CD14 is a ligand for the integrin α4β1

AbstractCell adhesion mediated by the integrin α4β1 plays a key role in many biological processes reflecting both the number and functional significance of α4β1 ligands. The lipopolysaccharide (LPS) receptor, CD14, is a GPI-linked cell surface glycoprotein with a wide range of reported functions and associations, some of which overlap with that of α4β1. This overlap led us to test the specific hypothesis that α4β1 and CD14 interact directly. Jurkat T cells (α4β1+) were found to adhere to a recombinant CD14-Fc protein via α4β1, whilst K562 cells (α4β1−) did not. However, stable reexpression of the α4-subunit conferred this ability. The adhesion of both cell types to CD14 displayed activation state-dependent binding very similar to the interaction of α4β1 with its prototypic ligand, VCAM-1. In solid-phase assays, CD14-Fc bound to affinity-purified α4β1 in a dose-dependent manner that was induced by activating anti-β1 mAbs. Finally, in related experiments, JY cells (α4β7+) were also found to attach to CD14-Fc in an α4-dependent manner. In summary, CD14 is a novel ligand for α4β1, exhibiting similar activation-state dependent binding characteristics as other α4β1 ligands. The biological relevance of this interaction will be the subject of further studies

Vinculin controls focal adhesion formation by direct interactions with talin and actin

Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs

Modulation of FAK and Src adhesion signaling occurs independently of adhesion complex composition

Integrin adhesion complexes (IACs) form mechanochemical connections between the extracellular matrix and actin cytoskeleton and mediate phenotypic responses via posttranslational modifications. Here, we investigate the modularity and robustness of the IAC network to pharmacological perturbation of the key IAC signaling components focal adhesion kinase (FAK) and Src. FAK inhibition using AZ13256675 blocked FAK(Y397) phosphorylation but did not alter IAC composition, as reported by mass spectrometry. IAC composition was also insensitive to Src inhibition using AZD0530 alone or in combination with FAK inhibition. In contrast, kinase inhibition substantially reduced phosphorylation within IACs, cell migration and proliferation. Furthermore using fluorescence recovery after photobleaching, we found that FAK inhibition increased the exchange rate of a phosphotyrosine (pY) reporter (dSH2) at IACs. These data demonstrate that kinase-dependent signal propagation through IACs is independent of gross changes in IAC composition. Together, these findings demonstrate a general separation between the composition of IACs and their ability to relay pY-dependent signals

Engineering Assessment of the Miocene Aquifer System in Coastal Georgia

Proceedings of the 2001 Georgia Water Resources Conference, April 26 and 27, 2001, Athens, Georgia.Under contract to the Georgia Geologic Survey, Golder Associates Inc. (Golder) performed an investigation to characterize the water-bearing properties of the Miocene aquifer system in coastal Georgia (also referred to as the upper and lower Brunswick aquifers). The investigation focussed on specific areas where a cap has been imposed restricting further withdrawals from the Upper Floridan Aquifer. At selected sites in Bryan, Chatham, Effingham and Glynn counties, Golder installed pumping wells and observation wells within the Miocene aquifer(s) and performed 72-hour pumping tests. Results of the investigation revealed that the lower Brunswick aquifer is absent at the sites in Chatham, Effingham and Bryan counties. Furthermore, the upper Brunswick aquifer at these locations is poorly developed, producing sustainable yields of only five to 15 gpm. However, there are Miocene wells within the Brunswick aquifer in Bryan County that produce upwards of 100 gpm, indicating significant variation in the hydraulic properties of the Miocene aquifer system in this area. Both the upper and lower Brunswick aquifers are present at the Glynn County site. While the upper Brunswick aquifer at this location is capable of producing only about 10 gpm, the lower Brunswick aquifer can produce a sustainable yield of over 300 gpm. In addition to providing water resource information to planners, developers and industry, the data produced from this investigation will also be incorporated into regional groundwater flow and transport models being developed by the USGS for management of groundwater resources throughout coastal Georgia.Sponsored and Organized by: U.S. Geological Survey, Georgia Department of Natural Resources, Natural Resources Conservation Service, The University of Georgia, Georgia State University, Georgia Institute of TechnologyThis book was published by the Institute of Ecology, The University of Georgia, Athens, Georgia 30602-2202. The views and statements advanced in this publication are solely those of the authors and do not represent official views or policies of The University of Georgia, the U.S. Geological Survey, the Georgia Water Research Institute as authorized by the Water Resources Research Act of 1990 (P.L. 101-397) or the other conference sponsors

Basement membrane ligands initiate distinct signalling networks to direct cell shape

Cells have evolved mechanisms to sense the composition of their adhesive microenvironment. Although much is known about general mechanisms employed by adhesion receptors to relay signals between the extracellular environment and the cytoskeleton, the nuances of ligand-specific signalling remain undefined. Here, we investigated how glomerular podocytes, and four other basement membrane-associated cell types, respond morphologically to different basement membrane ligands. We defined the composition of the respective adhesion complexes using mass spectrometry-based proteomics. On type IV collagen, all epithelial cell types adopted a round morphology, with a single lamellipodium and large adhesion complexes rich in actin-binding proteins. On laminin (511 or 521), all cell types attached to a similar degree but were polygonal in shape with small adhesion complexes enriched in endocytic and microtubule-binding proteins. Consistent with their distinctive morphologies, cells on type IV collagen exhibited high Rac1 activity, while those on laminin had elevated PKCα. Perturbation of PKCα was able to interchange morphology consistent with a key role for this pathway in matrix ligand-specific signalling. Therefore, this study defines the switchable basement membrane adhesome and highlights two key signalling pathways within the systems that determine distinct cell morphologies. Proteomic data are availableviaProteomeXchange with identifier PXD017913

Global analysis reveals the complexity of the human glomerular extracellular matrix.

The glomerulus contains unique cellular and extracellular matrix (ECM) components, which are required for intact barrier function. Studies of the cellular components have helped to build understanding of glomerular disease; however, the full composition and regulation of glomerular ECM remains poorly understood. We used mass spectrometry-based proteomics of enriched ECM extracts for a global analysis of human glomerular ECM in vivo and identified a tissue-specific proteome of 144 structural and regulatory ECM proteins. This catalog includes all previously identified glomerular components plus many new and abundant components. Relative protein quantification showed a dominance of collagen IV, collagen I, and laminin isoforms in the glomerular ECM together with abundant collagen VI and TINAGL1. Protein network analysis enabled the creation of a glomerular ECM interactome, which revealed a core of highly connected structural components. More than one half of the glomerular ECM proteome was validated using colocalization studies and data from the Human Protein Atlas. This study yields the greatest number of ECM proteins relative to previous investigations of whole glomerular extracts, highlighting the importance of sample enrichment. It also shows that the composition of glomerular ECM is far more complex than previously appreciated and suggests that many more ECM components may contribute to glomerular development and disease processes. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000456

Proteomic analysis of integrin-associated complexes from mesenchymal stem cells

PURPOSE: Multipotent mesenchymal stem cells (MSCs) have the capability to differentiate down adipocyte, osteocyte and chondrocyte lineages and as such offer a range of potential therapeutic applications. The composition and stiffness of the extracellular matrix (ECM) environment that surrounds cells dictates their transcriptional programme, thereby affecting stem cell lineage decision‐making. Cells sense force via linkages between themselves and their microenvironment, and this is transmitted by integrin receptors and associated adhesion signalling complexes. To identify regulators of MSC force sensing, we sought to catalogue MSC integrin‐associated adhesion complex composition. EXPERIMENTAL DESIGN: Adhesion complexes formed by MSCs plated on the ECM ligand fibronectin were isolated and characterised by MS. Identified proteins were interrogated by comparison to a literature‐based reference set of cell adhesion‐related components and using ontological and protein–protein interaction network analyses. RESULTS: Adhesion complex‐specific proteins in MSCs were identified that comprised predominantly cell adhesion‐related adaptors and actin cytoskeleton regulators. Furthermore, LIM domain‐containing proteins in MSC adhesion complexes were highlighted, which may act as force‐sensing components. CONCLUSION AND CLINICAL RELEVANCE: These data provide a valuable resource of information regarding the molecular connections that link integrins and adhesion signalling in MSCs, and as such may present novel opportunities for therapeutic intervention

Pancreatic ductal adenocarcinoma cells employ integrin α6β4 to form hemidesmosomes and regulate cell proliferation

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin α6β4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin β4 reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, suggesting a requirement for α6β4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC. Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827