Brock Katia, Alpha Kyle M, Brennan Grant, De Jong Ebbing P, Luke Elizabeth, Turner Christopher E
Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York, USA.
Proteomics Core Facility, State University of New York Upstate Medical University, Syracuse, New York, USA.
Cytoskeleton (Hoboken). 2025 Jan;82(1-2):12-31. doi: 10.1002/cm.21878. Epub 2024 May 27.
Focal adhesions serve as structural and signaling hubs, facilitating bidirectional communication at the cell-extracellular matrix interface. Paxillin and the related Hic-5 (TGFβ1i1) are adaptor/scaffold proteins that recruit numerous structural and regulatory proteins to focal adhesions, where they perform both overlapping and discrete functions. In this study, paxillin and Hic-5 were expressed in U2OS osteosarcoma cells as biotin ligase (BioID2) fusion proteins and used as bait proteins for proximity-dependent biotinylation in order to directly compare their respective interactomes. The fusion proteins localized to both focal adhesions and the centrosome, resulting in biotinylation of components of each of these structures. Biotinylated proteins were purified and analyzed by mass spectrometry. The list of proximity interactors for paxillin and Hic-5 comprised numerous shared core focal adhesion proteins that likely contribute to their similar functions in cell adhesion and migration, as well as proteins unique to paxillin and Hic-5 that have been previously localized to focal adhesions, the centrosome, or the nucleus. Western blotting confirmed biotinylation and enrichment of FAK and vinculin, known interactors of Hic-5 and paxillin, as well as several potentially unique proximity interactors of Hic-5 and paxillin, including septin 7 and ponsin, respectively. Further investigation into the functional relationship between the unique interactors and Hic-5 or paxillin may yield novel insights into their distinct roles in cell migration.
粘着斑作为结构和信号枢纽，促进细胞与细胞外基质界面的双向通讯。桩蛋白和相关的Hic-5（TGFβ1i1）是衔接/支架蛋白，可将众多结构和调节蛋白募集到粘着斑，在那里它们发挥重叠和离散的功能。在本研究中，桩蛋白和Hic-5在U2OS骨肉瘤细胞中作为生物素连接酶（BioID2）融合蛋白表达，并用作邻近依赖性生物素化的诱饵蛋白，以便直接比较它们各自的相互作用组。融合蛋白定位于粘着斑和中心体，导致这些结构中每个结构的成分发生生物素化。对生物素化蛋白进行纯化并通过质谱分析。桩蛋白和Hic-5的邻近相互作用蛋白列表包含许多共享的核心粘着斑蛋白，这些蛋白可能有助于它们在细胞粘附和迁移中发挥相似的功能，以及桩蛋白和Hic-5特有的蛋白，这些蛋白先前已定位于粘着斑、中心体或细胞核。蛋白质免疫印迹证实了FAK和纽蛋白的生物素化和富集，它们是Hic-5和桩蛋白已知的相互作用蛋白，以及Hic-5和桩蛋白各自一些潜在独特的邻近相互作用蛋白，分别包括septin 7和ponsin。对独特相互作用蛋白与Hic-5或桩蛋白之间功能关系的进一步研究可能会对它们在细胞迁移中的不同作用产生新的见解。
