ABSTRACT
Fibroblasts transformed by the proto-oncogene Src form individual invadopodia that can spontaneously self-organize into large matrix-degrading superstructures called rosettes. However, the mechanisms by which the invadopodia can spatiotemporally reorganize their architecture is not well understood. Here, we show that Hic-5, a close relative of the scaffold protein paxillin, is essential for the formation and organization of rosettes in active Src-transfected NIH3T3 fibroblasts and cancer-associated fibroblasts. Live cell imaging, combined with domain-mapping analysis of Hic-5, identified critical motifs as well as phosphorylation sites that are required for the formation and dynamics of rosettes. Using pharmacological inhibition and mutant expression, we show that FAK kinase activity, along with its proximity to and potential interaction with the LD2,3 motifs of Hic-5, is necessary for rosette formation. Invadopodia dynamics and their coalescence into rosettes were also dependent on Rac1, formin, and myosin II activity. Superresolution microscopy revealed the presence of formin FHOD1 and INF2-mediated unbranched radial F-actin fibers emanating from invadopodia and rosettes, which may facilitate rosette formation. Collectively, our data highlight a novel role for Hic-5 in orchestrating the organization of invadopodia into higher-order rosettes, which may promote the localized matrix degradation necessary for tumor cell invasion.
Subject(s)
Cytoskeletal Proteins/metabolism , DNA-Binding Proteins/metabolism , Fibroblasts/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , LIM Domain Proteins/metabolism , Podosomes/metabolism , Protein Processing, Post-Translational , src-Family Kinases/genetics , Actins/metabolism , Actins/physiology , Animals , Cell Line, Transformed , Cytoskeletal Proteins/physiology , DNA-Binding Proteins/physiology , Fetal Proteins/metabolism , Fetal Proteins/physiology , Fibroblasts/physiology , Focal Adhesion Protein-Tyrosine Kinases/physiology , Formins/metabolism , Formins/physiology , LIM Domain Proteins/physiology , Mice , Myosin Type II/metabolism , Myosin Type II/physiology , NIH 3T3 Cells , Neuropeptides/metabolism , Neuropeptides/physiology , Phosphorylation , Podosomes/physiology , Rosette Formation , rac1 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/physiologyABSTRACT
The linearization of the stromal extracellular matrix (ECM) by cancer-associated fibroblasts (CAFs) facilitates tumor cell growth and metastasis. However, the mechanism by which the ECM is remodeled is not fully understood. Hic-5 (TGFß1i1), a focal adhesion scaffold protein, has previously been reported to be crucial for stromal ECM deposition and remodeling in vivo. Herein we show that CAFs lacking Hic-5 exhibit a significant reduction in the ability to form fibrillar adhesions, a specialized form of focal adhesion that promote fibronectin fibrillogenesis. Hic-5 was found to promote fibrillar adhesion formation through a newly characterized interaction with tensin1. Furthermore, Src-dependent phosphorylation of Hic-5 facilitated the interaction with tensin1 to prevent ß1 integrin internalization and trafficking to the lysosome. The interaction between Hic-5 and tensin1 was mechanosensitive, promoting fibrillar adhesion formation and fibronectin fibrillogenesis in a rigidity-dependent fashion. Importantly, this Src-dependent mechanism was conserved in three-dimensional (3D) ECM environments. Immunohistochemistry of tensin1 showed enrichment in CAFs in vivo, which was abrogated upon deletion of Hic-5. Interestingly, elevated Hic-5 expression correlates with reduced distant metastasis-free survival in patients with basal-like, HER2+ and grade 3 tumors. Thus, we have identified Hic-5 as a crucial regulator of ECM remodeling in CAFs by promoting fibrillar adhesion formation through a novel interaction with tensin1.
Subject(s)
Cell Adhesion , Extracellular Matrix/metabolism , Focal Adhesions/metabolism , Intracellular Signaling Peptides and Proteins/physiology , LIM Domain Proteins/physiology , Neoplasms/metabolism , Tensins/metabolism , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Cell Adhesion/genetics , Cells, Cultured , Extracellular Matrix/genetics , Extracellular Matrix/pathology , Focal Adhesions/genetics , Gene Knockdown Techniques , Humans , Infant, Newborn , Intracellular Signaling Peptides and Proteins/metabolism , LIM Domain Proteins/metabolism , Male , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Neoplasms/genetics , Neoplasms/pathology , Protein BindingABSTRACT
The Human Metabolome Database (HMDB) is a Web-based bioinformatic/cheminformatic resource with detailed information about human metabolites and metabolic enzymes. It can be used for fields of study including metabolomics, biochemistry, clinical chemistry, biomarker discovery, medicine, nutrition, and general education. In addition to its comprehensive literature-derived data, the HMDB contains an extensive collection of experimental metabolite concentration data for plasma, urine, CSF, and/or other biofluids The HMDB is fully searchable, with many tools for viewing, sorting and extracting metabolite names, chemical structures, biofluid concentrations, enzymes, genes, NMR or MS spectra, and disease information. Each metabolite entry in the HMDB contains an average of 90 separate data fields including a comprehensive compound description, names and synonyms, chemical structure information, physico-chemical data, reference NMR and MS spectra, normal and abnormal biofluid concentrations, tissue locations, disease associations, pathway information, enzyme data, gene sequence data, and SNP and mutation data, as well as extensive links to images, references and other public databases.
Subject(s)
Databases, Factual , Metabolome , Metabolomics/methods , Humans , Information Storage and Retrieval , User-Computer InterfaceABSTRACT
PlasMapper is a comprehensive web server that automatically generates and annotates high-quality circular plasmid maps. Taking only the plasmid/vector DNA sequence as input, PlasMapper uses sequence pattern matching and BLAST alignment to automatically identify and label common promoters, terminators, cloning sites, restriction sites, reporter genes, affinity tags, selectable marker genes, replication origins and open reading frames. PlasMapper then presents the identified features in textual form and as high-resolution, multicolored graphical output. The appearance and contents of the output can be customized in numerous ways using several supplied options. Further, PlasMapper images can be rendered in both rasterized (PNG and JPG) and vector graphics (SVG) formats to accommodate a variety of user needs or preferences. The images and textual output are of sufficient quality that they may be used directly in publications or presentations. The PlasMapper web server is freely accessible at http://wishart.biology.ualberta.ca/PlasMapper.
