Increased abundance of translation machinery in stem cell-derived neural progenitor cells from four schizophrenia patients.

The genetic and epigenetic factors contributing to risk for schizophrenia (SZ) remain unresolved. Here we demonstrate, for the first time, perturbed global protein translation in human-induced pluripotent stem cell (hiPSC)-derived forebrain neural progenitor cells (NPCs) from four SZ patients relative to six unaffected controls. We report increased total protein levels and protein synthesis, together with two independent sets of quantitative mass spectrometry evidence indicating markedly increased levels of ribosomal and translation initiation and elongation factor proteins, in SZ hiPSC NPCs. We posit that perturbed levels of global protein synthesis in SZ hiPSC NPCs represent a novel post-transcriptional mechanism that might contribute to disease progression.

Pyk2 regulates multiple signaling events crucial for macrophage morphology and migration.

The biological role of the protein tyrosine kinase, Pyk2, was explored by targeting the Pyk2 gene by homologous recombination. Pyk2-/- mice are viable and fertile, without overt impairment in development or behavior. However, the morphology and behavior of Pyk2-/- macrophages were impaired. Macrophages isolated from mutant mice failed to become polarized, to undergo membrane ruffling, and to migrate in response to chemokine stimulation. Moreover, the contractile activity in the lamellipodia of Pyk2-/- macrophages was impaired, as revealed by measuring the rearward movement toward the nucleus of fibronectin-coated beads on the lamellipodia in opposition to an immobilizing force generated by optical tweezers. Consistently, the infiltration of macrophages into a carageenan-induced inflammatory region was strongly inhibited in Pyk2-/- mice. In addition, chemokine stimulation of inositol (1, 4, 5) triphosphate production and Ca2+ release, as well as integrin-induced activation of Rho and phosphatidyl inositol 3 kinase, were compromised in Pyk2-/- macrophages. These experiments reveal a role for Pyk2 in cell signaling in macrophages essential for cell migration and function.

Selective regulation of integrin--cytoskeleton interactions by the tyrosine kinase Src.

Cell motility on extracellular-matrix (ECM) substrates depends on the regulated generation of force against the substrate through adhesion receptors known as integrins. Here we show that integrin-mediated traction forces can be selectively modulated by the tyrosine kinase Src. In Src-deficient fibroblasts, cell spreading on the ECM component vitronectin is inhibited, while the strengthening of linkages between integrin vitronectin receptors and the force-generating cytoskeleton in response to substrate rigidity is dramatically increased. In contrast, Src deficiency has no detectable effects on fibronectin-receptor function. Finally, truncated Src (lacking the kinase domain) co-localizes to focal-adhesion sites with alpha v but not with beta 1 integrins. These data are consistent with a selective, functional interaction between Src and the vitronectin receptor that acts at the integrin-cytoskeleton interface to regulate cell spreading and migration.

Cell migration as a five-step cycle.

The migration of cells over substrata is a fundamental and critical function that requires the co-ordination of several cellular processes which operate in a cycle. At the level of the light microscope, the cycle can be divided into five steps: (1) extension of the leading edge; (2) adhesion to matrix contacts; (3) contraction of the cytoplasm; (4) release from contact sites; and (5) recycling of membrane receptors from the rear to the front of the cell. Each step is dependent upon one or more cyclical biochemical processes. The development of many in vitro and subcellular assays for the fundamental biochemical processes involved has increased our understanding of each cycle dramatically in the last several years to include a definition of many of the protein and enzymic components, the role of the position of extracellular-matrix receptors on the cell, and the contribution of physical force. The next generation of questions are directed at resolving the roles of the many individual proteins in each step of the cell migration process. In this chapter we will examine each of the migration steps and discuss the biochemical mechanisms that may underlie them.

Cell migration: regulation of force on extracellular-matrix-integrin complexes.

Cell migration relies upon forces generated by the cell. Recent studies have provided new insights into the processes by which cells generate and regulate the forces applied to extracellular matrix (ECM)-bound integrins and have led us to the working model described here. In this model, ECM binding to integrins in the front of lamellipodia causes those integrins to attach to the rearward-moving cytoskeleton. Integrin-cytoskeleton attachments in the front are strengthened as a result of ECM rigidity, enabling the cell to pull itself forward. The reduction in contact area at the rear compared with that at the lamellipodium concentrates the traction forces in the rear on fewer integrin-ECM bonds, facilitating release. In such a model, cell pathfinding and motility can be influenced by ECM rigidity.

Mutational evidence for control of cell adhesion through integrin diffusion/clustering, independent of ligand binding.

Previous studies have shown that integrin alpha chain tails make strong positive contributions to integrin-mediated cell adhesion. We now show here that integrin alpha4 tail deletion markedly impairs static cell adhesion by a mechanism that does not involve altered binding of soluble vascular cell adhesion molecule 1 ligand. Instead, truncation of the alpha4 cytoplasmic domain caused a severe deficiency in integrin accumulation into cell surface clusters, as induced by ligand and/ or antibodies. Furthermore, alpha4 tail deletion also significantly decreased the membrane diffusivity of alpha4beta1, as determined by a single particle tracking technique. Notably, low doses of cytochalasin D partially restored the deficiency in cell adhesion seen upon alpha4 tail deletion. Together, these results suggest that alpha4 tail deletion exposes the beta1 cytoplasmic domain, leading to cytoskeletal associations that apparently restrict integrin lateral diffusion and accumulation into clusters, thus causing reduced static cell adhesion. Our demonstration of integrin adhesive activity regulated through receptor diffusion/clustering (rather than through altered ligand binding affinity) may be highly relevant towards the understanding of inside-out signaling mechanisms for beta1 integrins.

Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages.

To move forward, migrating cells must generate traction forces through surface receptors bound to extracellular matrix molecules coupled to a rigid structure. We investigated whether cells sample and respond to the rigidity of the anchoring matrix. Movement of beads coated with fibronectin or an anti-integrin antibody was restrained with an optical trap on fibroblasts to mimic extracellular attachment sites of different resistance. Cells precisely sense the restraining force on fibronectin beads and respond by a localized, proportional strengthening of the cytoskeleton linkages, allowing stronger force to be exerted on the integrins. This strengthening was absent or transient with antibody beads, but restored with soluble fibronectin. Hence, ligand binding site occupancy was required. Finally, phenylarsine oxide inhibited strengthening of cytoskeletal linkages, indicating a role for dephosphorylation. Thus, the strength of integrin-cytoskeleton linkages is dependent on matrix rigidity and on its biochemical composition. Matrix rigidity may, therefore, serve as a guidance cue in a process of mechanotaxis.

Ligand binding regulates the directed movement of beta1 integrins on fibroblasts.

To enable cells to crawl, adhesion receptors such as integrins must bind to extracellular molecules and simultaneously interact with force-generating components of the cytoskeleton. We show here that the binding of extracellular ligand in living cells induces the attachment of beta1 integrins to the retrograde-moving cytoskeleton. Unliganded integrins are not associated with the rearward-moving cytoskeleton: gold particles attached to beta1 integrin by a monoclonal antibody diffuse in the membrane. However, addition of soluble RGD peptide (single-letter amino-acid code) or the use of fibronectin-coated gold particles causes the attachment of integrins to the rearward-moving cytoskeleton. Deletion of the beta1 cytoplasmic tail blocks cytoskeletal attachment. The directed movement of integrins in response to ligand indicates that ligand binding is the critical step in regulating organized receptor movement on the cell surface and the migration of adherent cells.

TAG-1 can mediate homophilic binding, but neurite outgrowth on TAG-1 requires an L1-like molecule and beta 1 integrins.

Subsets of axons in the embryonic nervous system transiently express the glycoprotein TAG-1, a member of the subfamily of immunoglobulin (Ig)-like proteins that contain both C2 class Ig and fibronectin type III domains. TAG-1 is attached to the cell surface by a glycosylphosphatidylinositol linkage and is secreted by neurons. In vitro studies have shown that substrate-bound TAG-1 promotes neurite outgrowth. We have examined the nature of axonal receptors that mediate the neurite-outgrowth promoting properties of TAG-1. Although TAG-1 can mediate homophilic binding, neurite outgrowth on a substrate of TAG-1 does not depend on the presence of TAG-1 on the axonal surface. Instead, neurite outgrowth on TAG-1 is inhibited by polyclonal antibodies directed against L1 and, independently, by polyclonal and monoclonal antibodies against beta 1-containing integrins. These results provide evidence that TAG-1 can interact with cell surfaces in both a homophilic and heterophilic manner and suggest that neurite extension on TAG-1 requires the function of both integrins and an L1-like molecule.

Fine specificity mapping and topography of an isozyme-specific epitope of the Na,K-ATPase catalytic subunit.

An isozyme-specific domain of the catalytic subunit of the Na,K-ATPase has been identified using a monoclonal antibody, McK1. The antibody's specificity was confirmed by its ability to stain proteolytic fingerprints of the Na,K-ATPase. The antibody recognized the alpha I isozyme of the rat Na,K-ATPase, but not the alpha II or alpha III isozymes. It recognized native and sodium dodecyl sulfate-denatured Na,K-ATPase and specifically stained basolateral membranes of the renal tubule. It bound to rat alpha I with highest affinity, but also cross-reacted with mouse, monkey, and human alpha I. It did not cross-react with sheep, pig, chicken, Torpedo, or dog alpha I. Fine specificity mapping was used to deduce the most likely antibody binding sites, based on comparison of eight amino acid sequences from cDNA clones. Two potential binding sites were found at widely separated locations. Limited tryptic digestion of the native enzyme was then used to demonstrate that the binding site was close to the N-terminal end of the Na,K-ATPase. The binding site is predicted to include the following essential amino acid sequence: Asp-Lys-Lys-Ser-Lys-Lys in rat alpha I or Asp-Lys-Lys-Gly-Lys-Lys in human alpha I. The antibody was found to bind to opened, but not to sealed right-side-out vesicles isolated from the rat renal medulla, demonstrating that the N-terminal end of the Na,K-ATPase is exposed at the interior of the cell.