The Cytoskele­ton and Cell Migra­tion

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)

Movie

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.

Related Media

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.

Related Pub­li­ca­tions

• 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.