9. Lamellipodium in 3D
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, see following section) using the emerging technique of electron tomography (Lucic et al., Ann. Rev. Biochem 74, 833-865, 2005; McIntosch et al., Trends Cell Biol. 15, 43-51: 2005: see also the end of this section).
To obtain an electron tomogram, images of a sample are taken at multiple angles of tilt in the range of +/- 70deg, 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. Figure 9-1 and the accompanying zoom video show 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. The section of the tomogram in Fig. 9-1 shows a stack of 3 slices. Fig. 9-2 shows the front region of the same lamellipodium, with 40 slices combined, simulating the view that would be obtained in a 2D projection of the whole network. A 3D model of the network is obtained by tracking filaments manually or automatically through the tomogram slices.
Figure 9-1. 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).
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Figure 9-2. Front region of lamellipodium from Fig.9-1 with 40 slices of the tomogram combined to give an overview of the network.
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., J. Cell Sci. 125, 2775-2785, 2012- Download Pdffileadmin/conferences/Videotour_CellMotility/NEW_2013/JCS_Vinzenz_et.al._supp.mat.pdf; see also ppt presentation at the end of this section)
Figure 9-3. 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 (Fig. 9-4).
(movie 2.8Mb) Click here to watch movie
Figure 9-4. Electron tomogram and model of the protruding lamellipodium in Fig 9-3 (position 1). 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).
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Figure 9-5. Electron tomogram of the treadmilling lamellipodium in Fig 9-3 (position 2). Otherwise as for Fig. 9-4. The tomogram scan and tracked filaments show how subsets of actin filaments linked by branch junctions (red points) make up the lamellipodium network.
(movie 7.5Mb) Click here to watch movie
Structure of actin branch junctions in vivo
Actin branch junctions
For increased resolution of actin branch junctions (see audio-visual presentation), tomograms were obtained of lamellipodia supported by thin carbon films spanning holes in thicker Quantifoil films.
Figure 9-6. Video zoom of an electron tomogram section of part of a lamellipodium cytoskeleton of a NIH3T3 cell grown on a perforated Quantifoil grid coated additionally with a thin carbon film. The cytoskeleton was stained with sodium silicotungstate. Such samples were used to obtain the 2.9nm resolution model of the in vivo branch (Vinzenz et al., 2012). Branch junctions are encircled in blue.
(Movie 28Mb) Click on picture to watch movie
Figure 9-7. A, gallery of branch junctions selected from tomograms of negatively stained 3T3 cell cytoskeletons. B, examples of multiple branch junctions in close proximity; otherwise as in A. C. branch junctions in tomograms of 3T3 cell cytoskeletons embedded in vitreous ice. D, Structure obtained from image averaging of branch junctions in negatively stained cytoskeletons, with the molecular model of actin and the Arp2/3 complex superimposed.
(see also video Figure 9-8).
Figure 9-8. Structure obtained from image averaging 654 branch junctions in negatively stained cytoskeletons as in Fig. 9-7 and corresponding to supplementary video S6 in Vinzenz et al., (J. Cell Science, 2012)
The average electron density in grey shows the branch junction at 2.9nm resolution. Actin and known components of the Arp2/3 complex have been fitted into the density, as follows: mother actin filament (white), daughter actin filament (light pink), Arp2 (golden), Arp3 (violet), ArpC1 (turquoise), ArpC2 (yellow), ArpC3 (red), ArpC4 (light green) and ArpC5 (purple).
(Movie 21Mb) Click on picture to watch movie
Initiation of a lamellipodium
When in culture, B16 melanoma cells sometimes form holes in their cytoplasm that are subsequently sealed via the induction of lamellipodia (Fig. 9.9).
Figure 9-9. Video of spontaneous holes in GFP-actin B16 cell. Click here to watch movie
Video shows a cultured melanoma cell that was expressing GFP-actin (courtesy of Klemens Rottner).
Holes can also be induced in the cytoplasm of cultured cells with a microneedle and these are also repaired by way of lamellipodium formation. This model system was exploited to determine the initial steps in lamellipodia induction.
Figure 9-10. Video in phase contrast on the left shows the injury of the cytoplasm of a fibroblast cell by a microneedle and the subsequent repair of the hole. Video on the right shows a similar experiment with a B16 melanoma cell pressing GFP-actin that was injured in two positions during the video sequence. Note the repair of both lesions via the induction of lamellipodia.
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Figure 9-11. By fixing cells briefly after wounding and then processing them for electron tomography we were able to monitor the structural changes in the actin cytoskeleton during lamellipodia formation. This figure shows a slice (15nm thick) of an electron tomogram of a hole in a B16 melanoma cell captured around 20sec after wounding of the cytoplasm with a microneedle. A typical lamellipodium network is observed to invade the wounded area. Filaments running parallel to the base of the lamellipodium (indicated by black arrow) mark the original perimeter of the hole immediately after wounding.
Figure 9-12. Cytoplasmic wounding by a micropipette and the first steps in repair are illustrated in this diagram. Immediately after wounding, actin filaments pre-existing in the cytoplasm accumulate below the membrane initially receding at the wound perimeter. These filaments then serve as platforms for lamellipodia induction (Figs 9-11 and 9-13).
Figure 9-13. The first steps in lamellipodia formation were captured by fixing cells a few seconds after wounding the cytoplasm with a micropipette. The figure shows sections of electron tomograms of cytoplasmic wound repair in a mouse 3T3 fibroblast (A,B) and a fish keratocyte (C,D). Actin filaments initiating lamellipodia formation are seen to originate via branching from the filaments accumulated in small bundles parallel to the wound edge (black arrows). For more details see Vinzenz et al., (2012).
Figure 9-14. Mathematical simulation of one scenario of lamellipodium induction (courtesy of Christoph Winkler). Actin filaments in a bundle parallel to the cell edge serve as platforms for actin filament branching to generate a lamellipodium network. Branch sites are depicted in yellow. Protein complexes involved in tethering and elongation of actin filaments at the leading cell membrane are represented as green cylinders. (For more details see Vinzenz et al., 2012).
(Movie 3.9Mb) Click on picture to watch movie
Audio-visual presentation in one file: Download (133MB)
The electron tomograms presented can be downloaded from this site and analyzed using IMOD software: bio3d.colorado.edu/imod/
