FIG 4 .

Palladin generates L. monocytogenes actin-rich clouds and comet tails in the absence of the Arp2/3 complex. (A) Wild-type and Arpc2^−/− cells transfected with GFP-palladin or GFP were infected with L. monocytogenes and then fixed and stained with a rabbit polyclonal antibody targeting p34 (blue) to visualize the Arp2/3 complex and Alexa 350-phalloidin (red) to visualize actin. The top row shows wild-type cells containing intracellular bacteria with actin comet tails enriched with palladin and p34. The second row shows cells depleted of the Arp2/3 complex containing intracellular bacteria with actin comet tails enriched with palladin. The third row shows wild-type cells containing intracellular bacteria with actin comet tails enriched with p34 but not GFP. The bottom row shows cells depleted of the Arp2/3 complex containing clustered intracellular bacteria without F-actin at their surface. Open arrowheads within insets (enlargement of boxed regions) indicate individual intracellular bacteria with actin comet tails, while solid arrowheads point to additional intracellular bacteria with actin comet tails. Scale bars, 10 μm and 2 μm (insets). (B) Typical phase-contrast and fluorescent images of actin clouds and comet tails formed in reaction mixtures containing Arp2/3 (top), full-length palladin (middle), or palladin-Ig3 (bottom). Actin clouds and comet tails can be generated by either Arp2/3 or palladin but not the actin-binding domain of palladin. Open arrowheads indicate bacteria, while solid arrowheads point to actin clouds or comet tails. Scale bar, 2 μm. (C) Actin polymerization induced by full-length palladin, Arp2/3, and ActA. Spontaneous assembly reactions were performed by simultaneous addition of actin (5% pyrene labeled, primed with 1 mM EGTA and 0.1 mM MgCl₂), palladin (10 µM), Arp2/3 (0.37 µM), and ActA (0.3 µM) as indicated. Polymerization was monitored by measuring an increase in fluorescence intensity of pyrene actin in the following reactions: actin alone (gray dashed line), actin with Arp2/3 (gray solid line), actin with palladin (black dashed line), and actin with both palladin and ActA (black solid line).