This report describes a congenital myopathy and major loss of thymic lymphocytes in ankyrin-B (−/−) mice as well as dramatic alterations in intracellular localization of key components of the Ca2+ homeostasis machinery in ankyrin-B (−/−) striated muscle and thymus. The sacoplasmic reticulum (SR) and SR/T-tubule junctions are apparently preserved in a normal distribution in ankyrin-B (−/−) skeletal muscle based on electron microscopy and the presence of a normal pattern of triadin and dihydropyridine receptor. Therefore, the abnormal localization of SR/ER Ca ATPase (SERCA) and ryanodine receptors represents a defect in intracellular sorting of these proteins in skeletal muscle. Extrapolation of these observations suggests defective targeting as the basis for abnormal localization of ryanodine receptors, IP3 receptors and SERCA in heart, and of IP3 receptors in the thymus of ankyrin-B (−/−) mice. Mis-sorting of SERCA 2 and ryanodine receptor 2 in ankyrin-B (−/−) cardiomyocytes is rescued by expression of 220-kD ankyrin-B, demonstrating that lack of the 220-kD ankyrin-B polypeptide is the primary defect in these cells. Ankyrin-B is associated with intracellular vesicles, but is not colocalized with the bulk of SERCA 1 or ryanodine receptor type 1 in skeletal muscle. These data provide the first evidence of a physiological requirement for ankyrin-B in intracellular targeting of the calcium homeostasis machinery of striated muscle and immune system, and moreover, support a catalytic role that does not involve permanent stoichiometric complexes between ankyrin-B and targeted proteins. Ankyrin-B is a member of a family of adapter proteins implicated in restriction of diverse proteins to specialized plasma membrane domains. Similar mechanisms involving ankyrins may be essential for segregation of functionally defined proteins within specialized regions of the plasma membrane and within the Ca2+ homeostasis compartment of the ER.

Close homolog of L1 (CHL1) is a member of the L1 family of cell adhesion molecules expressed by subpopulations of neurons and glia in the central and peripheral nervous system. It promotes neurite outgrowth and neuronal survival in vitro. This study describes a novel function for CHL1 in potentiating integrin-dependent cell migration toward extracellular matrix proteins. Expression of CHL1 in HEK293 cells stimulated their haptotactic migration toward collagen I, fibronectin, laminin, and vitronectin substrates in Transwell assays. CHL1-potentiated cell migration to collagen I was dependent on ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins, as shown with function blocking antibodies. Potentiated migration relied on the early integrin signaling intermediates c-Src, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase. Enhancement of migration was disrupted by mutation of a potential integrin interaction motif Asp-Gly-Glu-Ala (DGEA) in the sixth immunoglobulin domain of CHL1, suggesting that CHL1 functionally interacts with ␤ 1 integrins through this domain. CHL1 was shown to associate with ␤ 1 integrins on the cell surface by antibody-induced co-capping. Through a cytoplasmic domain sequence containing a conserved tyrosine residue (Phe-Ile-Gly-Ala-Tyr), CHL1 recruited the actin cytoskeletal adapter protein ankyrin to the plasma membrane, and this sequence was necessary for promoting integrin-dependent migration to extracellular matrix proteins. These results support a role for CHL1 in integrin-dependent cell migration that may be physiologically important in regulating cell migration in nerve regeneration and cortical development.

Dynamic modulation of adhesion provided by anchorage of axonal receptors with the cytoskeleton contributes to attractant or repellent responses that guide axons to topographic targets in the brain. The neural cell adhesion molecule L1 engages the spectrin-actin cytoskeleton through reversible linkage of its cytoplasmic domain to ankyrin. To investigate a role for L1 association with the cytoskeleton in topographic guidance of retinal axons to the superior colliculus, a novel mouse strain was generated by genetic knock-in that expresses an L1 point mutation (Tyr1229His) abolishing ankyrin binding. Axon tracing revealed a striking mistargeting of mutant ganglion cell axons from the ventral retina, which express high levels of ephrinB receptors, to abnormally lateral sites in the contralateral superior colliculus, where they formed multiple ectopic arborizations. These axons were compromised in extending interstitial branches in the medial direction, a normal response to the high medial to low lateral SC gradient of ephrinB1. Furthermore, ventral but not dorsal L1(Y1229H) retinal cells were impaired for ephrinB1-stimulated adhesion through ␤1 integrins in culture. The retinocollicular phenotype of the L1(Tyr1229His) mutant provides the first evidence that L1 regulates topographic mapping of retinal axons through adhesion mediated by linkage to the actin cytoskeleton and functional interaction with the ephrinB/EphB targeting system.