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.

Related Pub­li­ca­tions

  • Rouiller, I., Xu, X. P., Amann, K. J., Egile, C., Nick­ell, S., Nicas­tro, D., Li, R., Pol­lard, T. D., Volk­mann, N., Hanein, D. (2008). The struc­tural basis of actin fil­a­ment branch­ing by the Arp2/3 com­plex. J Cell Biol. 180: 887895 NCBI PubMed
  • Vinzenz, M., Nemethova, M., Schur, F., Mueller, J., Narita, A., et al. (2012) Actin branch­ing in the ini­ti­a­tion and main­te­nance of lamel­lipo­dia. J Cell Sci. 125: 27752785. NCBI PubMed
  • Xu, X. P., Rouiller, I., Slaugh­ter, B. D., Egile, C., Kim, E., Unruh, J. R., Fan, X., Pol­lard, T. D., Li, R., Hanein, D., Volk­mann, N. (2012). Three-dimensional recon­struc­tions of Arp2/3 com­plex with bound nucle­ation pro­mot­ing fac­tors. EMBO J. 31:23647. NCBI PubMed