FIG 3 .

Palladin compensates for Arp2/3 complex functional defects during the intracellular motility of L. monocytogenes. (A) Still frames from movies of HeLa cells transfected with mKate-LifeAct alone to visualize actin or cotransfected with mKate-LifeAct and GFP-tagged wild-type palladin, palladinK15/18/51A, or palladinFPAA constructs and infected with wild-type L. monocytogenes for at least 8 h. Bacterial movement halts immediately and comet tails disintegrate following perfusion of CK-666 (100 μM) in cells with endogenous levels of palladin. Bacterial movement and comet tails are unaffected in cells overexpressing palladin variants in the presence of CK-666 (100 μM). Open arrowheads indicate individual bacteria. Elapsed time is shown in minutes and seconds. Scale bar, 5 μm. (B and C) The effect of CK-666 (100 μM) on the instantaneous (B) and average (C) speeds of L. monocytogenes in cells with endogenous levels of palladin (red) or overexpressing GFP-tagged wild-type palladin (blue), palladinK15/18/51A (yellow), or palladinFPAA (purple) constructs. The origin of the axes indicates the final motility rate measured prior to injection of CK-666 in panel B. Injection of CK-666 significantly decreases bacterial motility rates, except in cells overexpressing wild-type palladin. The mean speed rates (C) of intracellular L. monocytogenes pre-CK-666 injection and post-CK-666 injection are as follows: 8.990 and 0.279 μm/min in mKate-LifeAct-expressing cells, 7.989 and 8.409 μm/min in wild-type palladin-expressing cells, 6.946 and 4.175 μm/min in palladinK15/18/51A-expressing cells, and 1.177 and 0.566 μm/min in palladinFPAA-expressing cells. **, P < 0.001; *, P < 0.01.