RESUMO
Viral replication and movement are intimately linked; however, the molecular mechanisms regulating the transition between replication and subsequent movement remain largely unknown. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein promotes viral replication and movement by interacting with the αa replicase and TGB1 movement proteins. Here, we found that γb is palmitoylated at Cys-10, Cys-19, and Cys-60 in Nicotiana benthamiana, which supports BSMV infection. Intriguingly, non-palmitoylated γb is anchored to chloroplast replication sites and enhances BSMV replication, whereas palmitoylated γb protein recruits TGB1 to the chloroplasts and forms viral replication-movement intermediate complexes. At the late stages of replication, γb interacts with NbPAT15 and NbPAT21 and is palmitoylated at the chloroplast periphery, thereby shifting viral replication to intracellular and intercellular movement. We also show that palmitoylated γb promotes virus cell-to-cell movement by interacting with NbREM1 to inhibit callose deposition at the plasmodesmata. Altogether, our experiments reveal a model whereby palmitoylation of γb directs a dynamic switch between BSMV replication and movement events during infection.
Assuntos
Lipoilação , Vírus de Plantas , Nicotiana/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
BACKGROUND: Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMß2) is a leukocyte integrin essential for firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration. We aimed to elucidate the molecular mechanism of PDI control of Mac-1 affinity for ICAM-1 during neutrophil migration under fluid shear. METHODS: Neutrophils isolated from whole blood were perfused over microfluidic chips coated with ICAM-1. Colocalization of Mac-1 and PDI on neutrophils was visualized by fluorescently labeled antibodies and confocal microscopy. The redox state of Mac-1 disulfide bonds was mapped by differential cysteine alkylation and mass spectrometry. Wild-type or disulfide mutant Mac-1 was expressed recombinantly in Baby Hamster Kidney cells to measure ligand affinity. Mac-1 conformations were measured by conformation-specific antibodies and molecular dynamics simulations. Neutrophils crawling on immobilized ICAM-1 were measured in presence of oxidized or reduced PDI, and the effect of PDI inhibition using isoquercetin on neutrophil crawling on inflamed endothelial cells was examined. Migration indices in the X- and Y-direction were determined and the crawling speed was calculated. RESULTS: PDI colocalized with high-affinity Mac-1 at the trailing edge of stimulated neutrophils when crawling on ICAM-1 under fluid shear. PDI cleaved 2 allosteric disulfide bonds, C169-C176 and C224-C264, in the ßI domain of the ß2 subunit, and cleavage of the C224-C264 disulfide bond selectively controls Mac-1 disengagement from ICAM-1 under fluid shear. Molecular dynamics simulations and conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the ßI domain. This allosterically alters the exposure of an αI domain epitope associated with a shift of Mac-1 to a lower-affinity state. These molecular events promote neutrophil motility in the direction of flow at high shear stress. Inhibition of PDI by isoquercetin reduces neutrophil migration in the direction of flow on endothelial cells during inflammation. CONCLUSIONS: Shear-dependent PDI cleavage of the neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.
Assuntos
Molécula 1 de Adesão Intercelular , Antígeno de Macrófago 1 , Humanos , Antígeno de Macrófago 1/fisiologia , Adesão Celular/fisiologia , Células Endoteliais , Inflamação , Movimento Celular/fisiologia , NeutrófilosRESUMO
The function of dopamine receptor D2 (D2R) is well associated with sperm motility; however, the physiological role of D2R present on testicular cells remains elusive. The aim of the present study is to delineate the function of testicular D2R. Serum dopamine levels were found to decrease with age, whereas testicular D2R expression increased. In rat testicular sections, D2R immunolabeling was observed in interstitial cells, spermatogonia, spermatocytes and mature elongated spermatids, whereas tyrosine hydroxylase immunolabeling was selectively detected in Leydig cells. In vitro seminiferous tubule culture following bromocriptine (D2R agonist) treatment resulted in decreased cAMP levels. Microarray identified 1077 differentially expressed genes (511 up-regulated, 566 down-regulated). The majority of differentially expressed genes were present in post-meiotic cells including early and late spermatids, and sperm. Gene ontology elucidated processes related to extra-cellular matrix to be enriched and was supported by differential expression of various collagens and laminins, thereby indicating a role of dopamine in extra-cellular matrix integrity and transport of spermatids across the seminiferous epithelium. Gene ontology and enrichment map also highlighted cell/sperm motility to be significantly enriched. Therefore, genes involved in sperm motility functions were further validated by RT-qPCR. Seven genes (Akap4, Ccnyl1, Iqcf1, Klc3, Prss55, Tbc1d21, Tl18) were significantly up-regulated, whereas four genes (Dnah1, Dnah5, Clxn, Fsip2) were significantly down-regulated by bromocriptine treatment. The bromocriptine-stimulated reduction in seminiferous tubule cyclic AMP and associated changes in spermatid gene expression suggests that dopamine regulates both spermatogenesis and spermiogenesis within the seminiferous epithelium, and spermatozoa motility following spermiation, as essential processes for fertility.
Assuntos
Motilidade dos Espermatozoides , Testículo , Ratos , Animais , Masculino , Testículo/metabolismo , Bromocriptina/metabolismo , Dopamina/farmacologia , Sêmen , Espermatozoides/metabolismo , Espermátides/metabolismo , Espermatogênese/genética , Receptores Dopaminérgicos/metabolismoRESUMO
Orthotospoviruses, the plant-infecting bunyaviruses, cause serious diseases in agronomic crops and pose major threats to global food security. The family of Tospoviridae contains more than 30 members that are classified into two geographic groups, American-type and Euro/Asian-type orthotospovirus. However, the genetic interaction between different species and the possibility, during mixed infections, for transcomplementation of gene functions by orthotospoviruses from different geographic groups remains underexplored. In this study, minireplicon-based reverse genetics (RG) systems have been established for Impatiens necrotic spot virus (INSV) (an American-type orthotospovirus) and for Calla lily chlorotic spot virus and Tomato zonate spot virus (CCSV and TZSV) (two representative Euro/Asian orthotospoviruses). Together with the earlier established RG system for Tomato spotted wilt virus (TSWV), a type species of the Orthotospovirus American-clade, viral replicase/movement proteins were exchanged and analyzed on interspecies transcomplementation. Whereas the homologous RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) protein supported the replication of orthotospoviruses from both geographic groups, heterologous combinations of RdRp from one group and N from the other group were unable to support the replication of viruses from both groups. Furthermore, the NSm movement protein (MP), from both geographic groups of orthotospoviruses, was able to transcomplement heterologous orthotospoviruses or a positive-strand Cucumber mosaic virus (CMV) in their movement, albeit with varying efficiency. MP from Rice stripe tenuivirus (RSV), a plant-infecting bunyavirus that is distinct from orthotospoviruses, or MP from CMV also moves orthotospoviruses. Our findings gain insights into the genetic interaction/reassortant potentials for the segmented plant orthotospoviruses. IMPORTANCE Orthotospoviruses are agriculturally important negative-strand RNA viruses and cause severe yield-losses on many crops worldwide. Whereas the emergence of new animal-infecting bunyaviruses is frequently associated with genetic reassortants, this issue remains underexposed with the plant-infecting orthotospovirus. With the development of reverse genetics systems for orthotospoviruses from different geographic regions, the interspecies/intergroup replication/movement complementation between American- and Euro/Asian-type orthotospoviruses were investigated. Genomic RNAs from American orthotospoviruses can be replicated by the RdRp and N from those of Euro/Asia-group orthotospoviruses, and vice versa. However, their genomic RNAs cannot be replicated by a heterologous combination of RdRp from one geographic group and N from another geographic group. Cell-to-cell movement of viral entity is supported by NSm from both geographic groups, with highest efficiency by NSm from viruses belonging to the same group. Our findings provide important insights into the genetic interaction and exchange ability of viral gene functions between different species of orthotospovirus.
Assuntos
Genética Reversa , Tospovirus , Replicação Viral , Animais , Genética Reversa/métodos , RNA Polimerase Dependente de RNA , Tospovirus/genética , Estados Unidos , Replicação Viral/genética , RNA Viral/genética , Proteínas do Nucleocapsídeo/genéticaRESUMO
BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by progressive distal pulmonary artery (PA) obstruction, leading to right ventricular hypertrophy and failure. Exacerbated intracellular calcium (Ca2+) signaling contributes to abnormalities in PA smooth muscle cells (PASMCs), including aberrant proliferation, apoptosis resistance, exacerbated migration, and arterial contractility. Store-operated Ca2+ entry is involved in Ca2+ homeostasis in PASMCs, but its properties in PAH are unclear. METHODS: Using a combination of Ca2+ imaging, molecular biology, in vitro, ex vivo, and in vivo approaches, we investigated the roles of the Orai1 SOC channel in PA remodeling in PAH and determined the consequences of pharmacological Orai1 inhibition in vivo using experimental models of pulmonary hypertension (PH). RESULTS: Store-operated Ca2+ entry and Orai1 mRNA and protein were increased in human PASMCs (hPASMCs) from patients with PAH (PAH-hPASMCs). We found that MEK1/2 (mitogen-activated protein kinase kinase 1/2), NFAT (nuclear factor of activated T cells), and NFκB (nuclear factor-kappa B) contribute to the upregulation of Orai1 expression in PAH-hPASMCs. Using small interfering RNA (siRNA) and Orai1 inhibitors, we found that Orai1 inhibition reduced store-operated Ca2+ entry, mitochondrial Ca2+ uptake, aberrant proliferation, apoptosis resistance, migration, and excessive calcineurin activity in PAH-hPASMCs. Orai1 inhibitors reduced agonist-evoked constriction in human PAs. In experimental rat models of PH evoked by chronic hypoxia, monocrotaline, or Sugen/hypoxia, administration of Orai1 inhibitors (N-{4-[3,5-bis(Trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide [BTP2], 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline [JPIII], or 5J4) protected against PH. CONCLUSIONS: In human PAH and experimental PH, Orai1 expression and activity are increased. Orai1 inhibition normalizes the PAH-hPASMCs phenotype and attenuates PH in rat models. These results suggest that Orai1 should be considered as a relevant therapeutic target for PAH.
Assuntos
Compostos de Anilina , Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Tiadiazóis , Animais , Humanos , Ratos , Compostos de Anilina/uso terapêutico , Calcineurina/metabolismo , Cálcio/metabolismo , Proliferação de Células/genética , Células Cultivadas , Hipertensão Pulmonar/tratamento farmacológico , Hipóxia/metabolismo , MAP Quinase Quinase 1/metabolismo , Monocrotalina/toxicidade , Miócitos de Músculo Liso/metabolismo , Proteína ORAI1 , Artéria Pulmonar/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Tiadiazóis/metabolismoRESUMO
Cell-cell adhesion plays a vital role in the development and maintenance of multicellular organisms. One of its functions is regulation of cell migration, such as occurs, e.g. during embryogenesis or in cancer. In this work, we develop a versatile multiscale approach to modelling a moving self-adhesive cell population that combines a careful microscopic description of a deterministic adhesion-driven motion component with an efficient mesoscopic representation of a stochastic velocity-jump process. This approach gives rise to mesoscopic models in the form of kinetic transport equations featuring multiple non-localities. Subsequent parabolic and hyperbolic scalings produce general classes of equations with non-local adhesion and myopic diffusion, a special case being the classical macroscopic model proposed in Armstrong et al. (J Theoret Biol 243(1): 98-113, 2006). Our simulations show how the combination of the two motion effects can unfold. Cell-cell adhesion relies on the subcellular cell adhesion molecule binding. Our approach lends itself conveniently to capturing this microscopic effect. On the macroscale, this results in an additional non-linear integral equation of a novel type that is coupled to the cell density equation.
Assuntos
Desenvolvimento Embrionário , Adesão Celular , Movimento Celular , Difusão , CinéticaRESUMO
New neurons are generated in the postnatal rodent hypothalamus, with a subset of tanycytes in the third ventricular (3V) wall serving as neural stem/progenitor cells. However, the precise stem cell niche organization, the intermediate steps and the endogenous regulators of postnatal hypothalamic neurogenesis remain elusive. Quantitative lineage-tracing in vivo revealed that conditional deletion of fibroblast growth factor 10 (Fgf10) from Fgf10-expressing ß-tanycytes at postnatal days (P)4-5 results in the generation of significantly more parenchymal cells by P28, composed mostly of ventromedial and dorsomedial neurons and some glial cells, which persist into adulthood. A closer scrutiny in vivo and ex vivo revealed that the 3V wall is not static and is amenable to cell movements. Furthermore, normally ß-tanycytes give rise to parenchymal cells via an intermediate population of α-tanycytes with transient amplifying cell characteristics. Loss of Fgf10 temporarily attenuates the amplification of ß-tanycytes but also appears to delay the exit of their α-tanycyte descendants from the germinal 3V wall. Our findings suggest that transience of cells through the α-tanycyte domain is a key feature, and Fgf10 is a negative regulator of postnatal hypothalamic neurogenesis.
Assuntos
Fator 10 de Crescimento de Fibroblastos/metabolismo , Hipotálamo/citologia , Hipotálamo/metabolismo , Neurogênese/fisiologia , Animais , Movimento Celular/fisiologia , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Feminino , Fator 10 de Crescimento de Fibroblastos/genética , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismoRESUMO
Molecular farming technology using transiently transformed Nicotiana plants offers an economical approach to the pharmaceutical industry to produce an array of protein targets including vaccine antigens and therapeutics. It can serve as a desirable alternative approach for those proteins that are challenging or too costly to produce in large quantities using other heterologous protein expression systems. However, since cost metrics are such a critical factor in selecting a production host, any system-wide modifications that can increase recombinant protein yields are key to further improving the platform and making it applicable for a wider range of target molecules. Here, we report on the development of a new approach to improve target accumulation in an established plant-based expression system that utilizes viral-based vectors to mediate transient expression in Nicotiana benthamiana. We show that by engineering the host plant to support viral vectors to spread more effectively between host cells through plasmodesmata, protein target accumulation can be increased by up to approximately 60%.
Assuntos
Vírus do Mosaico do Tabaco , Proteínas Recombinantes/genética , Plantas Geneticamente Modificadas/metabolismo , Vírus do Mosaico do Tabaco/genética , Nicotiana/genética , Transporte Proteico , Vetores GenéticosRESUMO
BACKGROUND: Migration of human aortic smooth muscle cells (HASMCs) contributes to the pathogenesis of atherosclerosis. This study aims to functionally characterize long noncoding RNA TPRG1-AS1 (tumor protein p63 regulated 1, antisense 1) in HASMCs and reveal the underlying mechanism of TPRG1-AS1 in HASMCs migration, neointima formation, and subsequent atherosclerosis. METHODS: The expression of TPRG1-AS1 in atherosclerotic plaques was verified a series of in silico analysis and quantitative real-time polymerase chain reaction analysis. Northern blot, rapid amplification of cDNA ends and Sanger sequencing were used to determine its full length. In vitro transcription-translation assay was used to investigate the protein-coding capacity of TPRG1-AS1. RNA fluorescent in situ hybridization was used to confirm its subcellular localization. Loss- and gain-of-function studies were used to investigate the function of TPRG1-AS1. Furthermore, the effect of TPRG1-AS1 on the pathological response was evaluated in carotid balloon injury model, wire injury model, and atherosclerosis model, respectively. RESULTS: TPRG1-AS1 was significantly increased in atherosclerotic plaques. TPRG1-AS1 did not encode any proteins and its full length was 1279nt, which was bona fide a long noncoding RNA. TPRG1-AS1 was mainly localized in cytoplasmic and perinuclear regions in HASMCs. TPRG1-AS1 directly interacted with MYH9 (myosin heavy chain 9) protein in HASMCs, promoted MYH9 protein degradation through the proteasome pathway, hindered F-actin stress fiber formation, and finally inhibited HASMCs migration. Vascular smooth muscle cell-specific transgenic overexpression of TPRG1-AS1 significantly reduced neointima formation, and attenuated atherosclerosis in apolipoprotein E knockout (Apoe-/-) mice. CONCLUSIONS: This study demonstrated that TPRG1-AS1 inhibited HASMCs migration through interacting with MYH9 protein and consequently suppressed neointima formation and atherosclerosis.
Assuntos
Aterosclerose , MicroRNAs , Placa Aterosclerótica , RNA Longo não Codificante , Humanos , Camundongos , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Músculo Liso Vascular/metabolismo , Neointima/metabolismo , Placa Aterosclerótica/metabolismo , Actinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , DNA Complementar/metabolismo , DNA Complementar/farmacologia , Hibridização in Situ Fluorescente , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Proliferação de Células , Miócitos de Músculo Liso/metabolismo , Movimento Celular , Aterosclerose/genética , Aterosclerose/prevenção & controle , Aterosclerose/metabolismo , MicroRNAs/genética , Proteínas do Citoesqueleto/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas/metabolismoRESUMO
BACKGROUND: ABCA1 (ATP-binding cassette transporter A1) mediates cholesterol efflux to apo AI to maintain cellular cholesterol homeostasis. The current study aims to investigate whether T-cell-specific deletion of ABCA1 modulates the phenotype/function of T cells and the development of atherosclerosis. METHODS: Mice with T-cell-specific deletion of ABCA1 on low-density lipoprotein receptor knockout (Ldlr-/-) background (Abca1CD4-/CD4-Ldlr-/-) were generated by multiple steps of (cross)-breedings among Abca1flox/flox, CD4-Cre, and Ldlr-/- mice. RESULTS: Deletions of ABCA1 greatly suppressed cholesterol efflux to apo AI but slightly reduced membrane lipid rafts on T cells probably due to the upregulation of ABCG1. Moreover, ABCA1 deficiency impaired TCR (T-cell receptor) signaling and inhibited the survival and proliferation of T cells as well as the formation of effector memory T cells. Despite the comparable levels of plasma total cholesterol after Western-type diet feeding, Abca1CD4-/CD4-Ldlr-/- mice showed significantly attenuated arterial accumulations of T cells and smaller atherosclerotic lesions than Abca1+/+Ldlr-/-controls, which were associated with reduced surface CCR5 (CC motif chemokine receptor 5) and CXCR3 (CXC motif chemokine receptor 3), decreased antiapoptotic Bcl-2 (B-cell lymphoma 2) and Bcl-xL (B-cell lymphoma extra-large), and hampered abilities to produce IL (interleukin)-2 and IFN (interferon)-γ by ABCA1-deficient T cells. CONCLUSIONS: ABCA1 is essential for T-cell cholesterol homeostasis. Deletion of ABCA1 in T cells impairs TCR signaling, suppresses the survival, proliferation, differentiation, and function of T cells, thereby providing atheroprotection in vivo.
Assuntos
Apolipoproteína A-I , Aterosclerose , Camundongos , Animais , Camundongos Knockout , Transportadores de Cassetes de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/genética , Aterosclerose/genética , Aterosclerose/prevenção & controle , Aterosclerose/patologia , Colesterol , Receptores de Antígenos de Linfócitos T , Receptores de QuimiocinasRESUMO
The use of oncolytic viruses as cancer treatment has received considerable attention in recent years, however the spatial dynamics of this viral infection is still poorly understood. We present here a stochastic agent-based model describing infected and uninfected cells for solid tumours, which interact with viruses in the absence of an immune response. Two kinds of movement, namely undirected random and pressure-driven movements, are considered: the continuum limit of the models is derived and a systematic comparison between the systems of partial differential equations and the individual-based model, in one and two dimensions, is carried out. In the case of undirected movement, a good agreement between agent-based simulations and the numerical and well-known analytical results for the continuum model is possible. For pressure-driven motion, instead, we observe a wide parameter range in which the infection of the agents remains confined to the center of the tumour, even though the continuum model shows traveling waves of infection; outcomes appear to be more sensitive to stochasticity and uninfected regions appear harder to invade, giving rise to irregular, unpredictable growth patterns. Our results show that the presence of spatial constraints in tumours' microenvironments limiting free expansion has a very significant impact on virotherapy. Outcomes for these tumours suggest a notable increase in variability. All these aspects can have important effects when designing individually tailored therapies where virotherapy is included.
Assuntos
Modelos Biológicos , Vírus Oncolíticos , Conceitos Matemáticos , Movimento (Física)RESUMO
The microscope has revolutionized the understanding of an organism's structural details and cellular functions. With the invention of highly evolved microscopes, the diagnosis and treatment of diseases has gained momentum. Technology has immensely helped demonstrate cellular events like phagocytosis, cell movement, cell division, etc. with enhanced temporal and spatial resolution. One of these advanced inventions is the fluorescent microscope which has enabled scanning through various physiological activities of the cell. A fluorescence microscope uses the property of fluorescence to create an image. In addition to visualizing the structural details of the cells, a fluorescence microscope also aids in witnessing cellular activities. With an immunofluorescence microscope, cellular antigens can be localized. This chapter highlights the basics of microscopy, types of microscopes, principles, and types of fluorescence microscopes, and recent advances in microscopy and its application.
Assuntos
Fagocitose , Microscopia de Fluorescência/métodos , Movimento CelularRESUMO
Dilated cardiomyopathy (DCM) with left ventricular non-compaction (LVNC) is a primary myocardial disease leading to contractile dysfunction, progressive heart failure, and excessive risk of sudden cardiac death. Using whole-exome sequencing to investigate a possible genetic cause of DCM with LVNC in a consanguineous child, a homozygous nucleotide change c.1532G>A causing p.Arg511His in PHACTR2 was found. The missense change can affect the binding of PHACTR2 to actin by eliminating the hydrogen bonds between them. The amino acid change does not change PHACTR2 localization to the cytoplasm. The patient's fibroblasts showed a decreased globular to fibrillary actin ratio compared to the control fibroblasts. The re-polymerization of fibrillary actin after treatment with cytochalasin D, which disrupts the actin filaments, was slower in the patient's fibroblasts. Finally, the patient's fibroblasts bridged a scar gap slower than the control fibroblasts because of slower and indirect movement. This is the first report of a human variation in this PHACTR family member. The knock-out mouse model presented no significant phenotype. Our data underscore the importance of PHACTR2 in regulating the monomeric actin pool, the kinetics of actin polymerization, and cell movement, emphasizing the importance of actin regulation for the normal function of the human heart.
Assuntos
Actinas , Cardiomiopatia Dilatada , Criança , Animais , Camundongos , Humanos , Actinas/genética , Actinas/metabolismo , Cardiomiopatia Dilatada/metabolismo , Citoesqueleto de Actina/metabolismo , Fenótipo , Morte Súbita Cardíaca/etiologia , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genéticaRESUMO
To move from cell to cell through plasmodesmata, many plant viruses require the concerted action of two or more movement proteins (MPs) encoded by transport gene modules of virus genomes. A tetra-cistron movement block (TCMB) is a newly discovered transport module comprising four genes. TCMB encodes three proteins, which are similar to MPs of the transport module known as the "triple gene block", and a protein unrelated to known viral MPs and containing a double-stranded RNA (dsRNA)-binding domain similar to that found in a family of cell proteins, including AtDRB4 and AtHYL1. Here, the latter TCMB protein, named vDRB for virus dsRNA-binding protein, is shown to bind both dsRNA and single-stranded RNA in vitro. In a turnip crinkle virus-based assay, vDRB exhibits the properties of a viral suppressor of RNA silencing (VSR). In the context of potato virus X infection, vDRB significantly decreases the number and size of "dark green islands", regions of local antiviral silencing, supporting the VSR function of vDRB. Nevertheless, vDRB does not exhibit the VSR properties in non-viral transient expression assays. Taken together, the data presented here indicate that vDRB is an RNA-binding protein exhibiting VSR functions in the context of viral infection.
RESUMO
Replication and movement are two critical steps in plant virus infection. Recent advances in the understanding of the architecture and subcellular localization of virus-induced inclusions and the interactions between viral replication complex (VRC) and movement proteins (MPs) allow for the dissection of the intrinsic relationship between replication and movement, which has revealed that recruitment of VRCs to the plasmodesma (PD) via direct or indirect MP-VRC interactions is a common strategy used for cell-to-cell movement by most plant RNA viruses. In this review, we summarize the recent advances in the understanding of virus-induced inclusions and their roles in virus replication and cell-to-cell movement, analyze the advantages of such coreplicational movement from a viral point of view and discuss the possible mechanical force by which MPs drive the movement of virions or viral RNAs through the PD. Finally, we highlight the missing pieces of the puzzle of viral movement that are especially worth investigating in the near future.
Assuntos
Proteínas do Movimento Viral em Plantas , Vírus de RNA , Plasmodesmos , RNA de Plantas , Nicotiana , Replicação ViralRESUMO
Vascular smooth muscle cells (VSMCs) contribute to the deposition of extracellular matrix proteins (ECMs), including Type IV collagen, in the vessel wall. ECMs coordinate communication among different cell types, but mechanisms underlying this communication remain unclear. Our previous studies have demonstrated that X-box binding protein 1 (XBP1) is activated and contributes to VSMC phenotypic transition in response to vascular injury. In this study, we investigated the participation of XBP1 in the communication between VSMCs and vascular progenitor cells (VPCs). Immunofluorescence and immunohistology staining revealed that Xbp1 gene was essential for type IV collagen alpha 1 (COL4A1) expression during mouse embryonic development and vessel wall ECM deposition and stem cell antigen 1-positive (Sca1+)-VPC recruitment in response to vascular injury. The Western blot analysis elucidated an Xbp1 gene dose-dependent effect on COL4A1 expression and that the spliced XBP1 protein (XBP1s) increased protease-mediated COL4A1 degradation as revealed by Zymography. RT-PCR analysis revealed that XBP1s in VSMCs not only upregulated COL4A1/2 transcription but also induced the occurrence of a novel transcript variant, soluble type IV collagen alpha 1 (COL4A1s), in which the front part of exon 4 is joined with the rear part of exon 42. Chromatin-immunoprecipitation, DNA/protein pulldown and in vitro transcription demonstrated that XBP1s binds to exon 4 and exon 42, directing the transcription from exon 4 to exon 42. This leads to transcription complex bypassing the internal sequences, producing a shortened COL4A1s protein that increased Sca1+-VPC migration. Taken together, these results suggest that activated VSMCs may recruit Sca1+-VPCs via XBP1s-mediated COL4A1s secretion, leading to vascular injury repair or neointima formation.
Assuntos
Comunicação Celular , Movimento Celular , Colágeno Tipo IV/metabolismo , Músculo Liso Vascular/fisiologia , Células-Tronco/fisiologia , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Colágeno Tipo IV/genética , Humanos , Camundongos , Músculo Liso Vascular/citologia , Transdução de Sinais , Células-Tronco/citologia , Proteína 1 de Ligação a X-Box/genéticaRESUMO
RNA transport and localization represent important post-transcriptional mechanisms to determine the subcellular localization of protein synthesis. Plants have the capacity to transport messenger (m)RNA molecules beyond the cell boundaries through plasmodesmata and over long distances in the phloem. RNA viruses exploit these transport pathways to disseminate their infections and represent important model systems to investigate RNA transport in plants. Here, we present an in vivo plant RNA-labeling system based on the Escherichia coli RNA-binding protein BglG. Using the detection of RNA in mobile RNA particles formed by viral movement protein (MP) as a model, we demonstrate the efficiency and specificity of mRNA detection by the BglG system as compared with MS2 and λN systems. Our observations show that MP mRNA is specifically associated with MP in mobile MP particles but hardly with MP localized at plasmodesmata. MP mRNA is clearly absent from MP accumulating along microtubules. We show that the in vivo BglG labeling of the MP particles depends on the presence of the BglG-binding stem-loop aptamers within the MP mRNA and that the aptamers enhance the coprecipitation of BglG by MP, thus demonstrating the presence of an MP:MP mRNA complex. The BglG system also allowed us to monitor the cell-to-cell transport of the MP mRNA, thus linking the observation of mobile MP mRNA granules with intercellular MP mRNA transport. Given its specificity demonstrated here, the BglG system may be widely applicable for studying mRNA transport and localization in plants.
Assuntos
Proteínas de Bactérias , RNA Mensageiro/ultraestrutura , RNA de Plantas/ultraestrutura , Proteínas de Ligação a RNA , Escherichia coli , Proteínas de Escherichia coli , Proteínas de Fluorescência Verde , Imunoprecipitação , Microscopia de Fluorescência , Epiderme Vegetal/metabolismo , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Nicotiana/genéticaRESUMO
BACKGROUND: Activin receptor-like kinase 1 (ALK1) is an endothelial transmembrane serine threonine kinase receptor for BMP family ligands that plays a critical role in cardiovascular development and pathology. Loss-of-function mutations in the ALK1 gene cause type 2 hereditary hemorrhagic telangiectasia, a devastating disorder that leads to arteriovenous malformations. Here, we show that ALK1 controls endothelial cell polarization against the direction of blood flow and flow-induced endothelial migration from veins through capillaries into arterioles. METHODS: Using Cre lines that recombine in different subsets of arterial, capillary-venous, or endothelial tip cells, we show that capillary-venous Alk1 deletion was sufficient to induce arteriovenous malformation formation in the postnatal retina. RESULTS: ALK1 deletion impaired capillary-venous endothelial cell polarization against the direction of blood flow in vivo and in vitro. Mechanistically, ALK1-deficient cells exhibited increased integrin signaling interaction with vascular endothelial growth factor receptor 2, which enhanced downstream YAP/TAZ nuclear translocation. Pharmacologic inhibition of integrin or YAP/TAZ signaling rescued flow migration coupling and prevented vascular malformations in Alk1-deficient mice. CONCLUSIONS: Our study reveals ALK1 as an essential driver of flow-induced endothelial cell migration and identifies loss of flow-migration coupling as a driver of arteriovenous malformation formation in hereditary hemorrhagic telangiectasia disease. Integrin-YAP/TAZ signaling blockers are new potential targets to prevent vascular malformations in patients with hereditary hemorrhagic telangiectasia.
Assuntos
Malformações Arteriovenosas , Células Endoteliais , Telangiectasia Hemorrágica Hereditária , Fator A de Crescimento do Endotélio Vascular , Animais , Humanos , Malformações Arteriovenosas/metabolismo , Movimento Celular/fisiologia , Células Endoteliais/metabolismo , Telangiectasia Hemorrágica Hereditária/mortalidade , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Malformações Vasculares/metabolismo , CamundongosRESUMO
South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite begomovirus, whose genome comprises DNA-A (encodes six genes) and DNA-B (encodes BC1 cell-to-cell movement and BV1 nuclear shuttle proteins) components. A few secondary and tertiary structural and physicochemical characteristics of partial but not full-length begomovirus proteins have been elucidated to date. The full-length codon-optimised SACMV BC1 gene was cloned into a pET-28a (+) expression vector and transformed into expression host cells E. coli BL21 (DE3). The optimal expression of the full-length BC1-encoded movement protein (MP) was obtained via induction with 0.25 mM IPTG at an OD600 of ~0.45 at 37 °C for four hours. Denatured protein fractions (dialysed in 4 M urea), passed through an IMAC column, successfully bound to the nickel resin, and eluted using 250 mM imidazole. The protein was refolded using stepwise dialysis. The molecular weight of MP was confirmed to be 35 kDa using SDS-PAGE. The secondary structure of SACMV MP presented as predominantly ß-strands. An ANS (1-anilino-8-naphthalene sulphonate)-binding assay confirmed that MP possesses hydrophobic pockets with the ability to bind ligands such as ANS (8-anilino-1-naphthalenesulphonic acid). A 2' (3')-N-methylanthraniloyl-ATP (mant-ATP) assay showed binding of mant-ATP to MP and indicated that, while hydrophobic pockets are present, MP also exhibits hydrophilic regions. Intrinsic tryptophan fluorescence indicated a significant conformational change in the denatured form of BC1 in the presence of ATP. In addition, a phosphatase assay showed that MP possessed ATPase activity.
RESUMO
FOXL2 and ESR2 are key transcriptional regulators in ovarian granulosa cells. To explore their transcriptional roles and their interplay, we have depleted Foxl2 and Esr2 in mouse primary granulosa cells to assess their ability to bind their targets and/or to modulate gene expression and cellular functions. We show that FOXL2 is involved in a large number of regulatory actions essential for the maintenance of granulosa cell fate. A parallel ChIP-seq analysis showed that FOXL2 mainly binds to sites located in intergenic regions quite far from its targets. A bioinformatic analysis demonstrated that FOXL2-activated genes were enriched in peaks associated with the H3K27ac mark, whereas FOXL2-repressed genes were not, suggesting that FOXL2 can activate transcription through binding to enhancer sites. We also identified about 500 deregulated genes upon Esr2 silencing, of which one third are also targets of FOXL2. We provide evidence showing that both factors modulate, through a coherent feed-forward loop, a number of common targets. Many of the FOXL2/ESR2 targets are involved in cell motility and, consistently, granulosa cells depleted for either Foxl2 or Esr2 exhibit decreased migration, invasion and adhesion. This effect is paralleled by the depletion of their target Phactr1, involved in actin cytoskeleton dynamics. Our analysis expands the number of direct and indirect transcriptional targets of both FOXL2 and ESR2, which deserve investigation in the context of adult-type granulosa cell tumors whose molecular diagnostic hallmark is the presence of the C134W FOXL2 pathogenic variant.