RESUMO
Borderline ovarian tumor is a non-invasive lesion with an excellent prognosis. Here we report a case of 48-year-old woman with distinctive clinical presentation of metastasis of ovarian adenocarcinoma, which was an microinvasive component of a serous borderline tumor. On initial diagnosis patient did not present any clinical manifestation of ovarian tumor. Histological examination of resected ovary showed typical features of the serous borderline tumor with one very diminutive focus of invasive serous adenocarcinoma 4mm in diameter. This exceptional case shows that borderline tumors of ovary with any features of invasion could present an aggressive course with distant metastases.
Assuntos
Adenocarcinoma , Cistadenocarcinoma Seroso , Neoplasias Ovarianas , Lesões Pré-Cancerosas , Feminino , Humanos , Pessoa de Meia-Idade , PrognósticoRESUMO
Vascular endothelial growth factor (VEGF), a key angiogenic and permeability factor, plays an important role in new blood vessel formation. However, abnormal VEGF-induced VEGFR2 signaling leads to hyperpermeability. We have shown previously that Rap1, best known for promoting cell adhesion and vessel stability, is a critical regulator of VEGFR2-mediated angiogenic and shear-stress EC responses. To determine the role of Rap1 role in endothelial barrier dynamics, we examined vascular permeability in EC-specific Rap1A- and Rap1B-knockout mice, cell-cell junction remodeling and EC monolayer resistivity in Rap1-deficient ECs under basal, inflammatory or elevated VEGF conditions. Deletion of either Rap1 isoform impaired de novo adherens junction (AJ) formation and recovery from LPS-induced barrier disruption in vivo However, only Rap1A deficiency increased permeability in ECs and lung vessels. Interestingly, Rap1B deficiency attenuated VEGF-induced permeability in vivo and AJ remodeling in vitro Therefore, only Rap1A is required for the maintenance of normal vascular integrity. Importantly, Rap1B is the primary isoform essential for normal VEGF-induced EC barrier dissolution. Deletion of either Rap1 isoform protected against hyper permeability in the STZ-induced diabetes model, suggesting clinical implications for targeting Rap1 in pathologies with VEGF-induced hyperpermeability.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Endotélio Vascular/fisiologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Adesão Celular , Linhagem Celular , Diabetes Mellitus Experimental/induzido quimicamente , Endotélio Vascular/efeitos dos fármacos , Feminino , Humanos , Junções Intercelulares/metabolismo , Masculino , Camundongos , Camundongos Knockout , Neovascularização Fisiológica , Transdução de SinaisRESUMO
Traditional medicinal plants are an important source of active compounds with potential antimutagenic activity. Polyscias filicifolia Bailey (Araliaceae) is a South Asian traditional herb used as an adaptogenic and cardiac drug. Extracts of P. filicifolia contain a wide range of biologically active compounds like phenolic acids and triterpenoid saponins. In the present study. antigenotoxic potential of three naturally occurring phenolic acids and extracts of P. filicifolia growing in vitro with the addition of elicitors was evaluated against direct (4-nitroquinoline-N-oxide (4NQO) and mitomycin C (MMC)) and indirect mutagens (2-aminoanthracene (2AA)). The evaluation was made using a bacterial umu-test. Moreover, the ability to prevent photogenotoxicity induced by chlorpromazine (CPZ) under UVA irradiation was measured. The phytochemical profiling of examined extracts revealed the presence of numerous compounds with the prevelance of chlorogenic, caffeic, and ferulic acid derivatives; however, saponin fractions were also determined. The antioxidant potential of extracts strictly correlated with their composition. The tested extracts exhibited high antigenotoxic activity if the assay was performed with 2AA and metabolic activation. Moreover, the extracts slightly decreased the MMC-induced genotoxicity. However, an increase of the genotoxic effect was observed in the assay performed with 4NQO. In addition, photo-antigenotoxic activity was observed. In our study, phenolic acids exhibited lower activity than the extracts.
Assuntos
Antimutagênicos/farmacologia , Antioxidantes/farmacologia , Araliaceae/química , Dano ao DNA , Extratos Vegetais/farmacologia , Brotos de Planta/química , Animais , Antimutagênicos/química , Antioxidantes/química , Clorpromazina/efeitos adversos , Clorpromazina/farmacologia , Masculino , Mitomicina/efeitos adversos , Mitomicina/farmacologia , Extratos Vegetais/química , Ratos , Ratos Sprague-DawleyRESUMO
DOCK7 (dedicator of cytokinesis 7) is a guanidine nucleotide exchange factor (GEF) for Rac1 GTPase that is involved in neuronal polarity and axon generation as well in Schwann cell differentiation and myelination. Recently, we identified DOCK7 as the binding partner of unconventional myosin VI (MVI) in neuronal-lineage PC12 cells and postulated that this interaction could be important in vivo [Majewski et al. (2012) Biochem Cell Biol., 90:565-574]. Herein, we found that MVI-DOCK7 interaction takes also place in other cell lines and demonstrated that MVI cargo domain via its RRL motif binds to DOCK7 C-terminal M2 and DHR2 domains. In MVI knockdown cells, lower Rac1 activity and a decrease of DOCK7 phosphorylation on Tyr1118 were observed, indicating that MVI could contribute to DOCK7 activity. MVI and DOCK7 co-localization was maintained during NGF-stimulated PC12 cell differentiation and observed also in the outgrowths. Also, during differentiation an increase in phosphorylation of DOCK7 as well as of its downstream effector JNK kinase was detected. Interestingly, overexpression of GFP-tagged MVI cargo domain (GFP-GT) impaired protrusion formation indicating that full length protein is important for this process. Moreover, a transient increase in Rac activity observed at 5min of NGF-stimulated differentiation of PC12 cells (overexpressing either GFP or GFP-MVI) was not detected in cells overexpressing the cargo domain. These data indicate that MVI-DOCK7 interaction could have functional implications in the protrusion outgrowth, and full length MVI seems to be important for delivery and maintenance of DOCK7 along the protrusions, and exerting its GEF activity.
Assuntos
Extensões da Superfície Celular/efeitos dos fármacos , Proteínas Ativadoras de GTPase/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Fator de Crescimento Neural/farmacologia , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Extensões da Superfície Celular/metabolismo , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina , Células HEK293 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Cadeias Pesadas de Miosina/genética , Neurônios/metabolismo , Células PC12 , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Decreased nitric oxide (NO) bioavailability underlies a number of cardiovascular pathologies, including hypertension. The shear stress exerted by flowing blood is the main determinant of NO release. Rap1 promotes integrin- and cadherin-mediated signaling. Here, we show that Rap1 is a critical regulator of NO production and endothelial function. Rap1 deficiency in murine endothelium attenuates NO production and diminishes NO-dependent vasodilation, leading to endothelial dysfunction and hypertension, without deleterious effects on vessel integrity. Mechanistically, Rap1 is activated by shear stress, promotes the formation of the endothelial mechanosensing complex-comprised of PECAM-1, VE-cadherin and VEGFR2- and downstream signaling to NO production. Our study establishes a novel paradigm for Rap1 as a regulator of mechanotransduction.
Assuntos
Endotélio/metabolismo , Mecanotransdução Celular , Óxido Nítrico/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Pressão Sanguínea , Permeabilidade Capilar/genética , Humanos , Hipertensão/genética , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Óxido Nítrico Sintase Tipo III/metabolismo , Especificidade de Órgãos/genética , Transdução de Sinais , Vasodilatação/genética , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
The important role of unconventional myosin VI (MVI) in skeletal and cardiac muscle has been recently postulated (Karolczak et al. in Histochem Cell Biol 139:873-885, 2013). Here, we addressed for the first time a role for this unique myosin motor in myogenic cells as well as during their differentiation into myotubes. During myoblast differentiation, the isoform expression pattern of MVI and its subcellular localization underwent changes. In undifferentiated myoblasts, MVI-stained puncti were seen throughout the cytoplasm and were in close proximity to actin filaments, Golgi apparatus, vinculin-, and talin-rich focal adhesion as well as endoplasmic reticulum. Colocalization of MVI with endoplasmic reticulum was enhanced during myotube formation, and differentiation-dependent association was also seen in sarcoplasmic reticulum of neonatal rat cardiomyocytes (NRCs). Moreover, we observed enrichment of MVI in myotube regions containing acetylcholine receptor-rich clusters, suggesting its involvement in the organization of the muscle postsynaptic machinery. Overexpression of the H246R MVI mutant (associated with hypertrophic cardiomyopathy) in myoblasts and NRCs caused the formation of abnormally large intracellular vesicles. MVI knockdown caused changes in myoblast morphology and inhibition of their migration. On the subcellular level, MVI-depleted myoblasts exhibited aberrations in the organization of actin cytoskeleton and adhesive structures as well as in integrity of Golgi apparatus and endoplasmic reticulum. Also, MVI depletion or overexpression of H246R mutant caused the formation of significantly wider or aberrant myotubes, respectively, indicative of involvement of MVI in myoblast differentiation. The presented results suggest an important role for MVI in myogenic cells and possibly in myoblast differentiation.
Assuntos
Desenvolvimento Muscular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/fisiologia , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Adesão Celular , Diferenciação Celular , Linhagem Celular , Movimento Celular , Forma Celular , Citoplasma/metabolismo , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Camundongos , Mioblastos/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Cadeias Pesadas de Miosina/química , Ratos , Retículo Sarcoplasmático/metabolismoRESUMO
Myosin VI (MVI) is a unique unconventional motor moving backwards on actin filaments. In non-muscle cells, it is involved in cell migration, endocytosis and intracellular trafficking, actin cytoskeleton dynamics, and possibly in gene transcription. An important role for MVI in striated muscle functioning was suggested in a report showing that a point mutation (H236R) within the MVI gene was associated with cardiomyopathy (Mohiddin et al., J Med Genet 41:309-314, 2004). Here, we have addressed MVI function in striated muscle by examining its expression and distribution in rat hindlimb skeletal muscle. We found that MVI was present predominantly at the muscle fiber periphery, and it was also localized within muscle nuclei. Analysis of both the hindlimb and cardiac muscle longitudinal sections revealed ~3 µm striation pattern, corresponding to the sarcoplasmic reticulum. Moreover, MVI was detected in the sarcoplasmic reticulum fractions isolated from skeletal and cardiac muscle. The protein also localized to the postsynaptic region of the neuromuscular junction. In denervated muscle, the defined MVI distribution pattern was abolished and accompanied by significant increase in its amount in the muscle fibers. In addition, we have identified several novel potential MVI-binding partners, which seem to aid our observations that in striated muscle MVI could be involved in postsynaptic trafficking as well as in maintenance of and/or transport within the sarcoplasmic reticulum and non-sarcomeric cytoskeleton.
Assuntos
Núcleo Celular/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Junção Neuromuscular/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Denervação , Feminino , Membro Posterior , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/química , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/análise , Ligação Proteica , Ratos , Ratos Wistar , Membranas Sinápticas/metabolismoRESUMO
Vascular endothelial growth factor (VEGF) acting through VEGF receptor 2 (VEGFR2) on endothelial cells (ECs) is a key regulator of angiogenesis, a process essential for wound healing and tumor metastasis. Rap1a and Rap1b, 2 highly homologous small G proteins, are both required for angiogenesis in vivo and for normal EC responses to VEGF. Here we sought to determine the mechanism through which Rap1 promotes VEGF-mediated angiogenesis. Using lineage-restricted Rap1-knockout mice we show that Rap1-deficiency in endothelium leads to defective angiogenesis in vivo, in a dose-dependent manner. Using ECs obtained from Rap1-deficient mice we demonstrate that Rap1b promotes VEGF-VEGFR2 kinase activation and regulates integrin activation. Importantly, the Rap1b-dependent VEGF-VEGFR2 activation is in part mediated via integrin α(v)ß(3). Furthermore, in an in vivo model of zebrafish angiogenesis, we demonstrate that Rap1b is essential for the sprouting of intersomitic vessels, a process known to be dependent on VEGF signaling. Using 2 distinct pharmacologic VEGFR2 inhibitors we show that Rap1b and VEGFR2 act additively to control angiogenesis in vivo. We conclude that Rap1b promotes VEGF-mediated angiogenesis by promoting VEGFR2 activation in ECs via integrin α(v)ß(3). These results provide a novel insight into the role of Rap1 in VEGF signaling in ECs.
Assuntos
Integrina alfaVbeta3/metabolismo , Neovascularização Fisiológica , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Peixe-Zebra , Proteínas rap de Ligação ao GTP/genética , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
Myosin VI (MVI), the only known myosin that walks towards the minus end of actin filaments, is involved in several processes such as endocytosis, cell migration, and cytokinesis. It may act as a transporting motor or a protein engaged in actin cytoskeleton remodelling via its binding partners, interacting with its C-terminal globular tail domain. By means of pull-down technique and mass spectrometry, we identified Dock7 (dedicator of cytokinesis 7) as a potential novel MVI-binding partner in neurosecretory PC12 cells. Dock7, expressed mainly in neuronal cells, is a guanine nucleotide exchange factor (GEF) for small GTPases, Rac1 and Cdc42, which are the major regulators of actin cytoskeleton. MVI-Dock7 interaction was further confirmed by co-immunoprecipitation of endogenous MVI complexed with Dock7. In addition, MVI and Dock7 colocalized in interphase and dividing cells. We conclude that in PC12 cells MVI-Dock7 complexes may function at different cellular locations during the entire cell cycle. Of note, MVI and Dock7 colocalized in primary culture hippocampal neurons also, predominantly in the outgrowths. We hypothesize that this newly identified interaction between MVI and Dock7 may help explain a mechanism for MVI-dependent regulation of actin cytoskeleton organization.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Neurônios/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Sítios de Ligação , Imunoprecipitação , Microscopia Confocal , Cadeias Pesadas de Miosina/genética , Células PC12 , Ratos , Espectrometria de Massas em Tandem , Proteínas rho de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Trophoblastic differentiation in primary urothelial carcinoma of the prostate is extremely rare. An increased level of ß-subunit human chorionic gonadotropin in serum in urothelial carcinoma is detected in approximately 30% of cases. To our knowledge, increased concentration of ß-subunit human chorionic gonadotropin in serum in prostatic urothelial carcinoma has never been reported and its clinical significance is not evaluated yet. CASE REPORT: Here we present the case of a 67-year-old European patient who was admitted to the hospital with hematuria, dysuria, and enlarged painful testis. Ultrasonographic examination of the testis did not reveal any focal lesion. Magnetic resonance imaging of the pelvis showed a tumor of 62 mm diameter mainly located in the posterior part of the prostatic gland. A pathological examination from cystoscopy biopsy allowed us to set the diagnosis of high-grade invasive urothelial carcinoma with trophoblastic differentiation. The patient received neoadjuvant treatment. Nonetheless, after a short period of disease stabilization, he developed progression and brain metastasis. He died 9 months after diagnosis. During the disease course, his ß-human chorionic gonadotropin level was measured repeatedly and analyzed in relation to disease progression. The level of serum ß-human chorionic gonadotropin corresponded with the therapy response; it was at its lowest during stabilization and the highest in the metastatic stage. CONCLUSION: Our case study provides the first report of urothelial cancer of the prostate, with a concomitant increase of ß-subunit human chorionic gonadotropin level with testis enlargement. Besides its rarity, it constitutes an interesting observation of increasing ß-subunit human chorionic gonadotropin concentration with concomitant disease progression.
Assuntos
Carcinoma de Células de Transição , Neoplasias da Bexiga Urinária , Idoso , Carcinoma de Células de Transição/diagnóstico por imagem , Carcinoma de Células de Transição/patologia , Gonadotropina Coriônica , Gonadotropina Coriônica Humana Subunidade beta , Progressão da Doença , Humanos , Masculino , Pelve , Próstata , Neoplasias da Bexiga Urinária/patologiaRESUMO
Amebin [formerly termed as ApABP-FI; Sobczak et al. (2007) Biochem. Cell Biol. 85] is encoded in Amoeba proteus by two transcripts, 2672-nt and 1125-nt. A product of the shorter transcript (termed as C-amebin), comprising C-terminal 375 amino-acid-residue fragment of amebin, has been expressed and purified as the recombinant GST-fusion protein. GST-C-amebin bound both to monomeric and filamentous actin. The binding was Ca(2+)-independent and promoted filament bundling, as revealed with the transmission electron microscopy. GST-C-amebin significantly decreased MgATPase activity of rabbit skeletal muscle acto-S1. Removal with endoproteinase ArgC of a positively charged C-terminal region of GST-amebin containing KLASMWEQ sequence abolished actin-binding and bundling as well as the ATPase-inhibitory effect of C-amebin, indicating that this protein region was involved in the interaction with actin. Microinjection of amoebae with antibody against C-terminus of amebin significantly affected amoebae morphology, disturbed cell polarization and transport of cytoplasmic granules as well as blocked migration. These data indicate that amebin may be one of key regulators of the actin-cytoskeleton dynamics and actin-dependent motility in A. proteus.
Assuntos
Citoesqueleto de Actina/metabolismo , Amoeba/química , Amoeba/fisiologia , Miosinas/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/fisiologia , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestrutura , Sequência de Aminoácidos , Amoeba/genética , Animais , Técnicas In Vitro , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Movimento/fisiologia , Complexos Multiproteicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Fragmentos de Peptídeos/ultraestrutura , Ligação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/ultraestrutura , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/ultraestruturaRESUMO
Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments and is believed to play distinct role(s) than other myosins. We addressed a role of this unique motor in secretory PC12 cells, derived from rat adrenal medulla pheochromocytoma using cell lines with reduced MVI synthesis (produced by means of siRNA). Decrease of MVI expression caused severe changes in cell size and morphology, and profound defects in actin cytoskeleton organization and Golgi structure. Also, significant inhibition of cell migration as well as cell proliferation was observed. Flow cytometric analysis revealed that MVI-deficient cells were arrested in G0/G1 phase of the cell cycle but did not undergo increased senescence as compared with control cells. Also, neither polyploidy nor aneuploidy were detected. Surprisingly, no significant effect on noradrenaline secretion was observed. These data indicate that in PC12 cells MVI is involved in cell migration and proliferation but is not crucial for stimulation-dependent catecholamine release.
Assuntos
Citoesqueleto de Actina/metabolismo , Catecolaminas/metabolismo , Movimento Celular/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Animais , Transporte Biológico , Pontos de Checagem do Ciclo Celular/fisiologia , Proliferação de Células , Tamanho Celular , Citometria de Fluxo , Complexo de Golgi/metabolismo , Cadeias Pesadas de Miosina/genética , Células PC12 , RatosRESUMO
Growth media composition is a critical factor influencing the yield of bacterial exopolysaccharides (EPSs), which have attracted the interest of researchers around the world due to their diverse physicochemical and biological properties. This work presents the optimization of media for EPS synthesis by three Lactobacillus rhamnosus strains, namely LOCK 0943, LOCK 0935, and OM-1. The optimized media led to a more than 13-fold increase in EPS yield for L. rhamnosus LOCK 0943 (from 85 to 1138.2 mg/L), an almost 9-fold increase for L. rhamnosus LOCK 0935 (from 103.67 to 900 mg/L), and a more than 7-fold increase for L. rhamnosus OM-1 (from 133.67 to 987.84 mg/L) as compared to cultures in standard MRS medium (de Man, Rogosa, and Sharpe). It has been found that the main medium-related determinant of EPS synthesis by the studied L. rhamnosus strains are the carbon source-in this case, it was fructose and sucrose.
Assuntos
Meios de Cultura/química , Lacticaseibacillus rhamnosus , Polissacarídeos Bacterianos/biossíntese , Carbono/metabolismo , Lacticaseibacillus rhamnosus/crescimento & desenvolvimento , Lacticaseibacillus rhamnosus/metabolismoRESUMO
Myosins, actin-dependent molecular motors are expressed in almost all eukaryotic cells where are engaged in a panoply of cellular processes such as muscle contraction, cell migration and adhesion, intracellular trafficking, cytokinesis, endocytosis and secretion. In recent years a number of reports has been published revealing nuclear localization of myosins as well as actin and actin-binding proteins. Namely, nuclear form of myosin IC (NMI), myosin VI, myosin XVIB and myosin XVIIIB were found in nucleus. NMI and myosin VI seem to be involved in gene transcription and possibly intranuclear transport. In this paper, current knowledge on the role of myosin motors in nucleus has been presented.
Assuntos
Núcleo Celular/metabolismo , Miosinas/metabolismo , Actinas/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Humanos , Proteínas dos Microfilamentos/metabolismo , Contração Muscular/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Transcrição Gênica/fisiologiaRESUMO
Myosin VI (MVI) is a unique actin-based motor protein moving towards the minus end of actin filaments, in the opposite direction than other known myosins. Besides well described functions of MVI in endocytosis and maintenance of Golgi apparatus, there are few reports showing its involvement in transcription. We previously demonstrated that in neurosecretory PC12 cells MVI was present in the cytoplasm and nucleus, and its depletion caused substantial inhibition of cell migration and proliferation. Here, we show an increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain. These signals seem to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import (ivermectin) and export (leptomycin B). In nuclei of stimulated cells, MVI colocalized with active RNA polymerase II, BrUTP-containing transcription sites and transcription factor SP1 as well as SC35 and PML proteins, markers of nuclear speckles and PML bodies, respectively. Mass spectrometry analysis of samples of a GST-pull-down assay with the MVI tail domain as a "bait" identified several new potential MVI binding partners. Among them are proteins involved in transcription and post-transcriptional processes. We confirmed interaction of MVI with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and nucleolin, proteins involved in pre-mRNA binding and transport, and nucleolar function, respectively. Our data provide an insight into mechanisms of involvement of MVI in nuclear processes via interaction with nuclear proteins and support a notion for important role(s) for MVI in gene expression.
Assuntos
Núcleo Celular/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Sistemas Neurossecretores/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Humanos , Ivermectina/farmacologia , Cadeias Pesadas de Miosina/antagonistas & inibidores , Células PC12 , Ratos , NucleolinaRESUMO
Myosin VI (MVI) is a unique motor protein moving towards the minus end of actin filaments unlike other known myosins. Its important role has recently been postulated for striated muscle and myogenic cells. Since MVI functions through interactions of C-terminal globular tail (GT) domain with tissue specific partners, we performed a search for MVI partners in myoblasts and myotubes using affinity chromatography with GST-tagged MVI-GT domain as a bait. A kinase anchoring protein 9 (AKAP9), a regulator of PKA activity, was identified by means of mass spectrometry as a possible MVI interacting partner both in undifferentiated and differentiating myoblasts and in myotubes. Coimmunoprecipitation and proximity ligation assay confirmed that both proteins could interact. MVI and AKAP9 colocalized at Rab5 containing early endosomes. Similarly to MVI, the amount of AKAP9 decreased during myoblast differentiation. However, in MVI-depleted cells, both cAMP and PKA levels were increased and a change in the MVI motor-dependent AKAP9 distribution was observed. Moreover, we found that PKA phosphorylated MVI-GT domain, thus implying functional relevance of MVI-AKAP9 interaction. We postulate that this novel interaction linking MVI with the PKA pathway could be important for targeting AKAP9-PKA complex within cells and/or providing PKA to phosphorylate MVI tail domain.
Assuntos
Proteínas de Ancoragem à Quinase A/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Desenvolvimento Muscular/genética , Cadeias Pesadas de Miosina/metabolismo , Proteínas de Ancoragem à Quinase A/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Diferenciação Celular/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Endossomos/genética , Endossomos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/genética , Ligação Proteica , RNA Interferente Pequeno , Transdução de SinaisRESUMO
Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin and anti-VEGFR2 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.
Assuntos
Técnicas de Cultura de Células/métodos , Células Endoteliais/citologia , Pulmão/citologia , Animais , Animais Recém-Nascidos , Anticorpos/química , Antígenos CD/química , Antígenos CD/imunologia , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/imunologia , Humanos , Camundongos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/química , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologiaRESUMO
Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments. Here, MVI, but not myosins IB or IIB, was detected in chromaffin granules isolated from bovine medulla and found to be tightly associated with the granule apical surface. MVI also localized to secretory granules within rat pheochromocytoma PC12 cells as well as to the Golgi apparatus, endoplasmic reticulum and clathrin-coated pits. Notably, it was also found in the nucleus. RT-PCR revealed that MVI splice variants with a large insert (LI), characteristic of polarized cells, were barely detectable in PC12 cells, whereas variants with a small insert (SI) were the major isoforms. The presented data indicate that MVI in adrenal medulla cells is engaged in secretory vesicle trafficking within the cytoplasm and possibly also involved in transport within the nucleus.
Assuntos
Medula Suprarrenal/metabolismo , Núcleo Celular/metabolismo , Células Cromafins/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Vesículas Secretórias/metabolismo , Transporte Ativo do Núcleo Celular , Processamento Alternativo , Animais , Bovinos , Cadeias Pesadas de Miosina/genética , Miosina Tipo I/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Células PC12 , RatosRESUMO
Recently, we found a 130-kDa myosin VI immunoanalog in amoeba, which bound to actin in an ATP-sensitive manner and in migrating amoebae colocalized to filamentous actin and dynamin II-containing vesicular structures. To further characterize this protein, we assessed its involvement in amoeba pinocytosis and phagocytosis. Confocal immunofluorescence microscopy and electron microscopy of immunogold-stained cells revealed that, in pinocytotic and phagocytotic amoebae, the myosin VI immunoanalog was visible throughout the cells, including pinocytotic channels and pinocytotic vesicles as well as phagosomes and emerging phagocytic cups. Blocking endogenous protein with anti-porcine myosin VI antibody (introduced into cells by means of microinjection) caused severe defects in pinocytosis and phagocytosis. In comparison with control cells, the treated amoebae formed ~75% less pinocytotic channels and phagocytosed ~65% less Tetrahymena cells. These data indicate that the myosin VI immunoanalog has an important role in pinocytosis and phagocytosis in Amoeba proteus (Pal.).
Assuntos
Amoeba/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Fagocitose/fisiologia , Pinocitose/fisiologia , Isoformas de Proteínas/metabolismo , Amoeba/citologia , Animais , Imuno-Histoquímica , Cadeias Pesadas de Miosina/genética , Isoformas de Proteínas/genética , Sus scrofaRESUMO
A novel 120 kDa actin-binding protein (ApABP-F1) was found in Amoeba proteus. It was distributed throughout the cytoplasm, mainly in the subplasma membrane and perinuclear-nuclear areas, enriched in actin. The full-length cDNA of ApABP consisted of 2672 nucleotides with an open reading frame of 878 amino acids, giving a ~95 kDa protein with a theoretical pI value of 5.11. It had a novel domain organization pattern: the N terminus (residues 1-104) contained 1 calponin-homology (CH) domain, followed by only 1 region that was homologous to the filamin repeat (FR, residues 209-324), and a central region (residues 344-577) exhibiting a very high probability of coiled-coil formation, probably engaged in the observed protein dimerization. A phylogenetic tree constructed for CH domains from 25 various proteins revealed that the CH domain of ApABP was most related to that of the hypothetical mouse KIAA0903-like protein, whereas not much relationship to either filamins or the gelation factor (ABP-120) of Dictyostelium discoideum and Entamoeba histolytica was found.