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1.
BMJ Case Rep ; 13(12)2020 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-33318253

RESUMO

A 4-year-old girl was referred to the geneticist with a history of ataxia associated with intention tremor of the hands, strabismus and hypermetropy. Her symptoms presented about 2 years earlier with inability to walk unaided and lower limbs hypotonia. Cognitive functions were normal. Brain MRI showed a cerebellar and vermian hypoplasia with enlargement of both the cerebrospinal fluid spaces and the IV brain ventricle. Family history was unremarkable. A genetic screening using a 42-gene panel for hereditary ataxia/spastic paraparesis identified a de novo c.1438C>T - p.(Arg480Trp) missense change in the SPTBN2 gene (NM_006946.2). This variant is reported to be associated with congenital ataxia, later evolving into ataxia and intellectual disability. This case further supports the existence of a specific SPTBN2 p.(Arg480Trp)-associated phenotype, with a de novo recurrence of this variant in the heterozygous state.


Assuntos
Encéfalo/patologia , Espectrina/genética , Ataxias Espinocerebelares/genética , Pré-Escolar , Feminino , Humanos , Deficiência Intelectual , Imagem por Ressonância Magnética , Mutação de Sentido Incorreto , Fenótipo , Espectrina/metabolismo
2.
Nat Commun ; 11(1): 5108, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037189

RESUMO

The spectrin-based membrane skeleton is a major component of the cell cortex. While expressed by all metazoans, its dynamic interactions with the other cortex components, including the plasma membrane or the acto-myosin cytoskeleton, are poorly understood. Here, we investigate how spectrin re-organizes spatially and dynamically under the membrane during changes in cell mechanics. We find spectrin and acto-myosin to be spatially distinct but cooperating during mechanical challenges, such as cell adhesion and contraction, or compression, stretch and osmolarity fluctuations, creating a cohesive cortex supporting the plasma membrane. Actin territories control protrusions and contractile structures while spectrin territories concentrate in retractile zones and low-actin density/inter-contractile regions, acting as a fence that organize membrane trafficking events. We unveil here the existence of a dynamic interplay between acto-myosin and spectrin necessary to support a mesoscale organization of the lipid bilayer into spatially-confined cortical territories during cell mechanoresponse.


Assuntos
Actomiosina/metabolismo , Membrana Celular/metabolismo , Espectrina/metabolismo , Actinas/metabolismo , Animais , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose/fisiologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Camundongos , Microscopia Confocal , Células NIH 3T3 , Espectrina/genética , Estresse Mecânico
3.
Mol Cell Biol ; 40(17)2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601107

RESUMO

Fodrin and its erythroid cell-specific isoform spectrin are actin-associated fibrous proteins that play crucial roles in the maintenance of structural integrity in mammalian cells, which is necessary for proper cell function. Normal cell morphology is altered in diseases such as various cancers and certain neuronal disorders. Fodrin and spectrin are two-chain (αß) molecules that are encoded by paralogous genes and share many features but also demonstrate certain differences. Fodrin (in humans, typically a heterodimer of the products of the SPTAN1 and SPTBN1 genes) is expressed in nearly all cell types and is especially abundant in neuronal tissues, whereas spectrin (in humans, a heterodimer of the products of the SPTA1 and SPTB1 genes) is expressed almost exclusively in erythrocytes. To fulfill a role in such a variety of different cell types, it was anticipated that fodrin would need to be a more versatile scaffold than spectrin. Indeed, as summarized here, domains unique to fodrin and its regulation by Ca2+, calmodulin, and a variety of posttranslational modifications (PTMs) endow fodrin with additional specific functions. However, how fodrin structural variations and misregulated PTMs may contribute to the etiology of various cancers and neurodegenerative diseases needs to be further investigated.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Actinas/metabolismo , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , Células Eritroides/metabolismo , Humanos , Neurônios/metabolismo , Espectrina/metabolismo , Espectrina/fisiologia , Relação Estrutura-Atividade
4.
Proc Natl Acad Sci U S A ; 117(25): 14119-14126, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513720

RESUMO

Proteins synthesized in the cell can begin to fold during translation before the entire polypeptide has been produced, which may be particularly relevant to the folding of multidomain proteins. Here, we study the cotranslational folding of adjacent domains from the cytoskeletal protein α-spectrin using force profile analysis (FPA). Specifically, we investigate how the cotranslational folding behavior of the R15 and R16 domains are affected by their neighboring R14 and R16, and R15 and R17 domains, respectively. Our results show that the domains impact each other's folding in distinct ways that may be important for the efficient assembly of α-spectrin, and may reduce its dependence on chaperones. Furthermore, we directly relate the experimentally observed yield of full-length protein in the FPA assay to the force exerted by the folding protein in piconewtons. By combining pulse-chase experiments to measure the rate at which the arrested protein is converted into full-length protein with a Bell model of force-induced rupture, we estimate that the R16 domain exerts a maximal force on the nascent chain of ∼15 pN during cotranslational folding.


Assuntos
Dobramento de Proteína , Espectrina/química , Escherichia coli , Simulação de Dinâmica Molecular , Biossíntese de Proteínas , Domínios Proteicos , Espectrina/genética , Espectrina/metabolismo
5.
Cancer Res ; 80(9): 1819-1832, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32127355

RESUMO

RING-finger E3 ligases are instrumental in the regulation of inflammatory cascades, apoptosis, and cancer. However, their roles are relatively unknown in TGFß/SMAD signaling. SMAD3 and its adaptors, such as ß2SP, are important mediators of TGFß signaling and regulate gene expression to suppress stem cell-like phenotypes in diverse cancers, including hepatocellular carcinoma (HCC). Here, PJA1, an E3 ligase, promoted ubiquitination and degradation of phosphorylated SMAD3 and impaired a SMAD3/ß2SP-dependent tumor-suppressing pathway in multiple HCC cell lines. In mice deficient for SMAD3 (Smad3 +/-), PJA1 overexpression promoted the transformation of liver stem cells. Analysis of genes regulated by PJA1 knockdown and TGFß1 signaling revealed 1,584 co-upregulated genes and 1,280 co-downregulated genes, including many implicated in cancer. The E3 ligase inhibitor RTA405 enhanced SMAD3-regulated gene expression and reduced growth of HCC cells in culture and xenografts of HCC tumors, suggesting that inhibition of PJA1 may be beneficial in treating HCC or preventing HCC development in at-risk patients.Significance: These findings provide a novel mechanism regulating the tumor suppressor function of TGFß in liver carcinogenesis.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Regulação para Baixo , Deleção de Genes , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Xenoenxertos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologia , Fosforilação , RNA Interferente Pequeno , Proteínas Smad/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/deficiência , Proteína Smad3/genética , Espectrina/genética , Espectrina/metabolismo , Células-Tronco/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Regulação para Cima , Sequenciamento Completo do Exoma
6.
Cell Mol Biol Lett ; 25: 3, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32042281

RESUMO

Background: Precise coordination of cytoskeletal components and dynamic control of cell adhesion and migration are required for crucial cell processes such as differentiation and morphogenesis. We investigated the potential involvement of αII-spectrin, a ubiquitous scaffolding element of the membrane skeleton, in the adhesion and angiogenesis mechanism. Methods: The cell models were primary human umbilical vein endothelial cells (HUVECs) and a human dermal microvascular endothelial cell line (HMEC-1). After siRNA- and shRNA-mediated knockdown of αII-spectrin, we assessed its expression and that of its partners and adhesion proteins using western blotting. The phenotypes of the control and spectrin-depleted cells were examined using immunofluorescence and video microscopy. Capillary tube formation was assessed using the thick gel Matrigel matrix-based method and a microscope equipped with a thermostatic chamber and a Nikon Biostation System camera. Results: Knockdown of αII-spectrin leads to: modified cell shape; actin cytoskeleton organization with the presence of peripheral actin patches; and decreased formation of stress fibers. Spectrin deficiency affects cell adhesion on laminin and fibronectin and cell motility. This included modification of the localization of adhesion molecules, such as αVß3- and α5-integrins, and organization of adhesion structures, such as focal points. Deficiency of αII-spectrin can also affect the complex mechanism of in vitro capillary tube formation, as demonstrated in a model of angiogenesis. Live imaging revealed that impairment of capillary tube assembly was mainly associated with a significant decrease in cell projection length and stability. αII-spectrin depletion is also associated with significantly decreased expression of three proteins involved in capillary tube formation and assembly: VE-cadherin, MCAM and ß3-integrin. Conclusion: Our data confirm the role of αII-spectrin in the control of cell adhesion and spreading. Moreover, our findings further support the participation of αII-spectrin in capillary tube formation in vitro through control of adhesion molecules, such as integrins. This indicates a new function of αII-spectrin in angiogenesis.


Assuntos
Citoesqueleto de Actina/metabolismo , Capilares/metabolismo , Adesão Celular/fisiologia , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Espectrina/metabolismo , Antígenos CD/metabolismo , Antígeno CD146/metabolismo , Caderinas/metabolismo , Capilares/crescimento & desenvolvimento , Adesão Celular/genética , Diferenciação Celular/genética , Movimento Celular/genética , Forma Celular , Células Endoteliais/citologia , Fibronectinas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Cadeias alfa de Integrinas/metabolismo , Integrina beta3/metabolismo , Laminina/metabolismo , Morfogênese/genética , Morfogênese/fisiologia , Neovascularização Fisiológica/genética , RNA Interferente Pequeno , Espectrina/deficiência , Espectrina/genética , Fibras de Estresse/metabolismo
7.
Sci Rep ; 10(1): 2917, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076054

RESUMO

Fluorescent nanoscopy approaches have been used to characterize the periodic organization of actin, spectrin and associated proteins in neuronal axons and dendrites. This membrane-associated periodic skeleton (MPS) is conserved across animals, suggesting it is a fundamental component of neuronal extensions. The nanoscale architecture of the arrangement (190 nm) is below the resolution limit of conventional fluorescent microscopy. Fluorescent nanoscopy, on the other hand, requires costly equipment and special analysis routines, which remain inaccessible to most research groups. This report aims to resolve this issue by using protein-retention expansion microscopy (pro-ExM) to reveal the MPS of axons. ExM uses reagents and equipment that are readily accessible in most neurobiology laboratories. We first explore means to accurately estimate the expansion factors of protein structures within cells. We then describe the protocol that produces an expanded specimen that can be examined with any fluorescent microscopy allowing quantitative nanoscale characterization of the MPS. We validate ExM results by direct comparison to stimulated emission depletion (STED) nanoscopy. We conclude that ExM facilitates three-dimensional, multicolor and quantitative characterization of the MPS using accessible reagents and conventional fluorescent microscopes.


Assuntos
Axônios/metabolismo , Microscopia de Fluorescência/métodos , Espectrina/metabolismo , Animais , Calibragem , Membrana Celular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Ratos Wistar , Reprodutibilidade dos Testes
8.
Nat Commun ; 11(1): 133, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919407

RESUMO

Neurons are subjected to strain due to body movement and their location within organs and tissues. However, how they withstand these forces over the lifetime of an organism is still poorly understood. Here, focusing on touch receptor neuron-epidermis interactions using Caenorhabditis elegans as a model system, we show that UNC-70/ß-spectrin and TBC-10, a conserved GTPase-activating protein, function non-cell-autonomously within the epidermis to dynamically maintain attachment of the axon. We reveal that, in response to strain, UNC-70/ß-spectrin and TBC-10 stabilize trans-epidermal hemidesmosome attachment structures which otherwise become lost, causing axonal breakage and degeneration. Furthermore, we show that TBC-10 regulates axonal attachment and maintenance by inactivating RAB-35, and reveal functional conservation of these molecules with their vertebrate orthologs. Finally, we demonstrate that ß-spectrin functions in this context non-cell-autonomously. We propose a model in which mechanically resistant epidermal attachment structures are maintained by UNC-70/ß-spectrin and TBC-10 during movement, preventing axonal detachment and degeneration.


Assuntos
Axônios/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Espectrina/metabolismo , Estresse Fisiológico/fisiologia , Animais , Citoesqueleto/fisiologia , Epiderme/metabolismo , Hemidesmossomos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
9.
J Biol Chem ; 295(1): 191-211, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31776189

RESUMO

Epithelial adherens junctions (AJs) and tight junctions (TJs) undergo disassembly and reassembly during morphogenesis and pathological states. The membrane-cytoskeleton interface plays a crucial role in junctional reorganization. Protein 4.1R (4.1R), expressed as a diverse array of spliceoforms, has been implicated in linking the AJ and TJ complex to the cytoskeleton. However, which specific 4.1 isoform(s) participate and the mechanisms involved in junctional stability or remodeling remain unclear. We now describe a role for epithelial-specific isoforms containing exon 17b and excluding exon 16 4.1R (4.1R+17b) in AJs. 4.1R+17b is exclusively co-localized with the AJs. 4.1R+17b binds to the armadillo repeats 1-2 of ß-catenin via its membrane-binding domain. This complex is linked to the actin cytoskeleton via a bispecific interaction with an exon 17b-encoded peptide. Exon 17b peptides also promote fodrin-actin complex formation. Expression of 4.1R+17b forms does not disrupt the junctional cytoskeleton and AJs during the steady-state or calcium-dependent AJ reassembly. Overexpression of 4.1R-17b forms, which displace the endogenous 4.1R+17b forms at the AJs, as well as depletion of the 4.1R+17b forms both decrease junctional actin and attenuate the recruitment of spectrin to the AJs and also reduce E-cadherin during the initial junctional formation of the AJ reassembly process. Expressing 4.1R+17b forms in depleted cells rescues junctional localization of actin, spectrin, and E-cadherin assembly at the AJs. Together, our results identify a critical role for 4.1R+17b forms in AJ assembly and offer additional insights into the spectrin-actin-4.1R-based membrane skeleton as an emerging regulator of epithelial integrity and remodeling.


Assuntos
Junções Aderentes/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Actinas/metabolismo , Processamento Alternativo , Animais , Sítios de Ligação , Caderinas/metabolismo , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/genética , Cães , Humanos , Células Madin Darby de Rim Canino , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Espectrina/metabolismo , beta Catenina/química , beta Catenina/metabolismo
10.
J Cell Physiol ; 235(1): 17-25, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31206681

RESUMO

SPTBN1 is a dynamic intracellular nonpleckstrin homology-domain protein, functioning as a transforming growth factor-ß signal transducing adapter protein which is necessary to form Smad3/Smad4 complex. Recently SPTBN1 is considered to be associated with many kinds of cancers. SPTBN1 expression and function differ between different tumor states or types. This review summarizes the recent advances in the expression patterns of SPTBN1 in cancers, and in understanding the mechanisms by which SPTBN1 affects the occurrence, progression, and metastasis of cancer. Identifying SPTBN1 expression and function in cancers will contribute to the clinical diagnosis and treatment of cancer and the investigation of anticancer drugs.


Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias/metabolismo , Espectrina/metabolismo , Antineoplásicos , Humanos , Neoplasias/classificação , Espectrina/genética
11.
J Biol Chem ; 294(49): 18881-18897, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31653705

RESUMO

Scavenger receptor class A member 1 (SCARA1 or CD204) is an immune receptor highly expressed on macrophages. It forms homotrimers on the cell surface and plays important roles in regulating immune responses via its involvement in multiple pathways. However, both the structure and the functional roles of SCARA1 are not fully understood. Here, we determined the crystal structure of the C-terminal SRCR domain of SCARA1 at 1.8 Å resolution, revealing its Ca2+-binding site. Results from cell-based assays revealed that SCARA1 can recognize dead cells, rather than live cells, specifically through its SRCR domain and in a Ca2+-dependent manner. Furthermore, by combining MS and biochemical assays, we found that cellular spectrin is the binding target of SCARA1 on dead cells and that the SRCR domain of SCARA1 recognizes the SPEC repeats of spectrin in the presence of Ca2+ We also found that macrophages can internalize dead cells or debris from both erythrocytes and other cells through the interaction between SCARA1 and spectrin, suggesting that SCARA1 could function as a scavenging receptor that recognizes dead cells. These results suggest that spectrin, which is one of the major components of the cytoskeleton, acts as a cellular marker that enables the recognition of dead cells by the immune system.


Assuntos
Proteínas de Choque Térmico/metabolismo , Receptores Depuradores Classe A/metabolismo , Espectrina/metabolismo , Animais , Endocitose/fisiologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Células HEK293 , Humanos , Células Jurkat , Espectrometria de Massas , Camundongos , Microscopia Confocal , Células NIH 3T3 , Ligação Proteica , Células RAW 264.7
12.
Traffic ; 20(12): 932-942, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31569283

RESUMO

Fenestrae are open transmembrane pores that are a structural hallmark of healthy liver sinusoidal endothelial cells (LSECs). Their key role is the transport of solutes and macromolecular complexes between the sinusoidal lumen and the space of Disse. To date, the biochemical nature of the cytoskeleton elements that surround the fenestrae and sieve plates in LSECs remain largely elusive. Herein, we took advantage of the latest developments in atomic force imaging and super-resolution fluorescence nanoscopy to define the organization of the supramolecular complex(es) that surround the fenestrae. Our data revealed that spectrin, together with actin, lines the inner cell membrane and provided direct structural support to the membrane-bound pores. We conclusively demonstrated that diamide and iodoacetic acid (IAA) affect fenestrae number by destabilizing the LSEC actin-spectrin scaffold. Furthermore, IAA induces rapid and repeatable switching between the open vs closed state of the fenestrae, indicating that the spectrin-actin complex could play an important role in controlling the pore number. Our results suggest that spectrin functions as a key regulator in the structural preservation of the fenestrae, and as such, it might serve as a molecular target for altering transendothelial permeability.


Assuntos
Actinas/metabolismo , Membrana Celular/ultraestrutura , Células Endoteliais/ultraestrutura , Fígado/ultraestrutura , Espectrina/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Membrana Celular/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Fígado/irrigação sanguínea , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica , Imagem Individual de Molécula
14.
Cytoskeleton (Hoboken) ; 76(6): 383-397, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31397976

RESUMO

Spectrin, the major protein of the erythrocyte membrane skeleton has canonically been thought to only serve a structural function. We have previously described a novel chaperone-like property of spectrin and also hypothesized that the chaperone activity and binding of a hydrophobic ligand, Prodan are localized in the self-association domain. Here we probe the location and molecular origin of the chaperone activity of multi-domain spectrin using a selection of individual recombinant spectrin domains, which we have characterized using intrinsic tryptophan fluorescence and CD spectroscopy to show their identity to native spectrin. Aggregation assays using insulin, ADH, α- and ß-globin as well as enzyme refolding assays using alkaline phosphatase and α-glucosidase show that the chaperone activity is not only localized in the self-association domain but is a generalized property of spectrin domains. This is to our understanding, a unique feature in the case of modular multi-repeat proteins, possibly implicating that the large family of "spectrin-repeat" domain containing proteins may also have chaperone like property. Substrate selectivity of chaperone activity as evidenced by the preferential protection of α- over ß-globin chains is seen; which has implications in hemoglobin diseases. Moreover, enzyme-refolding assays also indicate alternate modes of chaperone action. We propose that the molecular origin of chaperone activity resides in the surface exposed hydrophobic patches of the spectrin domains as shown by ANS (1-anilinonaphthalene-8-sulfonic acid) and Prodan (6-propionyl-2[dimethylamino]-naphthalene) binding. We also show that Prodan does indeed have a unique binding site on spectrin located at the self-association domain.


Assuntos
Eritrócitos/metabolismo , Chaperonas Moleculares/metabolismo , Espectrina/metabolismo , Fosfatase Alcalina/metabolismo , Anisotropia , Corantes Fluorescentes/metabolismo , Humanos , Ligantes , Ligação Proteica , Domínios Proteicos , Espectrina/química , Espectrometria de Fluorescência , alfa-Glucosidases/metabolismo
15.
Neuroscience ; 415: 147-160, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31369718

RESUMO

Stroke is a major life-threatening and disabling disease with a restricted therapeutic approach. Bone marrow stromal cells (BMSCs) possess proliferative ability and a multi-directional differentiation potential, and secrete a range of trophic/growth factors that can protect neurons after cerebral ischemia/reperfusion. Transient receptor potential canonical (TRPC) is a family of non-selective channels permeable to Ca2+, with several functions including neuronal survival. Over-expression of TRPC6, a subtype of the TRPC family, was shown to protect neurons against cerebral ischemia/reperfusion injury. However, it remains unclear whether over-expression of TRPC6 in BMSCs can further reduce brain injury after ischemia/reperfusion. In the present study, we report that over-expression of TRPC6 via a CRISPR-based synergistic activation mediator in BMSCs provided a greater reduction of brain injury in a rat model of ischemia/reperfusion. Further, the improved neurofunctional outcomes were associated with increased TRPC6 and brain derived neurotrophic factor expression levels. Overall, these data suggest that TRPC6 over-expressing BMSCs may be a promising therapeutic agent for ischemic stroke.


Assuntos
Neuroproteção/genética , Traumatismo por Reperfusão/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Isquemia Encefálica/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Calpaína/metabolismo , Sobrevivência Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Masculino , Células-Tronco Mesenquimais , Modelos Animais , Neurônios/metabolismo , Ratos , Ratos Wistar , Espectrina/metabolismo , Canais de Cátion TRPC/genética , Regulação para Cima
16.
Bone ; 128: 112056, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31376534

RESUMO

Vibration at high frequency has been demonstrated to be anabolic for bone and embedded osteocytes. The response of osteocytes to vibration is frequency-dependent, but the mechanism remains unclear. Our previous computational study using an osteocyte finite element model has predicted a resonance effect involving in the frequency-dependent response of osteocytes to vibration. However, the cellular spontaneous vibratory motion of osteocytes has not been confirmed. In the present study, the cellular vibratory motions (CVM) of osteocytes were recorded by a custom-built digital holographic microscopy and quantitatively analyzed. The roles of ATP and spectrin network in the CVM of osteocytes were studied. Results showed the MLO-Y4 osteocytes displayed dynamic vibratory motions with an amplitude of ~80 nm, which is relied both on the ATP content and spectrin network. Spectrum analysis showed several frequency peaks in CVM of MLO-Y4 osteocytes at 30 Hz, 39 Hz, 83 Hz and 89 Hz. These peak frequencies are close to the commonly used effective frequencies in animal training and in-vitro cell experiments, and show a correlation with the computational predictions of the osteocyte finite element model. These results implicate that osteocytes are dynamic and the cellular dynamic motion is involved in the cellular mechanotransduction of vibration.


Assuntos
Trifosfato de Adenosina/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Espectrina/metabolismo , Animais , Linhagem Celular , Camundongos
17.
Science ; 365(6456): 929-934, 2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31467223

RESUMO

Actin, spectrin, and related molecules form a membrane-associated periodic skeleton (MPS) in neurons. The function of the MPS, however, remains poorly understood. Using super-resolution imaging, we observed that G protein-coupled receptors (GPCRs), cell adhesion molecules (CAMs), receptor tyrosine kinases (RTKs), and related signaling molecules were recruited to the MPS in response to extracellular stimuli, resulting in colocalization of these molecules and RTK transactivation by GPCRs and CAMs, giving rise to extracellular signal-regulated kinase (ERK) signaling. Disruption of the MPS prevented such molecular colocalizations and downstream ERK signaling. ERK signaling in turn caused calpain-dependent MPS degradation, providing a negative feedback that modulates signaling strength. These results reveal an important functional role of the MPS and establish it as a dynamically regulated platform for GPCR- and CAM-mediated RTK signaling.


Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Neurônios/enzimologia , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Espectrina/metabolismo , Animais , Antígeno CD56/metabolismo , Calpaína/metabolismo , Moléculas de Adesão Celular/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Imagem Molecular , Cultura Primária de Células , Transporte Proteico , Proteólise , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo
18.
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
19.
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
20.
Proc Natl Acad Sci U S A ; 116(31): 15686-15695, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31209033

RESUMO

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/genética
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