An ankyrin-based mechanism for functional organization of dystrophin and dystroglycan.

beta-dystroglycan (DG) and the dystrophin-glycoprotein complex (DGC) are localized at costameres and neuromuscular junctions in the sarcolemma of skeletal muscle. We present evidence for an ankyrin-based mechanism for sarcolemmal localization of dystrophin and beta-DG. Dystrophin binds ankyrin-B and ankyrin-G, while beta-DG binds ankyrin-G. Dystrophin and beta-DG require ankyrin-G for retention at costameres but not delivery to the sarcolemma. Dystrophin and beta-DG remain intracellular in ankyrin-B-depleted muscle, where beta-DG accumulates in a juxta-TGN compartment. The neuromuscular junction requires ankyrin-B for localization of dystrophin/utrophin and beta-DG and for maintenance of its postnatal morphology. A Becker muscular dystrophy mutation reduces ankyrin binding and impairs sarcolemmal localization of dystrophin-Dp71. Ankyrin-B also binds to dynactin-4, a dynactin subunit. Dynactin-4 and a subset of microtubules disappear from sarcolemmal sites in ankyrin-B-depleted muscle. Ankyrin-B thus is an adaptor required for sarcolemmal localization of dystrophin, as well as dynactin-4.

ßII-spectrin promotes mouse brain connectivity through stabilizing axonal plasma membranes and enabling axonal organelle transport.

ßII-spectrin is the generally expressed member of the ß-spectrin family of elongated polypeptides that form micrometer-scale networks associated with plasma membranes. We addressed in vivo functions of ßII-spectrin in neurons by knockout of ßII-spectrin in mouse neural progenitors. ßII-spectrin deficiency caused severe defects in long-range axonal connectivity and axonal degeneration. ßII-spectrin-null neurons exhibited reduced axon growth, loss of actin-spectrin-based periodic membrane skeleton, and impaired bidirectional axonal transport of synaptic cargo. We found that ßII-spectrin associates with KIF3A, KIF5B, KIF1A, and dynactin, implicating spectrin in the coupling of motors and synaptic cargo. ßII-spectrin required phosphoinositide lipid binding to promote axonal transport and restore axon growth. Knockout of ankyrin-B (AnkB), a ßII-spectrin partner, primarily impaired retrograde organelle transport, while double knockout of ßII-spectrin and AnkB nearly eliminated transport. Thus, ßII-spectrin promotes both axon growth and axon stability through establishing the actin-spectrin-based membrane-associated periodic skeleton as well as enabling axonal transport of synaptic cargo.

Neurodevelopmental mutation of giant ankyrin-G disrupts a core mechanism for axon initial segment assembly.

Giant ankyrin-G (gAnkG) coordinates assembly of axon initial segments (AISs), which are sites of action potential generation located in proximal axons of most vertebrate neurons. Here, we identify a mechanism required for normal neural development in humans that ensures ordered recruitment of gAnkG and ß4-spectrin to the AIS. We identified 3 human neurodevelopmental missense mutations located in the neurospecific domain of gAnkG that prevent recruitment of ß4-spectrin, resulting in a lower density and more elongated pattern for gAnkG and its partners than in the mature AIS. We found that these mutations inhibit transition of gAnkG from a closed configuration with close apposition of N- and C-terminal domains to an extended state that is required for binding and recruitment of ß4-spectrin, and normally occurs early in development of the AIS. We further found that the neurospecific domain is highly phosphorylated in mouse brain, and that phosphorylation at 2 sites (S1982 and S2619) is required for the conformational change and for recruitment of ß4-spectrin. Together, these findings resolve a discrete intermediate stage in formation of the AIS that is regulated through phosphorylation of the neurospecific domain of gAnkG.

ANK2 autism mutation targeting giant ankyrin-B promotes axon branching and ectopic connectivity.

Giant ankyrin-B (ankB) is a neurospecific alternatively spliced variant of ANK2, a high-confidence autism spectrum disorder (ASD) gene. We report that a mouse model for human ASD mutation of giant ankB exhibits increased axonal branching in cultured neurons with ectopic CNS axon connectivity, as well as with a transient increase in excitatory synapses during postnatal development. We elucidate a mechanism normally limiting axon branching, whereby giant ankB localizes to periodic axonal plasma membrane domains through L1 cell-adhesion molecule protein, where it couples microtubules to the plasma membrane and prevents microtubule entry into nascent axon branches. Giant ankB mutation or deficiency results in a dominantly inherited impairment in selected communicative and social behaviors combined with superior executive function. Thus, gain of axon branching due to giant ankB-deficiency/mutation is a candidate cellular mechanism to explain aberrant structural connectivity and penetrant behavioral consequences in mice as well as humans bearing ASD-related ANK2 mutations.

Ankyrin-G regulated epithelial phenotype is required for mouse lens morphogenesis and growth.

Epithelial cell polarity, adhesion, proliferation, differentiation and survival are essential for morphogenesis of various organs and tissues including the ocular lens. The molecular mechanisms regulating the lens epithelial phenotype however, are not well understood. Here we investigated the role of scaffolding protein ankyrin-G (AnkG) in mouse lens development by conditional suppression of AnkG expression using the Cre-LoxP recombination approach. AnkG, which serves to link integral membrane proteins to the spectrin/actin cytoskeleton, was found to distribute predominantly to the lateral membranes of lens epithelium with several isoforms of the protein being detected in the mouse lens. Conditional deficiency of AnkG impaired mouse lens morphogenesis starting from embryonic stage E15.5, with neonatal (P1) AnkG cKO lenses exhibiting overt abnormalities in shape, size, epithelial cell height, sheet length and lateral membrane assembly together with defective fiber cell orientation relative to lenses from littermate AnkG floxed or Cre expressing mice. Severe disruptions in E-cadherin/ß-catenin-based adherens junctions, and the membrane organization of spectrin-actin cytoskeleton, ZO-1, connexin-50 and Na+-K+-ATPase were noted in AnkG deficient lenses, along with detection in lens epithelium of α-smooth muscle actin, a marker of epithelial to mesenchymal transition. Moreover, lens epithelial cell proliferation and survival were severely compromised while differentiation appears to be normal in AnkG deficient mouse lenses. Collectively, these results indicate that AnkG regulates establishment of the epithelial phenotype via lateral membrane assembly, stabilization of E-cadherin-based cell-cell junctions, polarity and membrane organization of transport and adhesion proteins and the spectrin-actin skeleton, and provide evidence for an obligatory role for AnkG in lens morphogenesis and growth.

Cell-autonomous adiposity through increased cell surface GLUT4 due to ankyrin-B deficiency.

Obesity typically is linked to caloric imbalance as a result of overnutrition. Here we propose a cell-autonomous mechanism for adiposity as a result of persistent cell surface glucose transporter type 4 (GLUT4) in adipocytes resulting from impaired function of ankyrin-B (AnkB) in coupling GLUT4 to clathrin-mediated endocytosis. Adipose tissue-specific AnkB-KO mice develop obesity and progressive pancreatic islet dysfunction with age or high-fat diet (HFD). AnkB-deficient adipocytes exhibit increased lipid accumulation associated with increased glucose uptake and impaired endocytosis of GLUT4. AnkB binds directly to GLUT4 and clathrin and promotes their association in adipocytes. AnkB variants that fail to restore normal lipid accumulation and GLUT4 localization in adipocytes are present in 1.3% of European Americans and 8.4% of African Americans, and are candidates to contribute to obesity susceptibility in humans.

Giant ankyrin-G stabilizes somatodendritic GABAergic synapses through opposing endocytosis of GABAA receptors.

GABAA-receptor-based interneuron circuitry is essential for higher order function of the human nervous system and is implicated in schizophrenia, depression, anxiety disorders, and autism. Here we demonstrate that giant ankyrin-G (480-kDa ankyrin-G) promotes stability of somatodendritic GABAergic synapses in vitro and in vivo. Moreover, giant ankyrin-G forms developmentally regulated and cell-type-specific micron-scale domains within extrasynaptic somatodendritic plasma membranes of pyramidal neurons. We further find that giant ankyrin-G promotes GABAergic synapse stability through opposing endocytosis of GABAA receptors, and requires a newly described interaction with GABARAP, a GABAA receptor-associated protein. We thus present a new mechanism for stabilization of GABAergic interneuron synapses and micron-scale organization of extrasynaptic membrane that provides a rationale for studies linking ankyrin-G genetic variation with psychiatric disease and abnormal neurodevelopment.

Giant ankyrin-G: a critical innovation in vertebrate evolution of fast and integrated neuronal signaling.

Axon initial segments (AISs) and nodes of Ranvier are sites of clustering of voltage-gated sodium channels (VGSCs) in nervous systems of jawed vertebrates that facilitate fast long-distance electrical signaling. We demonstrate that proximal axonal polarity as well as assembly of the AIS and normal morphogenesis of nodes of Ranvier all require a heretofore uncharacterized alternatively spliced giant exon of ankyrin-G (AnkG). This exon has sequence similarity to I-connectin/Titin and was acquired after the first round of whole-genome duplication by the ancestral ANK2/ANK3 gene in early vertebrates before development of myelin. The giant exon resulted in a new nervous system-specific 480-kDa polypeptide combining previously known features of ANK repeats and ß-spectrin-binding activity with a fibrous domain nearly 150 nm in length. We elucidate previously undescribed functions for giant AnkG, including recruitment of ß4 spectrin to the AIS that likely is regulated by phosphorylation, and demonstrate that 480-kDa AnkG is a major component of the AIS membrane "undercoat' imaged by platinum replica electron microscopy. Surprisingly, giant AnkG-knockout neurons completely lacking known AIS components still retain distal axonal polarity and generate action potentials (APs), although with abnormal frequency. Giant AnkG-deficient mice live to weaning and provide a rationale for survival of humans with severe cognitive dysfunction bearing a truncating mutation in the giant exon. The giant exon of AnkG is required for assembly of the AIS and nodes of Ranvier and was a transformative innovation in evolution of the vertebrate nervous system that now is a potential target in neurodevelopmental disorders.

Ankyrin-G Inhibits Endocytosis of Cadherin Dimers.

Dynamic regulation of endothelial cell adhesion is central to vascular development and maintenance. Furthermore, altered endothelial adhesion is implicated in numerous diseases. Therefore, normal vascular patterning and maintenance require tight regulation of endothelial cell adhesion dynamics. However, the mechanisms that control junctional plasticity are not fully understood. Vascular endothelial cadherin (VE-cadherin) is an adhesive protein found in adherens junctions of endothelial cells. VE-cadherin mediates adhesion through trans interactions formed by its extracellular domain. Trans binding is followed by cis interactions that laterally cluster the cadherin in junctions. VE-cadherin is linked to the actin cytoskeleton through cytoplasmic interactions with ß- and α-catenin, which serve to increase adhesive strength. Furthermore, p120-catenin binds to the cytoplasmic tail of cadherin and stabilizes it at the plasma membrane. Here we report that induced cis dimerization of VE-cadherin inhibits endocytosis independent of both p120 binding and trans interactions. However, we find that ankyrin-G, a protein that links membrane proteins to the spectrin-actin cytoskeleton, associates with VE-cadherin and inhibits its endocytosis. Ankyrin-G inhibits VE-cadherin endocytosis independent of p120 binding. We propose a model in which ankyrin-G associates with and inhibits the endocytosis of VE-cadherin cis dimers. Our findings support a novel mechanism for regulation of VE-cadherin endocytosis through ankyrin association with cadherin engaged in lateral interactions.

Ankyrin-B directs membrane tethering of periaxin and is required for maintenance of lens fiber cell hexagonal shape and mechanics.

Periaxin (Prx), a PDZ domain protein expressed preferentially in myelinating Schwann cells and lens fibers, plays a key role in membrane scaffolding and cytoarchitecture. Little is known, however, about how Prx is anchored to the plasma membrane. Here we report that ankyrin-B (AnkB), a well-characterized adaptor protein involved in linking the spectrin-actin cytoskeleton to integral membrane proteins, is required for membrane association of Prx in lens fibers and colocalizes with Prx in hexagonal fiber cells. Under AnkB haploinsufficiency, Prx accumulates in the soluble fraction with a concomitant loss from the membrane-enriched fraction of mouse lenses. Moreover, AnkB haploinsufficiency induced age-dependent disruptions in fiber cell hexagonal geometry and radial alignment and decreased compressive stiffness in mouse lenses parallel to the changes observed in Prx null mouse lens. Both AnkB- and Prx-deficient mice exhibit disruptions in membrane organization of the spectrin-actin network and the dystrophin-glycoprotein complex in lens fiber cells. Taken together, these observations reveal that AnkB is required for Prx membrane anchoring and for maintenance of lens fiber cell hexagonal geometry, membrane skeleton organization, and biomechanics.

Ankyrin-G coordinates intercalated disc signaling platform to regulate cardiac excitability in vivo.

RATIONALE: Nav1.5 (SCN5A) is the primary cardiac voltage-gated Nav channel. Nav1.5 is critical for cardiac excitability and conduction, and human SCN5A mutations cause sinus node dysfunction, atrial fibrillation, conductional abnormalities, and ventricular arrhythmias. Further, defects in Nav1.5 regulation are linked with malignant arrhythmias associated with human heart failure. Consequently, therapies to target select Nav1.5 properties have remained at the forefront of cardiovascular medicine. However, despite years of investigation, the fundamental pathways governing Nav1.5 membrane targeting, assembly, and regulation are still largely undefined. OBJECTIVE: Define the in vivo mechanisms underlying Nav1.5 membrane regulation. METHODS AND RESULTS: Here, we define the molecular basis of an Nav channel regulatory platform in heart. Using new cardiac-selective ankyrin-G(-/-) mice (conditional knock-out mouse), we report that ankyrin-G targets Nav1.5 and its regulatory protein calcium/calmodulin-dependent kinase II to the intercalated disc. Mechanistically, ßIV-spectrin is requisite for ankyrin-dependent targeting of calcium/calmodulin-dependent kinase II-δ; however, ßIV-spectrin is not essential for ankyrin-G expression. Ankyrin-G conditional knock-out mouse myocytes display decreased Nav1.5 expression/membrane localization and reduced INa associated with pronounced bradycardia, conduction abnormalities, and ventricular arrhythmia in response to Nav channel antagonists. Moreover, we report that ankyrin-G links Nav channels with broader intercalated disc signaling/structural nodes, as ankyrin-G loss results in reorganization of plakophilin-2 and lethal arrhythmias in response to ß-adrenergic stimulation. CONCLUSIONS: Our findings provide the first in vivo data for the molecular pathway required for intercalated disc Nav1.5 targeting/regulation in heart. Further, these new data identify the basis of an in vivo cellular platform critical for membrane recruitment and regulation of Nav1.5.

An Adaptable Spectrin/Ankyrin-Based Mechanism for Long-Range Organization of Plasma Membranes in Vertebrate Tissues.

Ankyrins are membrane-associated proteins that together with their spectrin partners are responsible for micron-scale organization of vertebrate plasma membranes, including those of erythrocytes, excitable membranes of neurons and heart, lateral membrane domains of columnar epithelial cells, and striated muscle. Ankyrins coordinate functionally related membrane transporters and cell adhesion proteins (15 protein families identified so far) within plasma membrane compartments through independently evolved interactions of intrinsically disordered sequences with a highly conserved peptide-binding groove formed by the ANK repeat solenoid. Ankyrins are coupled to spectrins, which are elongated organelle-sized proteins that form mechanically resilient arrays through cross-linking by specialized actin filaments. In addition to protein interactions, cellular targeting and assembly of spectrin/ankyrin domains also critically depend on palmitoylation of ankyrin-G by aspartate-histidine-histidine-cysteine 5/8 palmitoyltransferases, as well as interaction of beta-2 spectrin with phosphoinositide lipids. These lipid-dependent spectrin/ankyrin domains are not static but are locally dynamic and determine membrane identity through opposing endocytosis of bulk lipids as well as specific proteins. A partnership between spectrin, ankyrin, and cell adhesion molecules first emerged in bilaterians over 500 million years ago. Ankyrin and spectrin may have been recruited to plasma membranes from more ancient roles in organelle transport. The basic bilaterian spectrin-ankyrin toolkit markedly expanded in vertebrates through gene duplications combined with variation in unstructured intramolecular regulatory sequences as well as independent evolution of ankyrin-binding activity by ion transporters involved in action potentials and calcium homeostasis. In addition, giant vertebrate ankyrins with specialized roles in axons acquired new coding sequences by exon shuffling. We speculate that early axon initial segments and epithelial lateral membranes initially were based on spectrin-ankyrin-cell adhesion molecule assemblies and subsequently served as "incubators," where ion transporters independently acquired ankyrin-binding activity through positive selection.

A single divergent exon inhibits ankyrin-B association with the plasma membrane.

Vertebrate ankyrin-B and ankyrin-G exhibit divergent subcellular localization and function despite their high sequence and structural similarity and common origin from a single ancestral gene at the onset of chordate evolution. Previous studies of ankyrin family diversity have focused on the C-terminal regulatory domain. Here, we identify an ankyrin-B-specific linker peptide connecting the ankyrin repeat domain to the ZU52-UPA module that inhibits binding of ankyrin-B to membrane protein partners E-cadherin and neurofascin 186 and prevents association of ankyrin-B with epithelial lateral membranes as well as neuronal plasma membranes. The residues of the ankyrin-B linker required for autoinhibition are encoded by a small exon that is highly divergent between ankyrin family members but conserved in the ankyrin-B lineage. We show that the ankyrin-B linker suppresses activity of the ANK repeat domain through an intramolecular interaction, likely with a groove on the surface of the ANK repeat solenoid, thereby regulating the affinities between ankyrin-B and its binding partners. These results provide a simple evolutionary explanation for how ankyrin-B and ankyrin-G have acquired striking differences in their plasma membrane association while maintaining overall high levels of sequence similarity.

E-cadherin polarity is determined by a multifunction motif mediating lateral membrane retention through ankyrin-G and apical-lateral transcytosis through clathrin.

We report a highly conserved motif in the E-cadherin juxtamembrane domain that determines apical-lateral polarity by conferring both restricted mobility at the lateral membrane and transcytosis of apically mis-sorted protein to the lateral membrane. Mutations causing either increased lateral membrane mobility or loss of apical-lateral transcytosis result in partial mis-sorting of E-cadherin in Madin-Darby canine kidney cells. However, loss of both activities results in complete loss of polarity. We present evidence that residues required for restricted mobility mediate retention at the lateral membrane through interaction with ankyrin-G, whereas dileucine residues conferring apical-lateral transcytosis act through a clathrin-dependent process and function in an editing pathway. Ankyrin-G interaction with E-cadherin is abolished by the same mutations resulting in increased E-cadherin mobility. Clathrin heavy chain knockdown and dileucine mutation of E-cadherin both cause the same partial loss of polarity of E-cadherin. Moreover, clathrin knockdown causes no further change in polarity of E-cadherin with dileucine mutation but does completely randomize E-cadherin mutants lacking ankyrin-binding. Dileucine mutation, but not loss of ankyrin binding, prevented transcytosis of apically mis-sorted E-cadherin to the lateral membrane. Finally, neurofascin, which binds ankyrin but lacks dileucine residues, exhibited partial apical-lateral polarity that was abolished by mutation of its ankyrin-binding site but was not affected by clathrin knockdown. The polarity motif thus integrates complementary activities of lateral membrane retention through ankyrin-G and apical-lateral transcytosis of mis-localized protein through clathrin. Together, the combination of retention and editing function to ensure a high fidelity steady state localization of E-cadherin at the lateral membrane.

Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice.

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.

Cysteine 70 of ankyrin-G is S-palmitoylated and is required for function of ankyrin-G in membrane domain assembly.

Ankyrin-G (AnkG) coordinates protein composition of diverse membrane domains, including epithelial lateral membranes and neuronal axon initial segments. However, how AnkG itself localizes to these membrane domains is not understood. We report that AnkG remains on the plasma membrane in Madin-Darby canine kidney (MDCK) cells grown in low calcium, although these cells lack apical-basal polarity and exhibit loss of plasma membrane association of AnkG partners, E-cadherin and ß(2)-spectrin. We subsequently demonstrate using mutagenesis and mass spectrometry that AnkG is S-palmitoylated exclusively at Cys-70, which is located in a loop of the first ankyrin repeat and is conserved in the vertebrate ankyrin family. Moreover, C70A mutation abolishes membrane association of 190-kDa AnkG in MDCK cells grown in low calcium. C70A 190-kDa AnkG fails to restore biogenesis of epithelial lateral membranes in MDCK cells depleted of endogenous AnkG. In addition, C70A 270-kDa AnkG fails to cluster at the axon initial segment of AnkG-depleted cultured hippocampal neurons and fails to recruit neurofascin as well as voltage-gated sodium channels. These effects of C70A mutation combined with evidence for its S-palmitoylation are consistent with a requirement of palmitoylation for targeting and function of AnkG in membrane domain biogenesis at epithelial lateral membranes and neuronal axon initial segments.

Mutation of conserved histidines alters tertiary structure and nanomechanics of consensus ankyrin repeats.

The conserved TPLH tetrapeptide motif of ankyrin repeats (ARs) plays an important role in stabilizing AR proteins, and histidine (TPLH)-to-arginine (TPLR) mutations in this motif have been associated with a hereditary human anemia, spherocytosis. Here, we used a combination of atomic force microscopy-based single-molecule force spectroscopy and molecular dynamics simulations to examine the mechanical effects of His → Arg substitutions in TPLH motifs in a model AR protein, NI6C. Our molecular dynamics results show that the mutant protein is less mechanically stable than the WT protein. Our atomic force microscopy results indicate that the mechanical energy input necessary to fully unfold the mutant protein is only half of that necessary to unfold the WT protein (53 versus 106 kcal/mol). In addition, the ability of the mutant to generate refolding forces is also reduced. Moreover, the mutant protein subjected to cyclic stretch-relax measurements displays mechanical fatigue, which is absent in the WT protein. Taken together, these results indicate that the His → Arg substitutions in TPLH motifs compromise mechanical properties of ARs and suggest that the origin of hereditary spherocytosis may be related to mechanical failure of ARs.

Spectrin- and ankyrin-based membrane domains and the evolution of vertebrates.

Spectrin and ankyrin are membrane skeletal proteins that contribute to mechanical support of plasma membranes and micron-scale organization of diverse membrane-spanning proteins. This chapter provides a plausible scenario for the evolution of ankyrin- and spectrin-based membrane domains with a focus on vertebrates. The analysis integrates recent phylogenetic information with functional analyses of spectrin and ankyrin in erythrocytes, axon initial segments and nodes of Ranvier in neurons, T-tubules and intercalated disks of cardiomyocytes, lateral membrane domains of epithelial cells, and costameres of striated muscle. A core spectrin-ankyrin mechanism for coordinating membrane-spanning proteins and mechanically stabilizing membrane bilayers was expanded in vertebrates by gene duplication events, insertion of giant alternately spliced exons of axonal ankyrins, and a versatile peptide-binding fold of ANK repeats that facilitated acquisition of new protein partners. Cell adhesion molecules (CAM), including dystroglycan, L1 CAM family members, and cadherins, are the earliest examples of membrane-spanning proteins with ankyrin-binding motifs and were all present in urochordates. In contrast, ion channels have continued to evolve ankyrin-binding sites in vertebrates. These considerations suggest a model where proto-domains formed through interaction of ankyrin and spectrin with CAMs. These proto-domains then became populated with ion channels that developed ankyrin-binding activity with selective pressure provided by optimization of physiological function. The best example is the axon initial segment where ankyrin-binding activity evolved sequentially and independently first in L1 CAMs, then in voltage-gated sodium channels, and finally in KCNQ2/3 channels, with the selective advantage of fast and precisely regulated signaling.

Mechanical anisotropy of ankyrin repeats.

Red blood cells are frequently deformed and their cytoskeletal proteins such as spectrin and ankyrin-R are repeatedly subjected to mechanical forces. While the mechanics of spectrin was thoroughly investigated in vitro and in vivo, little is known about the mechanical behavior of ankyrin-R. In this study, we combine coarse-grained steered molecular dynamics simulations and atomic force spectroscopy to examine the mechanical response of ankyrin repeats (ARs) in a model synthetic AR protein NI6C, and in the D34 fragment of native ankyrin-R when these proteins are subjected to various stretching geometry conditions. Our steered molecular dynamics results, supported by AFM measurements, reveal an unusual mechanical anisotropy of ARs: their mechanical stability is greater when their unfolding is forced to propagate from the N-terminus toward the C-terminus (repeats unfold at ~60 pN), as compared to the unfolding in the opposite direction (unfolding force ∼ 30 pN). This anisotropy is also reflected in the complex refolding behavior of ARs. The origin of this unfolding and refolding anisotropy is in the various numbers of native contacts that are broken and formed at the interfaces between neighboring repeats depending on the unfolding/refolding propagation directions. Finally, we discuss how these complex mechanical properties of ARs in D34 may affect its behavior in vivo.

Ankyrin-B interactions with spectrin and dynactin-4 are required for dystrophin-based protection of skeletal muscle from exercise injury.

Costameres are cellular sites of mechanotransduction in heart and skeletal muscle where dystrophin and its membrane-spanning partner dystroglycan distribute intracellular contractile forces into the surrounding extracellular matrix. Resolution of a functional costamere interactome is still limited but likely to be critical for understanding forms of muscular dystrophy and cardiomyopathy. Dystrophin binds a set of membrane-associated proteins (the dystrophin-glycoprotein complex) as well as γ-actin and microtubules and also is required to align sarcolemmal microtubules with costameres. Ankyrin-B binds to dystrophin, dynactin-4, and microtubules and is required for sarcolemmal association of these proteins as well as dystroglycan. We report here that ankyrin-B interactions with ß2 spectrin and dynactin-4 are required for localization of dystrophin, dystroglycan, and microtubules at costameres as well as protection of muscle from exercise-induced injury. Knockdown of dynactin-4 in adult mouse skeletal muscle phenocopied depletion of ankyrin-B and resulted in loss of sarcolemmal dystrophin, dystroglycan, and microtubules. Moreover, mutations of ankyrin-B and of dynactin-4 that selectively impaired binary interactions between these proteins resulted in loss of their costamere-localizing activity and increased muscle fiber fragility as a result of loss of costamere-associated dystrophin and dystroglycan. In addition, costamere-association of dynactin-4 did not require dystrophin but did depend on ß2 spectrin and ankyrin-B, whereas costamere association of ankyrin-B required ß2 spectrin. Together, these results are consistent with a functional hierarchy beginning with ß2 spectrin recruitment of ankyrin-B to costameres. Ankyrin-B then interacts with dynactin-4 and dystrophin, whereas dynactin-4 collaborates with dystrophin in coordinating costamere-aligned microtubules.