RESUMO
We show that overexpression of Polo-like kinase 4 (Plk4) in human cells induces centrosome amplification through the simultaneous generation of multiple procentrioles adjoining each parental centriole. This provided an opportunity for dissecting centriole assembly and characterizing assembly intermediates. Critical components were identified and ordered into an assembly pathway through siRNA and localized through immunoelectron microscopy. Plk4, hSas-6, CPAP, Cep135, gamma-tubulin, and CP110 were required at different stages of procentriole formation and in association with different centriolar structures. Remarkably, hSas-6 associated only transiently with nascent procentrioles, whereas Cep135 and CPAP formed a core structure within the proximal lumen of both parental and nascent centrioles. Finally, CP110 was recruited early and then associated with the growing distal tips, indicating that centrioles elongate through insertion of alpha-/beta-tubulin underneath a CP110 cap. Collectively, these data afford a comprehensive view of the assembly pathway underlying centriole biogenesis in human cells.
Assuntos
Centríolos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Centríolos/ultraestrutura , Humanos , Microscopia Imunoeletrônica , Modelos BiológicosRESUMO
Primary cilia (PC) function as microtubule-based sensory antennae projecting from the surface of many eukaryotic cells. They play important roles in mechano- and chemosensory perception and their dysfunction is implicated in developmental disorders and severe diseases. The basal body that functions in PC assembly is derived from the mature centriole, a component of the centrosome. Through a small interfering RNA screen we found several centrosomal proteins (Ceps) to be involved in PC formation. One newly identified protein, Cep164, was indispensable for PC formation and hence characterized in detail. By immunogold electron microscopy, Cep164 could be localized to the distal appendages of mature centrioles. In contrast to ninein and Cep170, two components of subdistal appendages, Cep164 persisted at centrioles throughout mitosis. Moreover, the localizations of Cep164 and ninein/Cep170 were mutually independent during interphase. These data implicate distal appendages in PC formation and identify Cep164 as an excellent marker for these structures.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Cílios/metabolismo , Anticorpos/farmacologia , Proteínas de Transporte/ultraestrutura , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/ultraestrutura , Linhagem Celular Tumoral , Estruturas do Núcleo Celular/efeitos dos fármacos , Estruturas do Núcleo Celular/metabolismo , Centríolos/efeitos dos fármacos , Cílios/efeitos dos fármacos , Humanos , Proteínas dos Microtúbulos , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismoRESUMO
The basic helix-loop-helix TAL-1/SCL essential for hematopoietic development is also required during vascular development for embryonic angiogenesis. We reported that TAL-1 acts positively on postnatal angiogenesis by stimulating endothelial morphogenesis. Here, we investigated the functional consequences of TAL-1 silencing in human primary endothelial cells. We found that TAL-1 knockdown caused the inhibition of in vitro tubulomorphogenesis, which was associated with a dramatic reduction in vascular endothelial cadherin (VE-cadherin) at intercellular junctions. Consistently, silencing of TAL-1 as well as of its cofactors E47 and LMO2 down-regulated VE-cadherin at both the mRNA and the protein level. Endogenous VE-cadherin transcription could be activated in nonendothelial HEK-293 cells by the sole concomitant ectopic expression of TAL-1, E47, and LMO2. Transient transfections in human primary endothelial cells derived from umbilical vein (HUVECs) demonstrated that VE-cadherin promoter activity was dependent on the integrity of a specialized E-box associated with a GATA motif and was maximal with the coexpression of the different components of the TAL-1 complex. Finally, chromatin immunoprecipitation assays showed that TAL-1 and its cofactors occupied the VE-cadherin promoter in HUVECs. Together, these data identify VE-cadherin as a bona fide target gene of the TAL-1 complex in the endothelial lineage, providing a first clue to TAL-1 function in angiogenesis.
Assuntos
Antígenos CD/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Caderinas/biossíntese , Proteínas de Ligação a DNA/fisiologia , Células Endoteliais/metabolismo , Metaloproteínas/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição TCF/fisiologia , Regulação para Cima , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Antígenos CD/genética , Sequência de Bases , Caderinas/genética , Células Cultivadas , Colágeno , Combinação de Medicamentos , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Humanos , Proteínas com Domínio LIM , Laminina , Dados de Sequência Molecular , Neovascularização Fisiológica , Regiões Promotoras Genéticas , Ligação Proteica , Proteoglicanas , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Proteína 1 Semelhante ao Fator 7 de TranscriçãoRESUMO
The TAL-1/SCL gene encodes a basic helix-loop-helix (bHLH) transcription factor essential for primitive hematopoiesis and for adult erythroid and megakaryocytic development. Activated transcription of TAL-1 as a consequence of chromosomal rearrangements is associated with a high proportion of human T cell acute leukemias, showing that appropriate control of TAL-1 is crucial for the formation and subsequent fate of hematopoietic cells. Hence, the knowledge of the mechanisms, which govern the pattern of TAL-1 expression in hematopoiesis, is of great interest. We previously described a silencer in the 3'-untranslated region of human TAL-1, the activity of which is mediated through binding of a tissue-specific 40 kDa nuclear protein to a new DNA recognition motif, named tal-RE. Here, we show that tal-RE-binding activity, high in immature human hematopoietic progenitors is down regulated upon erythroid and megakaryocytic differentiation. This expression profile helped us to identify that PU.1/Spi-1 binds to the tal-RE sequences in vitro and occupies the TAL-1 silencer in vivo. By expressing a mutant protein containing only the ETS domain of PU.1 in human erythroleukemic HEL cells, we demonstrated that PU.1 mediates the transcriptional repression activity of the silencer. We found that ectopic PU.1 is not able to induce silencing activity in PU.1-negative Jurkat T cells, indicating that PU.1 activity, although necessary, is not sufficient to confer transcriptional repression activity to the TAL-1 silencer. Finally, we showed that the silencer is also active in TAL-1-negative myeloid HL60 cells that express PU.1 at high levels. In summary, our study shows that PU.1, in addition to its positive role in TAL-1 expression in early hematopoietic progenitors, may also act as a mediator of TAL-1 silencing in some hematopoietic lineages.
Assuntos
Inativação Gênica , Proteínas Proto-Oncogênicas/fisiologia , Transativadores/fisiologia , Ubiquitina-Proteína Ligases/genética , Linhagem Celular Tumoral , Regulação para Baixo , Hematopoese/genética , Humanos , Leucemia/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Elementos de Resposta , Transativadores/metabolismo , Fatores de Transcrição , Ativação Transcricional , TransfecçãoRESUMO
Replication and transcription machineries progress at high speed on the same DNA template, which inevitably causes traffic accidents. Problems are not only caused by frontal collisions between polymerases, but also by cotranscriptional R-loops. These RNA-DNA hybrids induce genomic instability by blocking fork progression and could be implicated in the development of cancer.
Assuntos
Replicação do DNA/genética , DNA/genética , Instabilidade Genômica , Transcrição Gênica/genética , Animais , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Modelos Genéticos , RNA/genética , RNA/metabolismo , Recombinação Genética , Moldes GenéticosRESUMO
The mammalian circadian clock plays a fundamental role in the liver by regulating fatty acid, glucose, and xenobiotic metabolism. Impairment of this rhythm has been shown to lead to diverse pathologies, including metabolic syndrome. Currently, it is supposed that the circadian clock regulates metabolism mostly by regulating expression of liver enzymes at the transcriptional level. Here, we show that the circadian clock also controls hepatic metabolism by synchronizing a secondary 12 hr period rhythm characterized by rhythmic activation of the IRE1alpha pathway in the endoplasmic reticulum. The absence of circadian clock perturbs this secondary clock and provokes deregulation of endoplasmic reticulum-localized enzymes. This leads to impaired lipid metabolism, resulting in aberrant activation of the sterol-regulated SREBP transcription factors. The resulting aberrant circadian lipid metabolism in mice devoid of the circadian clock could be involved in the appearance of the associated metabolic syndrome.
Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Endorribonucleases/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismoRESUMO
Centrioles function as the major components of centrosomes, which organize microtubule (MT) arrays in proliferating cells, and as basal bodies for primary cilia formation in quiescent cells. Centrioles and basal bodies are structurally similar, barrel-shaped organelles composed of MTs. In proliferating cells, two new centrioles, termed procentrioles, form during the S phase of the cell cycle in close proximity to the proximal ends of the two preexisting parental centrioles, often at a near-orthogonal angle. Considerable progress has been made toward understanding the biogenesis of centrioles, but the mechanisms that determine their lengths remain unknown. Here we show that overexpression of the centriolar protein CPAP in human cells enhances the accumulation of centriolar tubulin, leading to centrioles of strikingly increased length. Consistent with earlier work, we also find that elongated MT structures can be induced by depletion of the distal end-capping protein CP110 from centrioles. Importantly, though, these structures differ from genuine primary cilia. We thus propose that CPAP and CP110 play antagonistic roles in determining the extent of tubulin addition during centriole elongation, thereby controlling the length of newly formed centrioles.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Centríolos/metabolismo , Centríolos/ultraestrutura , Humanos , Proteínas Associadas aos Microtúbulos/genética , Fosfoproteínas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
BACKGROUND: Centrioles are microtubule-based cylindrical structures composed of nine triplet tubules and are required for the formation of the centrosome, flagella and cilia. Despite theirs importance, centriole biogenesis is poorly understood. Centrosome duplication is initiated at the G1/S transition by the sequential recruitment of a set of conserved proteins under the control of the kinase Plk4. Subsequently, the procentriole is assembled by the polymerization of centriolar tubules via an unknown mechanism involving several tubulin paralogs. METHODOLOGY/PRINCIPAL FINDINGS: Here, we developed a cellular assay to study centrosome duplication and procentriole stability based on its sensitivity to the microtubule-depolymerizing drug nocodazole. By using RNA interference experiments, we show that the stability of growing procentrioles is regulated by the microtubule-stabilizing protein CAP350, independently of hSAS-6 and CPAP which initiate procentriole growth. Furthermore, our analysis reveals the critical role of centriolar tubule stability for an efficient procentriole growth. CONCLUSIONS/SIGNIFICANCE: CAP350 belongs to a new class of proteins which associate and stabilize centriolar tubules to control centriole duplication.