Cells were anchored on 4

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Cells were anchored on 4.5 interactions between cells via their extracellular domain. velocity. In general, does not exceed 10% of = 80 pN. The extrusion force is thus simply proportional to the flow velocity anchors the cell to the bead held by the micropipette (Fig.?1). As the flow is stopped, the tube retracts and the cell is pulled back to the bead. This process was repeated for cycles of applied hydrodynamic flow at identical and constant velocity is plotted against time for each extrusion cycle in Fig.?2 = 1, the extrusion profile was frequently approximately linear. With increasing increased progressively with (Fig.?2, and affected the integrity of cortex/membrane coupling over a limited cell surface area proportional to the surface of extruded tube of length (Fig.?3 for FR rates () and SR rates () for S180 ( 0.001, ?? 0.01, and ? 0.06. The red asterisk marks the statistical difference between the FR and SR of the same cycle. The black and gray asterisks mark the statistical difference between values of the cycle with and of cycle for S180 (is smaller but proportional to (Fig.?2 and in between HR and RR regimes for cycles 1 and 2. Error bars represent the standard CAB39L errors (120 cells tested). We further assessed the role of the cytoplasmic domain of cadherins in the development of the HR regime by studying cells that produced a mutant form of cadherin with a deletion in the cytoplasmic domain (Ecad-cyto cells) anchored on specific antibody-coated beads. The tube extrusion dynamics of these cells did not differ significantly from those of nonspecifically anchored cells (Fig.?2, and interval. The development of FR may be accounted for by the incomplete reestablishment of membrane-cytoskeleton binding during the 30-s interval between extrusion cycles. Moreover, as shown in Fig.?2, and 5 (see Fig.?2, and transition length with indicates that membrane/cortex coupling is affected in two ways over successive extrusion cycles: 1), the membrane cytoskeleton binder density in the proximal region gradually decreases (see Fig.?6 remains constant during all the cycles = (see Fig.?7). It is clear that falls by a large factor after the first extrusion and less for the following extrusions. As decreases, the friction on the binders decreases and the tear out is reduced or stopped. Open in a separate window Figure 7 Plot of PCI-27483 the ratio (normalized binder density; is the density of binders for cycle = 30 s, we also performed extrusions at higher values. For time intervals of 2 min between extrusion cycles, extrusion did not become easier over successive cycles, as reported for = 30 s (Fig.?S2), indicating that the membrane reattaches to the cortex within 2 min. Role of specific adhesion The strengthening of Ecad-mediated cell-cell contact has been shown to PCI-27483 reach a plateau at 40 min (46). We allowed cell-bead contact to develop for 40 min before performing the extrusion experiments, to prevent excessive scattering of results PCI-27483 due to the development of cell-bead adhesion complexes. Extrusion cycles with a higher flow velocity (= 500 4.2 30 nm (= 4is the membrane curvature modulus of order 50 kT (30),), we obtain a surface area 0.4 = = 500?= 200 nN, the intercellular adhesion energy can be estimated at 8 mN/m using de Gennes’s formula = is the cell radius. It has been shown that the cadherin-cytoskeleton coupling is crucial (46). is divided by a factor of 50?for mutated cadherins (Ecad-cyto cell doublets) (see (50)). By contrast, the extrusion velocity for the RR regime was in the same range PCI-27483 as that for PL-mediated anchored cells (Fig.?4 and Fig.?S4). We detected an increase in with increasing and (53), where is the cortical tension. Using 3 10?5 Nm?1, a typical value for cortical tension (51,52), and = 2 on the order of 104 Pa (54,55) and thickness on the order of 0.5 Eh3/9 (56) for an incompressible layer). Open in a separate window Figure 9 Videomicrographs (remains constant. We also found that Ecad adhesion at the extrusion site altered cytoskeleton/membrane coupling, increasing resistance to membrane flow and tube extrusion. However, this effect was spatially limited to the cell/bead contact area, consistent with fine spatial regulation of.