Figure 4. Effects of mis-localization of Arp2 mRNA in cell migration.

A. Expression of exogenous Arp2 in Arp2-siRNA treated CEFs. Western blotting shows expression of siRNA-resistant rescue localizing Arp2 (HA-tagged, right lane) in comparison to localizing wild-type Arp2 (also HA-tagged, not resistant to Arp2-siRNA, left lane). B & C. Expression of exogenous Arp2 proteins in CEFs transfected with localizing Arp2 (B) or mis-localizing Arp2 (C) rescue construct. Note that to better show the localization of the exogenous Arp2 protein, these cells were fixed at 16 hours after transfection. HA-tagged exogenous Arp2 proteins were detected with immunofluorescence staining (green). Arrow points to protrusion-localized protein whereas arrow head points to perinuclearly localized protein. Scale bar: 10 μm. D. Protein and mRNA levels in Arp2-rescue cells detected by Western blotting and RT-PCR, respectively. 1. localizing rescue, 2. mis-localizing rescue. * exogenous Dia1 (fused with GFP or mCherry). Bottom panel shows exogenous HA-Arp2 mRNA (20 cycles of PCR). E–G. Montage of still images of representative Arp2-KD cell rescued with localizing Arp2 (E), localizing Arp2 plus Dia1 (F) and mis-localizing Arp2 (G) (also see corresponding supplementary movies 4–6). H–K, quantitative comparison of single cell migration parameters (see Materials and Methods for details). Results of analysis of 2-hour time-lapse movies of Arp2-KD cells rescued with 1. localizing (44 cells), 2. localizing plus Dia1 (58 cells) or 3. mis-localizing (36 cells) from three independent experiments. Similar to Figure 2, L–N show cell migration tracks of all the analyzed cells in H–K and tracks highlighted in red represent cells with directionality value < 0.5. Note that for live cell migration experiments, mCherry replaced GFP in the rescue plasmids as marker for the transfected cells.