Focal adhesion-based cell migration is differentially regulated in vivo versus in vitro by Paxillin phosphorylation

Abstract

SummaryFocal adhesions are important subcellular structures that physically link the cell to the extracellular matrix (ECM), thus facilitating efficient cell migration. Although in vitro cell culture studies have provided a wealth of information regarding focal adhesion biology, it is critical to understand how focal adhesions are dynamically regulated in their native environment. We developed a zebrafish transplantation system in which we could efficiently visualize focal adhesion structures during single cell migration in vivo with high-resolution live cell imaging. By comparing focal adhesions between this in vivo system and the traditional in vitro cell culture model, we show differential regulation of a core focal adhesion protein, Paxillin. We find that a key site of phosphoregulation on Paxillin, tyrosine 118 (Y118), exhibits reduced phosphorylation in migrating cells in vivo in both zebrafish and mouse melanoma models, contrary to the pivotal role for this phosphorylation event in cell culture studies. Furthermore, direct modulation of this residue by site directed mutagenesis leads to opposite cell migration phenotypes in vivo versus in vitro in both migrating cancer cells and macrophages. Unexpectedly, expression of a non-phosphorylatable version of Y118-Paxillin promotes cell migration in vivo, despite inhibiting cell migration in the in vitro cell culture conditions. To further understand the mechanism of this regulation, we find that the upstream kinase, focal adhesion kinase (FAK), is downregulated in cells in vivo, and that cells expressing non-phosphorylatable Y118-Paxillin exhibit increased interactions between Paxillin and CRKII, an adaptor protein known to promote cell migration signaling. Collectively, our findings provide significant new insight into how focal adhesions are regulated in cells migrating in their native environment.

An evolutionary and physiological perspective on cell-substrate adhesion machinery for cell migration

“…These punctae formed at cell protrusion sites and dispersed when the protrusions retracted ( Barros-Becker et al, 2017 ). Work from our own lab using the same-species transplantation of highly migratory zebrafish melanoma cells expressing fluorescently labeled Paxillin showed that Paxillin co-localizes with actin to form dynamic focal adhesion structures at sites of cell-ECM contact during single cell migration ( Xue et al, 2022 ). Taken together, these studies suggest that focal adhesion structures form during in vivo single cell migration.…”