RESUMO
Spherocytosis is one of the most common inherited disorders, yet presents with a wide range of clinical severity. While several genes have been found mutated in patients with spherocytosis, the molecular basis for the variability in severity of haemolytic anaemia is not entirely understood. To identify candidate proteins involved in haemolytic anaemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in red blood cells (RBCs) from normal and ß-adducin (Add2) knock-out mice. We detected seven proteins that were decreased and 48 proteins that were increased in ß-adducin null RBC ghosts. Since haemolytic anaemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Among the 48 proteins increased in Add2 knockout RBCs, only 11 were also increased in reticulocytes. Of the proteins decreased in Add2 knockout RBCs, α-adducin showed the greatest intensity difference, followed by SLC9A1, the sodium-hydrogen exchanger previously termed NHE1. We verified these mass spectrometry results by immunoblot. This is the first example of SLC9A1deficiency in haemolytic anaemia and suggests new insights into the mechanisms leading to fragile RBCs.
Assuntos
Proteínas de Transporte de Cátions/deficiência , Eritrócitos/metabolismo , Proteínas dos Microfilamentos/deficiência , Animais , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte de Cátions/sangue , Proteínas do Citoesqueleto , Membrana Eritrocítica/metabolismo , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/sangue , Proteômica/métodos , Reticulócitos/metabolismo , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/sangueRESUMO
Hemolytic anemia is one of the most common inherited disorders. To identify candidate proteins involved in hemolytic anemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in RBCs from normal and beta-adducin (Add2) knock-out mice. We detected 7 proteins that were decreased and 48 proteins that were increased in the beta-adducin knock-out RBC ghost. Since hemolytic anemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Label-free analysis identified 47 proteins that were increased in the reticulocyte-enriched samples and 21 proteins that were decreased. Among the proteins increased in Add2 knockout RBCs, only 11 were also found increased in reticulocytes. Among the proteins decreased in Add2 knockout RBCs, beta- and alpha-adducin showed the greatest intensity difference, followed by NHE-1 (Slc9a1), the sodium-hydrogen exchanger. We verified these mass spectrometry results by immunoblot. This is the first example of a deficiency of NHE-1 in hemolytic anemia and suggests new insights into the mechanisms leading to fragile RBCs. Our use of label-free comparative proteomics to make this discovery demonstrates the usefulness of this approach as opposed to metabolic or chemical isotopic labeling of mice.
Assuntos
Anemia Hemolítica/genética , Proteínas de Ligação a Calmodulina , Proteínas de Transporte de Cátions/genética , Eritrócitos/metabolismo , Fragilidade Osmótica/genética , Fenil-Hidrazinas/efeitos adversos , Isoformas de Proteínas/genética , Proteômica/métodos , Reticulócitos/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Sequência de Aminoácidos , Anemia Hemolítica/induzido quimicamente , Anemia Hemolítica/metabolismo , Anemia Hemolítica/patologia , Animais , Western Blotting , Proteínas de Ligação a Calmodulina/deficiência , Proteínas de Ligação a Calmodulina/genética , Proteínas de Transporte de Cátions/deficiência , Modelos Animais de Doenças , Contagem de Eritrócitos , Membrana Eritrocítica/genética , Membrana Eritrocítica/metabolismo , Eritrócitos/citologia , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Fenil-Hidrazinas/farmacologia , Isoformas de Proteínas/metabolismo , Contagem de Reticulócitos , Reticulócitos/citologia , Trocador 1 de Sódio-Hidrogênio , Espectrometria de Massas em TandemRESUMO
In the red blood cell (RBC), adducin is present primarily as tetramers of alpha- and beta-subunits at spectrin-actin junctions, or junctional complexes. Mouse RBCs also contain small amounts of gamma-adducin. Platelets contain alpha- and gamma-adducin only. Adducin functions as a barbed-end actin capping protein to regulate actin filament length and recruits spectrin to the ends of actin filaments. To further define adducin's role in vivo, we generated alpha-adducin knockout mice. alpha-Adducin is absent in all tissues examined in homozygous null mice. In RBCs, beta- and gamma-adducin are also absent, indicating that alpha-adducin is the limiting subunit in tetramer formation at the spectrin-actin junction. Similarly, gamma-adducin is absent in alpha-null platelets. alpha-Adducin-null mice display compensated hemolytic anemia with features characteristic of RBCs in hereditary spherocytosis (HS), including spherocytes with significant loss of surface area, decreased mean corpuscular volume (MCV), cell dehydration, and increased osmotic fragility. Platelets maintain their normal discoid shape, and bleeding times are normal. alpha-Adducin-null mice show growth retardation at birth and throughout adulthood. Approximately 50% develop lethal communicating hydrocephalus with striking dilation of the lateral, third, and fourth ventricles. These data indicate that adducin plays a role in RBC membrane stability and in cerebrospinal fluid homeostasis.
Assuntos
Anemia Hemolítica Congênita/metabolismo , Proteínas do Citoesqueleto/metabolismo , Hidrocefalia/metabolismo , Esferócitos/metabolismo , Actinas/genética , Actinas/metabolismo , Anemia Hemolítica Congênita/genética , Animais , Plaquetas/metabolismo , Proteínas do Citoesqueleto/genética , Deleção de Genes , Hidrocefalia/genética , Hidrocefalia/patologia , Camundongos , Camundongos Knockout , Fragilidade Osmótica/genética , Estrutura Quaternária de Proteína , Espectrina/genética , Espectrina/metabolismo , Esferócitos/patologia , Esferocitose Hereditária/genética , Esferocitose Hereditária/metabolismo , Esferocitose Hereditária/patologiaRESUMO
Previous research has shown that glycolytic enzymes (GEs) exist as multienzyme complexes on the inner surface of human erythrocyte membranes. Because GE binding sites have been mapped to sequences on the membrane protein, band 3, that are not conserved in other mammalian homologs, the question arose whether GEs can organize into complexes on other mammalian erythrocyte membranes. To address this, murine erythrocytes were stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, lactate dehydrogenase, and pyruvate kinase and analyzed by confocal microscopy. GEs were found to localize to the membrane in oxygenated erythrocytes but redistributed to the cytoplasm upon deoxygenation, as seen in human erythrocytes. To identify membrane proteins involved in GE assembly, erythrocytes from mice lacking each of the major erythrocyte membrane proteins were examined for GE localization. GEs from band 3 knockout mice were not membrane associated but distributed throughout the cytoplasm, regardless of erythrocyte oxygenation state. In contrast, erythrocytes from mice lacking alpha-spectrin, ankyrin, protein 4.2, protein 4.1, beta-adducin, or dematin headpiece exhibited GEs bound to the membrane. These data suggest that oxygenation-dependent assembly of GEs on the membrane could be a general phenomenon of mammalian erythrocytes and that stability of these interactions depends primarily on band 3.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Membrana Eritrocítica/metabolismo , Glicólise/fisiologia , Sequência de Aminoácidos , Animais , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Proteína 1 de Troca de Ânion do Eritrócito/genética , Sequência de Bases , DNA Complementar/genética , Frutose-Bifosfato Aldolase/sangue , Gliceraldeído-3-Fosfato Desidrogenases/sangue , L-Lactato Desidrogenase/sangue , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Complexos Multienzimáticos/sangue , Oxigênio/sangue , Fosfofrutoquinases/sangue , Piruvato Quinase/sangue , Homologia de Sequência de AminoácidosRESUMO
Primary sinonasal lymphomas are a rare type of non-Hodgkin lymphoma (NHL) with an overall incidence of about 1% of all head and neck cancers. Diffuse large B-cell lymphoma (DLBCL) is the most common type of NHL and it most commonly occurs in elderly men. The diagnosis of such a lymphoma is difficult because of its varied presentation, which usually occurs late after a significant mass effect has taken place. Symptoms vary significantly, from simply nasal obstruction or epistaxis to varied cranial neuropathies or the confusion seen with central nervous system (CNS) spread. Patients may present with a large orbital mass and proptosis. Therefore, emphasis should be placed on earlier detection by using appropriate imaging modalities to reveal such masses. A biopsy is necessary to confirm the diagnosis. The prognosis is scored by the International Prognostic Index. Staging scans with whole-body computed tomography (CT) with contrast and positron emission tomography-fluorodeoxyglucose (PET-FDG) are required to determine other areas of involvement. Treatment is with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) with the case-dependent use of intrathecal chemoprophylaxis (methotrexate) to prevent CNS spread. Here, we present a case series of two patients who were found to have a primary sinonasal lymphoma of the DLBCL type. In both cases, the presenting symptoms were vague. A high index of suspicion is required to diagnose NHL early on, which portends the best chance of a successful outcome. This article seeks to emphasize the role of including primary sinonasal lymphoma as a differential in the presentation of unrelenting cranial neuropathies or facial mass.
RESUMO
High-density oligonucleotide microarrays were used to compare gene expression profiles from uncultured CD34+/CD38lo cells and culture-derived megakaryocytes (MKs). As previously published, three replicate microarray data sets from three different sources of organ donor marrow were analyzed using the software program Rosetta Resolver. After setting a stringent p value of Assuntos
Plaquetas/metabolismo
, Dinamina III/biossíntese
, Regulação da Expressão Gênica/fisiologia
, Células Progenitoras de Megacariócitos/metabolismo
, Megacariócitos/metabolismo
, ADP-Ribosil Ciclase 1
, Actinas/metabolismo
, Animais
, Antígenos CD34
, Plaquetas/ultraestrutura
, Membrana Celular/metabolismo
, Membrana Celular/ultraestrutura
, Células Cultivadas
, Citoplasma/metabolismo
, Citoplasma/ultraestrutura
, Citoesqueleto/metabolismo
, Citoesqueleto/ultraestrutura
, Feminino
, Sangue Fetal/citologia
, Sangue Fetal/metabolismo
, Perfilação da Expressão Gênica
, Humanos
, Hidrólise
, Masculino
, Células Progenitoras de Megacariócitos/ultraestrutura
, Megacariócitos/ultraestrutura
, Glicoproteínas de Membrana
, Camundongos
, Subunidade p45 do Fator de Transcrição NF-E2/metabolismo
, Nucleotídeos/metabolismo
, Análise de Sequência com Séries de Oligonucleotídeos
, Tubulina (Proteína)/metabolismo
RESUMO
New approaches are needed for understanding and treating acute myeloid leukemia (AML). MicroRNAs (miRs) are important regulators of gene expression in all cells and disruption of their normal expression can lead to changes in phenotype of a cell, in particular the emergence of a leukemic clone. We collected peripheral blood samples from 10 adult patients with newly diagnosed AML, prior to induction chemotherapy, and 9 controls. Two and a half ml of whole blood was collected in Paxgene RNA tubes. MiRNA was purified using RNeasy mini column (Qiagen). We sequenced approximately 1000 miRs from each of 10 AML patients and 9 controls. In subset analysis, patients with NPM1 and FLT3 mutations showed the greatest number of miRNAs (63) with expression levels that differed from control with adjusted p-value of 0.05 or less. Some of these miRs have been described previously in association with leukemia, but many are new. Our approach of global sequencing of miRs as opposed to microarray analysis removes the bias regarding which miRs to assay and has demonstrated discovery of new associations of miRs with AML. Another strength of our approach is that sequencing miRs is specific for the 5p or 3p strand of the gene, greatly narrowing the proposed target genes to study further. Our study provides new information about the molecular changes that lead to evolution of the leukemic clone and offers new possibilities for monitoring relapse and developing new treatment strategies.
Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Mieloide Aguda/genética , MicroRNAs/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/sangue , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , Mutação , Proteínas Nucleares/genética , Nucleofosmina , Análise de Sequência de RNA/métodos , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
The adducin family of proteins interacts with the actin cytoskeleton and the plasma membrane in a calcium- and cAMP-dependent manner. Thus, adducins may be involved in changes in cytoskeletal organization resulting from synaptic stimulation. beta-Adducin knock-out mice were examined in physiological and behavioral paradigms related to synaptic plasticity to elucidate the role the adducin family plays in processes underlying learning and memory. In situ hybridization for alpha- and beta-adducin demonstrates that these mRNAs are found throughout the brain, with high levels of expression in the hippocampus. Schaffer collateral-CA1 tetanic long-term potentiation decayed rapidly in acute hippocampal slices from beta-adducin knock-out mice, although baseline spine morphology and postsynaptic density were normal. Interestingly, the input-output relationship was significantly increased in hippocampal slices from beta-adducin knock-out mice. Furthermore, beta-adducin knock-out mice were impaired in performance of fear conditioning and the water maze paradigm. The current results indicate that beta-adducin may play an important role in the cellular mechanisms underlying activity-dependent synaptic plasticity associated with learning and memory.
Assuntos
Actinas/metabolismo , Proteínas de Ligação a Calmodulina/fisiologia , Citoesqueleto/metabolismo , Deficiências da Aprendizagem/genética , Transtornos da Memória/genética , Proteínas do Tecido Nervoso/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Aprendizagem da Esquiva/fisiologia , Proteínas de Ligação a Calmodulina/deficiência , Proteínas de Ligação a Calmodulina/genética , Condicionamento Clássico/fisiologia , Citoesqueleto/ultraestrutura , Dendritos/ultraestrutura , Eletrochoque , Medo/fisiologia , Feminino , Reação de Congelamento Cataléptica/fisiologia , Giro do Cíngulo/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Deficiências da Aprendizagem/fisiopatologia , Masculino , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes Neurológicos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/genética , Núcleo Accumbens/metabolismo , RNA Mensageiro/biossínteseRESUMO
OBJECTIVE: The first objective of this study was to examine the differences in levels of adducin (ADD1, ADD2, ADD3) mRNA expression during human erythropoiesis. The second objective was to determine whether the rapid induction of ADD2 expression could be attributed to a novel erythroid-specific promoter. METHODS: Expression of mRNA was quantified using real-time RT-PCR. Primary erythroid precursors were isolated from normal human bone marrow using fluorescence-activated cell sorting. Two model systems were compared: CD34(+) hematopoietic stem cells induced to differentiate with erythropoietin and HEL cells induced to differentiate with hemin. 5'RACE analysis was performed using primary human erythroblasts as starting material. RESULTS: All three adducin genes showed different patterns of expression during erythropoietic differentiation of cultured CD34(+) stem cells. Levels of ADD3 mRNA were higher than levels of ADD2 mRNA at early stages of erythropoiesis. Expression of ADD2 was induced to very high levels (100 times baseline) in erythropoietin-stimulated cultures. 5'RACE analysis identified a novel starting exon and putative erythroid promoter for ADD2. CONCLUSION: These results suggest that expression of each adducin gene is regulated in a gene-specific manner during erythropoiesis. The early expression of ADD3 suggests that it may have a role in erythroblasts but is replaced by ADD2 in later stages of erythropoiesis. The very high levels of expression of ADD2 suggest that its promoter may be useful for directing erythroid-specific gene expression.
Assuntos
Proteínas de Ligação a Calmodulina/genética , Proteínas do Citoesqueleto/genética , Eritropoese/genética , Regulação Neoplásica da Expressão Gênica , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Antígenos CD/análise , Antígenos CD34/análise , Sequência de Bases , Biópsia por Agulha , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Linhagem Celular Tumoral , Éxons , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Íntrons , Leucemia Eritroblástica Aguda , Dados de Sequência Molecular , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Structural changes at synapses are associated with long-term facilitation (LTF) of synaptic transmission between sensory and motor neurons in Aplysia. We have cloned a cDNA encoding Aplysia adducin (ApADD), the Aplysia homolog of mammalian adducins that are regulatory components of the membrane cytoskeleton. ApADD is recovered in the particulate fraction of nervous system extracts and is localized predominantly in the submembraneous region of Aplysia neurons. ApADD is phosphorylated in vitro by protein kinase C (PKC) at a site homologous to the in vivo PKC phosphorylation site in mammalian adducins. Phosphorylation of ApADD at this site is also detected in vivo in the intact Aplysia nervous system and is increased 18 hr after serotonin-induced LTF. In contrast, there is no change in phosphorylation during short-term facilitation or 1 hr after initial LTF induction. Thus, ApADD is modulated specifically with later phases of LTF and provides an attractive candidate protein that contributes to structural changes accompanying long-lasting synaptic alteration.
Assuntos
Aplysia/fisiologia , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/metabolismo , Proteína Quinase C/metabolismo , Transmissão Sináptica , Sequência de Aminoácidos , Animais , Aplysia/metabolismo , Proteínas de Ligação a Calmodulina/genética , Clonagem Molecular , Sequência Consenso , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Humanos , Cinética , Mamíferos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Neurônios Motores/fisiologia , Sistema Nervoso/metabolismo , Plasticidade Neuronal , Neurônios/química , Neurônios Aferentes/fisiologia , Fosforilação , Proteína Quinase C/fisiologia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Serotonina/farmacologiaAssuntos
Proteínas de Ligação a Calmodulina/metabolismo , Neoplasias/metabolismo , Proteínas de Ligação a Calmodulina/genética , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Peso Molecular , Neoplasias/genética , Especificidade de Órgãos , FosforilaçãoRESUMO
The diuretic hormone aedeskinin-III is known to increase the paracellular Cl- conductance in Malpighian (renal) tubules of the mosquito Aedes aegypti via a G protein-coupled receptor. The increase serves the blood-meal-initiated diuresis and is associated with elevated levels of Ca2+ and phosphorylated adducin in the cytosol of tubule. In the present study we have cloned adducin in Aedes Malpighian tubules and investigated its physiological roles. Immunolabeling experiments are consistent with the association of adducin with the cortical cytoskeleton, especially near the apical brush border of the tubule. An antibody against phosphorylated adducin revealed the transient phosphorylation of adducin 2 min after stimulating tubules with aedeskinin-III. The PKC inhibitor bisindolylmaleimide-I blocked the phosphorylation of adducin as well as the electrophysiological and diuretic effects of aedeskinin-III. Bisindolylmaleimide-I also inhibited fluid secretion in control tubules. Phorbol 12-myristate 13-acetate increased phosphorylated adducin levels in Malpighian tubules, but it inhibited fluid secretion. Thus, the phosphorylation of adducin by PKC alone is insufficient to trigger diuretic rates of fluid secretion; elevated levels of intracellular Ca2+ may also be required. The above results suggest that the phosphorylation of adducin, which is known to destabilize the cytoskeleton, may (1) facilitate the traffic of transporters into the apical brush border supporting diuretic rates of cation secretion and (2) destabilize proteins in the septate junction thereby enabling paracellular anion (Cl-) secretion at diuretic rates. Moreover, PKC and the phosphorylation of adducin play a central role in control and diuretic tubules, consistent with the dynamic behavior of both transcellular and paracellular transport pathways.
RESUMO
Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and ß- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1â»/â» erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1â»/â» erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Eritrocítica/metabolismo , Actinas/química , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Deleção de Genes , Camundongos , Fosforilação , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Tempo , Transcriptoma , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Dynamin 3 (DNM3) is a member of a family of motor proteins that participate in a number of membrane rearrangements such as cytokinesis, budding of transport vesicles, phagocytosis, and cell motility. Recently, DNM3 was implicated as having a role in megakaryocyte (MK) development. To further investigate the functional role of DNM3 during megakaryocytopoiesis, we introduced sequence-specific short hairpin RNAs (shRNAs) into developing MKs. The results showed that knockdown of DNM3 inhibited a stage of MK development that involved progenitor amplification. This was evident by significant decreases in the number of colony forming unit-megakaryocytes, the total number of nucleated cells, and the number of CD41(+) and CD61(+) MKs produced in culture. Using a styrl membrane dye to quantify the demarcation membrane system (DMS) of terminally differentiated MKs, we found that DNM3 co-localized with the DMS and that DNM3 lentiviral shRNAs precluded the formation of the DMS. Knockdown of dynamin 3 in murine MKs also caused a decrease in the number of morphologically large MKs and the overall size of large MKs was decreased relative to controls. MK protein lysates were used in overlay blots to show that both DNM3 and actin bind to nonmuscle myosin IIA (MYH9). Consistent with these observations, immunofluorescence studies of MKs and proplatelet processes showed co-localization of DNM3 with MYH9. Overall, these studies demonstrate that DNM3 not only participates in MK progenitor amplification, but is also involved in cytoplasmic enlargement and the formation of the DMS.
Assuntos
Dinamina III/metabolismo , Trombopoese , Actinas/metabolismo , Animais , Sequência de Bases , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Tamanho Celular , DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Indóis/farmacologia , Lentivirus/genética , Megacariócitos/citologia , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Camundongos , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/metabolismo , Ploidias , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Sulfonamidas/farmacologia , Trombopoese/efeitos dos fármacosRESUMO
OBJECTIVE: A missense mutation in the microtubule-associated serine/threonine-like kinase gene (MASTL, FLJ14813) on human chromosome 10 was previously linked to a novel form of autosomal dominant inherited thrombocytopenia in a single pedigree. The mutation results in an amino acid change from glutamic acid at position 167 to aspartic acid and segregates perfectly with thrombocytopenic individuals within this extended family. The phenotype is characterized by mild thrombocytopenia with an average platelet count of 60,000 platelets per microliter of blood. We wanted to determine the expression and localization of MASTL, as well as its role in developing thrombocytes using an in vivo model system. MATERIALS AND METHODS: Northern blot analysis allowed us to examine expression patterns. Morpholino knockdown assays in zebrafish (Danio rerio) were employed to determine in vivo contribution to thrombocyte development. Transient expression in baby hamster kidney cells resulted in localization of both the wild-type and E167D mutant forms of MASTL kinase to the nucleus. RESULTS: Northern blot analysis indicates that MASTL messenger RNA is restricted in its expression to hematopoietic and cancer cell lines. A transient knockdown of MASTL in zebrafish results in deficiency of circulating thrombocytes. Transient expression of recombinant MASTL kinase in vitro demonstrates localization to the nucleus. CONCLUSIONS: Functional studies presented here demonstrate a direct relationship between transient knockdown of MASTL kinase gene expression and reduction of circulating thrombocytes in zebrafish. This transient knockdown of MASTL in zebrafish correlates with a decrease in the expression of the thrombopoietin receptor, c-mpl, and the CD41 platelet adhesion protein, GpIIb, but has no effect on essential housekeeping zebrafish gene, EF1alpha.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Trombocitopenia/etiologia , Animais , Plaquetas/enzimologia , Plaquetas/metabolismo , Plaquetas/patologia , Contagem de Células , Linhagem da Célula , Ativação Enzimática , Perfilação da Expressão Gênica , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/fisiologia , Mutação de Sentido Incorreto , Glicoproteína IIb da Membrana de Plaquetas/genética , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/fisiologia , RNA Mensageiro/análise , Receptores de Trombopoetina/genética , Trombocitopenia/enzimologia , Trombocitopenia/genética , Peixe-ZebraRESUMO
A large-scale analysis of proteins involved in host-cell signalling pathways was performed using chlamydia-infected murine cells in order to identify host proteins that are differentially activated or localized following infection. Two proteins whose distribution was altered in Chlamydia trachomatis-infected cells relative to mock-infected cells were the actin-binding protein adducin and the regulatory kinase Raf-1. Immunoblot analysis with antibodies to both phosphorylated and non-phosphorylated forms of these proteins demonstrated that the abundance of each protein was markedly reduced in the cytosolic fraction of C. trachomatis- and Chlamydophila caviae-infected cells, but the total cellular protein abundance remained unaffected by infection. Fluorescence microscopy of chlamydia-infected cells using anti-alpha-adducin antibodies demonstrated labelling at or near the chlamydial inclusion membrane. Treatment of infected cells with nocodazole or cytochalasin D did not affect alpha-adducin that was localized to the margins of the inclusion. The demonstration of alpha-adducin and Raf-1 redistribution within cells infected by different chlamydiae provides novel opportunities for analysis of host-pathogen interactions in this system.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Chlamydia trachomatis/fisiologia , Chlamydophila/patogenicidade , Interações Hospedeiro-Patógeno , Proteínas Proto-Oncogênicas c-raf/metabolismo , Chlamydia trachomatis/patogenicidade , Chlamydophila/classificação , Chlamydophila/fisiologia , Chlamydophila pneumoniae/patogenicidade , Chlamydophila pneumoniae/fisiologia , Células HeLa , Humanos , Immunoblotting , Corpos de Inclusão/metabolismoRESUMO
Dematin and adducin are actin-binding proteins of the erythrocyte "junctional complex." Individually, they exert modest effects on erythrocyte shape and membrane stability, and their homologues are expressed widely in non-erythroid cells. Here we report generation and characterization of double knock-out mice lacking beta-adducin and the headpiece domain of dematin. The combined mutations result in altered erythrocyte morphology, increased membrane instability, and severe hemolysis. Peripheral blood analysis shows evidence of severe hemolytic anemia with reduced number of erythrocytes/hematocrit/hemoglobin and an approximately 12-fold increase in the number of circulating reticulocytes. The presence of a variety of misshapen and fragmented erythrocytes correlates with increased osmotic fragility and reduced in vivo life span. Despite the apparently normal protein composition of the mutant erythrocyte membrane, the retention of the spectrin-actin complex in the membrane under low ionic strength conditions is significantly reduced by the double mutation. Atomic force microscopy reveals an increase in grain size and a decrease in filament number of the mutant membrane cytoskeleton, although the volume parameter is similar to wild type erythrocytes. Aggregated, disassembled, and irregular features are visualized in the mutant membrane, consistent with the presence of large protein aggregates. Importantly, purified dematin binds to the stripped inside-out vesicles in a saturable manner, and dematin-membrane binding is abolished upon pretreatment of membrane vesicles with trypsin. Together, these results reveal an essential role of dematin and adducin in the maintenance of erythrocyte shape and membrane stability, and they suggest that the dematin-membrane interaction could link the junctional complex to the plasma membrane in erythroid cells.
Assuntos
Actinas/metabolismo , Anemia Hemolítica/sangue , Proteínas Sanguíneas/genética , Proteínas de Ligação a Calmodulina/genética , Membrana Eritrocítica/patologia , Deleção de Genes , Fragilidade Osmótica/genética , Fosfoproteínas/genética , Espectrina/metabolismo , Actinas/fisiologia , Anemia Hemolítica/genética , Animais , Proteínas Sanguíneas/deficiência , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/fisiologia , Proteínas de Ligação a Calmodulina/sangue , Proteínas de Ligação a Calmodulina/deficiência , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/ultraestrutura , Humanos , Camundongos , Camundongos Knockout , Microscopia de Força Atômica , Fosfoproteínas/deficiência , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Espectrina/fisiologiaRESUMO
Adducins are a family of cytoskeletal proteins encoded by 3 genes (alpha, beta, and gamma). Platelets express alpha and gamma adducins, in contrast to red blood cells that express alpha and beta adducins. During platelet activation with thrombin, calcium ionophore A23187, or phorbol 12-myristate 13-acetate, alpha and gamma adducins were phosphorylated by protein kinase C (PKC) as detected by an antibody specific for a phosphopeptide sequence in the highly conserved carboxy terminus. Platelet activation also led to adducin proteolysis; inhibition by calpeptin suggests that the protease was calpain. The kinase inhibitor staurosporine inhibited PKC phosphorylation of adducin and also inhibited proteolysis of adducin. Experiments with recombinant alpha adducin demonstrated that the PKC-phosphorylated form was proteolyzed at a significantly faster rate than the unphosphorylated form. The concentration of adducin in platelets was estimated at 6 microM, similar to the concentration of capping protein. Fractionation of platelets into high-speed supernatant (cytosol) and pellet (membrane and cytoskeleton) revealed a shift of PKC-phosphorylated adducin to the cytosol during platelet activation. Platelet aggregation detected turbidometrically was decreased in the presence of staurosporine and was completely inhibited by calpeptin. Thrombin-induced changes in morphology were assessed by confocal microscopy with fluorescein phalloidin and were not prevented by staurosporine or calpeptin. Our results suggest that regulation of adducin function by PKC and calpain may play a role in platelet aggregation.