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.

Types 1 and 2 diabetes affect some 380 million people worldwide. Both result ultimately from a deficiency of functional pancreatic insulin-producing beta cells. Beta cells proliferate in humans during a brief temporal window beginning around the time of birth, with peak beta cell labeling indices achieving approximately 2% in first year of life1-4. In embryonic life and after early childhood, beta cell replication rates are very low. While beta cell expansion seems an obvious therapeutic approach to beta cell deficiency, adult human beta cells have proven recalcitrant to such efforts1-8. Hence, there remains an urgent need for diabetes therapeutic agents that can induce regeneration and expansion of adult human beta cells in vivo or ex vivo. Here, we report the results of a high-throughput small molecule screen (HTS) revealing a novel class of human beta cell mitogenic compounds, analogues of the small molecule, harmine. We also define dual specificity tyrosine-regulated kinase-1a (DYRK1A) as the likely target of harmine, and the Nuclear Factors of activated T-cells (NFAT) family of transcription factors as likely mediators of human beta cell proliferation as well as beta cell differentiation. These observations suggest that harmine analogues (“harmalogs”) may have unique therapeutic promise for human diabetes therapy. Enhancing potency and beta cell specificity are important future challenges.

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 Ca 2؉ 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.