When in cul­ture, B16 melanoma cells some­times form holes in their cyto­plasm that are sub­se­quently sealed via the induc­tion of lamel­lipo­dia:


Video of spon­ta­neous holes in GFP-actin B16 cell. 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.


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. (Right) Video 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.

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.

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 (fig­ures before and after).

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,B) a mouse 3T3 fibrob­last and (C,D) a fish ker­a­to­cyte. 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).

Related Pub­li­ca­tions

  • 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