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ABSTRACT: Radiotherapy is one of the major treatment modalities for malignancies. However, cells surviving irradiation often display high levels of invasiveness. This study shows that irradiation-tolerant lung adenocarcinoma demonstrates high invasive capability depending on dephosphorylation of the myosin regulatory light chain (MRLC). In a collagen gel overlay condition, low-invasive subclones of lung adenocarcinoma (A549P-3) showed a round morphology and diphosphorylation of MRLC. In contrast, irradiation-tolerant A549P-3 cells (A549P-3IR) displayed high invasiveness and a lower level of MRLC diphosphorylation. In addition, inhibition of MRLC phosphatase activity decreased the invasive activity. These findings suggest that A549P-3IR cells acquire high invasiveness through MRLC dephosphorylation.
FEBS letters 02/2013; · 3.54 Impact Factor
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ABSTRACT: We investigated the dynamics of the cortical cytoskeleton in living cells by analyzing the motion of the endogenous components of the cytoskeleton using scanning probe microscopy (SPM). We performed molecular characterization of the microgranules visualized by SPM in living cells and analyzed the motion of these microgranules via particle tracking. Simultaneous SPM and epifluorescence microscopy observations showed that the microgranules recruited not only actin but also cortactin, which can bind to actin filaments. This indicates condensation of actin filaments at microgranules, leading us to identify them as "cytoskeletal microdomains". High-speed SPM observation and particle-tracking analysis showed that these cytoskeletal microdomains exhibit random walk-like diffusive fluctuations over a timescale of seconds. Inhibition of the molecular motor myosin II, which drives actin filaments, led to subdiffusive fluctuations of the microdomains. These results can be explained by longitudinal sliding of actin filaments stochastically driven by myosin II and the bending motion of the actin filaments in the absence of sliding. Analysis of the cytoskeletal microdomains thus revealed the intrinsic dynamics of the cortical cytoskeleton.
Acta biomaterialia 06/2011; 7(10):3766-72. · 3.98 Impact Factor
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ABSTRACT: Filamentous actin and myosin-II are major determinants of cell mechanics and are tightly regulated by a small guanosine triphosphatase, RhoA, and its downstream effectors. We examined the effects of constitutively active mutants of RhoA effectors, which have not been reported before, on cortical stiffness of living cells by using scanning probe microscopy, fluorescence microscopy, and truncated mutants of RhoA effectors labeled with a fluorescent protein. Our data indicated that expression of a constitutively active mutant of Dia1, a formin-family actin polymerizer, enhanced cortical stiffness and increased actin filament quantity in cells. Furthermore, expression of a constitutively active mutant of Rho-associated coiled-coil kinase, a myosin-II activator, softened the cell cortex but increased myosin-II activity. Our findings provide new insights into anomalous mechanics of cells, which is a topic of current interest in a variety of biological research fields.
Biochemical and Biophysical Research Communications 11/2010; 403(3-4):363-7. · 2.48 Impact Factor
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ABSTRACT: The morphology of chromosomes (certain rod-shaped structures) is highly reproducible despite the high condensation of chromatin fibers (∼1 mm) into chromosomes (∼1 μm). However, the mechanism underlying the condensation of chromatin fibers into chromosomes is unclear. We assume that investigation of the internal structure of chromosomes will aid in elucidating the condensation process. In order to observe the detailed structure of a chromosome, we stretched a human chromosome by using a micromanipulator and observed its morphology along the stretched region by scanning probe microscopy (SPM). We found that the chromosome consisted of some fibers that were thicker than chromatin fibers. The found fiber was composed of approximately 90-nm-wide beads that were linked linearly. To explore the components of the fiber, we performed immunofluorescence staining of the stretched chromosome. Fluorescence signals of topoisomerase (Topo) IIα, which is known to interact with and support chromatin fibers, and DNA were detected both on the found fiber and beads. Furthermore, after micrococcal nuclease and trypsin treatments, the fibers were found to be mechanically supported by proteins. These results suggest that chromosome comprises an intermediate structure between chromatin fibers and chromosomes.
Biochemical and Biophysical Research Communications 09/2010; 400(1):181-6. · 2.48 Impact Factor
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ABSTRACT: Radiotherapy is one of the effective therapies used for treating various malignant tumors. However, the emergence of tolerant cells after irradiation remains problematic due to their high metastatic ability, sometimes indicative of poor prognosis. In this study, we showed that subcloned human lung adenocarcinoma cells (A549P-3) that are irradiation-tolerant indicate high invasive activity in vitro, and exhibit an integrin beta1 activity-dependent migratory pattern. In collagen gel overlay assay, majority of the A549P-3 cells displayed round morphology and low migration activity, whereas a considerable number of A549P-3IR cells surviving irradiation displayed a spindle morphology and high migration rate. Blocking integrin beta1 activity reduced the migration rate of A549P-3IR cells and altered the cell morphology allowing them to assume a round shape. These results suggest that the A549P-3 cells surviving irradiation acquire a highly invasive integrin beta1-dependent phenotype, and integrin beta1 might be a potentially effective therapeutic target in combination with radiotherapy.
Biochemical and Biophysical Research Communications 06/2010; 396(3):651-5. · 2.48 Impact Factor
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ABSTRACT: In this study, we aimed at improving the temporal resolution of scanning probe microscopy (SPM) for observing living cells by introducing soft cantilevers, low feedback-gain operations, and cantilever deflection imaging. We achieved visualization of the mechanical architecture in leading lamellae of living fibroblasts at a temporal resolution of around 10 s, which is higher than that of conventional contact-mode SPM. Time-lapse SPM could be used to monitor not only cytoskeletal dynamics but also the dynamics of numerous microgranules. Statistical analysis of microgranular motion revealed that the microgranules have superdiffusive behaviors and significant directional order of motion. We also found that the direction of their motion is correlated with the direction of growing actin stress fibers. The combination of SPM with fluorescence microscopy showed that vinculin, a component of cell-substratum adhesion sites, localizes at the microgranules. Our experimental data provides a new insight into the intracellular mechanical architecture and its structural dynamics, suggesting that high-speed live-cell SPM has great potential for investigating the structural origin of cellular dynamics.
Histochemie 10/2009; 133(1):59-67. · 2.59 Impact Factor
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ABSTRACT: 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.
Cell Motility and the Cytoskeleton 06/2009; 66(7):389-97. · 4.19 Impact Factor
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ABSTRACT: To find a new molecule that affects p53-dependent radiosensitivity.
A mouse sarcoma cell line, QRsP(p53+/+), was used. From this cell line, we established a radiosensitive clone and a radioresistant one. Colony assay, p53 gene transfer, a luciferase assay for p53 and p21, animal transplantation experiment, and DNA array analyses were performed.
Microarray showed marked reduction of a transcription factor, ATF5, both in vitro and in vivo for the radiosensitive clone. Interestingly, flow cytometric analysis demonstrated marked apoptosis for the radiosensitive clone by p53 cloned adenovirus infection. Luciferase reporter assay revealed that ATF5 suppressed the transactivational activity of p53 and p63. By ATF5 gene transfer, the radiosensitive clone regained resistance to both ionizing-radiation and Ad-p53 infection-induced cell death. Surprisingly, time-lapse cell migration observation revealed greater cell motility for ATF5-transfected radiosensitive clone.
It seems likely that ATF5 is a potent repressor of p53 and elevated expression of ATF5 in a tumor may relate to enhanced malignant phenotypes, such as radioresistance or greater cell motility.
Cell Structure and Function 02/2009; 34(1):17-22. · 2.29 Impact Factor
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ABSTRACT: The mechanical memory effect of single cells was reported in our recent study. In order to clarify this effect, various sequential stimuli of uniaxial deformation were applied to cells by deformable culture dishes and a deformation device, and the local stiffness distribution of single C2C12 myoblasts was visualized by scanning probe microscopy. Following a single step stretching, cellular stiffness first increased steeply and then gradually decreased for two hours. By a single step stretching 30 min after a long pulse-like deformation with a pulse duration of 30 min, the cells responded in the same way. On the other hand, they did not respond to a single step stretching 30 min after a short pulse-like deformation with a pulse duration of 0.5 min. These results indicated that cellular mechanical response to external deformation is affected strongly by a preceding deformation and that the duration time of the preceding deformation is an important factor in the change in mechanical response. We consider that the change in mechanical response contributes to a regulatory mechanism of cellular contractile force.
Archives of Histology and Cytology 01/2009; 72(4-5):227-34. · 0.57 Impact Factor
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ABSTRACT: Scanning probe microscopy (SPM) provides a range of strategies for studying biological phenomena due to its ability to image surfaces under liquids. However, some cellular events, such as cell migration, exceed the maximum measurable range of SPM. Recently, we have developed a wide range scanning probe microscope (WR-SPM) to investigate cellular events which exceed the range of the conventional SPM. In this review, we introduce the instrumentation of the WR-SPM, which can measure a sample for 400 µm in the xy directions and 23 µm in the z direction. We then show the application of the WR-SPM to studies of the stiffness response of epithelial cells to an external loading force and demonstrat that the stiffness of the epithelial cells increases under stretched conditions. We also showed the results on the mesh structure on the surface of a melanoma cell as well as the regulatory mechanism of the cellular contractile force by the combined use of topographical and mechanical modes of the WR-SPM. These findings indicate that the WR-SPM is very useful for studying the functions of a cell in relation to the surface structure and mechanical properties of that cell.
Archives of Histology and Cytology 01/2009; 72(4-5):235-43. · 0.57 Impact Factor
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ABSTRACT: Scanning probe microscopy (SPM) is especially attractive to biologists, because it has the advantage of obtaining three-dimensional
images of sample surfaces at high resolution not only in a vacuum but also in a nonvacuum (i. e., air or liquid) environment.
In addition to the visualization of biological structures, SPM has also been applied for measuring the physical properties
(i. e., viscoelasticity) of living cells, and as a manipulation tool in biomaterials. The present reviewdescribes the application
of SPM to biological fields for investigating the structure and function of biomaterials from biomolecules to living cells,
and shows that SPM has great potential for broadening and opening up new fields in biomedical research.
12/2007: pages 285-308;
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ABSTRACT: We have developed a new stretch device to investigate the biomechanical responses to an external loading force on a tissue-like material consisting of cells and a collagen gel. Collagen gel, a typical matrix found abundantly in the connective tissue, was attached to an elastic chamber that was precoated with a thin layer of collagen. Madin-Darby canine kidney cells that were cultured on the collagen gel were stretched in a uniaxial direction via deformation of the elastic chamber. Changes in the morphology and stiffness of the tissue-like structure were measured before and after the stretch using wide-range scanning probe microscopy (WR-SPM). The change in cellular morphology was heterogeneous, and there was a twofold increase in the intercellular junction due to the stretch. In addition to the WR-SPM measurements, this device enables observation of the spatial distribution of cytoskeletal proteins such as vimentin and alpha-catenin using immunofluorescent microscopy. We concluded that the stretch device we have reported in this paper is useful for measuring the mechanical response of a tissue-like material over a range of cell sizes when exposed to an external loading force.
Acta Biomaterialia 08/2007; 3(4):485-93. · 4.86 Impact Factor
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ABSTRACT: Scanning probe microscope (SPM) has been developed as a powerful tool for obtaining high resolution topographic images of biological samples in their natural aqueous environment. SPM can also be used to evaluate mechanical properties because its probe is physically in contact with the samples during measurement. To obtain cellular stiffness with SPM, we have proposed two methods: a force modulation mode and a force mapping mode. Considering the influence of the drag force of liquids, we have successfully improved the quantitative evaluation of cellular stiffness by using the force modulation mode. Experiments performed using the two methods revealed that the local stiffness of fibroblasts was not homogeneous on the cell surface but largely varied from point to point. It was revealed that spatial and temporal distributions of cellular stiffness originate in cytoskeletal distribution, mode of cellular migration, and intracellular contractile force.
Current Nanoscience 01/2007; 3(1):97-103. · 1.78 Impact Factor
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ABSTRACT: 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.
Journal of Cellular Physiology 01/2007; 209(3):726-31. · 3.87 Impact Factor
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ABSTRACT: Collective cell movement acts as an efficient strategy in many physiological events, including wound healing, embryonic development, and morphogenesis. We found that epithelial cells (Madin-Darby canine kidney cell) migrated collectively along one direction on a collagen gel substrate. Time-lapse images of Madin-Darby canine kidney cells cultured on type-I collagen gels and glass substrates were captured by phase contrast microscopy equipped with an incubation system. On the gel substrate, the directions of cell movement gradually converged on one direction as the number of cells increased, whereas the cells moved randomly on the glass substrate. We also observed "leader" cells, which extended large lamellae and were accompanied by many "follower" cells, migrating in the direction of oriented collagen fibers. The mean-squared displacement of each cell movement and the spatial correlation function calculated from the spatial distribution of cell velocity were obtained as functions of observation time. In the case of the gel substrate, the spatial correlation length increased gradually, representing the collectiveness of multicellular movement.
Biophysical Journal 04/2005; 88(3):2250-6. · 3.65 Impact Factor
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ABSTRACT: Stiffness responses of fibroblasts were measured by scanning probe microscopy, following elongation or compression by deformation of an elastic substrate by 8%. The cellular stiffness, reflecting intracellular tension acting along stress fibers, decreased or increased instantly in response to the elongating or compressing stimuli, respectively. After this rapid change, the fibroblasts gradually recovered to their initial stiffness during the following 2 h, and then stabilized. The cells did not show conspicuous changes in shape after the 8% deformation during the SPM measurements. Fluorescence examination for GFP-actin demonstrated that the structure of the stress fibers was not altered noticeably by this small degree of deformation. Treatment with Y-27632, to inhibit myosin phosphorylation and abrogate cellular contractility, eliminated the change in stiffness after the mechanical elongation. These results indicate that fibroblasts possess a mechanism that regulates intracellular tension along stress fibers to maintain the cellular stiffness in a constant equilibrium state.
Cell Motility and the Cytoskeleton 01/2005; 59(4):242-8. · 4.19 Impact Factor
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ABSTRACT: Scanning probe microscopy and immunofluorescence observations indicated that cellular stiffness was attributed to a contractile network structure consisting of stress fibers. We measured temporal variations in cellular stiffness when cellular contractility was regulated by dosing with lysophosphatidic acid or Y-27632. This experiment revealed a clear relation between cellular stiffness and contractility: Increases in contractility caused cells to stiffen. On the other hand, decreases in contractility reduced cellular stiffness. In both cases, not only the stiffness of the stress fibers but also that of the whole of the cell varied. Immunofluorescence observations of myosin II and vinculin indicated that the stiffness variations induced by the regulation of cellular contractility were mainly due to rearrangements of the contractile actin network on the dorsal surface. Taken together, our findings provide evidence that the actin cytoskeletal network and its contractility features provide and modulate the mechanical stability of adherent cells.
Experimental Cell Research 12/2004; 300(2):396-405. · 3.58 Impact Factor
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ABSTRACT: The inhomogeneous structure of agar gels was examined by means of mechanical-scanning probe microscopy. Several domains were observed in the elasticity images, while such domains could not be seen in the height images. The domain size decreased with increases in agar concentration. We found that the histograms of the logarithm of the local elastic modulus were described well by a single normal distribution. As the agar concentration increased, the peak values of the histograms increased, while the half-value width remained constant. These results imply that the gelation process of agar gels has a common mechanism, despite its complexity.
Journal of Electron Microscopy 02/2003; 52(3):277-81. · 1.31 Impact Factor
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ABSTRACT: Radiotherapy is one of the effective therapies used for treating various malignant tumors. However, the emergence of tolerant cells after irradiation remains problematic due to their high metastatic ability, sometimes indicative of poor prognosis. In this study, we showed that subcloned human lung adenocarcinoma cells (A549P-3) that are irradiation-tolerant indicate high invasive activity in vitro, and exhibit an integrin β1 activity-dependent migratory pattern. In collagen gel overlay assay, majority of the A549P-3 cells displayed round morphology and low migration activity, whereas a considerable number of A549P-3IR cells surviving irradiation displayed a spindle morphology and high migration rate. Blocking integrin β1 activity reduced the migration rate of A549P-3IR cells and altered the cell morphology allowing them to assume a round shape. These results suggest that the A549P-3 cells surviving irradiation acquire a highly invasive integrin β1-dependent phenotype, and integrin β1 might be a potentially effective therapeutic target in combination with radiotherapy.
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ABSTRACT: We improved the force modulation mode with scanning probe microscopy (SPM) in order to make a quantitative evaluation of the viscoelasticity of living cells. Taking account of the viscosity of liquid medium, the vibration frequency of the cantilever was selected to be 500 Hz, and analysis of cantilever vibration was adopted for evaluation of the viscoelasticity of the samples. Consequently, we have succeeded in determining viscoelasticity distribution on living cells. The values of Young's modulus and the coefficient of viscosity vary from 10 to 50 kPa and from 20 to 40 Pa·s on a cell, depending on its internal cellular structure.
