiological condition. The nuclear portion of the cellular surface is about 10 times softer than the surroundings. Stiffer fibers are confirmed in the elastic images. In order to investigate origin of the softer nuclear portion and the stiffer fibers, we fixed the identical cells imaged by the AFM, and carried out immunofluorescence observation for three types of cytoskeletal filaments--actin filaments, microtubules, and intermediate filaments, using confocal laser scanning microscopy (CLSM). A comparison between the AFM and the CLSM images revealed that the elasticity of the cells was concerned not only with the distribution of actin network, but also with intermediate filaments, whereas microtubules had no large effect on the measured elasticity.

Leader cells regulate collective cell migration via Rac activation in the downstream signaling of integrin β1 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 β1, 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 β1, 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 β1, and PI3K are upregulated in leader cells and drive collective cell migration.

Differential localization of non-muscle myosin II isoforms and phosphorylated regulatory light chains in human MRC-5 fibroblasts

We investigated the localization of non‐muscle myosin II isoforms and mono‐ (at serine 19) and diphosphorylated (at serine 19 and threonine 18) regulatory light chains (RLCs) in motile and non‐motile MRC‐5 fibroblasts. In migrating cells, myosin IIA localized to the lamella and throughout the posterior region. Myosin IIB colocalized with myosin IIA to the posterior region except at the very end. Diphosphorylated RLCs were detected in the restricted region where myosin IIA was enriched. In non‐motile cells, myosin IIA was enriched in peripheral stress fibers with diphosphorylated RLCs, but myosin IIB was not. Our results suggest that myosin IIA may be highly activated by diphosphorylation of RLCs and primarily involved in cell migration.

Three-dimensional characterization of interior structures of exocytotic apertures of nerve cells using atomic force microscopy

We examined the interior structure of exocytotic apertures in synaptic vesicles of neuroblastoma x glioma hybrid cells using atomic force microscopy. The atomic force microscopy detected apertures of 50-100nm in diameter at various depths within the varicosities of these cells. We were also able to image a regular radial pattern on the wall and lump-like structures at the bottom of these apertures. In contrast, scanning electron microscopy could only detect the apertures but not the fine details of their interior. The cells examined here exhibited the same electrophysiological properties and expression of synaptophysin and syntaxin 1 as presynaptic terminals, as studied by various electrophysiological and imaging techniques. Our results indicate that atomic force microscopy allows three-dimensional viewing of the fine structures located inside exocytotic apertures in nerve cells.

Diphosphorylation of the myosin regulatory light chain enhances the tension acting on stress fibers in fibroblasts

Regulation of the contractile force is crucial for cell migration, cell proliferation, and maintenance of cell morphology. Phosphorylation of the myosin II regulatory light chain (MRLC) is involved in these processes. To show whether the diphosphorylation of MRLC increases the tension acting on stress fibers, changes in the stiffness of fibroblasts expressing wild‐type MRLC and a mutant type, which cannot be diphosphorylated, on treatment with lysophosphatidic acid (LPA) were examined by a mechanical‐scanning probe microscope (M‐SPM). The LPA treatment increased cellular stiffness in the wild‐type MRLC expressing cells, while it had no effect on the mutated cells. Immunostaining showed that LPA stimulation induced the diphosphorylation of MRLC. These results suggest that the diphosphorylation of MRLC enhances the tension acting on stress fibers. J. Cell. Physiol. 209: 726–731, 2006.

Conducting thin films of α-(BEDT-TTF)2I3 by evaporation method

Abstract Thin films have been prepared by using the charge-transfer complex (BEDT-TTF) iodide as the raw material by a vacuum evaporation method. The electric conductivity in the film plane was as high as 2 S cm at room temperature. The conductivity measurement and X-ray examinations for as-grown specimens revealed that the films were composed of highly c-axis oriented α-(BEDT-TTF)2I3. Examinations on annealed specimens also provided another proof for the above conclusion.

Comparing microscopic with macroscopic elastic properties of polymer gel

Measurements of the local elastic modulus of agar gels obtained with atomic force microscope (AFM) force mapping were compared with values obtained by the tensile creep method. The observed spatial distributions of the local elastic modulus over the gel surface in AFM elastic images clearly corresponded to the network structure of agar fibers observed both in AFM topographic and scanning electron microscope (SEM) images. Both peak and average values of distribution functions in the histograms of local elastic modulus increase monotonically with the agar concentration. Values obtained by AFM force mapping were found to be proportional to values obtained from creep experiments.

Regulation of cellular contractile force in response to mechanical stretch by diphosphorylation of myosin regulatory light chain via RhoA signaling cascade.

Fibroblasts regulate their contractile force in response to external stretch; however, the detailed mechanism by which the force is regulated is unclear. Here, we show that diphosphorylation and dephosphorylation of myosin regulatory light chain (MRLC) are involved in the stretch-induced force response. Cellular stiffness, which reflects the cellular contractile force, under external stretch was measured by mechanical-scanning probe microscopy. Fibroblasts (NIH-3T3) expressing green fluorescent protein (GFP)-tagged mutant-type MRLC (MRLC(T18A)-GFP), which cannot be diphosphorylated, did not show any stretch-induced stiffness response, whereas the stiffness in cells expressing GFP-tagged wild-type MRLC (MRLC(WT)-GFP) increased immediately after the stretch and subsequently decreased after 1-2 h. Urea-PAGE western blot analysis showed that the proportion of diphosphorylated MRLC (PP-MRLC) transiently increased after the stretch and decreased after 1-2 h. Dominant-negative RhoA (RhoA(N19))-expressing cells did not show the stiffness response to the stretch, whereas wild-type RhoA-expressing cells did. It was concluded that the cellular force response originates in the stretch-induced diphosphorylation and dephosphorylation of MRLC and is regulated via the RhoA signaling cascade.

Comparative Atomic Force and Scanning Electron Microscopy for Fine Structural Images of Nerve Cells

Although we can routinely obtain fine structural images of cells by atomic force microscopy (AFM), the adequacy and reliability of morphological information acquired from these AFM images remain to be examined. In this report, we compared images of the fine structures of nerve cells as observed by both AFM and scanning electron microscopy (SEM). Although AFM revealed the structure of the top views of cells in greater detail than SEM, their side structures were better observed by SEM. The linear structures in the neural processes detected only by AFM were confirmed, by immunofluorescence staining, to be reflections of the cytoskeletal structures located beneath the cell membrane. These differences between the AFM and the SEM images reflected the characteristics of the detection systems and methods used for sample preparation. Therefore, these results revealed that more detailed information on cell morphology can be obtained by using both AFM and SEM to advantage.

Lung Cancer Cells That Survive Ionizing Radiation Show Increased Integrin α2β1- and EGFR-Dependent Invasiveness

Ionizing radiation (IR)-enhanced tumor invasiveness is emerging as a contributor to the limited benefit of radiotherapy; however, its mechanism is still unclear. We previously showed that subcloned lung adenocarcinoma A549 cells (P cells), which survived 10 Gy IR (IR cells), acquired high invasiveness in vitro. Here, we tried to identify the mechanism by which IR cells increase their invasiveness by examining altered gene expression and signaling pathways in IR cells compared with those in P cells. To simulate the microenvironment in vivo, cells were embedded in a three-dimensional (3D) collagen type I gel, in which the IR cells were elongated, while the P cells were spherical. The integrin expression pattern was surveyed, and expression levels of the integrin α2 and β1 subunits were significantly elevated in IR cells. Knockdown of α2 expression or functional blockade of integrin α2β1 resulted in a round morphology of IR cells, and abrogated their invasion in the collagen matrix, suggesting the molecule’s essential role in cell spread and invasion in 3D collagen. Epidermal growth factor receptor (EGFR) also presented enhanced expression and activation in IR cells. Treatment with EGFR tyrosine kinase inhibitor, PD168393, decreased the ratio of elongated cells and cell invasiveness. Signaling molecules, including extracellular signal-regulated kinase-1/2 (Erk1/2) and Akt, exhibited higher activation in IR cells. Inhibition of Akt activation by treating with phosphoinositide 3-kinase (PI3K) inhibitor LY294002 decreased IR cell invasion, whereas inhibition of Erk1/2 activation by mitogen-activated protein kinase kinase (MEK) inhibitor U0126 did not. Our results show that integrin α2β1 and EGFR cooperatively promote higher invasiveness of IR-survived lung cancer cells, mediated in part by the PI3K/Akt signaling pathway, and might serve as alternative targets in combination with radiotherapy.