LIMK activity is required for path generation in collective invasion. (A) To confirm that MDA-MB-231 strands invading 3D matrix were comprised of cell collectives, fixation and staining of F-actin (green) and DNA (red) revealed multiple cells within strands. Bar, 20 µM. (B) Separate pools of MDA-MB-231 cells were labeled either with RFP or GFP, then RFP-expressing cells were transfected either with NT or LIMK1/2 siRNA targeting duplexes as indicated. Untreated GFP-expressing cells were combined with siRNA-transfected RFP-expressing cells and the mixture assayed for 3D matrix invasion. Strands were equally led by GFP-expressing or NT-transfected RFP-expressing cells. Bar, 20 µM. (C) LIMK1/2 knockdown blocked the occurrence of leading RFP-expressing cells, although RFP-labeled cells were still observed after GFP-expressing cells. Bar, 20 µM. (D) 3D reconstructions of confocal image stacks were assembled with Volocity software, and used to assess the incidence of GFP or RFP cells leading collective invasion strands. Although there was no difference when cells were transfected with NT siRNA, LIMK1/2 knockdown significantly reduced the ability of RFP-expressing cells to lead collective invasion (average ± SEM, n = 4). (E) When the identity of the cell immediately after the leading cell was determined, there were no differences in the NT or LIMK1/2 siRNA-transfected RFP-expressing cells to follow in the second position (average ± SEM, n = 5). (F) Although there was no difference when cells were transfected with NT siRNA, MMP9 knockdown significantly reduced the ability of RFP-expressing cells to lead collective invasion (average ± SEM, n = 3).