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Introduction: Deletion or mutation of members of the spectrin gene family contributes to many neurologic and neuropsychiatric disorders. While each spectrinopathy may generate distinct neuropathology, the study of ßI spectrin's role (Sptb) in the brain has been hampered by the hematologic consequences of its loss. Methods: Jaundiced mice (ja/ja) that lack ßI spectrin suffer a rapidly fatal hemolytic anemia. We have used exchange transfusion of newborn ja/ja mice to blunt their hemolytic pathology, enabling an examination of ßI spectrin deficiency in the mature mouse brain by ultrastructural and biochemical analysis. Results: ßI spectrin is widely utilized throughout the brain as the ßIΣ2 isoform; it appears by postnatal day 8, and concentrates in the CA1,3 region of the hippocampus, dentate gyrus, cerebellar granule layer, cortical layer 2, medial habenula, and ventral thalamus. It is present in a subset of dendrites and absent in white matter. Without ßI spectrin there is a 20% reduction in postsynaptic density size in the granule layer of the cerebellum, a selective loss of ankyrinR in cerebellar granule neurons, and a reduction in the level of the postsynaptic adhesion molecule NCAM. While we find no substitution of another spectrin for ßI at dendrites or synapses, there is curiously enhanced ßIV spectrin expression in the ja/ja brain. Discussion: ßIΣ2 spectrin appears to be essential for refining postsynaptic structures through interactions with ankyrinR and NCAM. We speculate that it may play additional roles yet to be discovered. [ABSTRACT FROM AUTHOR]

Objective To investigate whether the effects of venlafaxine on major depression disorder is associated with ankyrin G. Methods Breed Synapsin-Cre1 and Ankyrin3-floxed mice (Ank3 cKO mice). Ank3 cKO mice and wild type mice were randomly divided into model and control groups. All mice in model group and control group were orally administrated with venlafaxine(1 g/L) or the solvent(normal saline, NS) with the volume of 200 μL,respectively. Depression-related behaviors were examined by sucrose preference test (SPT) and Y maze test. The level of ankyrin G and PSD95 in the cortex of four groups were detected by Western blot. The level of ankyrin G and MAP2 in the in hippocampus of four groups were detected by immunohistochemistry method. ResultsCompared with wt-saline group, the cKO mice in saline showed a significantly decreased preference of sucrose (P＜0.001) and low spontaneous alteration(P＜0.05). Compared with cKO control ones, the venlafaxine model cKO mice showed remarkably increased preference of sucrose(P＜0.001) and more spontaneous alteration(P＜0.05). The level of ankyrin G and PSD-95 in the cortex of venlafaxine cKO mice was much higher than that in control mice(P＜0.01)The level of ankyrin G and MAP2 in the hippocampus of venlafaxine cKO mice were much higher than those of control mice(P＜0.05). Conclusions Venlafaxine alleviates the depression symptoms caused by knocking down Ank3. The mechanism of depression treatment by venlafaxine is potentially associated with levitating ankyrin G level in prefrontal cortex and hippocampus.

Objective To investigate whether the effects of venlafaxine on major depression disorder is associated with ankyrin G. Methods Breed Synapsin-Cre1 and Ankyrin3-floxed mice (Ank3 cKO mice). Ank3 cKO mice and wild type mice were randomly divided into model and control groups. All mice in model group and control group were orally administrated with venlafaxine(1 g/L) or the solvent(normal saline, NS) with the volume of 200 μL,respectively. Depression-related behaviors were examined by sucrose preference test (SPT) and Y maze test. The level of ankyrin G and PSD95 in the cortex of four groups were detected by Western blot. The level of ankyrin G and MAP2 in the in hippocampus of four groups were detected by immunohistochemistry method. ResultsCompared with wt-saline group, the cKO mice in saline showed a significantly decreased preference of sucrose (P＜0.001) and low spontaneous alteration(P＜0.05). Compared with cKO control ones, the venlafaxine model cKO mice showed remarkably increased preference of sucrose(P＜0.001) and more spontaneous alteration(P＜0.05). The level of ankyrin G and PSD-95 in the cortex of venlafaxine cKO mice was much higher than that in control mice(P＜0.01)The level of ankyrin G and MAP2 in the hippocampus of venlafaxine cKO mice were much higher than those of control mice(P＜0.05). Conclusions Venlafaxine alleviates the depression symptoms caused by knocking down Ank3. The mechanism of depression treatment by venlafaxine is potentially associated with levitating ankyrin G level in prefrontal cortex and hippocampus. [ABSTRACT FROM AUTHOR]

IntroductionDeletion or mutation of members of the spectrin gene family contributes to many neurologic and neuropsychiatric disorders. While each spectrinopathy may generate distinct neuropathology, the study of βΙ spectrin’s role (Sptb) in the brain has been hampered by the hematologic consequences of its loss.MethodsJaundiced mice (ja/ja) that lack βΙ spectrin suffer a rapidly fatal hemolytic anemia. We have used exchange transfusion of newborn ja/ja mice to blunt their hemolytic pathology, enabling an examination of βΙ spectrin deficiency in the mature mouse brain by ultrastructural and biochemical analysis.ResultsβΙ spectrin is widely utilized throughout the brain as the βΙΣ2 isoform; it appears by postnatal day 8, and concentrates in the CA1,3 region of the hippocampus, dentate gyrus, cerebellar granule layer, cortical layer 2, medial habenula, and ventral thalamus. It is present in a subset of dendrites and absent in white matter. Without βΙ spectrin there is a 20% reduction in postsynaptic density size in the granule layer of the cerebellum, a selective loss of ankyrinR in cerebellar granule neurons, and a reduction in the level of the postsynaptic adhesion molecule NCAM. While we find no substitution of another spectrin for βΙ at dendrites or synapses, there is curiously enhanced βΙV spectrin expression in the ja/ja brain.DiscussionβΙΣ2 spectrin appears to be essential for refining postsynaptic structures through interactions with ankyrinR and NCAM. We speculate that it may play additional roles yet to be discovered.

Variants in the high confident autism spectrum disorder (ASD) gene ANK2 target both ubiquitously expressed 220 kDa ankyrin-B and neurospecific 440 kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3 A (Sema 3 A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.

Background: Hereditary spherocytosis (HS) is characterized by spherocytes on the peripheral smear and heterogeneous clinical presentation (mild, moderate, moderate/severe, and severe) depending upon the severity of hemolytic anemia, jaundice, and splenomegaly. The underlying cause is defects in various red blood cell membrane proteins due to pathogenic mutations. Genotype-phenotype correlation is known. Clinical Description: We report a consanguineous family with a healthy daughter, multiple pregnancy losses due to fetal hydrops, and the current daughter. She was diagnosed with fetal anemia at 22 weeks gestation, developed fetal hydrops, was born premature and developed respiratory distress, severe anemia, severe jaundice, and postnatal hemolysis. Management: The case was managed since intrauterine life till date by multispecialty coordination. Intrauterine transfusions were given during pregnancy. In the neonatal period, she received surfactant for respiratory distress syndrome, double volume exchange transfusion and phototherapy for the jaundice, and repeated blood transfusions for the hemolytic anemia, that is still continuing. Rational and sequential investigations were planned and a systematic approach used. She was diagnosed with severe HS due to a homozygous likely pathogenic variant in the SPTA1 gene (affecting formation of spectrin), and heterozygous variant of uncertain significance in the ANK1 gene (that encodes ankyrin). At 3 years she has transfusion dependent hemolytic anemia, moderate splenomegaly, and age appropriate growth and development. Conclusions: HS is a rare but manageable (albeit challenging) cause of nonimmune hydrops fetalis. Genetic diagnosis allows precise management and family counselling.

The ankyrin proteins (Ankyrin-R, Ankyrin-B, and Ankyrin-G) are a family of scaffolding, or membrane adaptor proteins necessary for the regulation and targeting of several types of ion channels and membrane transporters throughout the body. These include voltage-gated sodium, potassium, and calcium channels in the nervous system, heart, lungs, and muscle. At these sites, ankyrins recruit ion channels, and other membrane proteins, to specific subcellular domains, which are then stabilized through ankyrin’s interaction with the submembranous spectrin-based cytoskeleton. Several recent studies have expanded our understanding of both ankyrin expression and their ion channel binding partners. This review provides an updated overview of ankyrin proteins and their known channel and transporter interactions. We further discuss several potential avenues of future research that would expand our understanding of these important organizational proteins.

A novel function for the L1 cell adhesion molecule, which binds the actin adaptor protein Ankyrin was identified in constraining dendritic spine density on pyramidal neurons in the mouse neocortex. In an L1-null mouse mutant increased spine density was observed on apical but not basal dendrites of pyramidal neurons in diverse cortical areas (prefrontal cortex layer 2/3, motor cortex layer 5, visual cortex layer 4. The Ankyrin binding motif (FIGQY) in the L1 cytoplasmic domain was critical for spine regulation, as demonstrated by increased spine density and altered spine morphology in the prefrontal cortex of a mouse knock-in mutant (L1YH) harboring a tyrosine (Y) to histidine (H) mutation in the FIGQY motif, which disrupted L1-Ankyrin association. This mutation is a known variant in the human L1 syndrome of intellectual disability. L1 was localized by immunofluorescence staining to spine heads and dendrites of cortical pyramidal neurons. L1 coimmunoprecipitated with Ankyrin B (220 kDa isoform) from lysates of wild type but not L1YH forebrain. This study provides insight into the molecular mechanism of spine regulation and underscores the potential for this adhesion molecule to regulate cognitive and other L1-related functions that are abnormal in the L1 syndrome. [ABSTRACT FROM AUTHOR]

Hereditary Spherocytosis (HS) is a common cause of hemolytic anemia varying from mild to severe hemolysis due to defects in red cell membrane protein genes, namely ANK1, SPTB, SPTA1, SLC4A1, and EPB42. These genes are considerably very large spaning 40–50 exons making gene-by-gene analysis costly and laborious by conventional methods. In this study, we explored 26 HS patients harboring 21 ANK1 variants identified by next-generation sequencing (NGS), characteristics and spectrum of the detected ANK1variants were analyzed in this study. Clinically, all the HS patients showed moderate to severe transfusion-dependent hemolytic anemia, some requiring splenectomy. We identified 13 novel and 8 reported variants, mainly 9 frameshifts, 2 missense, 6 nonsense, and 4 splice site ANK1 variants, using NGS technology. Frameshifts were remarkably the most common variant type seen in Indian HS patients with ANK1 gene defects. We have also explored expression levels of red cell membrane ankyrin protein by flow cytometry in 14 HS patients with ANK1 gene defects and a significant reduction in ankyrin protein expression has been found. This report mainly illustrates the molecular and phenotypic heterogeneity of ANK1 variants causing HS in Indian patients. Ankyrin-1 mutations are a significant cause of loss of function in dominant HS in the Indian population. Comprehensive genetic and phenotypic evaluation assists in implementing the knowledge of genetic patterns and spectrum of ANK1 gene variants, providing molecular support for HS diagnosis. [ABSTRACT FROM AUTHOR]

[Display omitted] • Doublecortin-like kinase 1 (DCLK1) promotes spine development in mouse cortex. • An inducible mouse line was generated to delete DCLK1 isoforms from cortical pyramidal neurons. • DCLK1 binds the cell adhesion molecule NrCAM at its Ankyrin interaction motif. The L1 cell adhesion molecule NrCAM (Neuron-glia related cell adhesion molecule) functions as a co-receptor for secreted class 3 Semaphorins to prune subpopulations of dendritic spines on apical dendrites of pyramidal neurons in the developing mouse neocortex. The developing spine cytoskeleton is enriched in actin filaments, but a small number of microtubules have been shown to enter the spine apparently trafficking vesicles to the membrane. Doublecortin-like kinase 1 (DCLK1) is a member of the Doublecortin (DCX) family of microtubule-binding proteins with serine/threonine kinase activity. To determine if DCLK1 plays a role in spine remodeling, we generated a tamoxifen-inducible mouse line (Nex1Cre-ERT2: DCLK1flox/flox: RCE) to delete microtubule binding isoforms of DCLK1 from pyramidal neurons during postnatal stages of spine development. Homozygous DCLK1 conditional mutant mice exhibited decreased spine density on apical dendrites of pyramidal neurons in the prefrontal cortex (layer 2/3). Mature mushroom spines were selectively decreased upon DCLK1 deletion but dendritic arborization was unaltered. Mutagenesis and binding studies revealed that DCLK1 bound NrCAM at the conserved FIGQY1231 motif in the NrCAM cytoplasmic domain, a known interaction site for the actin-spectrin adaptor Ankyrin. These findings demonstrate in a novel mouse model that DCLK1 facilitates spine growth and maturation on cortical pyramidal neurons in the mouse prefrontal cortex. [ABSTRACT FROM AUTHOR]

Vertically transmitted (VT) microbial symbionts play a vital role in the evolution of their insect hosts. A longstanding question in symbiont research is what genes help promote long-term stability of vertically transmitted lifestyles. Symbiont success in insect hosts is due in part to expression of beneficial or manipulative phenotypes that favor symbiont persistence in host populations. In Spiroplasma, these phenotypes have been linked to toxin and virulence domains among a few related strains. However, these domains also appear frequently in phylogenetically distant Spiroplasma, and little is known about their distribution across the Spiroplasma genus. In this study, we present the complete genome sequence of the Spiroplasma symbiont of Drosophila atripex, a non-manipulating member of the Ixodetis clade of Spiroplasma, for which genomic data are still limited. We perform a genus-wide comparative analysis of toxin domains implicated in defensive and reproductive phenotypes. From 12 VT and 31 non-VT Spiroplasma genomes, ribosome-inactivating proteins (RIPs), OTU-like cysteine proteases (OTUs), ankyrins, and ETX/MTX2 domains show high propensity for VT Spiroplasma compared to non-VT Spiroplasma. Specifically, OTU and ankyrin domains can be found only in VT-Spiroplasma, and RIP domains are found in all VT Spiroplasma and three non-VT Spiroplasma. These domains are frequently associated with Spiroplasma plasmids, suggesting a possible mechanism for dispersal and maintenance among heritable strains. Searching insect genome assemblies available on public databases uncovered uncharacterized Spiroplasma genomes from which we identified several spaid-like genes encoding RIP, OTU, and ankyrin domains, suggesting functional interactions among those domain types. Our results suggest a conserved core of symbiont domains play an important role in the evolution and persistence of VT Spiroplasma in insects.

Abstract Notch signalling pathway plays a key role in metazoan biology by contributing to resolution of binary decisions in the life cycle of cells during development. Outcomes such as proliferation/differentiation dichotomy are resolved by transcriptional remodelling that follows a switch from Notchon to Notchoff state, characterised by dissociation of Notch intracellular domain (NICD) from DNA-bound RBPJ. Here we provide evidence that transitioning to the Notchoff state is regulated by heat flux, a phenomenon that aligns resolution of fate dichotomies to mitochondrial activity. A combination of phylogenetic analysis and computational biochemistry was utilised to disclose structural adaptations of Notch1 ankyrin domain that enabled function as a sensor of heat flux. We then employed DNA-based micro-thermography to measure heat flux during brain development, followed by analysis in vitro of the temperature-dependent behaviour of Notch1 in mouse neural progenitor cells. The structural capacity of NICD to operate as a thermodynamic sensor in metazoans stems from characteristic enrichment of charged acidic amino acids in β-hairpins of the ankyrin domain that amplify destabilising inter-residue electrostatic interactions and render the domain thermolabile. The instability emerges upon mitochondrial activity which raises the perinuclear and nuclear temperatures to 50 °C and 39 °C, respectively, leading to destabilization of Notch1 transcriptional complex and transitioning to the Notchoff state. Notch1 functions a metazoan thermodynamic sensor that is switched on by intercellular contacts, inputs heat flux as a proxy for mitochondrial activity in the Notchon state via the ankyrin domain and is eventually switched off in a temperature-dependent manner. Video abstract

A novel function for the L1 cell adhesion molecule, which binds the actin adaptor protein Ankyrin was identified in constraining dendritic spine density on pyramidal neurons in the mouse neocortex. In an L1-null mouse mutant increased spine density was observed on apical but not basal dendrites of pyramidal neurons in diverse cortical areas (prefrontal cortex layer 2/3, motor cortex layer 5, visual cortex layer 4. The Ankyrin binding motif (FIGQY) in the L1 cytoplasmic domain was critical for spine regulation, as demonstrated by increased spine density and altered spine morphology in the prefrontal cortex of a mouse knock-in mutant (L1YH) harboring a tyrosine (Y) to histidine (H) mutation in the FIGQY motif, which disrupted L1-Ankyrin association. This mutation is a known variant in the human L1 syndrome of intellectual disability. L1 was localized by immunofluorescence staining to spine heads and dendrites of cortical pyramidal neurons. L1 coimmunoprecipitated with Ankyrin B (220 kDa isoform) from lysates of wild type but not L1YH forebrain. This study provides insight into the molecular mechanism of spine regulation and underscores the potential for this adhesion molecule to regulate cognitive and other L1-related functions that are abnormal in the L1 syndrome.

The emerging and ongoing outbreak of human monkeypox (hMPX) in 2022 is a serious global threat. An understanding of the evolution of the monkeypox virus (MPXV) at the single-gene level may provide clues for exploring the unique aspects of the current outbreak: rapidly expanding and sustained human-to-human transmission. For the current investigation, alleles of 156 MPXV coding genes (which account for >95% of the genomic sequence) have been gathered from roughly 1,500 isolates, including those responsible for the previous outbreaks. Using a range of molecular evolution approaches, we demonstrated that intra-species homologous recombination has a negligible effect on MPXV evolution. Despite the fact that the majority of the MPXV genes (64.10%) were subjected to negative selection at the whole gene level, 10 MPXV coding genes (MPXVgp004, 010, 012, 014, 044, 098, 138, 178, 188, and 191) were found to have a total of 15 codons or amino acid sites that are known to evolve under positive Darwinian selection. Except for MPXVgp138, almost all of these genes encode proteins that interact with the host. Of these, five ankyrin proteins (MPXVgp004, 010, 012, 178, and 188) and one Bcl-2-like protein (MPXVgp014) are involved in poxviruses’ host range determination. We discovered that the majority (80%) of positive amino acid substitutions emerged several decades ago, indicating that these sites have been under constant selection pressure and that more adaptable alleles have been circulating in the natural reservoir. This finding was also supported by the minimum spanning networks of the gene alleles. The three positive amino acid substitutions (T/A426V in MPXVgp010, A423D in MPXVgp012, and S105L in MPXVgp191) appeared in 2019 or 2022, indicating that they would be crucial for the virus’ eventual adaptation to humans. Protein modeling suggests that positive amino acid substitutions may affect protein functions in a variety of ways. Further study should focus on revealing the biological effects of positive amino acid substitutions in the genes for viral adaptation to humans, virulence, transmission, and so on. Our study advances knowledge of MPXV’s adaptive mechanism and provides insights for exploring factors that are responsible for the unique aspects of the current outbreak.

Transmembrane channel-like (TMC) proteins are expressed throughout the animal kingdom and are thought to encode components of ion channels. Mammals express eight TMCs (mTMC1–8), two of which (mTMC1 and mTMC2) are subunits of mechanotransduction channels. C. elegans expresses two TMCs (TMC-1 and TMC-2), which mediate mechanosensation, egg laying, and alkaline sensing. The mechanisms by which nematode TMCs contribute to such diverse physiological processes and their functional relationship to mammalian mTMCs is unclear. Here, we show that association with accessory proteins tunes nematode TMC-1 to divergent sensory functions. In addition, distinct TMC-1 domains enable touch and alkaline sensing. Strikingly, these domains are segregated in mammals between mTMC1 and mTMC3. Consistent with these findings, mammalian mTMC1 can mediate mechanosensation in nematodes, while mTMC3 can mediate alkaline sensation. We conclude that sequence diversification and association with accessory proteins has led to the emergence of TMC protein complexes with diverse properties and physiological functions. [Display omitted] • Accessory proteins tune TMC-1 to different sensory functions • Distinct TMC1 domains mediate mechanosensation and alkaline sensing • Mammalian TMC-1 supports mechanosensation in nematodes • Mammalian TMC-3 supports alkaline sensing in nematodes Jiang et al. demonstrate that combinations of different accessory proteins tune nematode TMC-1 to different sensory functions. In addition, distinct structural domains of nematode TMC-1 mediate mechanosensation and alkaline sensing. These domains are segregated between mammalian mTMC1 and mTMC3, and mTMC1 supports mechanosensation in nematodes, while mTMC3 supports alkaline sensing. [ABSTRACT FROM AUTHOR]

Background/Aims: Mercury (Hg) is a heavy metal widespread in all environmental compartments as one of the most hazardous pollutants. Human exposure to this natural element is detrimental for several cellular types including erythrocytes (RBC) that accumulate Hg mainly bound to the SH groups of different cellular components, including protein cysteine residues. The cellular membrane represents a major target of Hg-induced damage in RBC with loss of physiological phospholipid asymmetry, due to phosphatidylserine (PS) exposure to the external membrane leaflet. To investigate Hg-induced cytotoxicity at the molecular level, the possible interaction of this heavy metal with RBC membrane proteins was investigated. Furthermore, Hg-induced alterations in band 3 protein (B3p) transport function, PS-exposing macrovesicle (MVs) formation and morphological changes were assessed. Methods: For this aim, human RBC were treated in vitro with different HgCl2 concentrations (range 10-40 M) and the electrophoretic profile of membrane proteins as well as the expression levels of Ankyrin and Flottilin-2 evaluated by SDS-PAGE and Western blot, respectively. The effect of alterations in these proteins on RBC morphology was evaluated by digital holographic microscopy and anionic transport efficiency of B3p was evaluated as sulphate uptake. Finally, PS-bearing MVs were quantified by annexin-V binding using FACS analysis. Results: Findings presented in this paper indicate that RBC exposure to HgCl2 induces modifications in the electrophoretic profile of membrane protein fraction. Furthermore, our study reveals the Hg induced alterations of specific membrane proteins, such as Ankyrin, a protein essential for membrane-cytoskeleton linkage and Flotillin-2, a major integral protein of RBC lipid rafts, likely responsible for decreased membrane stability and increased fragmentations. Accordingly, under the same experimental conditions, RBC morphological changes and PS-bearing MVs release are observed. Finally, RBC treatment significantly affects the B3p-mediated anionic transport, that we report reduced upon HgCl2 treatment in a dose dependent manner. Conclusion: Altogether, the findings reported in this paper confirm that RBC are particularly vulnerable to Hg toxic effect and provide new insight in the Hg-induced protein modification in human RBC affecting the complex biological system of cellular membrane. In particular, Hg could induce dismantle of vertical cohesion between the plasma membrane and cytoskeleton as well as destabilization of lateral linkages of functional domains. Consequently, decreased membrane deformability could impair RBC capacity to deal with the shear forces in the circulation increasing membrane fragmentations. Furthermore, findings described in this paper have also significant implication in RBC physiology, particularly related to gas exchanges. [ABSTRACT FROM AUTHOR]

Obscurin is a large scaffolding protein in striated muscle that maintains sarcolemmal integrity and aligns the sarcoplasmic reticulum with the underlying contractile machinery. Ankyrins are a family of adaptor proteins with some isoforms that interact with obscurin. Previous studies have examined obscurin interacting with individual ankyrins. In this study, we demonstrate that two different ankyrins interact with obscurin's carboxyl terminus via independent ankyrin-binding domains (ABDs). Using in-vitro binding assays, co-precipitation assays, and FLIM-FRET analysis, we show that obscurin interacts with small ankyrin 1.5 (sAnk1.5) and the muscle-specific ankyrin-G isoform (AnkG107). While there is no direct interaction between sAnk1.5 and AnkG107, obscurin connects the two ankyrins both in vitro and in cells. Moreover, AnkG107 recruits β-spectrin to this macromolecular protein complex and mutating obscurin's ABDs disrupts complex formation. To further characterize AnkG107 interaction with obscurin, we measure obscurin-binding to different AnkG107 isoforms expressed in the heart and find that the first obscurin-binding domain in AnkG107 principally mediates this interaction. We also find that AnkG107 does not bind to filamin-C and displays minimal binding to plectin-1 compared to obscurin. Finally, both sAnk1.5-GFP and AnkG107-CTD-RFP are targeted to the M-lines of ventricular cardiomyocytes and mutating their obscurin-binding domains disrupts the M-line localization of these ankyrin constructs. Altogether, these findings support a model in which obscurin can interact via independent binding domains with two different ankyrin protein complexes to target them to the sarcomeric M-line of ventricular cardiomyocytes. [Display omitted] • AnkG107 and sAnk1.5 bind obscurin via independent binding sites. • Obscurin forms a complex with AnkG107 and sAnk1.5 in vitro and in cells. • AnkG107 recruits β-spectrin to a complex of obscurin, sAnk1.5, and AnkG107. • Removing AnkG107's first obscurin-binding domain disrupts obscurin interaction. • AnkG107 obscurin-binding domains do not interact with filamin-C and minimally with plectin-1. [ABSTRACT FROM AUTHOR]

Plasmodium proteins are exported to the erythrocyte cytoplasm to create an environment that supports parasite replication. Although hundreds of proteins are predicted to be exported through Plasmodium export element (PEXEL)-dependent and -independent mechanisms, the functions of exported proteins are largely uncharacterized. In this study, we used a biochemical screening approach to identify putative exported P. falciparum proteins that bound to inside-out vesicles prepared from erythrocytes. Out of 69 P. falciparum PEXEL-motif proteins tested, 18 bound to inside-out vesicles (IOVs) in two or more independent assays. Using co-affinity purifications followed by mass spectrometry, pairwise co-purification experiments, and the split-luciferase assay, we identified 31 putative protein–protein interactions between erythrocyte cytoskeletal proteins and predicted exported P. falciparum proteins. We further showed that PF3D7_1401600 binds to the spectrin-binding domain of erythrocyte ankyrin via its MESA erythrocyte cytoskeleton binding (MEC) motif and to the N-terminal domains of ankyrin and 4.1R through a fragment that required an intact Plasmodium helical interspersed sub-telomeric (PHIST) domain. Introduction of PF3D7_1401600 into erythrocyte ghosts increased retention in the microsphiltration assay, consistent with previous data that reported a reduction of rigidity in red blood cells infected with PF3D7_1401600-deficient parasites. [ABSTRACT FROM AUTHOR]

Spectrins form a submembranous cytoskeleton proposed to confer strength and flexibility to neurons and to participate in ion channel clustering at axon initial segments (AIS) and nodes of Ranvier. Neuronal spectrin cytoskeletons consist of diverse β subunits and αII spectrin. Although αII spectrin is found in neurons in both axonal and somatodendritic domains, using proteomics, biochemistry, and superresolution microscopy, we show that αII and βIV spectrin interact and form a periodic AIS cytoskeleton. To determine the role of spectrins in the nervous system, we generated Sptan1f/f mice for deletion of CNS αII spectrin. We analyzed αII spectrin-deficient mice of both sexes and found that loss of αII spectrin causes profound reductions in all β spectrins. αII spectrin-deficient mice die before 1 month of age and have disrupted AIS and many other neurological impairments including seizures, disrupted cortical lamination, and widespread neurodegeneration. These results demonstrate the importance of the spectrin cytoskeleton both at the AIS and throughout the nervous system.SIGNIFICANCE STATEMENT Spectrin cytoskeletons play diverse roles in neurons, including assembly of excitable domains such as the axon initial segment (AIS) and nodes of Ranvier. However, the molecular composition and structure of these cytoskeletons remain poorly understood. Here, we show that αII spectrin partners with βIV spectrin to form a periodic cytoskeleton at the AIS. Using a new αII spectrin conditional knock-out mouse, we show that αII spectrin is required for AIS assembly, neuronal excitability, cortical lamination, and to protect against neurodegeneration. These results demonstrate the broad importance of spectrin cytoskeletons for nervous system function and development and have important implications for nervous system injuries and diseases because disruption of the spectrin cytoskeleton is a common molecular pathology.

Notch signalling pathway plays a key role in metazoan biology by contributing to resolution of binary decisions in the life cycle of cells during development. Outcomes such as proliferation/differentiation dichotomy are resolved by transcriptional remodelling that follows a switch from Notchon to Notchoff state, characterised by dissociation of Notch intracellular domain (NICD) from DNA-bound RBPJ. Here we provide evidence that transitioning to the Notchoff state is regulated by heat flux, a phenomenon that aligns resolution of fate dichotomies to mitochondrial activity. A combination of phylogenetic analysis and computational biochemistry was utilised to disclose structural adaptations of Notch1 ankyrin domain that enabled function as a sensor of heat flux. We then employed DNA-based micro-thermography to measure heat flux during brain development, followed by analysis in vitro of the temperature-dependent behaviour of Notch1 in mouse neural progenitor cells. The structural capacity of NICD to operate as a thermodynamic sensor in metazoans stems from characteristic enrichment of charged acidic amino acids in β-hairpins of the ankyrin domain that amplify destabilising inter-residue electrostatic interactions and render the domain thermolabile. The instability emerges upon mitochondrial activity which raises the perinuclear and nuclear temperatures to 50 °C and 39 °C, respectively, leading to destabilization of Notch1 transcriptional complex and transitioning to the Notchoff state. Notch1 functions a metazoan thermodynamic sensor that is switched on by intercellular contacts, inputs heat flux as a proxy for mitochondrial activity in the Notchon state via the ankyrin domain and is eventually switched off in a temperature-dependent manner. 4Fh46PNx-YRBNHf862t6mS Video abstract [ABSTRACT FROM AUTHOR]

A designed repeat scaffold protein (AnkGAG1D4) recognizing the human immunodeficiency virus-1 (HIV-1) capsid (CA) was formerly established with antiviral assembly. Here, we investigated the molecular mechanism of AnkGAG1D4 function during the late stages of the HIV-1 replication cycle. By applying stimulated emission-depletion (STED) microscopy, Gag polymerisation was interrupted at the plasma membrane. Disturbance of Gag polymerisation triggered Gag accumulation inside producer cells and trapping of the CD81 tetraspanin on the plasma membrane. Moreover, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) experiments were performed to validate the packaging efficiency of RNAs. Our results advocated that AnkGAG1D4 interfered with the Gag precursor protein from selecting HIV-1 and cellular RNAs for encapsidation into viral particles. These findings convey additional information on the antiviral activity of AnkGAG1D4 at late stages of the HIV-1 life cycle, which is potential for an alternative anti-HIV molecule. [ABSTRACT FROM AUTHOR]

Hereditary spherocytosis (HS) is the most frequently observed chronic non-immune hemolytic disorder caused by altered red cell membrane function. SPTB gene mutation is one of the most common causes of HS, but pathogenicity analyses and pathogenesis research on these mutations have not been widely conducted. In this study, a novel heterozygous mutation of the SPTB gene (c.1509_1518del; p.K503Nfs*67) was identified in a Chinese family with HS by whole-exome sequencing (WES) and was then confirmed by Sanger sequencing. Next, the pathogenicity and pathogenesis of this mutation were studied using peripheral blood. We found that this mutation disrupted the synthesis and localization of β-spectrin and weakened the interaction between β-spectrin and ankyrin, which may be caused by the nonsense-mediated mRNA degradation pathway. These changes lead to the transformation of discoid erythrocytes into spherocytes, resulting in hemolytic anemia. Therefore, we classified this novel mutation as a pathogenic mutation leading to loss-of-function of β-spectrin. It would be insightful to perform the same mutation test and to provide genetic counseling to other relatives of the proband. Our study increases the current understanding of the molecular mechanisms related to mutations in SPTB. [ABSTRACT FROM AUTHOR]

Dysfunction in sodium channels and their ankyrin scaffolding partners have both been implicated in neurodevelopmental disorders, including autism spectrum disorder (ASD). In particular, the genes SCN2A , which encodes the sodium channel Na V 1.2, and ANK2 , which encodes ankyrin-B, have strong ASD association. Recent studies indicate that ASD-associated haploinsufficiency in Scn2a impairs dendritic excitability and synaptic function in neocortical pyramidal cells, but how Na V 1.2 is anchored within dendritic regions is unknown. Here, we show that ankyrin-B is essential for scaffolding Na V 1.2 to the dendritic membrane of mouse neocortical neurons and that haploinsufficiency of Ank2 phenocopies intrinsic dendritic excitability and synaptic deficits observed in Scn2a +/− conditions. These results establish a direct, convergent link between two major ASD risk genes and reinforce an emerging framework suggesting that neocortical pyramidal cell dendritic dysfunction can contribute to neurodevelopmental disorder pathophysiology. • Ankyrin-B (Ank2) is expressed throughout neocortical pyramidal cell dendrites • Ankyrin-B scaffolds the sodium channel Na V 1.2 (Scn2a) to dendritic membranes • Haploinsufficiency in either Ank2 or Scn2a impairs dendritic excitability • Shared function suggests that dendritic dysfunction contributes to ASD etiology Autism spectrum disorder (ASD) is associated with dysfunction in hundreds of genes. How dysfunction in ASD-associated genes converges on shared biological mechanisms remains an open question. Here, Nelson et al. show that one ASD-associated gene, Ank2 , scaffolds another, Scn2a , in neocortical pyramidal cell dendrites, with shared effects on dendritic integration. [ABSTRACT FROM AUTHOR]

The knob-associated histidine-rich protein (KAHRP) plays a pivotal role in the pathophysiology of Plasmodium falciparum malaria by forming membrane protrusions in infected erythrocytes, which anchor parasite- encoded adhesins to the membrane skeleton. The resulting sequestration of parasitized erythrocytes in the microvasculature leads to severe disease. Despite KAHRP being an important virulence factor, its physical location within the membrane skeleton is still debated, as is its function in knob formation. Here, we show by super-resolution microscopy that KAHRP initially associates with various skeletal components, including ankyrin bridges, but eventually colocalizes with remnant actin junctions. We further present a 35 Å map of the spiral scaffold underlying knobs and show that a KAHRP-targeting nanoprobe binds close to the spiral scaffold. Single-molecule localization microscopy detected ~60 KAHRP molecules/knob. We propose a dynamic model of KAHRP organization and a function of KAHRP in attaching other factors to the spiral scaffold. [ABSTRACT FROM AUTHOR]

Abstract Hereditary spherocytosis (HS) is often misdiagnosed due to lack of specific diagnostic methods. Our study summarized clinical characteristics and described the diagnostic workflow for mild and moderate HS in Chinese individuals, using data from 20 adults, 8 of whom presented a familial history for HS. We used scanning electron microscopy (SEM) to diagnose HS. We observed reduced eosin maleimide fluorescence activity (5.50 mean channel fluorescence (MCF) units) in the 10 cases of HS, which differed significantly when compared with 10 normal adults (15.50 units), iron deficiency anemia (15.50 MCF units), and megaloblastic anemia (12.00 MCF units) values (P

Variants in the high confident autism spectrum disorder (ASD) gene ANK2 target both ubiquitously expressed 220 kDa ankyrin-B and neurospecific 440 kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3 A (Sema 3 A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.

Neuronal ankyrins cluster and link membrane proteins to the actin and spectrin-based cytoskeleton. Among the three vertebrate ankyrins, little is known about neuronal Ankyrin-R (AnkR). We report AnkR is highly enriched in Pv+ fast-spiking interneurons in mouse and human. We identify AnkR-associated protein complexes including cytoskeletal proteins, cell adhesion molecules (CAMs), and perineuronal nets (PNNs). We show that loss of AnkR from forebrain interneurons reduces and disrupts PNNs, decreases anxiety-like behaviors, and changes the intrinsic excitability and firing properties of Pv+ fast-spiking interneurons. These changes are accompanied by a dramatic reduction in Kv3.1b K+ channels. We identify a novel AnkR-binding motif in Kv3.1b, and show that AnkR is both necessary and sufficient for Kv3.1b membrane localization in interneurons and at nodes of Ranvier. Thus, AnkR regulates Pv+ fast-spiking interneuron function by organizing ion channels, CAMs, and PNNs, and linking these to the underlying β1 spectrin-based cytoskeleton.

A designed repeat scaffold protein (AnkGAG1D4) recognizing the human immunodeficiency virus-1 (HIV-1) capsid (CA) was formerly established with antiviral assembly. Here, we investigated the molecular mechanism of AnkGAG1D4 function during the late stages of the HIV-1 replication cycle. By applying stimulated emission-depletion (STED) microscopy, Gag polymerisation was interrupted at the plasma membrane. Disturbance of Gag polymerisation triggered Gag accumulation inside producer cells and trapping of the CD81 tetraspanin on the plasma membrane. Moreover, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) experiments were performed to validate the packaging efficiency of RNAs. Our results advocated that AnkGAG1D4 interfered with the Gag precursor protein from selecting HIV-1 and cellular RNAs for encapsidation into viral particles. These findings convey additional information on the antiviral activity of AnkGAG1D4 at late stages of the HIV-1 life cycle, which is potential for an alternative anti-HIV molecule.

Mammalian brain circuits are wired by dynamic formation and remodeling during development to produce a balance of excitatory and inhibitory synapses. Synaptic regulation is mediated by a complex network of proteins including immunoglobulin (Ig)- class cell adhesion molecules (CAMs), structural and signal-transducing components at the pre- and post-synaptic membranes, and the extracellular protein matrix. This review explores the current understanding of developmental synapse regulation mediated by L1 and NCAM family CAMs. Excitatory and inhibitory synapses undergo formation and remodeling through neuronal CAMs and receptor-ligand interactions. These responses result in pruning inactive dendritic spines and perisomatic contacts, or synaptic strengthening during critical periods of plasticity. Ankyrins engage neural adhesion molecules of the L1 family (L1-CAMs) to promote synaptic stability. Chondroitin sulfates, hyaluronic acid, tenascin-R, and linker proteins comprising the perineuronal net interact with L1-CAMs and NCAM, stabilizing synaptic contacts and limiting plasticity as critical periods close. Understanding neuronal adhesion signaling and synaptic targeting provides insight into normal development as well as synaptic connectivity disorders including autism, schizophrenia, and intellectual disability.

The development of a polarized neuron relies on the selective transport of proteins to axons and dendrites. Although it is well known that the microtubule cytoskeleton has a central role in establishing neuronal polarity, how its specific organization is established and maintained is poorly understood. Using the in vivo model system Caenorhabditis elegans, we found that the highly conserved UNC-119 protein provides a link between the membrane-associated Ankyrin (UNC-44) and the microtubule-associated CRMP (UNC-33). Together they form a periodic membrane-associated complex that anchors axonal and dendritic microtubule bundles to the cortex. This anchoring is critical to maintain microtubule organization by opposing kinesin-1 powered microtubule sliding. Disturbing this molecular complex alters neuronal polarity and causes strong developmental defects of the nervous system leading to severely paralyzed animals.

Abstract Background How regulatory networks incorporate additional components and how novel genes are functionally integrated into well-established developmental processes are two important and intertwined questions whose answers have major implications for understanding the evolution of development. We recently discovered a set of lineage-restricted genes with strong and specific expression patterns along the dorsal-ventral (DV) axis of the embryo of the wasp Nasonia that may serve as a powerful system for addressing these questions. We sought to both understand the evolutionary history of these genes and to determine their functions in the Nasonia DV patterning system. Results We have found that the novel DV genes are part of a large family of rapidly duplicating and diverging ankyrin domain-encoding genes that originated most likely by horizontal transfer from a prokaryote in a common ancestor of the wasp superfamily Chalcidoidea. We tested the function of those ankyrin-encoding genes expressed along the DV axis and found that they participate in early embryonic DV patterning. We also developed a new wasp model system (Melittobia) and found that some functional integration of ankyrin genes have been preserved for over 90 million years. Conclusions Our results indicate that regulatory networks can incorporate novel genes that then become necessary for stable and repeatable outputs. Even a modest role in developmental networks may be enough to allow novel or duplicate genes to be maintained in the genome and become fully integrated network components.

Alternative splicing is a powerful mechanism for molecular and functional diversification. In neurons, alternative splicing extensively controls various developmental steps as well as the plasticity and remodeling of neuronal activity in the adult brain. The axon initial segment (AIS) is the specialized compartment of proximal axons that initiates action potential (AP). At the AIS, the ion channels and cell adhesion molecules (CAMs) required for AP initiation are densely clustered via the scaffolding and cytoskeletal proteins. Notably, recent studies have elucidated that multiple AIS proteins are controlled by extensive alternative splicing in developing and adult brains. Here, we argue the potential role of dynamic regulation of alternative splicing in the development, specification, and functions of the AIS. In particular, we propose the novel concept that alternative splicing potentially modulates the structural and functional plasticity at the AIS.

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Hereditary spherocytosis (HS) is often misdiagnosed due to lack of specific diagnostic methods. Our study summarized clinical characteristics and described the diagnostic workflow for mild and moderate HS in Chinese individuals, using data from 20 adults, 8 of whom presented a familial history for HS. We used scanning electron microscopy (SEM) to diagnose HS. We observed reduced eosin maleimide fluorescence activity (5.50 mean channel fluorescence (MCF) units) in the 10 cases of HS, which differed significantly when compared with 10 normal adults (15.50 units), iron deficiency anemia (15.50 MCF units), and megaloblastic anemia (12.00 MCF units) values (P <.05). Next generation sequencing results revealed that 9 out of 10 patients were found to have mutations in the spectrin alpha chain (SPTB), anchor protein (ANK1), and SLC4A1 genes. These mutations were not reported in the Human Gene Mutation Database (HGMD), 1000 human genome, ExAC, and dbSNP147 databases. Splenectomy proved to be beneficial in alleviating HS symptoms in 10 cases. It was found that for the diagnosis of HS, SEM and next generation gene sequencing method proved to be more ideal than red blood cell membrane protein analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. [ABSTRACT FROM AUTHOR]

Ankyrins (encoded by ANK1/2/3 corresponding to Ankyrin-R/B/G or AnkR/B/G), via binding to spectrins, connect plasma membranes with actin cytoskeleton to maintain mechanical strengths and to modulate excitabilities of diverse cells such as neurons, muscle cells, and erythrocytes. Cellular and genetic evidences suggest that each isoform of ankyrins pairs with a specific β-spectrin in discrete subcellular membrane microdomains for distinct functions, although the molecular mechanisms underlying such ankyrin/β-spectrin pairings are unknown. In this study, we discover that a conserved and short extension N-terminal to the ZU5 N –ZU5 C –UPA tandem (exZZU) is critical for each ankyrin to bind to β-spectrins with high affinities. Structures of AnkB/G exZZU in complex with spectrin repeats13–15 of β2/β4-spectrins solved here reveal that the extension sequence of exZZU forms an additional β-strand contributing to the structural stability and enhanced affinity of each ZU5 N /spectrin repeat interaction. The complex structures further reveal that the UPA domain of exZZU directly participates in spectrin binding. Formation of the exZZU supramodule juxtaposes the ZU5 N and UPA domains for simultaneous interacting with spectrin repeats 14 and 15. However, our biochemical and structural investigations indicate that the direct and strong interactions between ankyrins and β-spectrins do not appear to determine their pairing specificities. Therefore, there likely exists additional mechanism(s) for modulating functional pairings between ankyrins and β-spectrins in cells. Unlabelled Image • A short N-terminal extension to the ZZU supramodule of ankyrins dramatically enhances the ankyrin/spectrin binding. • Structures of the ankyrin and spectrin complexes reveal the role of the extension in the spectrin/ankyrin complex formation. • The direct and strong interactions between ZZU supramodules of ankyrins and β-spectrins are not sufficient to determine their isoform-specific pairings. [ABSTRACT FROM AUTHOR]

Summary: Defects in various erythrocyte membrane proteins genes (ankyrin, band‐3, β‐ and α‐spectrin and protein 4·2) can cause hereditary spherocytosis (HS). This molecular heterogeneity of HS, together with co‐inherited genetic modifiers, results in marked phenotypic variability among patients. We studied the molecular spectrum and genotype‐phenotype correlations in 73 families (with 113 patients) with HS. Deleterious variants including nonsense (42%), deletions (18%), splice site (20%), missense (10%) and duplication/insertion (10%) were found in 47 patients. The variants detected included sporadic and dominantly‐inherited defects in ANK1 (53·2%), SPTB (36·2%) and SLC4A1 (4·2%). Compound heterozygous variants in SPTA1 (6·4%) showed autosomal recessive inheritance. Alpha‐spectrin variants were associated with severe anaemia and splenectomy alleviated symptoms. Co‐inherited glucose‐6‐phosphate dehydrogenase (G6PD) deficiency was found in 15%. G6PD variants (n = 5) led to greater transfusion requirements (1‐8 times) in males with HS. Homozygosity (41%) for the promoter variant of UGT1A1 (Gilbert syndrome) led to a significantly higher mean bilirubin level (126·54 µmol/l) with a higher frequency of cholelithiasis (30%) (P < 0·001). This first‐ever south Asian study on the molecular spectrum of HS found ANK1 and SPTB genes variants to be the commonest with inheritance being sporadic/dominant. Next‐generation sequencing provided a relatively sensitive and rapid tool for molecular diagnosis with a diagnostic yield of 64·4%. [ABSTRACT FROM AUTHOR]

Among the several tetratricopeptide (TPR) repeat-containing proteins encoded by the Arabidopsis thaliana genome, AtTPR10 exhibits an atypical structure with three TPR domain repeats at the C-terminus in addition to seven ankyrin (ANK) domain repeats at the N-terminus. However, the function of AtTPR10 remains elusive. Here, we investigated the biochemical function of AtTPR10. Bioinformatic analysis revealed that AtTPR10 expression is highly enhanced by heat shock compared with the other abiotic stresses, suggesting that AtTPR10 functions as a molecular chaperone to protect intracellular proteins from thermal stresses. Under the heat shock treatment, the chaperone activity of AtTPR10 increased significantly; this was accompanied by a structural switch from the low molecular weight (LMW) protein to a high molecular weight (HMW) complex. Analysis of two truncated fragments of AtTPR10 containing the TPR and ANK repeats showed that each domain exhibits a similar range of chaperone activity (approximately one-third of that of the native protein), suggesting that each domain cooperatively regulates the chaperone function of AtTPR10. Additionally, both truncated fragments of AtTPR10 underwent structural reconfiguration to form heat shock-dependent HMW complexes. Our results clearly demonstrate that AtTPR10 functions as a molecular chaperone in plants to protect intracellular targets from heat shock stress. [ABSTRACT FROM AUTHOR]

In striated muscles, the large scaffolding protein obscurin and a small SR-integral membrane protein sAnk1.5 control the retention of longitudinal SR across the sarcomere. How a complex of these proteins facilitates localization of longitudinal SR has yet to be resolved, but we hypothesize that obscurin interacts with a complex of sAnk1.5 proteins. To begin to address this hypothesis, we demonstrate that sAnk1.5 interacts with itself and identify two domains mediating self-association. Specifically, we show by co-precipitation and FLIM-FRET analysis that sAnk1.5 and another small AnkR isoform (sAnk1.6) interact with themselves and each other. We demonstrate that obscurin interacts with a complex of sAnk1.5 proteins and that this complex formation is enhanced by obscurin-binding. Using FLIM-FRET analysis, we show that obscurin interacts with sAnk1.5 alone and with sAnk1.6 in the presence of sAnk1.5. We find that sAnk1.5 self-association is disrupted by mutagenesis of residues Arg64-Arg69, residues previously associated with obscurin-binding. Molecular modeling of two interacting sAnk1.5 monomers facilitated the identification of Gly31-Val36 as an additional site of interaction, which was subsequently corroborated by co-precipitation and FLIM-FRET analysis. In closing, these results support a model in which sAnk1.5 forms large oligomers that interact with obscurin to facilitate the retention of longitudinal SR throughout skeletal and cardiac myocytes. • Small Ank1 isoforms 1.5, 1.6, and 1.9 interact with themselves and each other. • Amino acids Gly31-Val36 and Arg64-Arg69 regulate small Ank1 self-association. • Obscurin interacts with a complex of sAnk1.5 proteins. • Obscurin-binding enhances sAnk1.5 protein complex formation. • sAnk1.5 mediates obscurin interaction with sAnk1.6 in cells. [ABSTRACT FROM AUTHOR]

Alternative splicing is a powerful mechanism for molecular and functional diversification. In neurons, alternative splicing extensively controls various developmental steps as well as the plasticity and remodeling of neuronal activity in the adult brain. The axon initial segment (AIS) is the specialized compartment of proximal axons that initiates action potential (AP). At the AIS, the ion channels and cell adhesion molecules (CAMs) required for AP initiation are densely clustered via the scaffolding and cytoskeletal proteins. Notably, recent studies have elucidated that multiple AIS proteins are controlled by extensive alternative splicing in developing and adult brains. Here, we argue the potential role of dynamic regulation of alternative splicing in the development, specification, and functions of the AIS. In particular, we propose the novel concept that alternative splicing potentially modulates the structural and functional plasticity at the AIS. [ABSTRACT FROM AUTHOR]

Previously, a designed ankyrin repeat protein, AnkGAG1D4, was generated for intracellular targeting of the HIV-1 capsid domain. The efficiency was satisfactory in interfering with the HIV assembly process. Consequently, improved AnkGAG1D4 binding affinity was introduced by substituting tyrosine (Y) for serine (S) at position 45. However, the intracellular anti-HIV-1 activity of AnkGAG1D4-S45Y has not yet been validated. In this study, the performance of AnkGAG1D4 and AnkGAG1D4-S45Y in inhibiting wild-type HIV-1 and HIV-1 maturation inhibitor-resistant replication in SupT1 cells was evaluated. HIV-1 p24 and viral load assays were used to verify the biological activity of AnkGAG1D4 and AnkGAG1D4-S45Y as assembly inhibitors. In addition, retardation of syncytium formation in infected SupT1 cells was observed. Of note, the defense mechanism of both ankyrins did not induce the mutation of target amino acids in the capsid domain. The present data show that the potency of AnkGAG1D4-S45Y was superior to AnkGAG1D4 in interrupting either HIV-1 wild-type or the HIV maturation inhibitor-resistant strain.

The cardiac conduction system is an extended network of excitable tissue tasked with generation and propagation of electrical impulses to signal coordinated contraction of the heart. The fidelity of this system depends on the proper spatio-temporal regulation of ion channels in myocytes throughout the conduction system. Importantly, inherited or acquired defects in a wide class of ion channels has been linked to dysfunction at various stages of the conduction system resulting in life-threatening cardiac arrhythmia. There is growing appreciation of the role that adapter and cytoskeletal proteins play in organizing ion channel macromolecular complexes critical for proper function of the cardiac conduction system. In particular, members of the ankyrin and spectrin families have emerged as important nodes for normal expression and regulation of ion channels in myocytes throughout the conduction system. Human variants impacting ankyrin/spectrin function give rise to a broad constellation of cardiac arrhythmias. Furthermore, chronic neurohumoral and biomechanical stress promotes ankyrin/spectrin loss of function that likely contributes to conduction disturbances in the setting of acquired cardiac disease. Collectively, this review seeks to bring attention to the significance of these cytoskeletal players and emphasize the potential therapeutic role they represent in a myriad of cardiac disease states.

Background: Old human red blood cells (RBCs) have a reduced surface area with respect to young RBCs. If this decrease occurred through the release of vesicles similar to the spectrin-free vesicles that are shed in vitro under different experimental conditions or during storage, there would be no decrease of membrane-skeleton, but only of lipid bilayer surface area, during RBC ageing in vivo. However, we observed a decrease in spectrin and other membrane-skeletal proteins in old RBCs. Because RBCs contain components of the ubiquitin-proteasome system and other hydrolytic systems for protein degradation, we asked whether increased membrane-skeleton fragments could be detected in older RBCs. Methods: Four different anti-spectrin antibodies and an antibody anti-ubiquitin conjugates were used to analyse, by Western blotting, fragments of spectrin and other proteins in RBCs of different age separated in density gradients and characterized for their protein 4.1a/4.1b ratio as a cell age parameter. Results: spectrin fragments do exist in RBCs of all ages, they represent a minute fraction of all spectrin, are membrane-bound and not cytoplasmic and do not increase with cell age. Besides spectrin, other membrane-skeletal components decrease with cell age. Conclusion: Observed results challenge the commonly accepted view that decrease in cell membrane throughout RBC life in vivo occurs via the release of spectrin-free vesicles.

The structural integrity of synaptic connections critically depends on the interaction between synaptic cell adhesion molecules (CAMs) and the underlying actin and microtubule cytoskeleton. This interaction is mediated by giant Ankyrins, that act as specialized adaptors to establish and maintain axonal and synaptic compartments. In Drosophila, two giant isoforms of Ankyrin2 (Ank2) control synapse stability and organization at the larval neuromuscular junction (NMJ). Both Ank2-L and Ank2-XL are highly abundant in motoneuron axons and within the presynaptic terminal, where they control synaptic CAMs distribution and organization of microtubules. Here, we address the role of the conserved N-terminal ankyrin repeat domain (ARD) for subcellular localization and function of these giant Ankyrins in vivo. We used a P[acman] based rescue approach to generate deletions of ARD subdomains, that contain putative binding sites of interacting transmembrane proteins. We show that specific subdomains control synaptic but not axonal localization of Ank2-L. These domains contain binding sites to L1-family member CAMs, and we demonstrate that these regions are necessary for the organization of synaptic CAMs and for the control of synaptic stability. In contrast, presynaptic Ank2-XL localization only partially depends on the ARD but strictly requires the presynaptic presence of Ank2-L demonstrating a critical co-dependence of the two isoforms at the NMJ. Ank2-XL dependent control of microtubule organization correlates with presynaptic abundance of the protein and is thus only partially affected by ARD deletions. Together, our data provides novel insights into the synaptic targeting of giant Ankyrins with relevance for the control of synaptic plasticity and maintenance.

Postnatal regulation of dendritic spine formation and refinement in cortical pyramidal neurons is critical for excitatory/inhibitory balance in neocortical networks. Recent studies have identified a selective spine pruning mechanism in the mouse prefrontal cortex mediated by class 3 Semaphorins and the L1 cell adhesion molecules, neuron-glia related cell adhesion molecule, Close Homolog of L1, and L1. L1 cell adhesion molecules bind Ankyrin B, an actin-spectrin adaptor encoded by Ankyrin2, a high-confidence gene for autism spectrum disorder. In a new inducible mouse model (Nex1Cre-ERT2: Ank2flox: RCE), Ankyrin2 deletion in early postnatal pyramidal neurons increased spine density on apical dendrites in prefrontal cortex layer 2/3 of homozygous and heterozygous Ankyrin2-deficient mice. In contrast, Ankyrin2 deletion in adulthood had no effect on spine density. Sema3F-induced spine pruning was impaired in cortical neuron cultures from Ankyrin B-null mice and was rescued by re-expression of the 220 kDa Ankyrin B isoform but not 440 kDa Ankyrin B. Ankyrin B bound to neuron-glia related CAM at a cytoplasmic domain motif (FIGQY1231), and mutation to FIGQH inhibited binding, impairing Sema3F-induced spine pruning in neuronal cultures. Identification of a novel function for Ankyrin B in dendritic spine regulation provides insight into cortical circuit development, as well as potential molecular deficiencies in autism spectrum disorder., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Alzheimer's disease (AD) is the most common form of dementia and is characterized by intracellular neurofibrillary tangles of hyperphosphorylated Tau, including the 0N4R isoform and accumulation of extracellular amyloid beta (Aβ) plaques. However, less than 5% of AD cases are familial, with many additional risk factors contributing to AD including aging, lifestyle, the environment and epigenetics. Recent epigenome-wide association studies (EWAS) of AD have identified a number of loci that are differentially methylated in the AD cortex. Indeed, hypermethylation and reduced expression of the Ankyrin 1 (ANK1) gene in AD has been reported in the cortex in numerous different post-mortem brain cohorts. Little is known about the normal function of ANK1 in the healthy brain, nor the role it may play in AD. We have generated Drosophila models to allow us to functionally characterize Drosophila Ank2 , the ortholog of human ANK1 and to determine its interaction with human Tau and Aβ. We show expression of human Tau 0N4R or the oligomerizing Aβ 42 amino acid peptide caused shortened lifespan, degeneration, disrupted movement, memory loss, and decreased excitability of memory neurons with co-expression tending to make the pathology worse. We find that Drosophila with reduced neuronal Ank2 expression have shortened lifespan, reduced locomotion, reduced memory and reduced neuronal excitability similar to flies overexpressing either human Tau 0N4R or Aβ42. Therefore, we show that the mis-expression of Ank2 can drive disease relevant processes and phenocopy some features of AD. Therefore, we propose targeting human ANK1 may have therapeutic potential. This represents the first study to characterize an AD-relevant gene nominated from EWAS. [ABSTRACT FROM AUTHOR]

Rare genetic variants in ANK2 , which encodes ankyrin-B, are associated with neurodevelopmental disorders (NDDs); however, their pathogenesis is poorly understood. We find that mice with prenatal deletion in cortical excitatory neurons and oligodendrocytes (Ank2 −/−:Emx1-Cre), but not with adolescent deletion in forebrain excitatory neurons (Ank2 −/−:CaMKIIα-Cre), display severe spontaneous seizures, increased mortality, hyperactivity, and social deficits. Calcium imaging of cortical slices from Ank2 −/−:Emx1-Cre mice shows increased neuronal calcium event amplitude and frequency, along with network hyperexcitability and hypersynchrony. Quantitative proteomic analysis of cortical synaptic membranes reveals upregulation of dendritic spine plasticity-regulatory proteins and downregulation of intermediate filaments. Characterization of the ankyrin-B interactome identifies interactors associated with autism and epilepsy risk factors and synaptic proteins. The AMPA receptor antagonist, perampanel, restores cortical neuronal activity and partially rescues survival in Ank2 −/−:Emx1-Cre mice. Our findings suggest that synaptic proteome alterations resulting from Ank2 deletion impair neuronal activity and synchrony, leading to NDDs-related behavioral impairments. [Display omitted] • Ank2 -deficient mice show spontaneous seizures and increased mortality • Ank2 -deficient mice show increased neuronal excitability and network synchrony • Proteomic analysis reveals specific alterations in the postsynaptic proteome • AMPAR antagonist improves circuit deficits and survival endpoints in cKO mice Prenatal deletion of the Ank2 gene in mouse forebrain alters the synaptic proteome and enhances neuronal activity and synchronicity, leading to seizures and mortality. Normalization of neuronal and network activity through an AMPAR antagonist reduces hypersynchrony and lethality. [ABSTRACT FROM AUTHOR]