Junko Sunaga

Actomyosin contractility spatiotemporally regulates actin network dynamics in migrating cells

Coupling interactions among mechanical and biochemical factors are important for the realization of various cellular processes that determine cell migration. Although F-actin network dynamics has been the focus of many studies, it is not yet clear how mechanical forces generated by actomyosin contractility spatiotemporally regulate this fundamental aspect of cell migration. In this study, using a combination of fluorescent speckle microscopy and particle imaging velocimetry techniques, we perturbed the actomyosin system and examined quantitatively the consequence of actomyosin contractility on F-actin network flow and deformation in the lamellipodia of actively migrating fish keratocytes. F-actin flow fields were characterized by retrograde flow at the front and anterograde flow at the back of the lamellipodia, and the two flows merged to form a convergence zone of reduced flow intensity. Interestingly, activating or inhibiting actomyosin contractility altered network flow intensity and convergence, suggesting that network dynamics is directly regulated by actomyosin contractility. Moreover, quantitative analysis of F-actin network deformation revealed that the deformation was significantly negative and predominant in the direction of cell migration. Furthermore, perturbation experiments revealed that the deformation was a function of actomyosin contractility. Based on these results, we suggest that the actin cytoskeletal structure is a mechanically self-regulating system, and we propose an elaborate pathway for the spatiotemporal self-regulation of the actin cytoskeletal structure during cell migration. In the proposed pathway, mechanical forces generated by actomyosin interactions are considered central to the realization of the various mechanochemical processes that determine cell motility.

Observation of chondrocyte aggregate formation and internal structure on micropatterned fibroin-coated surface

Condensation/aggregation process of rabbit-derived chondrocytes on a fibroin-coated patterned substrate was observed to estimate initial aggregation process in fibroin sponge. Chondrocytes were seeded on array of 160 microm diameter pits in three densities: 5 cells/pit (2.5 x 10(4) cells/cm(2), LOW), 15 cells/pit (7.5 x 10(4) cells/cm(2), MID) and 25 cells/pit (12.5 x 10(4) cells/cm(2), HIGH). In the MID and HIGH groups, cells tended to form aggregates after 24 h after cell seeding. In the LOW group, cell aggregate were not seen in a majority of the pits. Observation of aggregates using confocal laser scanning microscope showed that the chondrocytes at the interface of the fibroin surface tended to extend to the surface, developing an extensive network of stress fibers throughout the cytoplasm. On the other hand, chondrocytes in the other part of the aggregates maintained spherical shape, and most of the actin was localized in the cell cortex as opposed to in stress fibers. These results suggest two functional structures in the aggregates, which may explain the good balance between the maintenance of their differentiated phenotype and proliferation rate in the fibroin sponge.

Analysis of Ca2+ response of osteocyte network by three-dimensional time-lapse imaging in living bone

Osteocytes form a three-dimensional (3D) cellular network within the mineralized bone matrix. The cellular network has important roles in mechanosensation and mechanotransduction related to bone homeostasis. We visualized the embedded osteocyte network in chick calvariae and observed the flow-induced Ca2+ signaling in osteocytes using 3D time-lapse imaging. In response to the flow, intracellular Ca2+ ([Ca2+]i) significantly increased in developmentally mature osteocytes in comparison with young osteocytes in the bone matrix. To investigate the differences in response between young and developmentally mature osteocytes in detail, we evaluated the expression of osteocyte-related genes using the osteocyte-like cell line MLO-Y4, which was 3D-cultured within type I collagen gels. We found that the c-Fos, Cx43, Panx3, Col1a1, and OCN mRNA levels significantly increased on day 15 in comparison with day 7. These findings indicate that developmentally mature osteocytes are more responsive to mechanical stress than young osteocytes and have important functions in bone formation and remodeling.