Cell polarization and directed migration play pivotal roles in diverse physiological and pathological processes. Herein, we identify new roles for paxillin-mediated HDAC6 inhibition in regulating key aspects of cell polarization in both two-dimensional and one-dimensional matrix environments. Paxillin, by modulating microtubule acetylation through HDAC6 regulation, was shown to control centrosome and Golgi reorientation toward the leading edge, a hallmark of cell polarization to ensure directed trafficking of promigratory factors. Paxillin was also required for pericentrosomal Golgi localization and centrosome cohesion, independent of its localization to, and role in, focal adhesion signaling. In addition, we provide evidence of an accumulation of paxillin at the centrosome that is dependent on focal adhesion kinase (FAK) and identify an important collaboration between paxillin and FAK signaling in the modulation of microtubule acetylation, as well as centrosome and Golgi organization and polarization. Finally, paxillin was also shown to be required for optimal anterograde vesicular trafficking to the plasma membrane. INTRODUCTIONThe establishment and maintenance of front-rear cell polarity and directed migration is critical during organism development, tissue remodeling, immune surveillance, and wound repair (Ridley et al., 2003). Disruption of cell polarization contributes to the progression of many diseases, including cardiovascular disease, neurodegenerative disorders, tissue fibrosis, and metastatic cancer (Lee and Vasioukhin, 2008). The microtubule (MT) network plays a crucial role in the maintenance of cell architecture and cell polarity. Furthermore, regulated trafficking of vesicles to the cell's leading edge during migration requires an asymmetric distribution of stable MTs (Etienne-Manneville, 2013), which serve as tracks to facilitate directed transport of vesicles containing promigratory factors such as integrins and matrix remodeling enzymes, including metalloproteinases (Mellman and Nelson, 2008;Petrie et al., 2009).The distribution of MTs is defined mainly by the activities of the centrosome and Golgi complex (Jaffe and Hall, 2005;Bornens, 2012). By nucleating and anchoring MTs, the centrosome and Golgi serve as the two principal MT-organizing centers (MTOCs) in animal cells (Rivero et al., 2009;Rios, 2014;Conduit et al., 2015;Sanders and Kaverina, 2015). Moreover, it has been shown that newly nucleated MTs can become stabilized by posttranslational modifications, such as acetylation on lysine 40 of tubulin (Chabin-Brion et al., 2001;Matsuyama et al., 2002;Matov et al., 2010). Of importance, disruption of MTs by drug treatment or depletion of endogenous MT stabilizers, such as RASSF1A or CLASPs, also perturbs Golgi complex integrity (Hoppeler-Lebel et al., 2007;Miller et al., 2009;Vinogradova et al., 2012;Arnette et al., 2014), as well as centrosome positioning and cohesion (Meraldi and Nigg, 2001;Burakov et al., 2003), further implicating MTs in the maintenance of Golgi and centrosome s...

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Distant organ metastasis is linked to poor prognosis during cancer progression. The expression level of the focal adhesion adapter protein paxillin varies among different human cancers, but its role in tumor progression is unclear. Herein, we utilize a newly generated PyMT mammary tumor mouse model with conditional paxillin ablation in breast tumor epithelial cells, combined with in vitro 3D tumor organoids invasion analysis and 2D calcium switch assays, to assess the roles for paxillin in breast tumor cell invasion. Paxillin had little effect on primary tumor initiation and growth but is critical for the formation of distant lung metastasis. In paxillin-depleted 3D tumor organoids, collective cell invasion was substantially perturbed. Two-dimensional cell culture revealed paxillin-dependent stabilization of adherens junctions (AJ). Mechanistically, paxillin is required for AJ assembly through facilitating E-cadherin endocytosis and recycling and HDAC6-mediated microtubule acetylation. Furthermore, Rho GTPase activity analysis and rescue experiments with a RhoA activator or Rac1 inhibitor suggest paxillin is potentially regulating the E-cadherin-dependent junction integrity and contractility through control of the balance of RhoA and Rac1 activities. Together, these data highlight new roles for paxillin in the regulation of cell-cell adhesion and collective tumor cell migration to promote the formation of distance organ metastases. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Focal adhesion (FA)-stimulated reorganization of the F-actin cytoskeleton regulates cellular size, shape, and mechanical properties. However, FA cross-talk with the intermediate filament cytoskeleton is poorly understood. Genetic ablation of the FA-associated scaffold protein Hic-5 in mouse cancer-associated fibroblasts (CAFs) promoted a dramatic collapse of the vimentin network, which was rescued following EGFP-Hic-5 expression. Vimentin collapse correlated with a loss of detergent-soluble vimentin filament precursors and decreased vimentin S72/S82 phosphorylation. Additionally, fluorescence recovery after photobleaching analysis indicated impaired vimentin dynamics. Microtubule (MT)-associated EB1 tracking and Western blotting of MT posttranslational modifications indicated no change in MT dynamics that could explain the vimentin collapse. However, pharmacological inhibition of the RhoGTPase Cdc42 in Hic-5 knockout CAFs rescued the vimentin collapse, while pan-formin inhibition with SMIFH2 promoted vimentin collapse in Hic-5 heterozygous CAFs. Our results reveal novel regulation of vimentin organization/dynamics by the FA scaffold protein Hic-5 via modulation of RhoGTPases and downstream formin activity.