Abstract
The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the coordinated regulation of actin dynamics. Palladin localizes to actin-rich protrusions and has a well-documented effect on metastasis of invasive cancers. However, its potential effects on actin dynamics have remained elusive. Here, we show that the C-terminal immunoglobulin-like domain of palladin (Ig3) that is directly responsible for actin binding and bundling also potently nucleates the formation of actin filaments in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation process of actin polymerization under both G- and F-actin buffer conditions. Furthermore palladin did not alter the critical concentration, and only had a modest effect on the rate of elongation of actin filaments. Therefore the increase in polymerization rate brought about by palladin can be attributed to a direct role for palladin in stabilizing the formation of actin nuclei. We also present evidence that nucleation is likely achieved by a mechanism involving actin-induced dimerization. In addition, we monitored actin polymerization in real-time using TIRF microscopy and found that palladin bundles the actin filaments while promoting polymerization. Finally, we examined whether the Ig3 domain of palladin is required for actin organization in a cellular context. In cells transfected with a full-length palladin construct containing either a deletion of the actin-binding domain or point mutations that disrupt actin-binding we observe dramatically altered cellular distributions of both palladin and actin, which suggests that this direct interaction with actin is critical for regulating cytoskeletal organization and dynamics. These observations define a new function for palladin and support an emerging view of actin-binding proteins that exhibit a dual cellular-nuclear localization and also participate in the regulation of the actin cytoskeleton architecture.
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