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  • Atomic force microscope accessory enables discovery

Jun 2008
David L. Shenkenberg

All animals with a backbone undergo an early developmental stage known as gastrulation, in which three distinct layers of cells form a sphere, and the layers are dubbed endoderm, mesoderm and ectoderm — according to whether the cells are in the inside, middle or outside of the sphere. But how do these layers form essentially the same way every time? Although forces such as adhesion and contraction have been implicated in the process, clear understanding has been prevented by a lack of reliable quantitative methods for measuring these forces.

Scientists at Dresden University of Technology in Germany, advised by Daniel J. Müller, now report reliable quantitative measurements of adhesion and surface tension of zebra fish cells that form the three layers during gastrulation. They worked in cooperation with Carl-Philipp Heisenberg’s group at Max Planck Institute for Molecular Cell Biology and Genetics, also in Dresden.

Even when scientists mix up the cells that form a ball called a gastrula during early vertebrate development, the cells sort into the three-layered ball again, shown here for two of the cell types, ectoderm (red) and mesoderm (green). Reprinted from Nature Cell Biology with permission of the researchers.

For both cell adhesion and tension measurements, the researchers used a JPK Instruments’ atomic force micro-scope (AFM) mounted on a Carl Zeiss inverted fluorescence microscope. They also used a special chamber that kept the fish cells alive and well for the experiments.

For adhesion measurements, they attached one zebra fish cell to the cantilever of the AFM and the other to the sample holder, both of which were on the microscope stage. Then they measured the adhesion between two cells by determining the force required to pull them apart. “The trick is turning the cell into a probe,” Müller said.

For this purpose, they also used JPK Instruments’ aptly named CellHesion module because it enables cell-stretching distances up to 100 μm. To do so, the module uses piezo elements to move the fluorescence microscope objective simultaneously with the sample holder to keep the microscope in focus. “Our resolution is one angstrom,” Müller said. His group and JPK Instruments previously developed both the module and the setup with Pierre-Henri Puech at Inserm in Marseille, France, as detailed in the June-July 2006 issue of Ultramicroscopy.

In the current zebra fish study, the researchers measured adhesion and found that endoderm and mesoderm cells bind more strongly to other cells than ectoderm cells do. They also found that adhesion among the cells is mediated by cadherins — proteins that, in other cases, are known to participate in adhesion.

In another part of the zebra fish study, they measured tension by indenting the cells with a tiny glass bead attached to an AFM cantilever. The use of the bead prevented damage to the cells. In these experiments, they assumed that the liquid-droplet model accurately describes the physics of the cell as a viscous fluid-filled bag with constant surface tension from interactions between actin and myosin in the cytoskeleton. They found that ectoderm has the highest tension, followed by mesoderm and endoderm.

To test whether differential tension is involved in sorting the cells into the three layers, the researchers mixed up the layers of cells. Normally, the cells sort into the three layers when they are mixed up this way. However, when the researchers inhibited actin and myosin activity, they found that tension decreased and that the cells could not sort into the three layers, suggesting that tension from actin and myosin determines the ability of these cells to sort into the three layers.

Because TGF-beta proteins are believed to influence the development and movements of these cells, the researchers added recombinant TGF-beta proteins to them. They found that tension decreased, leading to the conclusion that TGF-beta proteins regulate actin and myosin interactions that are crucial for the zebra fish cells to sort into the three layers. More experiments are needed to determine whether this phenomenon occurs in other animals with a backbone.

Nature Cell Biology, April 2008, pp. 429-436.

The intermolecular attraction between two surfaces, as between a substrate and a coating; it is an important factor in the durability of optical thin films.
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