RESUMO
BACKGROUND INFORMATION: Arpin, an Arp2/3 inhibitory protein, inhibits lamellipodial protrusions and cell migration. Arpin expression is lost in tumor cells of several cancer types. RESULTS: Here we analyzed expression levels of Arpin and various markers using Reverse Phase Protein Array (RPPA) in human mammary carcinomas. We found that Arpin protein levels were correlated with those of several DNA damage response markers. Arpin-null cells display enhanced clustering of double stand breaks (DSBs) when cells are treated with a DNA damaging agent, in line with a previously described role of the Arp2/3 complex in promoting DSB clustering for homologous DNA repair (HDR) in the nucleus. Using a specific HDR assay, we further showed that Arpin depletion increased HDR efficiency two-fold through its ability to inactivate the Arp2/3 complex. CONCLUSIONS: Arpin regulates both cell migration in the cytosol and HDR in the nucleus. SIGNIFICANCE: Loss of Arpin expression coordinates enhanced cell migration with up-regulated DNA repair, which is required when DNA damage is induced by active cell migration.
RESUMO
A hallmark of Wnt/ß-Catenin signaling is the extreme diversity of its transcriptional response, which varies depending on the cell and developmental context. What controls this diversity is poorly understood. In all cases, the switch from transcriptional repression to activation depends on a nuclear increase in ß-Catenin, which detaches the transcription factor T cell factor 7 like 1 (Tcf7l1) bound to Groucho (Gro) transcriptional co-repressors from its DNA-binding sites and transiently converts Tcf7/Lymphoid enhancer binding factor 1 (Lef1) into a transcriptional activator. One of the earliest and evolutionarily conserved functions of Wnt/ß-Catenin signaling is the induction of the blastopore lip organizer. Here, we demonstrate that the evolutionarily conserved BarH-like homeobox-2 (Barhl2) protein stabilizes the Tcf7l1-Gro complex and maintains the repressed expression of Tcf target genes by a mechanism that depends on histone deacetylase 1 (Hdac-1) activity. In this way, Barhl2 switches off the Wnt/ß-Catenin-dependent early transcriptional response, thereby limiting the formation of the organizer in time and/or space. This study reveals a novel nuclear inhibitory mechanism of Wnt/Tcf signaling that switches off organizer fate determination.
Assuntos
Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Organizadores Embrionários/metabolismo , Fatores de Transcrição TCF/metabolismo , beta Catenina/metabolismo , Animais , Feminino , Proteínas de Homeodomínio/genética , Imunoprecipitação , Hibridização In Situ , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Masculino , Proteínas do Tecido Nervoso/genética , Plasmídeos/genética , Fatores de Transcrição TCF/genética , Xenopus laevis , beta Catenina/genéticaRESUMO
BACKGROUND: Organizing centers are groups of specialized cells that secrete morphogens, thereby influencing development of their neighboring territories. Apoptosis is a form of programmed cell death reported to limit the size of organizers. Little is known about the identity of intracellular signals driving organizer cell death. Here we investigated in Xenopus the role of both the anti-apoptotic protein Myeloid-cell-leukemia 1 (Mcl1) and the cysteine proteases Caspase-3 and Caspase-7 in formation of the axial organizing center-the notochord-that derives from the Spemann organizer, and participates in the induction and patterning of the neuroepithelium. RESULTS: We confirm a role for apoptosis in establishing the axial organizer in early neurula. We show that the expression pattern of mcl1 is coherent with a role for this gene in early notochord development. Using loss of function approaches, we demonstrate that Mcl1 depletion decreases neuroepithelium width and increases notochord cells apoptosis, a process that relies on Caspase-7, and not on Caspase-3, activity. Our data provide evidence that Mcl1 protein levels physiologically control notochord cells' survival and that Caspase-7 is the executioner protease in this developmental process. CONCLUSIONS: Our study reveals new functions for Mcl1 and Caspase-7 in formation of the axial signalling center.
Assuntos
Caspase 7/biossíntese , Sobrevivência Celular , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Animais , Apoptose , Padronização Corporal/fisiologia , Caspase 3/biossíntese , Epitélio/metabolismo , Fertilização in vitro , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Humanos , Neurônios/metabolismo , Notocorda/metabolismo , Organizadores Embrionários/metabolismo , Biossíntese de Proteínas , Transdução de Sinais , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
An intramolecular Diels-Alder (IMDA) reaction efficiently accelerated by Schreiner's thiourea is reported, to build a functionalized cytochalasin scaffold (periconiasin series) for biological purposes. DFT calculation highlighted a unique multidentate cooperative hydrogen bonding in this catalysis. The deprotection end game afforded a collection of diverse structures and showed the peculiar reactivity of the Diels-Alder cycloadducts upon functionalization. Biological studies revealed strong cytotoxicity of a few compounds on breast cancer cell lines while actin polymerization is preserved.
Assuntos
Antineoplásicos/química , Citocalasinas/química , Citoesqueleto de Actina/efeitos dos fármacos , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Catálise , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cobre/química , Cristalografia por Raios X , Reação de Cicloadição , Citocalasinas/síntese química , Citocalasinas/farmacologia , Humanos , Ligação de Hidrogênio , Conformação Molecular , Paládio/química , Estereoisomerismo , Termodinâmica , Tioureia/químicaRESUMO
While apneas are associated with multiple pathological and fatal conditions, the underlying molecular mechanisms remain elusive. We report that a mutated form of the transcription factor Mafa (Mafa4A) that prevents phosphorylation of the Mafa protein leads to an abnormally high incidence of breath holding apneas and death in newborn Mafa4A/4A mutant mice. This apneic breathing is phenocopied by restricting the mutation to central GABAergic inhibitory neurons and by activation of inhibitory Mafa neurons while reversed by inhibiting GABAergic transmission centrally. We find that Mafa activates the Gad2 promoter in vitro and that this activation is enhanced by the mutation that likely results in increased inhibitory drives onto target neurons. We also find that Mafa inhibitory neurons are absent from respiratory, sensory (primary and secondary) and pontine structures but are present in the vicinity of the hypoglossal motor nucleus including premotor neurons that innervate the geniohyoid muscle, to control upper airway patency. Altogether, our data reveal a role for Mafa phosphorylation in regulation of GABAergic drives and suggest a mechanism whereby reduced premotor drives to upper airway muscles may cause apneic breathing at birth.
Assuntos
Apneia , Neurônios Motores , Animais , Fatores de Transcrição Maf Maior , Camundongos , Neurônios Motores/fisiologia , Fosforilação , Regiões Promotoras GenéticasRESUMO
Dendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the capping protein for unclear reasons. Here, we show that the WASH complex interacts with dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins.
RESUMO
The actin cytoskeleton generates and senses forces. Here we report that branched actin networks from the cell cortex depend on ARPC1B-containing Arp2/3 complexes and that they are specifically monitored by type I coronins to control cell cycle progression in mammary epithelial cells. Cortical ARPC1B-dependent branched actin networks are regulated by the RAC1/WAVE/ARPIN pathway and drive lamellipodial protrusions. Accordingly, we uncover that the duration of the G1 phase scales with migration persistence in single migrating cells. Moreover, cortical branched actin more generally determines S-phase entry by integrating soluble stimuli such as growth factors and mechanotransduction signals, ensuing from substratum rigidity or stretching of epithelial monolayers. Many tumour cells lose this dependence for cortical branched actin. But the RAC1-transformed tumour cells stop cycling upon Arp2/3 inhibition. Among all genes encoding Arp2/3 subunits, ARPC1B overexpression in tumours is associated with the poorest metastasis-free survival in breast cancer patients. Arp2/3 specificity may thus provide diagnostic and therapeutic opportunities in cancer.
Assuntos
Actinas/metabolismo , Neoplasias da Mama/genética , Ciclo Celular , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Quimioterapia Adjuvante , Estudos de Coortes , Feminino , Humanos , Pessoa de Meia-Idade , RNA Mensageiro/genéticaRESUMO
Cancer cells often express differentiation programs unrelated to their tissue of origin, although the contribution of these aberrant phenotypes to malignancy is poorly understood. An aggressive subgroup of medulloblastoma, a malignant pediatric brain tumor of the cerebellum, expresses a photoreceptor differentiation program normally expressed in the retina. We establish that two photoreceptor-specific transcription factors, NRL and CRX, are master regulators of this program and are required for tumor maintenance in this subgroup. Beyond photoreceptor lineage genes, we identify BCL-XL as a key transcriptional target of NRL and provide evidence substantiating anti-BCL therapy as a rational treatment opportunity for select MB patients. Our results highlight the utility of studying aberrant differentiation programs in cancer and their potential as selective therapeutic vulnerabilities.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Meduloblastoma/genética , Transativadores/genética , Animais , Diferenciação Celular/genética , Neoplasias Cerebelares/genética , Humanos , Camundongos Nus , Retina/patologia , Transcrição Gênica/genéticaRESUMO
Dorsal root ganglia proceed from the coalescence of cell bodies of sensory neurons, which have migrated dorsoventrally from the delaminating neural crest. They are composed of different neuronal subtypes with specific sensory functions, including nociception, thermal sensation, proprioception, and mechanosensation. In contrast to proprioceptors and thermonociceptors, little is known about the molecular mechanisms governing the early commitment and later differentiation into mechanosensitive neurons. This is mainly due to the absence of specific molecular markers for this particular cell type. Using knockout mice, we identified the bZIP transcription factor MafA as the first specific marker of a subpopulation of "early c-ret" positive neurons characterized by medium-to-large diameters. This marker will allow further functional characterization of these neurons.
Assuntos
Gânglios Espinais/embriologia , Fatores de Transcrição Maf Maior/genética , Mecanorreceptores/metabolismo , Crista Neural/embriologia , Proteínas Proto-Oncogênicas c-ret/biossíntese , Células Receptoras Sensoriais/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Tamanho Celular , Gânglios Espinais/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Marcadores Genéticos/genética , Fatores de Transcrição Maf Maior/biossíntese , Mecanorreceptores/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutagênese Insercional , Crista Neural/citologia , Proteínas Proto-Oncogênicas c-ret/genética , Células Receptoras Sensoriais/citologiaRESUMO
Like JUN and FOS, the Maf transcription factors belong to the AP1 family. Besides their established role in human cancer--overexpression of the large Maf genes promotes the development of multiple myeloma--they can display tumour suppressor-like activity in specific cellular contexts, which is compatible with their physiological role in terminal differentiation. However, their oncogenic activity relies mostly on the acquisition of new biological functions relevant to cell transformation, the most striking characteristic of Maf oncoproteins being their ability to enhance pathological interactions between tumour cells and the stroma.
Assuntos
Transformação Celular Neoplásica/genética , Fatores de Transcrição Maf Maior/genética , Neoplasias/genética , Regulação da Expressão Gênica , Genes Supressores de Tumor , Humanos , Fatores de Transcrição Maf Maior/fisiologia , Modelos Biológicos , Mieloma Múltiplo/genética , Processamento Pós-Transcricional do RNARESUMO
The Maf oncoproteins are b-Zip transcription factors of the AP-1 superfamily. They are involved in developmental, metabolic, and tumorigenic processes. Maf proteins are overexpressed in about 50% of human multiple myelomas. Here, we show that Maf-transforming activity is controlled by GSK-3-dependent phosphorylation and that phosphorylation by GSK-3 can increase the oncogenic activity of a protein. Using microarray analysis, we identify a gene-expression subprogram regulated by GSK-3-mediated Maf phosphorylation involved in extracellular matrix remodeling and relevant to cancer progression. We also demonstrate that GSK-3 triggers MafA sequential phosphorylation on residues S61, T57, T53, and S49, inducing its ubiquitination and degradation. Paradoxically, this phosphorylation increases MafA-transcriptional activity through the recruitment of the coactivator P/CAF. We further demonstrate that P/CAF protects MafA from ubiquitination and degradation, suggesting that, upon the release of the coactivator complex, MafA becomes polyubiquitinated and degraded to allow the response to terminate.
Assuntos
Transformação Celular Neoplásica , Quinase 3 da Glicogênio Sintase/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular , Galinhas , Chlorocebus aethiops , Humanos , Fatores de Transcrição Maf Maior/química , Fatores de Transcrição Maf Maior/genética , Dados de Sequência Molecular , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Processamento de Proteína Pós-Traducional , Ratos , Transcrição Gênica , Ubiquitinação , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
The success of the first meiotic division relies (among other factors) on the formation of bivalents between homologous chromosomes, the monopolar orientation of the sister kinetochores at metaphase I and the maintenance of centromeric cohesion until the onset of anaphase II. The meiotic cohesin subunit, Rec8 has been reported to be one of the key players in these processes, but its precise role in kinetochore orientation is still under debate. By contrast, much less is known about the other non-SMC cohesin subunit, Scc3. We report the identification and the characterisation of AtSCC3, the sole Arabidopsis homologue of Scc3. The detection of AtSCC3 in mitotic cells, the embryo lethality of a null allele Atscc3-2, and the mitotic defects of the weak allele Atscc3-1 suggest that AtSCC3 is required for mitosis. AtSCC3 was also detected in meiotic nuclei as early as interphase, and bound to the chromosome axis from early leptotene through to anaphase I. We show here that both AtREC8 and AtSCC3 are necessary not only to maintain centromere cohesion at anaphase I, but also for the monopolar orientation of the kinetochores during the first meiotic division. We also found that AtREC8 is involved in chromosome axis formation in an AtSPO11-1-independent manner. Finally, we provide evidence for a role of AtSPO11-1 in the stability of the cohesin complex.