Focal adhesions are structures that physically link the cell to the extracellular matrix for cell migration. Although 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 system to visualize focal adhesion structures during single-cell migration in vivo. We find that a key site of phosphoregulation (Y118) on Paxillin exhibits reduced phosphorylation in migrating cells in vivo compared to in vitro. Furthermore, expression of a non-phosphorylatable version of Y118-Paxillin increases focal adhesion disassembly and promotes cell migration in vivo, despite inhibiting cell migration in vitro. Using a mouse model, we further find that the upstream kinase, focal adhesion kinase, is downregulated in cells in vivo, and cells expressing non-phosphorylatable Y118-Paxillin exhibit increased activation of the CRKII-DOCK180/RacGEF pathway. Our findings provide significant new insight into the intrinsic regulation of focal adhesions in cells migrating in their native environment.

Cell-substrate adhesion is a critical aspect of many forms of cell migration. Cell adhesion to an extracellular matrix (ECM) generates traction forces necessary for efficient migration. One of the most well-studied structures cells use to adhere to the ECM is focal adhesions, which are composed of a multilayered protein complex physically linking the ECM to the intracellular actin cytoskeleton. Much of our understanding of focal adhesions, however, is primarily derived from in vitro studies in Metazoan systems. Though these studies provide a valuable foundation to the cell-substrate adhesion field, the evolution of cell-substrate adhesion machinery across evolutionary space and the role of focal adhesions in vivo are largely understudied within the field. Furthering investigation in these areas is necessary to bolster our understanding of the role cell-substrate adhesion machinery across Eukaryotes plays during cell migration in physiological contexts such as cancer and pathogenesis. In this review, we review studies of cell-substrate adhesion machinery in organisms evolutionary distant from Metazoa and cover the current understanding and ongoing work on how focal adhesions function in single and collective cell migration in an in vivo environment, with an emphasis on work that directly visualizes cell-substrate adhesions. Finally, we discuss nuances that ought to be considered moving forward and the importance of future investigation in these emerging fields for application in other fields pertinent to adhesion-based processes.

It is well known that melanoma lipid metabolism is regulated by intrinsic genetic and epigenetic changes in the cancer cells. However, recent work reveals that lipid metabolism is also controlled by the tumor microenvironment through the direct transfer of lipids from adipocytes to melanoma cells.

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.