Dynacortin contributes to cortical viscoelasticity and helps define the shape changes of cytokinesis. (9/2848)

During cytokinesis, global and equatorial pathways deform the cell cortex in a stereotypical manner, which leads to daughter cell separation. Equatorial forces are largely generated by myosin-II and the actin crosslinker, cortexillin-I. In contrast, global mechanics are determined by the cortical cytoskeleton, including the actin crosslinker, dynacortin. We used direct morphometric characterization and laser-tracking microrheology to quantify cortical mechanical properties of wild-type and cortexillin-I and dynacortin mutant Dictyostelium cells. Both cortexillin-I and dynacortin influence cytokinesis and interphase cortical viscoelasticity as predicted from genetics and biochemical data using purified dynacortin proteins. Our studies suggest that the regulation of cytokinesis ultimately requires modulation of proteins that control the cortical mechanical properties that establish the force-balance that specifies the shapes of cytokinesis. The combination of genetic, biochemical, and biophysical observations suggests that the cell's cortical mechanical properties control how the cortex is remodeled during cytokinesis.  (+info)

Forskolin promotes astroglial differentiation of human central neurocytoma cells. (10/2848)

Human central neurocytoma is a kind of the brain tumors that are usually found in anterior part of the lateral ventricles. In this study, we established conditions that allowed proliferation of neurocytoma cells culture and analyzed characteristics of neurocytoma cells in vitro. For in vitro, a condition that used for culturing neural stem cells and contained basic fibroblast growth factor (bFGF) provided high proliferation. RT-PCR analaysis showed that nestin was found in neurocytoma cells, indicating that the neurocytomas possess neural stem cell properties. Interestingly, treatment of neurocytoma cells with forskolin increased expression of glial fibrillary acidic protein with a concomitant decrease in the nestin expression. Forskolin also induced morphological changes of neurocytoma cells to adopt an astrocyte-like phenotype. The results suggest that neurocyotma cells may have properties of multipotent neural stem cells.  (+info)

Length control is determined by the pattern of cytoskeleton. (11/2848)

In our previous experiments with linear strips of adhesive substrate, we found that elongated cultured fibroblasts preserve their length regardless of cell width and the number of cytoplasmic processes. This constancy of length was called 'length control'. In contrast to fibroblasts, single cultured epitheliocytes have nearly discoid shape on the plane substrata and have no length-controlling mechanism: their length on the narrow strips of adhesive substrate increased significantly in comparison with the diameter on the plane substrate. These results suggested that control of length is cell specific. An alternative suggestion is that length control is associated not with the cell type but with the cell cytoskeletal pattern (namely, with epithelioid circular actin bundles or straight actin bundles). Experiments described in this paper were made to choose between these two suggestions. Mouse embryo fibroblasts spreading on the planar substrate first acquire discoid epithelioid shape with a circular actin bundle. Only later did they acquire a polarized shape with straight actin bundles. Polarized, fully spread fibroblasts temporarily acquire discoid epithelioid shape when treated with the Taxol, disorganizing microtubules. However, epithelial discoid cells can be transformed into elongated fibroblast-like cells by scatter factor (HGF/SF; a cytokine) and by agents inhibiting Rho kinase. These reversible transitions from fibroblastic to epithelioid shape and vice versa were accompanied by a corresponding disappearance and appearance of length control. Fibroblasts with stress fibers destroyed by the Rho-kinase inhibitor Y27632 became considerable longer on the adhesive strips than on the plane while retaining a near-polarized shape. Thus, length control is typical not of the cell origin but of the cell phenotype (i.e. for polarized cells with microtubules and intact actin cytoskeleton).  (+info)

Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. (12/2848)

BACKGROUND: For the investigation of the molecular mechanisms involved in neurite outgrowth and differentiation, accurate and reproducible segmentation and quantification of neuronal processes are a prerequisite. To facilitate this task, we developed a semiautomatic neurite tracing technique. This article describes the design and validation of the technique. METHODS: The technique was compared to fully manual delineation. Four observers repeatedly traced selected neurites in 20 fluorescence microscopy images of cells in culture, using both methods. Accuracy and reproducibility were determined by comparing the tracings to high-resolution reference tracings, using two error measures. Labor intensiveness was measured in numbers of mouse clicks required. The significance of the results was determined by a Student t-test and by analysis of variance. RESULTS: Both methods slightly underestimated the true neurite length, but the differences were not unanimously significant. The average deviation from the true neurite centerline was a factor 2.6 smaller with the developed technique compared to fully manual tracing. Intraobserver variability in the respective measures was reduced by a factor 6.0 and 23.2. Interobserver variability was reduced by a factor 2.4 and 8.8, respectively, and labor intensiveness by a factor 3.3. CONCLUSIONS: Providing similar accuracy in measuring neurite length, significantly improved accuracy in neurite centerline extraction, and significantly improved reproducibility and reduced labor intensiveness, the developed technique may replace fully manual tracing methods.  (+info)

Vascular endothelial-cadherin regulates cytoskeletal tension, cell spreading, and focal adhesions by stimulating RhoA. (13/2848)

Changes in vascular endothelial (VE)-cadherin-mediated cell-cell adhesion and integrin-mediated cell-matrix adhesion coordinate to affect the physical and mechanical rearrangements of the endothelium, although the mechanisms for such cross talk remain undefined. Herein, we describe the regulation of focal adhesion formation and cytoskeletal tension by intercellular VE-cadherin engagement, and the molecular mechanism by which this occurs. Increasing the density of endothelial cells to increase cell-cell contact decreased focal adhesions by decreasing cell spreading. This contact inhibition of cell spreading was blocked by disrupting VE-cadherin engagement with an adenovirus encoding dominant negative VE-cadherin. When changes in cell spreading were prevented by culturing cells on a micropatterned substrate, VE-cadherin-mediated cell-cell contact paradoxically increased focal adhesion formation. We show that VE-cadherin engagement mediates each of these effects by inducing both a transient and sustained activation of RhoA. Both the increase and decrease in cell-matrix adhesion were blocked by disrupting intracellular tension and signaling through the Rho-ROCK pathway. In all, these findings demonstrate that VE-cadherin signals through RhoA and the actin cytoskeleton to cross talk with cell-matrix adhesion and thereby define a novel pathway by which cell-cell contact alters the global mechanical and functional state of cells.  (+info)

A three-dimensional finite element model of an adherent eukaryotic cell. (14/2848)

Mechanical stimulation is known to cause alterations in the behaviour of cells adhering to a substrate. The mechanisms by which forces are transduced into biological responses within the cell remain largely unknown. Since cellular deformation is likely involved, further understanding of the biomechanical origins of alterations in cellular response can be aided by the use of computational models in describing cellular structural behaviour and in determining cellular deformation due to imposed loads of various magnitudes. In this paper, a finite element modelling approach that can describe the biomechanical behaviour of adherent eukaryotic cells is presented. It fuses two previous modelling approaches by incorporating, in an idealised geometry, all cellular components considered structurally significant, i.e. prestressed cytoskeleton, cytoplasm, nucleus and membrane components. The aim is to determine if we can use this model to describe the non-linear structural behaviour of an adherent cell and to determine the contribution of the various cellular components to cellular stability. Results obtained by applying forces (in the picoNewton range) to the model membrane nodes suggest a key role for the cytoskeleton in determining cellular stiffness. The model captures non-linear structural behaviours such as strain hardening and prestress effects (in the region of receptor sites), and variable compliance along the cell surface. The role of the cytoskeleton in stiffening a cell during the process of cell spreading is investigated by applying forces to five increasingly spread cell geometries. Parameter studies reveal that material properties of the cytoplasm (elasticity and compressibility) also have a large influence on cellular stiffness. The computational model of a single cell developed here is proposed as one that is sufficiently complex to capture the non-linear behaviours of the cell response to forces whilst not being so complex that the parameters cannot be specified. The model could be very useful in computing cellular structural behaviour in response to various in vitro mechanical stimuli (e.g. fluid flow, substrate strain), or for use in algorithms that attempt to simulate mechanobiological processes.  (+info)

Effects of cyclic longitudinal mechanical strain and dexamethasone on osteogenic differentiation of human bone marrow stromal cells. (15/2848)

The aim of the study was to investigate the effect of cyclic mechanical strain on differentiation markers in the presence or absence of dexamethasone. Human bone marrow stromal cells (BMSC) from seven donors (32.5+/-6.2 years) were cultivated with (D+) or without (D-) dexamethasone. A cyclic mechanical strain with an elongation of 2% (D+2; D-2) or 8% (D+8; D-8) was applied for three days with a stimulation time of three times two hours each day. Levels of alkaline phosphatase (ALP) and osteocalcin (OC) were compared after time intervals of four and seven days. mRNA expression of Collagen I, III and Cbfa1 was investigated after one, four, and seven days. ALP levels were significantly increased in the D+8 group after four and seven days (147.1+/-6.3%; p<0.05 and 168.6+/-6,5%; p<0.03) and in the D-8 group after 7 days (197.4+/-10.4; p<0.04). Cyclic strain had a significant influence on ALP-secretion (F=7.5; p<0.01). In the D-8 group there was a significant increase in OC secretion after 4 days (140.9+/-12.5%; p<0.05).; p<0.01). The effect of stretching was significantly stronger than that of dexamethasone (F=17.2 vs. 1.8). Collagen I (Col I) expression was upregulated in D+8 cultures after 4 days (215.0+/-53.3 p<0.04) and after seven days (166.7+/-55.7; p<0.04). Collagen III (Col III) expression was upregulated in D+2 and D+8 cultures after 4 days (200.7+/-16.3 and 185.9+/-12.7; p<0.04) and after seven days (154.4+/-10.1 and 118.8+/-16.4; p<0.04). There was a significant increase of Cbfa1 expression in D+8 cultures at all investigated time intervals (day 1: 105.5+/-3.7%; day 4: 104.7+/-3.0%; day 7: 104.4+/-2.1%; p<0.03). Stretching (F=20.0; p<0.01) was a stronger contributor to Cbfa-1 expression than dexamethasone (F=12.1; p<0.01). Cyclical mechanical stimulation with 8% elongation increases ALP and OC levels and upregulates Col I and III synthesis and Cbfa1 expression. In the short term, cyclical stretching is a stronger differentiation factor than dexamethasone. Cyclical stretching and dexamethasone both enhance the osteogenic commitment of hBMSC.  (+info)

Actin filaments play a permissive role in the inhibition of store-operated Ca2+ entry by extracellular ATP in rat brown adipocytes. (16/2848)

Stimulation of P2 receptors with micromolar concentration of ATP evokes a transient increase in [Ca2+]i (intracellular free Ca2+ concentration), primarily due to release of Ca2+ from intracellular stores; such stimulation also triggers almost complete suppression of thapsigargin-evoked sustained [Ca2+]i increase mediated through a store-operated Ca2+ entry pathway in rat brown adipocytes. We investigated the role of cytoskeletal actin in the inhibitory effect of the extracellular ATP on store-operated Ca2+ entry, using fura 2 fluorescence for continuous measurement of [Ca2+]i, and using Alexa fluor 488-phalloidin staining of actin. Disassembly of actin networks by cytochalasin D (1 microM) or latrunculin A (3 microM) prevented the inhibitory effect of ATP (10 microM) on the thapsigargin (100 nM)-evoked store-operated Ca2+ entry, without changing the effect of ATP in increasing [Ca2+]i. In normal cells, bath application of ATP induced a transient [Ca2+]i increase, consisting of a rapid increase (the rising phase) and the subsequent decrease (the declining phase) to a lower steady level despite the continued presence of the agonist. Disruption of actin assemblies did not significantly affect the rising phase, but prevented the declining phase. Cells incubated with 10 microM ATP for 4 min demonstrated marked accumulations of actin filaments at the cell periphery, showing protrusions at the cell surface; this actin-assembly process is mediated through P2 receptors. In cells treated with cytochalasin D or latrunculin A, extracellular ATP did not induce actin redistribution. These results suggest that the actin reorganization plays a role in ATP-induced inhibition of store-operated Ca2+ entry in rat brown adipocytes.  (+info)