RESUMO
During development, cycles of spatiotemporal remodeling of higher-order networks of actin filaments contribute to control cell fate specification and differentiation. Programs for controlling these dynamics are hard-wired into actin-regulatory proteins. The filamin family of actin-binding proteins exert crucial mechanotransduction and signaling functions in tissue morphogenesis. Filamin-B (FLNB) is a key player in chondrocyte progenitor differentiation for endochondral ossification. Biallelic loss-of-function mutations or gain-of-function mutations in FLNB cause two groups of skeletal disorders that can be attributed to either the loss of repressive function on TGF-ß signaling or a disruption in mechanosensory properties, respectively. In this Review, we highlight a unique family of vertebrate-specific short-lived filamin-binding proteins, the refilins (refilin-A and refilin-B), that modulate filamin-dependent actin crosslinking properties. Refilins are downstream TGF-ß effectors in epithelial cells. Double knockout of both refilin-A and refilin-B in mice results in precocious ossification of some axial skeletal elements, leading to malformations that are similar to those seen in FLNB-deficient mice. Based on these findings, we present a model summarizing the role of refilins in regulating the mechanosensory functions of FLNB during skeletal development. We also discuss the possible contribution of refilins to FLNB-related skeletal pathologies that are associated with gain-of-function mutations.
Assuntos
Citoesqueleto de Actina/metabolismo , Filaminas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Diferenciação Celular/fisiologia , HumanosRESUMO
The intracellular localization and shape of the nucleus plays a central role in cellular and developmental processes. In fibroblasts, nuclear movement and shape are controlled by a specific perinuclear actin network made of contractile actin filament bundles called transmembrane actin-associated nuclear (TAN) lines that form a structure called the actin cap. The identification of regulatory proteins associated with this specific actin cytoskeletal dynamic is a priority for understanding actin-based changes in nuclear shape and position in normal and pathological situations. Here, we first identify a unique family of actin regulators, the refilin proteins (RefilinA and RefilinB), that stabilize specifically perinuclear actin filament bundles. We next identify the actin-binding filamin A (FLNA) protein as the downstream effector of refilins. Refilins act as molecular switches to convert FLNA from an actin branching protein into one that bundles. In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes the perinuclear actin filament bundles forming the actin cap. Finally, we demonstrate that in epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during the epithelial-mesenchymal transition (EMT). Our studies open perspectives for further functional analyses of this unique actin-based network and shed light on FLNA function during development and in human syndromes associated with FLNA mutations.
Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Proteínas Contráteis/metabolismo , Proteínas dos Microfilamentos/metabolismo , Sequência de Aminoácidos , Animais , Astrocitoma/metabolismo , Astrocitoma/ultraestrutura , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/ultraestrutura , Dimerização , Transição Epitelial-Mesenquimal , Feminino , Filaminas , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Complexos Multiproteicos , Células NIH 3T3 , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genética , Deleção de SequênciaRESUMO
Ca(2+)-binding proteins of the S100 family participate in intracellular Ca(2+) signaling by binding to and regulating specific cellular targets in their Ca(2+)-loaded conformation. Because the information on specific cellular targets of different S100 proteins is still limited, we developed an affinity approach that selects for protein targets only binding to the physiologically active dimer of an S100 protein. Using this approach, we here identify IQGAP1 as a novel and dimer-specific target of S100P, a member of the S100 family enriched in the cortical cytoskeleton. The interaction between S100P and IQGAP1 is strictly Ca(2+)-dependent and characterized by a dissociation constant of 0.2 µM. Binding occurs primarily through the IQ domain of IQGAP1 and the first EF hand loop of S100P, thus representing a novel structural principle of S100-target protein interactions. Upon cell stimulation, S100P and IQGAP1 co-localize at or in close proximity to the plasma membrane, and complex formation can be linked to altered signal transduction properties of IQGAP1. Specifically, the EGF-induced tyrosine phosphorylation of IQGAP1 that is thought to function in assembling signaling intermediates at IQGAP1 scaffolds in the subplasmalemmal region is markedly reduced in cells overexpressing S100P but not in cells expressing an S100P mutant deficient in IQGAP1 binding. Furthermore, B-Raf binding to IQGAP1 and MEK1/2 activation occurring downstream of IQGAP1 in EGF-triggered signaling cascades are compromised at elevated S100P levels. Thus, S100P is a novel Ca(2+)-dependent regulator of IQGAP1 that can down-regulate the function of IQGAP1 as a signaling intermediate by direct interaction.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Neoplasias/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Calmodulina/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Dimerização , Células HeLa , Humanos , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fosforilação/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ativadoras de ras GTPase/química , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Gliomas are the most common primary brain tumor affecting human adults and remain a therapeutic challenge because cells of origin are still unknown. Here, we investigated the cellular origin of low-grade gliomas in a rat model based on transplacental exposure to N-ethyl-N-nitrosourea (ENU). Longitudinal magnetic resonance imaging coupled to immunohistological and immunocytochemical analyses were used to further characterize low-grade rat gliomas at different stages of evolution. We showed that early low-grade gliomas have characteristics of oligodendroglioma-like tumors and exclusively contain NG2-expressing slow dividing precursor cells, which express early markers of oligodendroglial lineage. These tumor-derived precursors failed to fully differentiate into oligodendrocytes and exhibited multipotential abilities in vitro. Moreover, a few glioma NG2+ cells are resistant to radiotherapy and may be responsible for tumor recurrence, frequently observed in humans. Overall, these findings suggest that transformed multipotent NG2 glial precursor cell may be a potential cell of origin in the genesis of rat ENU-induced oligodendroglioma-like tumors. This work may open up new perspectives for understanding biology of human gliomas.
Assuntos
Antígenos/análise , Neoplasias Encefálicas/induzido quimicamente , Etilnitrosoureia/toxicidade , Células-Tronco Neoplásicas/patologia , Oligodendroglioma/induzido quimicamente , Proteoglicanas/análise , Animais , Neoplasias Encefálicas/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Células-Tronco Neoplásicas/química , Oligodendroglioma/patologia , Ratos , Ratos Sprague-Dawley , Proteínas Ativadoras de ras GTPase/análiseRESUMO
In mammals, adipose tissue is an active secretory tissue that responds to mild hypothermia and as such is a genuine model to study molecular and cellular adaptive responses to cold-stress. A recent study identified a mammal-specific protein of the endoplasmic reticulum that is strongly induced in the inguinal subcutaneous white adipocyte upon exposure to cold, calsyntenin 3ß (CLSTN3ß). CLSTN3ß regulates sympathetic innervation of thermogenic adipocytes and contributes to adaptive non-shivering thermogenesis. The calcium- and zinc-binding S100B is a downstream effector in the CLSTN3ß pathways. We review, here, the literature on the transcriptional regulation of the S100b gene in adipocyte cells. We also rationalize the interactions of the S100B protein with its recognized or hypothesized intracellular (p53, ATAD3A, CYP2E1, AHNAK) and extracellular (Receptor for Advanced Glycation End products (RAGE), RPTPσ) target proteins in the context of adipocyte differentiation and adaptive thermogenesis. We highlight a chaperon-associated function for the intracellular S100B and point to functional synergies between the different intracellular S100B target proteins. A model of non-classical S100B secretion involving AHNAK/S100A10/annexin2-dependent exocytosis by the mean of exosomes is also proposed. Implications for related areas of research are noted and suggestions for future research are offered.
Assuntos
Adipócitos/metabolismo , Resposta ao Choque Frio , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Termogênese , Animais , Humanos , Subunidade beta da Proteína Ligante de Cálcio S100/genéticaRESUMO
The S100 genes encode a conserved group of 21 vertebrate-specific EF-hand calcium-binding proteins. Since their discovery in 1965, S100 proteins have remained enigmatic in terms of their cellular functions. In this review, we summarize the calcium- and zinc-binding properties of the dimeric S100B and S100A1 proteins and highlight data that shed new light on the extracellular and intracellular regulation and functions of S100B. We point out that S100B and S100A1 homodimers are not functionally interchangeable and that in a S100A1/S100B heterodimer, S100A1 acts as a negative regulator for the ability of S100B to bind Zn2+ . The Ca2+ and Zn2+ -dependent interactions of S100B with a wide array of proteins form the basis of its activities and have led to the derivation of some initial rules for S100B recognition of protein targets. However, recent findings have strongly suggested that these rules need to be revisited. Here, we describe a new consensus S100B binding motif present in intracellular and extracellular vertebrate-specific proteins and propose a new model for stable interactions of S100B dimers with full-length target proteins. A chaperone-associated function for intracellular S100B in adaptive cellular stress responses is also discussed. This review may help guide future studies on the functions of S100 proteins in general.
Assuntos
Cálcio/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Proteínas S100/metabolismo , Zinco/metabolismo , Animais , Humanos , Estrutura Molecular , Subunidade beta da Proteína Ligante de Cálcio S100/química , Proteínas S100/químicaRESUMO
Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell-cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca(2+)-dependent cell-cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2-specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.
Assuntos
Anexina A2/metabolismo , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas S100/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Anexina A2/antagonistas & inibidores , Anexina A2/genética , Adesão Celular/genética , Comunicação Celular/genética , Linhagem Celular Tumoral , Membrana Celular/ultraestrutura , Polaridade Celular/genética , Tamanho Celular/genética , Citosol/metabolismo , Citosol/ultraestrutura , Cães , Regulação para Baixo/genética , Células Epiteliais/ultraestrutura , Humanos , Junções Intercelulares/metabolismo , Junções Intercelulares/ultraestrutura , Substâncias Macromoleculares , Estrutura Terciária de Proteína/genética , RNA Interferente PequenoRESUMO
Human oligodendrogliomas are chemosensitive gliomas usually characterized by a loss of heterozygosity in the large distal regions of the short arm of chromosome 1 (1p LOH). Chemoresistant astrocytomas do not have this genetic signature, suggesting that the 1p arms may contain anti-oncogene and/or genes enabling chemoresistance. We have focused here on two human 1p-distal genes, ATAD 3A and ATAD 3B (1p36-33), and analyzed their gene products in normal human cell lines and tissues and in glioma-derived human cell lines. Using specific anti-peptide antibodies, we have found that ATAD 3A is ubiquitously expressed, whereas ATAD 3B is expressed in embryonic tissues, adult germinative zone and in astrocytoma cell lines but it is not expressed in oligodendroglioma cell lines or in the adult cortex. Furthermore, we have found that human glioma cell lines overexpressing or underexpressing ATAD 3A and ATAD 3B, show modified cell growth, anchorage-independent growth, and chemoresistance to doxorubicin and other genotoxic drugs. These results demonstrate the potential for ATAD 3B as a putative marker in discriminating astrocytomas from oligodendrogliomas. We also have shown that the loss of ATAD 3A/3B may be involved in the transformation pathway and the chemosensitivity of oligodendrogliomas.
Assuntos
Astrocitoma/genética , Neoplasias Encefálicas/genética , Cromossomos Humanos Par 1/genética , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Genes Supressores de Tumor/fisiologia , Oligodendroglioma/genética , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases , Animais , Astrocitoma/diagnóstico , Astrocitoma/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Células COS , Células CACO-2 , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Chlorocebus aethiops , Diagnóstico Diferencial , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Humanos , Proteínas de Membrana , Camundongos , Proteínas Mitocondriais , Mutação/genética , Células NIH 3T3 , Invasividade Neoplásica/genética , Oligodendroglioma/diagnóstico , Oligodendroglioma/metabolismo , Isoformas de Proteínas/genéticaRESUMO
In the germinative zone of the adult rodent brain, neural progenitors migrate into niches delimited by astrocyte processes and differentiate into neuronal precursors. In the present study, we report a modulating role for the scaffolding protein IQGAP1 in neural progenitor migration. We have identified IQGAP1 as a new marker of amplifying neural progenitor and neuronal precursor cells of the subventricular zone (SVZ) and the rostral migratory stream (RMS) in the adult mouse brain. To determine functions for IQGAP1 in neural progenitors, we compared the properties of neural progenitor cells from wild-type and Iqgap1-null mutant mice in vivo and in vitro. The in vivo studies reveal a delay in the transition of de novo neural progenitors into neuronal precursor cells in Iqgap1-null mice. In vitro, we demonstrated that IQGAP1 acts as a downstream effector in the vascular endothelial growth factor (VEGF)-dependent migratory response of neural progenitors that also impacts on their neuronal differentiation. The Rho-family GTPases cdc42/Rac1 and Lis1 are major partners of IQGAP1 in this migratory process. Finally, astrocytes of the neurogenic SVZ and RMS are shown to express VEGF. We propose that VEGF synthesized by astrocytes could be involved in the guidance of neural progenitors to neurogenic niches and that IQGAP1 is an effector of the VEGF-dependent migratory signal.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/fisiologia , Movimento Celular/fisiologia , Neurônios/citologia , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Ventrículos Cerebrais/citologia , Técnicas In Vitro , Camundongos , Camundongos Knockout , Neuropeptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Mutations in dysferlin cause limb girdle muscular dystrophy 2B, Miyoshi myopathy and distal anterior compartment myopathy. Dysferlin is proposed to play a role in muscle membrane repair. To gain functional insight into the molecular mechanisms of dysferlin, we have searched for dysferlin-interacting proteins in skeletal muscle. By coimmunoprecipitation coupled with mass spectrometry, we demonstrate that AHNAK interacts with dysferlin. We defined the binding sites in dysferlin and AHNAK as the C2A domain in dysferlin and the carboxyterminal domain of AHNAK by glutathione S-transferase (GST)-pull down assays. As expected, the N-terminal domain of myoferlin also interacts with the carboxyterminal domain of AHNAK. In normal skeletal muscle, dysferlin and AHNAK colocalize at the sarcolemmal membrane and T-tubules. In dysferlinopathies, reduction or absence of dysferlin correlates with a secondary muscle-specific loss of AHNAK. Moreover, in regenerating rat muscle, dysferlin and AHNAK showed a marked increase and cytoplasmic localization, consistent with the direct interaction between them. Our data suggest that dysferlin participates in the recruitment and stabilization of AHNAK to the sarcolemma and that AHNAK plays a role in dysferlin membrane repair process. It may also have significant implications for understanding the biology of AHNAK-containing exocytotic vesicles, "enlargosomes," in plasma membrane remodeling and repair.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Neoplasias/metabolismo , Regeneração/fisiologia , Animais , Proteínas de Transporte/metabolismo , Citoplasma/metabolismo , Disferlina , Feminino , Humanos , Imunoprecipitação , Espectrometria de Massas , Proteínas de Membrana/química , Camundongos , Proteínas Musculares/química , Músculo Esquelético/fisiologia , Mutação , Ratos , Ratos WistarRESUMO
The accurate identification and thorough characterization of tumorigenic cells in glioblastomas are essential to enhance our understanding of their malignant behavior and for the design of strategies that target this important cell population. We report here that, in rat brain, the scaffolding protein IQGAP1 is a marker of brain nestin+ amplifying neural progenitor cells. In a rat model of glioma, IQGAP1 also characterizes a subpopulation of nestin+ amplifying tumor cells in glioblastoma-like tumors but not in tumors with oligodendroglioma features. We next confirmed that IQGAP1 represents a new marker that may help to discriminate human glioblastoma from oligodendrogliomas. In human glioblastoma exclusively, IQGAP1 specifies a subpopulation of amplifying nestin+ cancer cells. Neoplastic IQGAP1+ cells from glioblastoma can be expanded in culture and possess all the characteristics of cancer stem-like progenitors. The similarities between amplifying neural progenitors and glioblastoma amplifying cancer cells may have significant implications for understanding the biology of glioblastoma.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteínas Ativadoras de ras GTPase/biossíntese , Animais , Humanos , Imuno-Histoquímica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neurônios/patologia , Ratos , Células-Tronco/patologiaRESUMO
In yeast, a sequence of physical and genetic interactions termed the endoplasmic reticulum (ER)-mitochondria organizing network (ERMIONE) controls mitochondria-ER interactions and mitochondrial biogenesis. Several functions that characterize ERMIONE complexes are conserved in mammalian cells, suggesting that a similar tethering complex must exist in metazoans. Recent studies have identified a new family of nuclear-encoded ATPases associated with diverse cellular activities (AAA+-ATPase) mitochondrial membrane proteins specific to multicellular eukaryotes, called the ATPase family AAA domain-containing protein 3 (ATAD3) proteins (ATAD3A and ATAD3B). These proteins are crucial for normal mitochondrial-ER interactions and lie at the heart of processes underlying mitochondrial biogenesis. ATAD3A orthologues have been studied in flies, worms, and mammals, highlighting the widespread importance of this gene during embryonic development and in adulthood. ATAD3A is a downstream effector of target of rapamycin (TOR) signalling in Drosophila and exhibits typical features of proteins from the ERMIONE-like complex in metazoans. In humans, mutations in the ATAD3A gene represent a new link between altered mitochondrial-ER interaction and recognizable neurological syndromes. The primate-specific ATAD3B protein is a biomarker of pluripotent embryonic stem cells. Through negative regulation of ATAD3A function, ATAD3B supports mitochondrial stemness properties.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Retículo Endoplasmático/enzimologia , Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Regulação Enzimológica da Expressão Gênica/fisiologia , Mamíferos , Proteínas de Membrana/genéticaRESUMO
Refilins (RefilinA and RefilinB) are members of a novel family of Filamin binding proteins that function as molecular switches to conformationally alter the Actin filament network into bundles. We show here that Refilins are extremely labile proteins. An N-terminal PEST/DSG(X)2-4S motif mediates ubiquitin-independent rapid degradation. A second degradation signal is localized within the C-terminus. Only RefilinB is protected from rapid degradation by an auto-inhibitory domain that masks the PEST/DSG(X)2-4S motif. Dual regulation of RefilinA and RefilinB stability was confirmed in rat brain NG2 precursor cells (polydendrocyte). Using loss- and gain-of-function approaches we show that in these cells, and in U373MG cells, Refilins contribute to the dynamics of lamellipodium protrusion by catalysing Actin bundle formation within the lamella Actin network. These studies extend the Actin bundling function of the Refilin-Filamin complex to dynamic regulation of cell membrane remodelling.
RESUMO
Mitochondria are dynamic organelles that alter their morphology in response to cellular signaling and differentiation through balanced fusion and fission. In this study, we found that the mitochondrial inner membrane ATPase ATAD3A interacted with ccdc56/MITRAC12/COA3, a subunit of the cytochrome oxidase (COX)-assembly complex. Overproduction of ccdc56 in HeLa cells resulted in fragmented mitochondrial morphology, while mitochondria were highly elongated in ccdc56-repressed cells by the defective recruitment of the fission factor Drp1. We also found that mild and chronic inhibition of COX led to mitochondrial elongation, as seen in ccdc56-repressed cells. These results indicate that ccdc56 positively regulates mitochondrial fission via regulation of COX activity and the mitochondrial recruitment of Drp1, and thus, suggest a novel relationship between COX assembly and mitochondrial morphology.
Assuntos
GTP Fosfo-Hidrolases/genética , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Mitocondriais/genética , Tamanho Mitocondrial/genética , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Dinaminas , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Immunoblotting , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Proteína Vermelha FluorescenteRESUMO
Here we report a detailed analysis of the expression and localization of the giant protein AHNAK in adult mouse tissues. We show that AHNAK is widely expressed in muscle cells, including cardiomyocytes, smooth muscle cells, skeletal muscle, myoepithelium, and myofibroblasts. AHNAK is also specifically expressed in epithelial cells of most lining epithelium, but is absent in epithelium with more specialized secretory or absorptive functions. In all adult tissues, the main localization of AHNAK is at the plasma membrane. A role for AHNAK in the specific organization and the structural support of the plasma membrane common to muscle and lining epithelium is discussed.
Assuntos
Proteínas de Membrana/metabolismo , Músculos/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Células Epiteliais/diagnóstico por imagem , Células Epiteliais/metabolismo , Epitélio/metabolismo , Epitélio/ultraestrutura , Immunoblotting , Imuno-Histoquímica , Camundongos , Músculos/citologia , Músculos/ultraestrutura , Especificidade de Órgãos , UltrassonografiaRESUMO
The S100 family consists of 19 members, which function as transducers of calcium signals in a tissue-specific manner. Upon calcium binding, the conformation of many S100 proteins changes dramatically. Several hydrophobic residues are exposed, allowing the S100 proteins to interact with their target proteins, and thereby to transduce calcium signals into specific biological responses. To further elucidate the exact contribution of the S100 calciproteins in the calcium signalling pathways, several groups have applied the yeast two-hybrid technology to identify putative target proteins for the various S100 calciproteins. Two-hybrid large screens using S100 proteins as baits have confirmed the biochemical and structural feature of S100, which enable them to form homodimers and the ability of some members to form specific heterodimers in vivo. Yeast two-hybrid investigations have allowed the identification of conserved hydrophobic residues and domains that are crucial for the stabilization of S100 homo- and heterodimers. Furthermore, this method clearly underlines that the homo- and heterodimerization mechanisms differ among the members of the S100 family. However, several lines of evidence strongly suggest that two-hybrid methodology is limited to the analysis of interactions that are calcium-independent, since no target proteins other than S100 family members themselves have been detected with this methodology.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas S100/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Dimerização , Motivos EF Hand , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas S100/química , Proteínas S100/genéticaRESUMO
S100A1 and S100B are induced by the SOX trio transcription factors (SOX9, SOX5, and SOX6) in chondrocytes, and inhibit their hypertrophic differentiation in culture. However, functional roles of S100A1 and S100B during in vivo skeletal development are yet to be determined. Here we show that mice deficient of both the S100a1 and S100b genes displayed normal skeletal growth from embryonic stage to adulthood. Although no compensatory upregulation of other S100 family members was observed in S100a1/S100b double mutants, the related S100a2, S100a4, S100a10, and S100a11 were expressed at similarly high levels to S100a1 and S100b in mouse primary chondrocytes. Furthermore, overexpression of these other S100 members suppressed the hypertrophic differentiation of chondrocytes in vitro as efficiently as S100A1 and S100B. Taken together, the present study demonstrates that S100A1 and S100B are dispensable for endochondral ossification during skeletal development, most likely because their deficiency may be masked by other S100 proteins which have similar functions to those of S100A1 and S100B.
Assuntos
Osteogênese/genética , Osteogênese/fisiologia , Subunidade beta da Proteína Ligante de Cálcio S100/genética , Proteínas S100/genética , Animais , Diferenciação Celular , Linhagem Celular , Condrócitos/citologia , Camundongos , Camundongos Knockout , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Proteínas S100/metabolismo , Regulação para CimaRESUMO
Here we report on the identification of a human pluripotent embryonic stem cell (hESC) specific mitochondrial protein that is re-expressed in cancer cells, ATAD3B. ATAD3B belongs to the AAA+ ATPase ATAD3 protein family of mitochondrial proteins specific to multicellular eukaryotes. Using loss- and gain-of-function approaches, we show that ATAD3B associates with the ubiquitous ATAD3A species, negatively regulates the interaction of ATAD3A with matrix nucleoid complexes and contributes to a mitochondria fragmentation phenotype. We conclude that ATAD3B is a negative regulator of ATAD3A and may function as an adaptor of mitochondrial homeostasis and metabolism in hESCs and cancer cells.
Assuntos
Adenosina Trifosfatases/metabolismo , Células-Tronco Embrionárias/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias/fisiopatologia , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Proteínas Mutantes/biossíntese , Proteínas Mutantes/genética , MutaçãoRESUMO
The Refilins (RefilinA and RefilinB) are a novel family of short-lived actin regulatory proteins that are expressed during changes in cellular phenotype such as epithelial to mesenchymal transition (EMT). The Refilins promote to the formation of actin- and myosin-rich perinuclear bundles that are characteristic of cellular phenotypic switches. In epithelial cells, RefilinB is up-regulated in response to TGF-ß stimulation and function in organization of apical perinuclear actin fibers during early stage of the EMT process1. In fibroblasts, RefilinB stabilizes perinuclear parallel actin bundles which resemble actin cap 2. Refilins bind and modulate the function of Filamin A (FLNA). Upon binding to Refilins, FLNA is capable of assembling actin filaments into parallel bundles, possibly by undergoing conformational changes at the C-terminal. Perinuclear actin structures determine nuclear shape, cell morphology, cell adhesion and possibly cell proliferation and gene regulation. Identifying the role of Refilins in organizing perinuclear actin networks provides additional insight in the process of intracellular mechanotransduction that regulate changes in cellular phenotype such as those observed during EMT.
RESUMO
Actin cytoskeleton dynamics lie at the heart of cell mechanosensing signaling. In fibroblast cells, two perinuclear acto-myosin structures, the actin cap and the transmembrane actin-associated nuclear (TAN) line, are components of a physical pathway transducing extracellular physical signals to changes in nuclear shape and movements. We recently demonstrated the existence of a previously uncharacterized third apical perinuclear actin organization in epithelial cells that forms during epithelial-mesenchymal transition (EMT) mediated by TGFß (TGFß). A common regulatory mechanism for these different perinuclear actin architectures has emerged with the identification of a novel family of actin bundling proteins, the Refilins. Here we provide updates on some characteristics of Refilin proteins, and we discuss potential function of the Refilins in cell mechanosensing signaling.