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1.
Biochim Biophys Acta Proteins Proteom ; 1867(11): 140267, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31470132

RESUMO

Spectrin, the major protein component of the erythrocyte membrane skeleton has chaperone like activity and is known to bind membrane phospholipids and hemoglobin. We have probed the chaperone activity of spectrin in presence of hemoglobin and phospholipid SUVs of different compositions to elucidate the effect of phospholipid/hemoglobin binding on chaperone function. It is seen that spectrin displays a preference for hemoglobin over other substrates leading to a decrease in chaperone activity in presence of hemoglobin. A competition is seen to exist between phospholipid binding and chaperone function of spectrin, in a dose dependent manner with the greatest extent of decrease being seen in case of phospholipid vesicles containing aminophospholipids e.g. PS and PE which may have implications in diseases like hereditary spherocytosis where mutation in spectrin is implicated in its detachment from cell membrane. To gain a clearer understanding of the chaperone like activity of spectrin under in-vivo like conditions we have investigated the effect of macromolecular crowders as well as phosphorylation and glycation states on chaperone activity. It is seen that the presence of non-specific, protein and non-protein macromolecular crowders do not appreciably affect chaperone function. Phosphorylation also does not affect the chaperone function unlike glycation which progressively diminishes chaperone activity. We propose a model where chaperone clients adsorb onto spectrin's surface and processes that bind to and occlude these surfaces decrease chaperone activity.


Assuntos
Membrana Eritrocítica/química , Hemoglobinas/química , Chaperonas Moleculares/química , Espectrina/química , Animais , Bovinos , Membrana Eritrocítica/metabolismo , Hemoglobinas/metabolismo , Chaperonas Moleculares/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Ovinos , Espectrina/metabolismo
2.
PLoS Biol ; 17(7): e3000369, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31299042

RESUMO

Cilia are remarkable cellular devices that power cell motility and transduce extracellular signals. To assemble a cilium, a cylindrical array of 9 doublet microtubules push out an extension of the plasma membrane. Membrane tension regulates cilium formation; however, molecular pathways that link mechanical stimuli to ciliogenesis are unclear. Using genome editing, we introduced hereditary elliptocytosis (HE)- and spinocerebellar ataxia (SCA)-associated mutations into the Caenorhabditis elegans membrane skeletal protein spectrin. We show that these mutations impair mechanical support for the plasma membrane and change cell shape. RNA sequencing (RNA-seq) analyses of spectrin-mutant animals uncovered a global down-regulation of ciliary gene expression, prompting us to investigate whether spectrin participates in ciliogenesis. Spectrin mutations affect intraflagellar transport (IFT), disrupt axonemal microtubules, and inhibit cilium formation, and the endogenous spectrin periodically distributes along cilia. Mammalian spectrin also localizes in cilia and regulates ciliogenesis. These results define a previously unrecognized yet conserved role of spectrin-based mechanical support for cilium biogenesis.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Membrana Celular/metabolismo , Cílios/genética , Mutação , Espectrina/genética , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cílios/metabolismo , Cílios/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Análise de Sequência de RNA , Espectrina/metabolismo
3.
Ann Hematol ; 98(8): 1813-1826, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31098739

RESUMO

Pregnant patients with ß-thalassemia are more likely to have progressive anemia which expose them to risk of adverse pregnancy outcomes, blood transfusion, and iron overload. Results from our previous study indicated that Colla corii asini (CCA, E'jiao), a natural ingredient of traditional Chinese medicine, could significantly increase hemoglobin level of pregnant women with ß- thalassemia, but the underlying molecular mechanism was unclear. Thus, we applied high-throughput transcriptome sequencing to study the transcriptomic change before and after the CCA treatment. Twenty eligible pregnant women were recruited and randomized to either the CCA treatment group or the blank control group in a 3:1 ratio. Patients in the treatment group orally received daily 15 g CCA powder for 4 weeks. We analyzed the therapeutic effect indexes and the transcriptomic change in subjects' peripheral blood before and after treatment. We found that ß CD 41-42(-TTCT)/ßA was the main genotype of the subjects. The regulatory impact of CCA treatment became more evident among the subjects of genotype ß CD 41-42(-TTCT)/ßA. Gene ontogenesis analysis revealed that the top five molecular functions of differentially expressed genes were involved in membrane functionality and cellular structure. We further identified two consistent upregulated genes ZNF471 and THOC5 in the effective treatment group, which were engaged in Kruppel-associated box (KRAB) domain-containing zinc-finger protein pathway and THOC5 pathway, respectively. Based on our current findings, we hypothesize that the anti-anemia effect of CCA on pregnant women with ß-thalassemia might be related to translation regulation of spectrin synthesis, membrane stability, and eventually prolonged the life span of erythrocytes.


Assuntos
Gelatina/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Fármacos Hematológicos/uso terapêutico , Medicina Tradicional Chinesa/métodos , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Talassemia beta/tratamento farmacológico , Administração Oral , Adulto , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas Nucleares/agonistas , Proteínas Nucleares/metabolismo , Gravidez , Proteômica/métodos , Proteínas Repressoras/agonistas , Proteínas Repressoras/metabolismo , Transdução de Sinais , Espectrina/genética , Espectrina/metabolismo , Transcriptoma/efeitos dos fármacos , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/patologia
4.
Orphanet J Rare Dis ; 14(1): 114, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31122244

RESUMO

BACKGROUND: Current diagnostic tests for hereditary spherocytosis (HS) focus on the detection of hemolysis or indirectly assessing defects of membrane protein, whereas direct methods to detect protein defects are complicated and difficult to implement. In the present study, we investigated the patterns of genetic variation associated with HS among patients clinically diagnosed with HS. METHODS: Multi-gene targeted sequencing of 43 genes (17 RBC membrane protein-encoding genes, 20 RBC enzyme-encoding genes, and six additional genes for the differential diagnosis) was performed using the Illumina HiSeq platform. RESULTS: Among 59 patients with HS, 50 (84.7%) had one or more significant variants in a RBC membrane protein-encoding genes. A total of 54 significant variants including 46 novel mutations were detected in six RBC membrane protein-encoding genes, with the highest number of variants found in SPTB (n = 28), and followed by ANK1 (n = 19), SLC4A1 (n = 3), SPTA1 (n = 2), EPB41 (n = 1), and EPB42 (n = 1). Concurrent mutations of genes encoding RBC enzymes (ALDOB, GAPDH, and GSR) were detected in three patients. UGT1A1 mutations were present in 24 patients (40.7%). Positive rate of osmotic fragility test was 86.8% among patients harboring HS-related gene mutations. CONCLUSIONS: This constitutes the first large-scaled genetic study of Korean patients with HS. We demonstrated that multi-gene target sequencing is sensitive and feasible that can be used as a powerful tool for diagnosing HS. Considering the discrepancies of clinical and molecular diagnoses of HS, our findings suggest that molecular genetic analysis is required for accurate diagnosis of HS.


Assuntos
Fragilidade Osmótica/fisiologia , Esferócitos/metabolismo , Esferocitose Hereditária/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Anquirinas/genética , Anquirinas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Criança , Pré-Escolar , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Feminino , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Lactente , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Mutação/genética , Fragilidade Osmótica/genética , Patologia Molecular , República da Coreia , Espectrina/genética , Espectrina/metabolismo , Esferocitose Hereditária/genética , Adulto Jovem
5.
J Therm Biol ; 81: 98-102, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30975429

RESUMO

Measuring the impedance of heated suspensions of erythrocytes and erythrocyte ghost membranes, two thermally-induced alterations are registered in the plasma membrane at TA (denaturation of spectrin with inducing temperature at 49,5 °C) and TG (hyperthermic activation of basal ion permeability with inducing temperature at 60.7 °C). In this study erythrocytes from 9 healthy patients and 15 patients with hemolytic anemia were studied and divided into four groups depending on their TA and TG top temperatures. The TA and TG of erythrocytes with hemoglobinopathy were the same as those of control erythrocytes while those of erythrocytes with membranopathy were significantly reduced. In erythrocytes with severe membranopathy, the TG was decreased by about 5 °C. In latter cells the normal value of TG was restored and the resistance to thermal haemolysis was increased by 90% after the specific stabilization of band 3 protein by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). Obtained results indicate the involvement of band 3 in the membrane alteration at TG and in the heat target responsible for thermal haemolysis.


Assuntos
Anemia Hemolítica/metabolismo , Anemia Hemolítica/patologia , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/patologia , Hemólise , Temperatura Alta , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Humanos , Espectrina/metabolismo
6.
Brain Dev ; 41(7): 630-633, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30898343

RESUMO

BACKGROUND: Spinocerebellar ataxia type 5 (SCA5), a dominant spinocerebellar ataxia is caused by spectrin beta nonerythrocytic 2 gene (SPTBN2) mutation. It typically consists of a slow progressive cerebellar ataxia with an onset principally in adulthood. Here, we report on the first Japanese patient with infantile-onset SCA5 associated with a novel heterozygous SPTBN2 mutation. CASE REPORT: The patient, a 6-year-old girl, developed delayed motor development and unsteady arm movement during infancy. She also showed gaze-evoked nystagmus, saccadic eye pursuit, dysarthria, dysmetria, intention tremor and mild intellectual disability. Brain MRI revealed moderate cerebellar atrophy and mild pontine atrophy. Comprehensive target capture sequencing to identify the causative gene identified a novel missense mutation in SPTBN2 (c.1309C

Assuntos
Espectrina/genética , Ataxias Espinocerebelares/genética , Degenerações Espinocerebelares/genética , Criança , Feminino , Humanos , Japão , Imagem por Ressonância Magnética , Mutação , Mutação de Sentido Incorreto , Espectrina/metabolismo , Ataxias Espinocerebelares/fisiopatologia
7.
Neuron ; 102(2): 358-372.e9, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30846310

RESUMO

Among the diverse interneuron subtypes in the neocortex, chandelier cells (ChCs) are the only population that selectively innervate pyramidal neurons (PyNs) at their axon initial segment (AIS), the site of action potential initiation, allowing them to exert powerful control over PyN output. Yet, mechanisms underlying their subcellular innervation of PyN AISs are unknown. To identify molecular determinants of ChC/PyN AIS innervation, we performed an in vivo RNAi screen of PyN-expressed axonal cell adhesion molecules (CAMs) and select Ephs/ephrins. Strikingly, we found the L1 family member L1CAM to be the only molecule required for ChC/PyN AIS innervation. Further, we show that L1CAM is required during both the establishment and maintenance of innervation, and that selective innervation of PyN AISs by ChCs requires AIS anchoring of L1CAM by the cytoskeletal ankyrin-G/ßIV-spectrin complex. Thus, our findings identify PyN-expressed L1CAM as a critical CAM required for innervation of neocortical PyN AISs by ChCs. VIDEO ABSTRACT.


Assuntos
Axônios/metabolismo , Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Células Piramidais/metabolismo , Animais , Anquirinas/metabolismo , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Efrinas/metabolismo , Camundongos , Neocórtex/citologia , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptor EphA1/metabolismo , Espectrina/metabolismo , Sinapses
8.
Ann Diagn Pathol ; 39: 86-91, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30798076

RESUMO

Spectrins are a group of cytoskeletal proteins which participate in many important cellular functions. It has been suggested that loss of spectrin isoforms may be associated with tumorigenesis of lymphoma, leukemia, gastric cancer and hepatocellular carcinoma (HCC). We recently reported that ßI spectrin expression was present in normal hepatocytes but lost in HCC cells, which suggested that spectrins may be helpful markers in diagnosis of HCC. In this study, using immunohistochemical staining, we further investigated the expression pattern of four spectrin isoforms (αII, ßI-III) on different benign and malignant liver tumors including focal nodular hyperplasia (FNH), hepatic adenoma (HA), HCC, and cholangiocarcinoma (CC). The results revealed that ßI spectrin was moderately to strongly positive in FNH and HA tissues, but was only weakly positive or lost in HCC cases and was weakly positive in all CC cases. In addition, the ßIII spectrin, majority of which was moderately positive in both FNH and HA tissues, was mostly lost in poorly differentiated HCC but remained at least moderately positive in most CC cases. These results suggest that spectrins ßI and ßIII may be used to differentiate well differentiated HCC from FNH or HA, and poorly differentiated HCC from CC, respectively.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Espectrina/metabolismo , Adenoma de Células Hepáticas/metabolismo , Adolescente , Adulto , Idoso , Neoplasias dos Ductos Biliares/metabolismo , Carcinoma Hepatocelular/metabolismo , Diferenciação Celular , Criança , Colangiocarcinoma/metabolismo , Feminino , Hiperplasia Nodular Focal do Fígado/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto Jovem
9.
Pediatr Neonatol ; 60(4): 435-440, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30559060

RESUMO

BACKGROUND: To investigate the clinical values of serum melatonin and αII spectrin cleavage products (SBDPs) in assessing the severity of brain injury in preterm infants. METHODS: Sixty-four premature infants in total were selected and classified into the brain injury group (BI, n = 30) and the non-brain injury group (CON, n = 34) according to cranial imaging examination. The serum melatonin and SBDPs were detected by ELISA. All the preterm infants were received NBNA testing at 40 weeks of corrected gestational age. RESULTS: The levels of melatonin and SBDPs in the BI group were significantly higher than the CON group (p < 0.05) and the levels in the infants with severe brain injury were significantly higher than those with mild brain injury (p < 0.05), as well as exhibiting a negative correlation with the NBNA score at 40 weeks of corrected gestational age (p < 0.05). CONCLUSIONS: Detecting melatonin and SBDPs has clinical value in diagnosing and assessing the severity of brain injury in preterm infants.


Assuntos
Encéfalo/diagnóstico por imagem , Infarto Cerebral/sangue , Hemorragia Cerebral Intraventricular/sangue , Hemorragias Intracranianas/sangue , Leucomalácia Periventricular/sangue , Melatonina/sangue , Espectrina/sangue , Lesões Encefálicas/sangue , Lesões Encefálicas/diagnóstico por imagem , Lesões Encefálicas/metabolismo , Lesões Encefálicas/fisiopatologia , Estudos de Casos e Controles , Infarto Cerebral/diagnóstico por imagem , Infarto Cerebral/metabolismo , Infarto Cerebral/fisiopatologia , Hemorragia Cerebral Intraventricular/diagnóstico por imagem , Hemorragia Cerebral Intraventricular/metabolismo , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Doenças do Prematuro/sangue , Doenças do Prematuro/diagnóstico por imagem , Doenças do Prematuro/metabolismo , Doenças do Prematuro/fisiopatologia , Hemorragias Intracranianas/diagnóstico por imagem , Hemorragias Intracranianas/metabolismo , Hemorragias Intracranianas/fisiopatologia , Leucomalácia Periventricular/diagnóstico por imagem , Leucomalácia Periventricular/metabolismo , Leucomalácia Periventricular/fisiopatologia , Imagem por Ressonância Magnética , Masculino , Índice de Gravidade de Doença , Espectrina/metabolismo
10.
Mol Neurobiol ; 56(5): 3069-3078, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30091036

RESUMO

Exercise increases the levels of neurogenic factors and enhances neurogenesis, memory, and learning. However, the molecular link between exercise and neurogenesis is not clear. The purpose of this study was to examine the effects of exercise intensity on cognitive function and protein expression in the hippocampus of old mice. To compare the effects of aerobic exercise intensity on cognition in old mice, we exposed 18-month-old mice to low- and moderate-intensity treadmill exercise for 4 weeks. Moderate-intensity exercise improved cognitive function in the old mice, while low-intensity exercise did not. To investigate the underlying mechanisms, two-dimensional electrophoresis was used to examine protein expression. Using peptide fingerprinting mass spectrometry, we identified 19 proteins that were upregulated in the hippocampus following exercise training, and seven of these proteins were normalized by the control value. Among them, the levels of 14-3-3 zeta and heat shock protein 70, which have been shown to be induced by exercise training and related to neurogenesis, were dramatically increased by moderate exercise. Hippocalcin, α-spectrin, ovarian tumor domain-containing ubiquitin aldehyde-binding protein 1 (otub1), mu-crystallin, serine racemase, and rho GDP dissociation inhibitor 1, which are related to neurogenesis, neuroprotection, and synaptic strength, were upregulated in the hippocampus by moderate exercise. In addition, we confirmed that neurogenic markers, including doublecortin and the neuronal nuclei antigen, and hippocalcin, otub1, and spectrin-α are potential molecular links between hippocampal neurogenesis and exercise in the elderly. Thus, these results showed that steady moderate-intensity exercise delayed the declines in cognitive function in the elderly through the activation of multiple factors.


Assuntos
Cognição , Cisteína Endopeptidases/metabolismo , Hipocalcina/metabolismo , Hipocampo/metabolismo , Neurogênese , Condicionamento Físico Animal , Espectrina/metabolismo , Regulação para Cima , Envelhecimento/metabolismo , Animais , Biomarcadores/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo
11.
Sci Adv ; 4(11): eaau8621, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30498783

RESUMO

Central neurons initiate action potentials (APs) in the axon initial segment (AIS), a compartment characterized by a high concentration of voltage-dependent ion channels and specialized cytoskeletal anchoring proteins arranged in a regular nanoscale pattern. Although the AIS was a key evolutionary innovation in neurons, the functional benefits it confers are not clear. Using a mutation of the AIS cytoskeletal protein ßIV-spectrin, we here establish an in vitro model of neurons with a perturbed AIS architecture that retains nanoscale order but loses the ability to maintain a high NaV density. Combining experiments and simulations, we show that a high NaV density in the AIS is not required for axonal AP initiation; it is, however, crucial for a high bandwidth of information encoding and AP timing precision. Our results provide the first experimental demonstration of axonal AP initiation without high axonal channel density and suggest that increasing the bandwidth of the neuronal code and, hence, the computational efficiency of network function, was a major benefit of the evolution of the AIS.


Assuntos
Potenciais de Ação , Segmento Inicial do Axônio/fisiologia , Citoesqueleto/metabolismo , Hipocampo/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Espectrina/metabolismo , Animais , Células Cultivadas , Camundongos , Canais de Potássio/metabolismo , Canais de Sódio/metabolismo
12.
Zhongguo Zhong Yao Za Zhi ; 43(20): 4125-4131, 2018 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-30486541

RESUMO

Drynariae Rhizoma has great significance in the clinical practice of osteoporosis treatment. Based on the perspective of integrative pharmacology, the study explored the mechanism of action of Drynariae Rhizoma in the treatment of osteoporosis. Six active components in Drynariae Rhizoma were obtained, mainly including glycosides and sterols. Taking the median of 2 times of "node connectivity" as the card value, the core node of the Chinese medicine target disease gene interaction network was selected. Based on this, three topological structural eigenvalues, such as "node connectivity" "node tightness" and "node connectivity" were calculated, thereby screening out four core targets of Drynariae Rhizoma treatment for osteoporosis, including thyroid parathyroid hormone 1 receptor (PTH1R), parathyroid hormone 2 receptor (PTH2R), calcitonin receptor gene (CALCR), and SPTBN1 gene (SPTBN1). Based on the gene ontology database GO and KEGG pathway database, the molecular function, intracellular localization, and biological reactions and pathways of proteins encoded by drug target genes were determined. Combined with enrichment calculation, it is predicted that osteoporosis may play a role in biosynthetic processes, such as circulatory system, nervous system, energy metabolism, prolactin signal pathway, GnRH signaling pathway, neurotrophic factor signaling pathway and other pathway. The conclusion of this study is certain with the existing research results, and the new target and new pathway could also be used as a theoretical basis for the further verification of osteoporosis.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Osteoporose/tratamento farmacológico , Polypodiaceae/química , Humanos , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptor Tipo 2 de Hormônio Paratireóideo/metabolismo , Receptores da Calcitonina/metabolismo , Rizoma/química , Espectrina/metabolismo
13.
J Clin Invest ; 128(12): 5561-5572, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30226828

RESUMO

Heart failure (HF) remains a major source of morbidity and mortality in the US. The multifunctional Ca2+/calmodulin-dependent kinase II (CaMKII) has emerged as a critical regulator of cardiac hypertrophy and failure, although the mechanisms remain unclear. Previous studies have established that the cytoskeletal protein ßIV-spectrin coordinates local CaMKII signaling. Here, we sought to determine the role of a spectrin-CaMKII complex in maladaptive remodeling in HF. Chronic pressure overload (6 weeks of transaortic constriction [TAC]) induced a decrease in cardiac function in WT mice but not in animals expressing truncated ßIV-spectrin lacking spectrin-CaMKII interaction (qv3J mice). Underlying the observed differences in function was an unexpected differential regulation of STAT3-related genes in qv3J TAC hearts. In vitro experiments demonstrated that ßIV-spectrin serves as a target for CaMKII phosphorylation, which regulates its stability. Cardiac-specific ßIV-spectrin-KO (ßIV-cKO) mice showed STAT3 dysregulation, fibrosis, and decreased cardiac function at baseline, similar to what was observed with TAC in WT mice. STAT3 inhibition restored normal cardiac structure and function in ßIV-cKO and WT TAC hearts. Our studies identify a spectrin-based complex essential for regulation of the cardiac response to chronic pressure overload. We anticipate that strategies targeting the new spectrin-based "statosome" will be effective at suppressing maladaptive remodeling in response to chronic stress.


Assuntos
Cardiomegalia/metabolismo , Insuficiência Cardíaca/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Espectrina/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomegalia/genética , Cardiomegalia/patologia , Fibrose , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Camundongos , Camundongos Transgênicos , Fosforilação , Fator de Transcrição STAT3/genética , Espectrina/genética
14.
Genes Cells ; 23(11): 974-981, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30175422

RESUMO

At the initial stage of carcinogenesis, transformation occurs in single cells within the epithelium. Recent studies have revealed that the newly emerging transformed cells are often apically eliminated from epithelial tissues. However, the underlying molecular mechanisms of this cancer preventive phenomenon still remain elusive. In this study, we first demonstrate that myosin-II accumulates in Src-transformed cells when they are surrounded by normal epithelial cells. Knock-down of the heavy chains of myosin-II substantially diminishes apical extrusion of Src cells, suggesting that accumulated myosin-II positively regulates the apical elimination of transformed cells. Furthermore, we have identified ß-spectrin as a myosin-II-binding protein under the coculture of normal and Src-transformed epithelial cells. ß-spectrin is also accumulated in Src cells that are surrounded by normal cells, and the ß-spectrin accumulation is regulated by myosin-II. Moreover, knock-down of ß-spectrin significantly suppresses apical extrusion of Src cells. Collectively, these results indicate that accumulation of the myosin-II-spectrin complex plays a positive role in apical extrusion of Src-transformed epithelial cells. Further elucidation of the molecular mechanisms of apical extrusion would lead to the establishment of a novel type of cancer preventive medicine.


Assuntos
Citoesqueleto de Actina/metabolismo , Transformação Celular Neoplásica/patologia , Células Epiteliais/patologia , Miosina Tipo II/metabolismo , Proteína Oncogênica pp60(v-src)/metabolismo , Espectrina/metabolismo , Animais , Comunicação Celular , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Cães , Células Epiteliais/metabolismo , Transdução de Sinais
15.
Mol Biol Cell ; 29(20): 2433-2449, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30091661

RESUMO

Disruption to the contractility of cells, including smooth muscle cells of the cardiovascular system and myoepithelial cells of the glandular epithelium, contributes to the pathophysiology of contractile tissue diseases, including asthma, hypertension, and primary Sjögren's syndrome. Cell contractility is determined by myosin activity and actomyosin network organization and is mediated by hundreds of protein-protein interactions, many directly involving actin. Here we use a candidate RNA interference screen of more than 100 Caenorhabditis elegans genes with predicted actin-binding and regulatory domains to identify genes that contribute to the contractility of the somatic gonad. We identify the spectrin cytoskeleton composed of SPC-1/α-spectrin, UNC-70/ß-spectrin, and SMA-1/ß heavy-spectrin as required for contractility and actin organization in the myoepithelial cells of the C. elegans spermatheca. We use imaging of fixed and live animals as well as tissue- and developmental-stage-specific disruption of the spectrin cytoskeleton to show that spectrin regulates the production of prominent central actin bundles and is required for maintenance of central actin bundles throughout successive rounds of stretch and contraction. We conclude that the spectrin cytoskeleton contributes to spermathecal contractility by promoting maintenance of the robust actomyosin bundles that drive contraction.


Assuntos
Actinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Espectrina/metabolismo , Espermatozoides/metabolismo , Actomiosina/metabolismo , Animais , Testes Genéticos , Masculino , Ovulação , Espermatozoides/citologia
16.
Cell Rep ; 24(6): 1512-1522, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30089262

RESUMO

Through three-dimensional STORM super-resolution microscopy, we resolve the spectrin-actin-based membrane cytoskeleton of neural stem cells (NSCs) and NSC-derived neurons, astrocytes, and oligodendrocytes. We show that undifferentiated NSCs are capable of forming patches of locally periodic, one-dimensional (1D) membrane cytoskeleton with ∼180 nm periodicity. Such periodic structures become increasingly ordered and long-ranging as the NSCs mature into terminally differentiated neuronal and glial cell types, and, during this process, distinct 1D periodic "strips" dominate the flat 2D membranes. Moreover, we report remarkable alignment of the periodic cytoskeletons between abutting cells at axon-axon and axon-oligodendrocyte contacts and identify two adhesion molecules, neurofascin and L1CAM, as candidates to drive this nanoscale alignment. We thus show that a conserved 1D periodic membrane cytoskeletal motif serves as a nanoscale scaffold and ruler to mediate the physical interactions between cell types of the NSC lineage.


Assuntos
Citoesqueleto de Actina/metabolismo , Células-Tronco Neurais/metabolismo , Espectrina/metabolismo , Linhagem da Célula , Humanos
17.
eNeuro ; 5(3)2018.
Artigo em Inglês | MEDLINE | ID: mdl-29963618

RESUMO

Nodes of Ranvier are unique regions where voltage-gated sodium channels are highly enriched to drive saltatory conduction. Genetic ablations in adult mice with loss of specific nodal proteins causes slow but progressive nodal deterioration associated with decreased nerve conduction and axonopathy. What has remained unaddressed is whether loss of nodal proteins at different time points in postnatal life follows similar timelines of nodal disorganization. Here we utilized simultaneous ablation of Neurofascin (NF186) and Ankyrin G (AnkG) in mice of both sexes at three specific time points. We report that concurrent ablation of these core nodal components at postnatal day 13 (P13) leads to accelerated nodal destabilization in comparison with P23, and this disorganization is even slower when ablated at P93. Ablation of NF186 with AnkG at P13 reduced the half-life of NF186 to 15 days compared to 1 month at P23, which increased to 2 months at P93, indicating increasing nodal stability. The half-life of AnkG at the nodes also increased with age but showed enhanced disappearance from the node in the absence of NF186, with a half-life of 3 days at P13 ablation. The nodal disorganization occurred in a sequential manner, with AnkG disappearing first from the nodal areas irrespective of the timing of ablation, and led to decreased nerve conduction and affected axonal health. Together, our studies reveal that nodes of Ranvier in myelinated axons continue to become more stable with age and suggest that nodal disorganization in adult human demyelinating disorders occurs slowly until neurological symptoms become evident.


Assuntos
Anquirinas/metabolismo , Axônios/metabolismo , Moléculas de Adesão Celular/metabolismo , Longevidade , Bainha de Mielina/metabolismo , Fatores de Crescimento Neural/metabolismo , Nós Neurofibrosos/metabolismo , Animais , Axônios/ultraestrutura , Feminino , Masculino , Camundongos , Camundongos Knockout , Bainha de Mielina/ultraestrutura , Condução Nervosa , Canais de Sódio/metabolismo , Espectrina/metabolismo
18.
J Neuroinflammation ; 15(1): 203, 2018 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-29996878

RESUMO

BACKGROUND: Epilepsy is a prevalent neurological disorder worldwide. It is characterized by an enduring predisposition to generate seizures and its development is accompanied by alterations in many cellular processes. Organotypic slice cultures represent a multicellular environment with the potential to assess biological mechanisms, and they are used as a starting point for refining molecules for in vivo studies. Here, we investigated organotypic slice cultures as a model of epilepsy. METHODS: We assessed, by electrophysiological recordings, the spontaneous activity of organotypic slices maintained under different culture protocols. Moreover, we evaluated, through molecular-based approaches, neurogenesis, neuronal death, gliosis, expression of proinflammatory cytokines, and activation of NLRP3 inflammasome (nucleotide-binding, leucine-rich repeat, pyrin domain) as biomarkers of neuroinflammation. RESULTS: We demonstrated that organotypic slices, maintained under a serum deprivation culture protocol, develop epileptic-like activity. Furthermore, throughout a comparative study with slices that do not depict any epileptiform activity, slices with epileptiform activity were found to display significant differences in terms of inflammation-related features, such as (1) increased neuronal death, with higher incidence in CA1 pyramidal neurons of the hippocampus; (2) activation of astrocytes and microglia, assessed through western blot and immunohistochemistry; (3) upregulation of proinflammatory cytokines, specifically interleukin-1ß (IL-1ß), interleukin-6, and tumor necrosis factor α, revealed by qPCR; and (4) enhanced expression of NLRP3, assessed by western blot, together with increased NLRP3 activation, showed by IL-1ß quantification. CONCLUSIONS: Thus, organotypic slice cultures gradually deprived of serum mimic the epileptic-like activity, as well as the inflammatory events associated with in vivo epilepsy. This system can be considered a new tool to explore the interplay between neuroinflammation and epilepsy and to screen potential drug candidates, within the inflammatory cascades, to reduce/halt epileptogenesis.


Assuntos
Anticonvulsivantes/uso terapêutico , Citocinas/metabolismo , Epilepsia/tratamento farmacológico , Epilepsia/patologia , Hipocampo/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Compostos de Boro/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Caspase 3/metabolismo , Meios de Cultura Livres de Soro/toxicidade , Citocinas/genética , Modelos Animais de Doenças , Epilepsia/induzido quimicamente , Epilepsia/complicações , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/etiologia , Gliose/patologia , Hipocampo/patologia , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neuropeptídeos/metabolismo , Técnicas de Cultura de Órgãos , Gravidez , Ratos , Ratos Sprague-Dawley , Espectrina/metabolismo
19.
J Neurosci ; 38(27): 6063-6075, 2018 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-29853631

RESUMO

Action potential conduction along myelinated axons depends on high densities of voltage-gated Na+ channels at the nodes of Ranvier. Flanking each node, paranodal junctions (paranodes) are formed between axons and Schwann cells in the peripheral nervous system (PNS) or oligodendrocytes in the CNS. Paranodal junctions contribute to both node assembly and maintenance. Despite their importance, the molecular mechanisms responsible for paranode assembly and maintenance remain poorly understood. ßII spectrin is expressed in diverse cells and is an essential part of the submembranous cytoskeleton. Here, we show that Schwann cell ßII spectrin is highly enriched at paranodes. To elucidate the roles of glial ßII spectrin, we generated mutant mice lacking ßII spectrin in myelinating glial cells by crossing mice with a floxed allele of Sptbn1 with Cnp-Cre mice, and analyzed both male and female mice. Juvenile (4 weeks) and middle-aged (60 weeks) mutant mice showed reduced grip strength and sciatic nerve conduction slowing, whereas no phenotype was observed between 8 and 24 weeks of age. Consistent with these findings, immunofluorescence microscopy revealed disorganized paranodes in the PNS and CNS of both postnatal day 13 and middle-aged mutant mice, but not in young adult mutant mice. Electron microscopy confirmed partial loss of transverse bands at the paranodal axoglial junction in the middle-aged mutant mice in both the PNS and CNS. These findings demonstrate that a spectrin-based cytoskeleton in myelinating glia contributes to formation and maintenance of paranodal junctions.SIGNIFICANCE STATEMENT Myelinating glia form paranodal axoglial junctions that flank both sides of the nodes of Ranvier. These junctions contribute to node formation and maintenance and are essential for proper nervous system function. We found that a submembranous spectrin cytoskeleton is highly enriched at paranodes in Schwann cells. Ablation of ßII spectrin in myelinating glial cells disrupted the paranodal cell adhesion complex in both peripheral and CNSs, resulting in muscle weakness and sciatic nerve conduction slowing in juvenile and middle-aged mice. Our data show that a spectrin-based submembranous cytoskeleton in myelinating glia plays important roles in paranode formation and maintenance.


Assuntos
Axônios/metabolismo , Citoesqueleto/metabolismo , Neuroglia/metabolismo , Espectrina/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , Nós Neurofibrosos
20.
Elife ; 72018 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-29901439

RESUMO

Assembly, maintenance and function of synaptic junctions depend on extracellular matrix (ECM) proteins and their receptors. Here we report that Tenectin (Tnc), a Mucin-type protein with RGD motifs, is an ECM component required for the structural and functional integrity of synaptic specializations at the neuromuscular junction (NMJ) in Drosophila. Using genetics, biochemistry, electrophysiology, histology and electron microscopy, we show that Tnc is secreted from motor neurons and striated muscles and accumulates in the synaptic cleft. Tnc selectively recruits αPS2/ßPS integrin at synaptic terminals, but only the cis Tnc/integrin complexes appear to be biologically active. These complexes have distinct pre- and postsynaptic functions, mediated at least in part through the local engagement of the spectrin-based membrane skeleton: the presynaptic complexes control neurotransmitter release, while postsynaptic complexes ensure the size and architectural integrity of synaptic boutons. Our study reveals an unprecedented role for integrin in the synaptic recruitment of spectrin-based membrane skeleton.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Cadeias alfa de Integrinas/metabolismo , Junção Neuromuscular/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas da Matriz Extracelular/genética , Cadeias alfa de Integrinas/genética , Microscopia Confocal , Neurônios Motores/metabolismo , Complexos Multiproteicos/metabolismo , Músculo Estriado/metabolismo , Espectrina/metabolismo , Sinapses/metabolismo , Transmissão Sináptica , Sinaptossomos/metabolismo
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