RESUMO
Microtubules (MTs) are crucial for both the establishment of cellular polarity and the progression of all mitotic phases leading to karyokinesis and cytokinesis. MT organization and spindle formation rely on the activity of γ-tubulin and associated proteins throughout the cell cycle. To date, the molecular mechanisms modulating γ-tubulin complex location remain largely unknown. In this work, two Arabidopsis thaliana proteins interacting with gamma-tubulin complex protein3 (GCP3), GCP3-interacting protein1 (GIP1) and GIP2, have been characterized. Both GIP genes are ubiquitously expressed in all tissues analyzed. Immunolocalization studies combined with the expression of GIP-green fluorescent protein fusions have shown that GIPs colocalize with γ-tubulin, GCP3, and/or GCP4 and reorganize from the nucleus to the prospindle and the preprophase band in late G2. After nuclear envelope breakdown, they localize on spindle and phragmoplast MTs and on the reforming nuclear envelope of daughter cells. The gip1 gip2 double mutants exhibit severe growth defects and sterility. At the cellular level, they are characterized by MT misorganization and abnormal spindle polarity, resulting in ploidy defects. Altogether, our data show that during mitosis GIPs play a role in γ-tubulin complex localization, spindle stability and chromosomal segregation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Transporte/metabolismo , Instabilidade Cromossômica , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , DNA Bacteriano/genética , Regulação da Expressão Gênica de Plantas , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Dados de Sequência Molecular , Mutagênese InsercionalRESUMO
Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence.
Assuntos
Doenças dos Bovinos/imunologia , Doenças dos Bovinos/patologia , Leucócitos/imunologia , Leucócitos/parasitologia , Theileria/patogenicidade , Theileriose/imunologia , Fator de Crescimento Transformador beta2/metabolismo , Animais , Apresentação de Antígeno , Biomarcadores/metabolismo , Western Blotting , Bovinos , Doenças dos Bovinos/metabolismo , Citoesqueleto/metabolismo , Perfilação da Expressão Gênica , Interações Hospedeiro-Parasita , Leucócitos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Theileriose/metabolismo , Theileriose/parasitologia , Transcrição Gênica , Fator de Crescimento Transformador beta2/genética , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
[This corrects the article DOI: 10.3389/fpls.2013.00480/full.].
RESUMO
During interphase, the microtubular cytoskeleton of cycling plant cells is organized in both cortical and perinuclear arrays. Perinuclear microtubules (MTs) are nucleated from γ-Tubulin Complexes (γ-TuCs) located at the surface of the nucleus. The molecular mechanisms of γ-TuC association to the nuclear envelope (NE) are currently unknown. The γ-TuC Protein 3 (GCP3)-Interacting Protein 1 (GIP1) is the smallest γ-TuC component identified so far. AtGIP1 and its homologous protein AtGIP2 participate in the localization of active γ-TuCs at interphasic and mitotic MT nucleation sites. Arabidopsis gip1gip2 mutants are impaired in establishing a fully functional mitotic spindle and exhibit severe developmental defects. In this study, gip1gip2 knock down mutants were further characterized at the cellular level. In addition to defects in both the localization of γ-TuC core proteins and MT fiber robustness, gip1gip2 mutants exhibited a severe alteration of the nuclear shape associated with an abnormal distribution of the nuclear pore complexes. Simultaneously, they showed a misorganization of the inner nuclear membrane protein AtSUN1. Furthermore, AtGIP1 was identified as an interacting partner of AtTSA1 which was detected, like the AtGIP proteins, at the NE. These results provide the first evidence for the involvement of a γ-TuC component in both nuclear shaping and NE organization. Functional hypotheses are discussed in order to propose a model for a GIP-dependent nucleo-cytoplasmic continuum.
RESUMO
Upregulation of the matrix metalloproteinase (MMP)-9 plays a central role in tumor progression and metastasis by stimulating cell migration, tumor invasion, and angiogenesis. To gain insights into MMP-9 expression, we investigated its epigenetic control in a reversible model of cancer that is initiated by infection with intracellular Theileria parasites. Gene induction by parasite infection was associated with trimethylation of histone H3K4 (H3K4me3) at the MMP-9 promoter. Notably, we found that the H3K4 methyltransferase SMYD3 was the only histone methyltransferase upregulated upon infection. SMYD3 is overexpressed in many types of cancer cells, but its contributions to malignant pathophysiology are unclear. We found that overexpression of SMYD3 was sufficient to induce MMP-9 expression in transformed leukocytes and fibrosarcoma cells and that proinflammatory phorbol esters further enhanced this effect. Furthermore, SMYD3 was sufficient to increase cell migration associated with MMP-9 expression. In contrast, RNA interference-mediated knockdown of SMYD3 decreased H3K4me3 modification of the MMP-9 promoter, reduced MMP-9 expression, and reduced tumor cell proliferation. Furthermore, SMYD3 knockdown also reduced cellular invasion in a zebrafish xenograft model of cancer. Together, our results define SMYD3 as an important new regulator of MMP-9 transcription, and they provide a molecular link between SMYD3 overexpression and metastatic cancer progression.
Assuntos
Epigênese Genética , Histona-Lisina N-Metiltransferase/genética , Metaloproteinase 9 da Matriz/genética , Neoplasias/genética , Regulação para Cima , Animais , Western Blotting , Bovinos , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Interações Hospedeiro-Parasita , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Metilação , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias/parasitologia , Neoplasias/patologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Theileria/fisiologia , Theileriose/genética , Theileriose/parasitologia , Transplante Heterólogo , Peixe-ZebraRESUMO
In higher plants, microtubules (MTs) show dynamic structural changes during cell cycle and development progression. A precise control of MT nucleation at dispersed sites is one way used to regulate the cytoskeletal organization. Some gamma-tubulin complex proteins (GCPs) were previously identified in Arabidopsis thaliana (At). They are directly involved in the nucleation process. Nevertheless, no additional player which may anchor the nucleating complex or regulate the nucleation activity has been found in plant cells so far. Therefore, our aim was the identification of Arabidopsis proteins interacting with MT nucleating complexes and particularly with AtGCP3. Performing a yeast two-hybrid screen, we discovered a new protein which we called AtGCP3 Interacting Protein 1 (AtGIP1). The possible role of this protein during the nucleation process is discussed.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Dados de Sequência Molecular , Ligação Proteica , Técnicas do Sistema de Duplo-HíbridoRESUMO
The Targeting Protein for Xklp2 (TPX2) is a central regulator of spindle assembly in vertebrate cells. The absence or excess of TPX2 inhibits spindle formation. We have defined a TPX2 signature motif that is present once in vertebrate sequences but twice in plants. Plant TPX2 is predominantly nuclear during interphase and is actively exported before nuclear envelope breakdown to initiate prospindle assembly. It localizes to the spindle microtubules but not to the interdigitating polar microtubules during anaphase or to the phragmoplast as it is rapidly degraded during telophase. We characterized the Arabidopsis thaliana TPX2-targeting domains and show that the protein is able to rescue microtubule assembly in TPX2-depleted Xenopus laevis egg extracts. Injection of antibodies to TPX2 into living plant cells inhibits the onset of mitosis. These results demonstrate that plant TPX2 already functions before nuclear envelope breakdown. Thus, plants have adapted nuclear-cytoplasmic shuttling of TPX2 to maintain proper spindle assembly without centrosomes.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , Parede Celular/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Membrana Nuclear/metabolismo , Fuso Acromático/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Ácidos Graxos Insaturados/farmacologia , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Alinhamento de Sequência , Análise de Sequência de Proteína , Nicotiana/citologia , Nicotiana/efeitos dos fármacos , Nicotiana/genética , Xenopus , alfa Carioferinas/metabolismoRESUMO
In higher plants, microtubules (MTs) are assembled in distinctive arrays in the absence of a defined organizing center. Three MT nucleation sites have been described: the nuclear surface, the cell cortex and cortical MT branch points. The Arabidopsis thaliana (At) genome contains putative orthologues encoding all the components of characterized mammalian nucleation complexes: gamma-tubulin and gamma-tubulin complex proteins GCP2 to GCP6. We have cloned the cDNA encoding AtGCP2, and show that gamma-tubulin, AtGCP2 and AtGCP3 are part of the same tandem affinity-purified complex and are present in a large membrane-associated complex. In addition, small soluble gamma-tubulin complexes of the size expected for a gamma-tubulin core complex are recruited to isolated nuclei. Using immunogold labelling, AtGCP3 is localized to both the nuclear envelope (NE) and the plasma membrane. To identify domains that could play a role in targeting complexes to these nucleation sites, truncated AtGCP2- and AtGCP3-green fluorescent protein fusion proteins were expressed in BY-2 cells. Several domains from AtGCP2 and AtGCP3 are capable of targeting fusions to the NE. We propose that regulated recruitment of soluble gamma-tubulin-containing complexes is responsible for nucleation at dispersed sites in plant cells and contributes to the formation and organization of the various MT arrays.