From images of lamellipodia obtained by electron tomography we were able to determine the structure of the actin branch junction at 2.9nm resolution. This was achieved by averaging over 650 branch junctions obtained from images of the type shown in this section.
Further examples of actin branches, in the case of a spontaneously protruding lamellipodium are shown in the figures below.
Video frames of the living cell up to the time of fixation by a mixture of Triton and glutaraldehyde. Electron tomography was performed on the region of lamellipodium boxed in the last frame of the movie (Fixed).
Electron tomogram sections of the lamellipodium region indicated in the figure above. (top) 7nm tomogram sections, (bottom) 19nm tomogram sections. The total thickness of the stained, dried lamellipodium was around 40nm. Branch junctions are highlighted in open circles. Lower scale indicates the distance from the lamellipodium front in microns.
For increased resolution of actin branch junctions, tomograms were obtained of lamellipodia supported by thin carbon films spanning holes in perforated films.
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.
(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. © 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 below).
Structure obtained from averaging 654 branch junctions in negatively stained cytoskeletons corresponding to supplementary video S6 in Vinzenz et al., 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).
The structure of the branch junction derived from lamellipodia networks compares very closely to the structure described for branches formed by mixing actin and the Arp2/3 complex in vitro.
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