Video 5.
Three-dimensional reconstructions of cells moving under agarose overlays of different strengths, and movement of cells under agarose containing fluorescent beads. Aggregation-competent Ax2 cells, expressing ABD-GFP as a reporter for F-actin, were attracted to move under agarose overlays of different strength by chemotaxis to cyclic-AMP. As the strength of the overlay increases, cells become more flattened, produce fewer dorsal projections, and use blebs instead of pseudopods to move (blebs and the scar left behind are very clear under 1 and 2% agarose). Cells are reconstructed from 0.38-µm sections taken by laser-scanning confocal microscopy (model LSM 780; Carl Zeiss) and reconstructed using the ImageJ 3D Viewer plug-in (National Institutes of Health). Bar, 10 µm. In the second part of the movie, similarly prepared cells are shown moving under 0.7% agarose containing red fluorescent, 0.45-µm diameter beads. The focal plane is set on the beads slightly above the cells, and when a cell moves under a bead, it dims and then brightens once the cell has passed, indicating that it is displaced out of and then back into the focal plane. This is consistent with the cells elastically deforming the agarose as they move. Taken by laser-scanning confocal microscopy (model LSM 780; Carl Zeiss) at 1 frame per second.