In order understand how actin filaments push in lamellipodia we need to know their three dimensional organization. This has recently been made possible (Vinzenz et al., 2012) using electron tomography (Lucic et al., 2005; McIntosh et al., 2005).
To obtain an electron tomogram, images of a sample are taken at multiple angles of tilt in the range of ± 70°, preferably around two orthogonal axes. The series of projected images are aligned with each other and the tomogram computed using tailored software. Because of the limited penetration of an electron beam (routine accelerating voltage 300kV), electron tomography is currently limited to specimens less than 0.5µm thick and we are fortunate that lamellipodia fall within this range. The next figure shows a section of a tomogram of a lamellipodium of a 3T3 cell contrasted by embedding in a heavy metal salt (sodium silicotungstate) in which the front and rear boundaries of the lamellipodia are included. Typically, a tomogram consists of 100–200 slices, each slice in this case being 0.746nm thick. This section of the tomogram shows a stack of 3 slices. A 3D model of the network is obtained by tracking filaments manually or automatically through the tomogram slices.
Tomogram section (2.2nm thick) of a lamellipodium from a NIH 3T3 cell, contrasted in negative stain. (For experimental details, see Vinzenz et al., 2012)
Actin branching in the initiation and maintenance of lamellipodia
We have used live cell imaging combined with electron tomography to reveal the 3D structure of lamellipodia in different stages of protrusion (Vinzenz et al., 2012; see also audiovisual presentation at the end of this section).
Video shows an NIH 3T3 cell that was expressing fluorescent actin (Lifeact-GFP) and L61 Rac that was also injected with L61Rac to induce wide lamellipodia. Electron tomography was performed on the regions marked with the squares, corresponding in position 1 to a protruding lamellipodium and in position 2 to a treadmilling lamellipodium at the time of fixation. The cell was fixed in a glutaraldehyde-detergent mixture to expose the cytoskeleton for electron tomography.
Electron tomogram and model of the protruding lamellipodium from the figure above. The cell was dried in a tungsten salt to produce a negative contrast in the electron microscope (actin filaments are light against a darker background). The video shows a z-scan through the tomogram and the tracked actin filament array in 3D. The thicker colored lines highlight examples of filament subsets linked by branch junctions (red points).
Electron tomogram of the treadmilling lamellipodium in the figures above. The tomogram scan and tracked filaments show how subsets of actin filaments linked by branch junctions (red points) make up the lamellipodium network.
The background and details of the publication by Vinzenz et al. (2012) are also presented in this audio-visual presentation.
The original electron tomograms are available for download and can be analyzed using the software package IMOD, available from the The Boulder Laboratory for 3-D Electron Microscopy of Cells, Colorado.
- Lucić, V., Förster, F., Baumeister, W. (2005). Structural studies by electron tomography: from cells to molecules. Annu Rev Biochem. 74: 833–65.
- McIntosh, R., Nicastro, D., Mastronarde, D. (2005). New views of cells in 3D: an introduction to electron tomography. Trends Cell Biol. 15(1): 43–51.
- Vinzenz, M., Nemethova, M., Schur, F., Mueller, J., Narita, A., Urban, E., Winkler, C., Schmeiser, C., Koestler, S.A., Rottner, K., Resch, G.P., Maeda, Y., Small, J.V. (2012): Actin branching in the initiation and maintenance of lamellipodia. J Cell Sci. 125(Pt 11): 2775–85.