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).