We have seen that the active pro­tru­sion of lamel­lipo­dia and filopo­dia (as well as the microspike bun­dles that span the lamel­lipodium with­out pro­trud­ing from the edge) results from the local induc­tion of actin poly­meri­sa­tion at the cell mem­brane. Exper­i­ments in which flu­o­res­cent actin was microin­jected into fibrob­lasts showed that actin is rapidly incor­po­rated into these periph­eral struc­tures and only much later into the inner parts of the cytoskele­ton. Con­sis­tent with this find­ing, stud­ies on the move­ments of filopo­dia and microspikes indi­cate that these struc­tures func­tion not only in pro­tru­sion, but also in seed­ing the for­ma­tion of con­trac­tile bun­dles in the main body of the cytoskele­ton, behind the pro­trud­ing zones. This can be appre­ci­ated in videos of fibrob­lasts and melanoma cells express­ing flu­o­res­cent actin and also flu­o­res­cent fascin to label filopo­dia.

Video of a fish fibrob­last express­ing mCherry-actin and GFP-fascin (to label filopo­dia). Note that some filopo­dia fold bi-laterally into the cell edge, even­tu­ally giv­ing rise to an actin bun­dle in the body of the cyto­plasm. From Nemethova et al., (2008).

After pro­tru­sion, filopo­dia can exhibit dif­fer­ent fates result­ing in the con­tri­bu­tion of fil­a­ments to the cytoskele­ton behind. They can fold lat­er­ally together with oppo­sitely polarised filopo­dia into the cell edge, they can fold upwards and back­wards into the cell body and can kink and frag­ment, leav­ing the frag­ments to flow into the cell body. All these activ­i­ties result in the deliv­ery to the cell body of fil­a­ments of mixed ori­en­ta­tion ready to inter­act with myosin fil­a­ments to form con­trac­tile arrays.

As indi­cated in the sec­tion on adhe­sion, filopo­dia can also con­tribute to the for­ma­tion of a con­trac­tile acto­myosin bun­dle by ini­ti­at­ing the for­ma­tion of a focal adhe­sion. In this case the filopodium makes con­tact with the matrix at a point along its length and relin­quishes the basal part to ini­ti­ate stress fibre assem­bly (see Sub­strate Adhe­sion).

Actin dynam­ics in a CAR fibrob­last trans­fected with mCherry-actin. Two pairs of filopo­dia, the top one of each pair marked with an arrow­head, per­sist as the cell front advances and become inte­grated into the stress fibre net­work of the lamella. The sites of kink­ing of the filopo­dia mark the point of sep­a­ra­tion of the filopo­dia from the advanc­ing front, except in one case for which a sec­ond filopo­dial exten­sion adds onto the first, giv­ing rise to a longer bun­dle in the lamella. Note also fold­ing and bend­ing of other filopo­dia into the lamella (Nemethova et al., 2008).

Schematic illus­tra­tion of the dif­fer­ent fates of filopo­dia. Inte­gra­tion of filopo­dia is cou­pled with the incor­po­ra­tion of myosin and actin cross-linkers into the bun­dle. Sin­gle fil­a­ments orig­i­nat­ing from the lamel­lipodium can also con­tribute to these bun­dles. For more details see Nemethova et al., 2008.

The bilat­eral move­ments of microspikes, com­mon in B16 melanoma cells, like­wise gives rise to bipo­lar assem­blies of actin that inte­grate with myosin at the base of the lamel­lipodium form­ing con­trac­tile arrays that flow rear­wards con­tribut­ing to an inte­grated con­trac­tile net­work.

Lat­eral flow of actin fil­a­ment bun­dles (microspikes and filopo­dia) in migrat­ing B16 melanoma cells. Actin was labeled with GFP in (a-d) and with mCherry in (e-g). In (f), the cell was also trans­fected with myosin-GFP: note the incor­po­ra­tion of myosin into the bun­dles that retreat behind the lamel­lipodium to pro­duce con­trac­tile arrays. (g) inhi­bi­tion of myosin with bleb­bis­tatin does not inhibit lat­eral flow: this is dri­ven by actin poly­mer­iza­tion. For more details see Koestler et al., 2008.

Build­ing retrac­tion assem­blies at the rear

How are con­trac­tile assem­blies of actin and myosin devel­oped at the cell rear? This is achieved by gen­er­at­ing pro­tru­sions around the entire cell periph­ery at some point in the migra­tion cycle. Cells do not move in straight lines, but in a zig-zag fash­ion, even when under­go­ing chemo­taxis. They form pro­tru­sions around the whole cell perime­ter at one time or another and it is the pole with per­sis­tant pro­tru­sion that deter­mines the move­ment direc­tion.

As we have seen above, the fold­ing of filpo­dia and microspikes into the lamella behind the lamel­lipodium con­tribute to the for­ma­tion of con­trac­tile arrays. Lamel­lipo­dia also fold upwards and back­wards to form “ruf­fles” (Aber­crom­bie et al., 1970) that move rear­wards and merge into the cell body. We sug­gest this is another way that the cell uses to deliver actin fil­a­ments into the cell body to seed the for­ma­tion of con­trac­tile arrays:

A hypo­thet­i­cal migrat­ing cell show­ing ruf­fles fold­ing rear­wards both at the front and tran­siently at the flanks and rear, con­tribut­ing fil­a­ments for the assem­bly of con­trac­tile arrays. From Small and Rot­tner (2010).

Related Pub­li­ca­tions

  • Aber­crom­bie, M., Heaysman, J .E., Pegrum, S. M. (1970). The loco­mo­tion of fibrob­lasts in cul­ture. II. “Ruf­fling”. Exp Cell Res. 60(3): 437444. NCBI PubMed
  • Koestler, S. A., Auinger, S., Vinzenz, M., Rot­tner, K., Small, J. V. (2008). Dif­fer­en­tially ori­ented pop­u­la­tions of actin fil­a­ments gen­er­ated in lamel­lipo­dia col­lab­o­rate in push­ing and paus­ing at the cell front. Nat Cell Biol. 10, 306313. PDF
  • Nemethova, M., Auinger, S., Small, J. V. (2008). Build­ing the actin cytoskele­ton: filopo­dia con­tribute to the con­struc­tion of con­trac­tile bun­dles in the lamella. J Cell Biol. 180, 12331244. PDF
  • Small, J. V., Rot­tner, K. (2010). Ele­men­tary cel­lu­lar processes dri­ven by actin assem­bly: Lamel­lipo­dia and filopo­dia. In: Car­lier M-F (ed.) Actin Based Motil­ity: Cel­lu­lar, Mol­e­c­u­lar and Phys­i­cal Aspects. Springer.