Scar/WAVE3 contributes to motility and plasticity of lamellipodial dynamics but not invasion in three dimensions

Abstract

The Scar (suppressor of cAMP receptor)/WAVE [WASP (Wiskott-Aldrich syndrome protein) verprolin homologous] complex plays a major role in the motility of cells by activating the Arp2/3 complex, which initiates actin branching and drives protrusions. Mammals have three Scar/WAVE isoforms, which show some tissue-specific expression, but their functions have not been differentiated. In the present study we show that depletion of Scar/WAVE3 in the mammalian breast cancer cells MDA-MB-231 results in larger and less dynamic lamellipodia. Scar/WAVE3-depleted cells move more slowly but more persistently on a two-dimensional matrix and they typically only show one lamellipod. However, Scar/WAVE3 appears to have no role in driving invasiveness in a three-dimensional Matrigel™ invasion assay or a three-dimensional collagen invasion assay, suggesting that lamellipodial persistence as seen in two-dimensions is not crucial in three-dimensional environments.

Figure 1. Reduction of endogenous Scar/WAVE3 by siRNA and shRNAmir in MDA-MB-231 cells

A) Two independent Scar/WAVE3 specific siRNA oligos (t Scar3 3-1 and t Scar3 3-3, where t= transient) were transfected into MDA-MB-231 cells and cell lysates were used to perform immunoblotting with Scar/WAVE3 and tubulin antisera. B) Cell lysates derived from MDA-MB-231 stable (st) Scar3 kd cells (1a, 1b, 1c, 2a and 2b) and cells expressing shRNAmir control construct were used for immunoblotting with Scar/Wave3 and tubulin antisera. C) Western blot analysis of Scar/Wave complex members of cell lysates from clone stScar3-2a and nt cells. The mean % intensities of stScar3-a compared to nt cells were as follows, for Scar3 25% +/− 8, Scar1 107% +/− 13, Scar2 100% +/− 5, Sra 88% +/− 10, Nap 105% +/− 4, Abi1 108% +/− 9, Abi2 95% +/− 13 and tubulin as control 110% +/− 5. The results represent 3 western blot experiments.

Figure 2. 2D time-lapse video microscopy of Scar/WAVE3 knockdown cells

Time-lapse video microscopy was performed on MDA-MB-231 cells plated on fibronectin and images were taken every 15 min for 19 hrs. Shown in A are a selection of stills at 0, 15, 30 and 45 min of MDA-MB-231 nt cells, Scar/Wave3 stable (st) and transient (t) MDA-MB-231 kd cells (clone 2a). See movies 1-3. B) Spider plots of tracking individual cells for 2.5 hrs. C) Quantification of spider plots depicting the speed and persistence of cells. Persistence represents the ratio between Euclidean distance and total distance travelled. Shown in each instance is the mean and SEM. Results represent 3 independent experiments. D and E) Analysis of time-lapse video microscopy performed on MDA-MB-231 nt and MDA-MB-231 Scar/WAVE3 stable kd cells (clone 2a) where images were taken every minute for 200 minutes. D) Average number of lamellipodia present at each time point. E) Average number of new protrusions formed at each time point. Results represent tracking of 10 cells. * represents a p value of < 0.02, ** p value of < 0.005 and *** p value of <0.0006 in the Student’s T-test.

Figure 3. Scar/WAVE3 knockdown cells on 3 D cell-derived matrices

Scar/WAVE3 MDA-MB-231 knockdown and control nt cells were plated onto 3D cell-derived matrices and video time-lapse microscopy was performed with images taken every 15 min for 19 hrs. See Movies 4 and 5. A) Still photos of MDA-MB-231 nt and stable Scar/WAVE3 knockdown cells (clone 2a) on CDMs. B) Quantification of the persistence and velocity of cells. Shown is the mean and SEM. C) Quantification of the shape of cells on CDM matrices. Value of 1 represents circular and 0 represents elongated. Shown is the mean and SEM. Results in A, B and C represent 3 independent experiments. * represents a p value of < 0.02 and *** represents value of <0.0006 in the Student’s T-test.

Figure 4. Scar/Wave3 MDA-MB-231 knockdown and control nt cells in Boyden chamber invasion assays, inverted invasion assays and collagen invasion assays

A) Boyden chamber invasion assays were performed on control, transient and stable Scar/WAVE kd MDA-MB-231 cells. Images show cells that have invaded and quantification of the mean invasion and SEM. B) Inverted invasion assays were performed on control, transient and stable Scar/WAVE kd MDA-MB-231 cells. Results show confocal microscopy images of invading cells and quantification of the mean % invasion and SEM. C) Haematoxylin and eosin stained paraffin sections and quantification of control and Scar/WAVE3 knockdown MDA-MB-231 cells in collagen plug invasion assays. Shown is the mean invasion with S.EM. The number of cells that invaded as colonies of more than 5 cells is also quantified. Data in A, B and C represents 3 individual experiments per value on the graphs. In C * represents a p value of < 0.02 and *** represents value of <0.0006 in the Student’s T-test.