Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro. Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility.

Palladin is an actin binding protein that is specifically upregulated in metastatic cancer cells but also co-localizes with actin stress fibers in normal cells and is critical for embryonic development as well as wound healing. Of nine isoforms present in humans, only the 90 kDa isoform of palladin, comprising three immunoglobulin (Ig) domains and one proline-rich region, is ubiquitously expressed. Previous work has also established that the Ig3 domain of palladin is the minimal binding site for F-actin. In this work, we compare functions of the 90 kDa isoform of palladin to the isolated actin binding domain. To understand the mechanism of action for how palladin can influence actin assembly, we monitored F-actin binding and bundling as well as actin polymerization, depolymerization, and copolymerization. We also provide initial evidence that 90 kDa palladin exists in a closed conformation that prevents binding by the Ig3 domain to G-actin as compared to the isolated domain. Understanding the role of palladin in regulating the actin cytoskeleton may help us develop means to prevent cancer cells from reaching the metastatic stage of cancer progression.

Palladin is an actin binding protein that is specifically upregulated in metastatic cancer cells but also colocalizes with actin stress fibers in normal cells and is critical for embryonic development as well as wound healing. Of nine isoforms present in humans, only the 90 kDa isoform of palladin, comprising three immunoglobulin (Ig) domains and one proline‐rich region, is ubiquitously expressed. Previous work has established that the Ig3 domain of palladin is the minimal binding site for F‐actin. In this work, we compare functions of the 90 kDa isoform of palladin to the isolated actin binding domain. To understand the mechanism of action for how palladin can influence actin assembly, we monitored F‐actin binding and bundling as well as actin polymerization, depolymerization, and copolymerization. Together, these results demonstrate that there are key differences between the Ig3 domain and full‐length palladin in actin binding stoichiometry, polymerization, and interactions with G‐actin. Understanding the role of palladin in regulating the actin cytoskeleton may help us develop means to prevent cancer cells from reaching the metastatic stage of cancer progression.

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