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

An in vitro analysis of neuronal survival in response to hormones and photoperiod in the HVc of the songbird Junco hyemalis

Thesis (M.S.) University of Alaska Fairbanks, 2003The ability of songbirds to sing is essential for their survival, proper reproductive behavior, and territorial establishment. Male and female juvenile passerine songbirds learn their song through the formation of a song template in their earliest days of life, first by listening to their parents, and then followed by auditory feedback against their own templates to crystallize their individual songs. However, in most passerine species, only the adult males actually sing on a seasonal basis with little to no singing during winter, followed by a phase of song production in the spring in correlation with increased plasma testosterone concentration and extended photoperiods. While the production of new neurons in the song system of adult males is continuous throughout the year, a counterbalancing turnover of these neurons must exist until the spring, when a three- to four-fold decrease in dying HVc (hyperstriatum pars ventralis caudale or higher vocal center) neurons in males initiates song production. We hypothesized that testosterone, under the influence of increased photoperiod, attenuates the rate of programmed cell death (apoptosis) of newly generated neurons migrating into the HVc song nucleus in the wild arctic songbird Junco hyemalis. Using an organotypic culture system, we examined the effect of testosterone and [beta]-estradiol on the degree of apoptosis in the HVc obtained from photo stimulated and non-photo stimulated male and female juncos. We employed a TUNEL assay and BrdU-labeling to detect and quantify apoptosis. We found that hormonal treatment with testosterone, and [beta]-estradiol in photostimulated birds only, extends the lifespan of cells within the HVc compared to controls, as shown by BrdU labeling, and decreasing apoptosis, as shown by TUNEL assay

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

Adhesion signalling complexes

SummaryIntercellular communication in metazoa not only requires autocrine, paracrine and exocrine signalling systems, but it also relies on the structural and positional information encoded in extracellular matrices (ECMs). Most cells in tissues are structurally and functionally integrated with their surrounding ECM in a highly organised manner involving thousands of dynamic connections. On the intracellular face of these linkages, adhesion receptors — principally integrins and syndecans — link the cytoskeleton to the plasma membrane and compartmentalise cytoplasmic signalling events, whereas at the extracellular face the same receptors direct and organise the deposition of the ECM itself. Adhesion receptors transduce mechanical force bidirectionally across the plasma membrane by tethering variably deformable ECMs to the contractile cytoskeleton (Figure 1), and they translate the topography and composition of the ECM into chemical signals that determine behaviour. The membrane-proximal functions of adhesion receptors in turn trigger distal processes within cells, such as alterations in the direction of cell movement and the regulation of gene transcription, and long-range effects outside cells, such as the construction of ECM networks and consequent shaping of higher-order tissue structure. Given the diverse and fundamental roles attributed to adhesion, it is understandable that adhesion receptor engagement has been reported to alter the flux through virtually all major signalling pathways

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

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