Catherine Barentin, Jean-Paul Rieu
Dictyostelium Discoideum (D.D.) is a widely used system for studying a variety of basic processes in development including cell-cell signaling, signal transduction, pattern formation and cell motility. Cells of D.D typically exist as independent soil-inhabiting amoebae that upon exhaustion of food supply aggregate to form a multicellular structure. A mound of cells is first formed, which then elongates vertically like a finger and finally crawls in a manner resembling the movement of a garden slug (thus called a "migrating slug"). Both aggregation and migration are mediated by extracellular cyclic AMP signal but the migration mechanisms (i.e., how each individual cell participate to the overall slug motion) are largely unknown. In collaboration with Y. Sawada (Tohoku Institute of Technology, Sendai, Japan), we found that slug velocity is proportional to slug length and that waves of movements are present within 2-dimensional Dictyostelium slugs (Rieu et al., 2003).
We recently succeeded in measuring the force exerted by the different parts of the slug using the flexible substrata method. The method lies on the observation with a two-channel confocal microscope of the deformations of a very soft elastomer substrate (Young's modulus ~ 5 kPa) with fluorescent beads embedded inside. It was applied to date to the measurement of mechanical forces exerted by fibroblasts or muscular cells but never of D.D. cells or multicellular systems.
Figure: (A) Image of a slug taken with the confocal microscope: transmission channel (grey) fluorescence channel (green beads) are superimposed. (B) Bead displacement field at a given time. (C) Bead displacement field averaged in the slug frame. (D) Stress Field calculated from (C). (E) Parallel component of the stress field averaged along slug width as a function of distance along slug axis.
Beads displacements are averaged in the slug frame. Calculation of forces per unit area (stresses) from such a bead displacement field is a very difficult computational task. We recently succeeded in this calculation by averaging the displacements in the slug frame (Fig. 2D). Contrary to the displacement field (Fig. 2C), forces are almost absent outside the slug area. This confirms the validity of the calculations. We are able to identify clearly separate friction areas in the tip and in the trail, and traction in the central prespore area (Fig. 2E). We also measure large perpendicular forces around slug boundary suggesting an important role of the sheath in the transmission of forces to the substrate.
Our current work is to measure the quantitative dependence of traction and friction forces with respect to slug velocity in order to answer to the old and contentious issue about the mechanisms of slug migration. We are also planning to extend the flexible substrata technique to single dictyostelium cells.