RESUMO
Spectrins are cytoskeletal proteins that are expressed ubiquitously in the mammalian nervous system. Pathogenic variants in SPTAN1, SPTBN1, SPTBN2 and SPTBN4, four of the six genes encoding neuronal spectrins, cause neurological disorders. Despite their structural similarity and shared role as molecular organizers at the cell membrane, spectrins vary in expression, subcellular localization and specialization in neurons, and this variation partly underlies non-overlapping disease presentations across spectrinopathies. Here, we summarize recent progress in discerning the local and long-range organization and diverse functions of neuronal spectrins. We provide an overview of functional studies using mouse models, which, together with growing human genetic and clinical data, are helping to illuminate the aetiology of neurological spectrinopathies. These approaches are all critical on the path to plausible therapeutic solutions.
Assuntos
Doenças do Sistema Nervoso , Espectrina , Animais , Camundongos , Humanos , Espectrina/genética , Espectrina/metabolismo , Doenças do Sistema Nervoso/genética , Neurônios/metabolismo , Membrana Celular/metabolismo , Mamíferos/metabolismoRESUMO
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.
Assuntos
Axônios/metabolismo , Encéfalo/metabolismo , Membrana Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Espectrina/metabolismo , Animais , Encéfalo/citologia , Membrana Celular/genética , Conectoma , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Espectrina/genéticaRESUMO
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.
Assuntos
Anquirinas/genética , Transtorno do Espectro Autista/genética , Molécula L1 de Adesão de Célula Nervosa/genética , Crescimento Neuronal , Neurônios/metabolismo , Sinapses/metabolismo , Processamento Alternativo , Animais , Anquirinas/metabolismo , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/fisiopatologia , Comportamento Animal , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Conectoma , Modelos Animais de Doenças , Função Executiva/fisiologia , Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Mutação , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neurônios/patologia , Cultura Primária de Células , Comportamento Social , Sinapses/patologiaRESUMO
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.
Assuntos
Adipócitos/metabolismo , Adiposidade/genética , Anquirinas/genética , Transportador de Glucose Tipo 4/genética , Glucose/metabolismo , Obesidade/genética , Adipócitos/patologia , Animais , Anquirinas/química , Anquirinas/metabolismo , Transporte Biológico , População Negra , Clatrina/genética , Clatrina/metabolismo , Dieta Hiperlipídica/efeitos adversos , Endocitose , Regulação da Expressão Gênica , Transportador de Glucose Tipo 4/metabolismo , Humanos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Camundongos Knockout , Modelos Moleculares , Mutação , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Ligação Proteica , Estrutura Secundária de Proteína , Transdução de Sinais , População BrancaRESUMO
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.
Assuntos
Adaptação Fisiológica , Anquirinas/metabolismo , Membrana Celular/metabolismo , Espectrina/metabolismo , Vertebrados , Animais , HumanosRESUMO
In a recent study, Ziak et al. employed precise sparse labeling and spatiotemporally controlled genetic manipulations to uncover novel regulators of axon branching of layer 2/3 mouse callosal projection neurons. The authors elucidated a cell-autonomous signaling pathway wherein glycogen synthase kinase 3ß (GSK3ß) phosphorylation of microtubule-associated protein 1B (MAP1B) restricts interstitial axon branching by modulating microtubule (MT) tyrosination status.
Assuntos
Axônios , Animais , Humanos , Camundongos , Axônios/fisiologia , Axônios/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Anquirinas/metabolismo , Evolução Biológica , Membrana Celular/metabolismo , Espectrina/metabolismo , Animais , Anquirinas/química , Axônios/metabolismo , Membrana Celular/química , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Espectrina/químicaRESUMO
Elevated plasma branched-chain amino acids (BCAAs) are strongly associated with obesity, insulin resistance (IR), and diabetes in humans and rodent models. However, the mechanisms of BCAA dysregulation and its systemic, organ, and cell-specific implications in the development of obesity and IR are not well understood. To gain mechanistic insight into the causes and effects of plasma BCAA elevations, we leveraged mouse models with high circulating BCAA levels prior to the onset of obesity and IR. Young mice lacking ankyrin-B in white adipose tissue (WAT) or bearing an ankyrin-B variant that causes age-driven metabolic syndrome exhibit downregulation of BCAA catabolism selectively in WAT and excess plasma BCAAs. Using cellular assays, we demonstrated that ankyrin-B promotes the surface localization of the amino acid transporter Asct2 in white adipocytes, and its deficit impairs BCAA uptake. Excess BCAA supplementation worsened glucose tolerance and insulin sensitivity across genotypes. In contrast, BCAA overconsumption only increased adiposity in control mice, implicating WAT utilization of BCAAs in their obesogenic effects. These results shed light into the mechanistic underpinnings of metabolic syndrome caused by ankyrin-B deficits and provide new evidence of the relevance of WAT in the regulation of systemic BCAA levels, adiposity, and glucose homeostasis.
RESUMO
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.
Assuntos
Anquirinas/genética , Orientação de Axônios/genética , Axônios/fisiologia , Semaforina-3A/genética , Transdução de Sinais/genética , Animais , Anquirinas/metabolismo , Camundongos , Semaforina-3A/metabolismoRESUMO
The highly polarized, typically very long, and nonmitotic nature of neurons present them with unique challenges in the maintenance of their homeostasis. This architectural complexity serves a rich and tightly controlled set of functions that enables their fast communication with neighboring cells and endows them with exquisite plasticity. The submembrane neuronal cytoskeleton occupies a pivotal position in orchestrating the structural patterning that determines local and long-range subcellular specialization, membrane dynamics, and a wide range of signaling events. At its center is the partnership between ankyrins and spectrins, which self-assemble with both remarkable long-range regularity and micro- and nanoscale specificity to precisely position and stabilize cell adhesion molecules, membrane transporters, ion channels, and other cytoskeletal proteins. To accomplish these generally conserved, but often functionally divergent and spatially diverse, roles these partners use a combinatorial program of a couple of dozens interacting family members, whose code is not fully unraveled. In a departure from their scaffolding roles, ankyrins and spectrins also enable the delivery of material to the plasma membrane by facilitating intracellular transport. Thus, it is unsurprising that deficits in ankyrins and spectrins underlie several neurodevelopmental, neurodegenerative, and psychiatric disorders. Here, I summarize key aspects of the biology of spectrins and ankyrins in the mammalian neuron and provide a snapshot of the latest advances in decoding their roles in the nervous system.
Assuntos
Anquirinas/metabolismo , Transporte Biológico/fisiologia , Neurônios/metabolismo , Espectrina/metabolismoRESUMO
Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the "tri-partite" synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons, where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes' normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.
Assuntos
Astrócitos/metabolismo , Espaço Intracelular/metabolismo , Organelas/metabolismo , Animais , Astrócitos/citologia , Transporte Biológico , Encéfalo/metabolismo , Células Cultivadas , Proteínas de Fluorescência Verde/metabolismo , CamundongosRESUMO
Lysosomes play a critical role in maintenance of the integrity of neuronal function, and mutations in genes that contribute to lysosome formation, transport, and activity are associated with neurodegenerative disorders. Recently, the multisubunit complex, BLOC-one-related complex (BORC), has been shown to be involved in positioning lysosomes within the cytoplasm, although the consequences of altered BORC function in adult animals have not been established. We show that a spontaneous truncation mutation in the mouse Borcs7 gene, identified through whole-genome sequencing followed by genetic complementation, results in progressive axonal dystrophy with dramatic impairment of motor function. Furthermore, mice homozygous for deletion of the entire Borcs7 coding sequence die shortly after birth, and neurons cultured from these animals show impaired centrifugal transport of lysosomes. This identifies BORCS7 as a central factor in axonal transport of lysosomes and a possible target for improving disease-related disturbances in this important function.
Assuntos
Transporte Axonal , Proteínas de Membrana Lisossomal/genética , Distrofias Neuroaxonais/genética , Animais , Feminino , Locomoção , Mutação com Perda de Função , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neurônios/metabolismoRESUMO
Endosomal membrane trafficking requires coordination between phosphoinositide lipids, Rab GTPases, and microtubule-based motors to dynamically determine endosome identity and promote long-range organelle transport. Here we report that ankyrin-B (AnkB), through integrating all three systems, functions as a critical node in the protein circuitry underlying polarized recycling of α5ß1-integrin in mouse embryonic fibroblasts, which enables persistent fibroblast migration along fibronectin gradients. AnkB associates with phosphatidylinositol 3-phosphate (PI3P)-positive organelles in fibroblasts and binds dynactin to promote their long-range motility. We demonstrate that AnkB binds to Rab GTPase Activating Protein 1-Like (RabGAP1L) and recruits it to PI3P-positive organelles, where RabGAP1L inactivates Rab22A, and promotes polarized trafficking to the leading edge of migrating fibroblasts. We further determine that α5ß1-integrin depends on an AnkB/RabGAP1L complex for polarized recycling. Our results reveal AnkB as an unexpected key element in coordinating polarized transport of α5ß1-integrin and likely of other specialized endocytic cargos.
Assuntos
Anquirinas/metabolismo , Complexo Dinactina/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Integrina alfa5beta1/metabolismo , Animais , Anquirinas/genética , Complexo Dinactina/genética , Endossomos/genética , Endossomos/metabolismo , Fibroblastos/metabolismo , Proteínas Ativadoras de GTPase/genética , Antígenos de Histocompatibilidade , Humanos , Integrina alfa5beta1/genética , Lipídeos/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Fosfatos de Fosfatidilinositol/metabolismo , Ligação ProteicaRESUMO
Rare functional variants of ankyrin-B have been implicated in human disease, including hereditary cardiac arrhythmia and type 2 diabetes (T2D). Here, we developed murine models to evaluate the metabolic consequences of these alterations in vivo. Specifically, we generated knockin mice that express either the human ankyrin-B variant R1788W, which is present in 0.3% of North Americans of mixed European descent and is associated with T2D, or L1622I, which is present in 7.5% of African Americans. Young AnkbR1788W/R1788W mice displayed primary pancreatic ß cell insufficiency that was characterized by reduced insulin secretion in response to muscarinic agonists, combined with increased peripheral glucose uptake and concomitantly increased plasma membrane localization of glucose transporter 4 (GLUT4) in skeletal muscle and adipocytes. In contrast, older AnkbR1788W/R1788W and AnkbL1622I/L1622I mice developed increased adiposity, a phenotype that was reproduced in cultured adipocytes, and insulin resistance. GLUT4 trafficking was altered in animals expressing mutant forms of ankyrin-B, and we propose that increased cell surface expression of GLUT4 in skeletal muscle and fatty tissue of AnkbR1788W/R1788W mice leads to the observed age-dependent adiposity. Together, our data suggest that ankyrin-B deficiency results in a metabolic syndrome that combines primary pancreatic ß cell insufficiency with peripheral insulin resistance and is directly relevant to the nearly one million North Americans bearing the R1788W ankyrin-B variant.
Assuntos
Adiposidade/genética , Envelhecimento , Anquirinas , Células Secretoras de Insulina , Síndrome Metabólica , Mutação de Sentido Incorreto , Envelhecimento/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Substituição de Aminoácidos , Animais , Anquirinas/genética , Anquirinas/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Masculino , Síndrome Metabólica/genética , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Camundongos , Camundongos Mutantes , Músculo Esquelético/metabolismo , Músculo Esquelético/patologiaRESUMO
Spinocerebellar ataxia type 5 (SCA5) is an autosomal dominant neurodegenerative disorder caused by mutations in the SPBTN2 gene encoding beta-III-spectrin. To investigate the molecular basis of SCA5, we established a series of transgenic Drosophila models that express human beta-III-spectrin or fly beta-spectrin proteins containing SCA5 mutations. Expression of the SCA5 mutant spectrin in the eye causes a progressive neurodegenerative phenotype, and expression in larval neurons results in posterior paralysis, reduced synaptic terminal growth, and axonal transport deficits. These phenotypes are genetically enhanced by both dynein and dynactin loss-of-function mutations. In summary, we demonstrate that SCA5 mutant spectrin causes adult-onset neurodegeneration in the fly eye and disrupts fundamental intracellular transport processes that are likely to contribute to this progressive neurodegenerative disease.