A Micromechanic Study of Cell Polarity and Plasma Membrane Cell Body Coupling in Dictyostelium

Abstract

We used micropipettes to aspirate leading and trailing edges of wild-type and mutant cells of Dictyostelium discoideum. Mutants were lacking either myosin II or talin, or both proteins simultaneously. Talin is a plasma membrane-associated protein important for the coupling between membrane and actin cortex, whereas myosin II is a cytoplasmic motor protein essential for the locomotion of Dictyostelium cells. Aspiration into the pipette occurred above a threshold pressure only. For all cells containing talin this threshold was significantly lower at the leading edge of an advancing cell as compared to its rear end, whereas we found no such difference in cells lacking talin. Wild-type and talin-deficient cells were able to retract from the pipette against an applied suction pressure. In these cells, retraction was preceded by an accumulation of myosin II in the tip of the aspirated cell lobe. Mutants lacking myosin II could not retract, even if the suction pressures were removed after aspiration. We interpreted the initial instability and the subsequent plastic deformation of the cell surface during aspiration in terms of a fracture between the cell plasma membrane and the cell body, which may involve destruction of part of the cortex. Models are presented that characterize the coupling strength between membrane and cell body by a surface energy sigma. We find sigma approximately 0.6(1.6) mJ/m(2) at the leading (trailing) edge of wild-type cells.

Model for Probing Membrane-Cortex Adhesion by Micropipette Aspiration and Fluctuation Spectroscopy

“…The asymmetric distribution of this small density of talin links seems to be enough to drive directed motion in amoebae (28). Similar observations are reported for zebrafish cells (31).…”

“…This relationship provides not only a rational explanation for the membrane unbinding for a variety of cell phenotypes where either the density of ligands or myosin activity is altered, but also a method to directly probe cortex activity by measuring the critical pressure needed to unbind the membrane. We refer to previous experimental results concerning the abrupt unbinding induced by micropipette suction to assess the validity of our model (12,28). To test the relationship among critical pressure, ligand density, and cortical tension, we would ideally need to measure the critical pressure for cells whose phenotype has been quantitatively altered.…”

“…Whenever the equilibrium stress exceeds the critical value s*, we expect the cell membrane to detach spontaneously. Micropipette aspiration (12,16,(27)(28)(29), among other techniques (11,30,31), allows us to apply pressure perturbations of controlled intensity and area. Pressure perturbations can be supplemented with perturbations on relevant cell parameters such as myosin activity and link or cortex density, by genetics (27)(28)(29) or direct drug treatment (10,11,31).…”

Active mechanical stabilization of the viscoplastic intracellular space of Dictyostelia cells by microtubule–actin crosstalk

“…The shift of the maximum of P(v) to lower velocities is explained in terms of an increase of the viscosity of the cytoplasm by knock out of myosin II. This increase of the viscosity of myosin II null mutants was observed in earlier studies [29,34].…”

Dictyostelium myosin II mechanochemistry promotes active behavior of the cortex on long time scales

“…From magnetic twisting rheometry, myosin II appears to increase cytoplasmic fluidity of a related amoeba, Entamoeba histolytica (36). Dictyostelium myosin II mutants appear to have greater mechanical resistance as measured by micropipette aspiration and magnetic rheometry (37,38), whereas other studies indicate that myosin II mutants have lower cortical tension (39,40). Thus, myosin II contributes to the passive mechanics of the cell in many ways; however, our study separates myosin II's roles in active and passive behaviors of the cell cortex.…”