RESUMO
Combining single cell experiments, population dynamics and theoretical methods of membrane mechanics, we put forward that the rate of cell proliferation in E. coli colonies can be regulated by modifiers of the mechanical properties of the bacterial membrane. Bacterial proliferation was modelled as mediated by cell division through a membrane constriction divisome based on FtsZ, a mechanically competent protein at elastic interaction against membrane rigidity. Using membrane fluctuation spectroscopy in the single cells, we revealed either membrane stiffening when considering hydrophobic long chain fatty substances, or membrane softening if short-chained hydrophilic molecules are used. Membrane stiffeners caused hindered growth under normal division in the microbial cultures, as expected for membrane rigidification. Membrane softeners, however, altered regular cell division causing persistent microbes that abnormally grow as long filamentous cells proliferating apparently faster. We invoke the concept of effective growth rate under the assumption of a heterogeneous population structure composed by distinguishable individuals with different FtsZ-content leading the possible forms of cell proliferation, from regular division in two normal daughters to continuous growing filamentation and budding. The results settle altogether into a master plot that captures a universal scaling between membrane rigidity and the divisional instability mediated by FtsZ at the onset of membrane constriction.
Assuntos
Membrana Celular/metabolismo , Proliferação de Células/fisiologia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Divisão Celular/fisiologia , Membrana Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Membranas/metabolismoRESUMO
Botryococcus braunii produce liquid hydrocarbons able to be processed into combustion engine fuels. Depending on the growing conditions, the cell doubling time can be up to 6 days or more, which is a slow growth rate in comparison with other microalgae. Few studies have analyzed the cell cycle of B. braunii. We did a bioinformatic comparison between the protein sequences for retinoblastoma and cyclin-dependent kinases from the A (Yamanaka) and B (Showa) races, with those sequences from other algae and Arabidopsis thaliana. Differences in the number of cyclin-dependent kinases and potential retinoblastoma phosphorylation sites between the A and B races were found. Some cyclin-dependent kinases from both races seemed to be phylogenetically more similar to A. thaliana than to other microalgae. Microscopic observations were done using several staining procedures. Race A colonies, but not race B, showed some multinucleated cells without chlorophyll. An active mitochondrial net was detected in those multinucleated cells, as well as being defined in polyphosphate bodies. These observations suggest differences in the cell division processes between the A and B races of B. braunii.
Assuntos
Sequência de Aminoácidos/genética , Divisão Celular/genética , Hidrocarbonetos/metabolismo , Microalgas/genética , Arabidopsis/genética , Ciclo Celular/genética , Linhagem da Célula/genética , Clorofila/genética , Simulação por Computador , Hidrocarbonetos/química , Microalgas/crescimento & desenvolvimento , Fotossíntese/genéticaRESUMO
BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide; it is the fourth leading cause of death in the world and the third in Brazil. Mutations in the APC, DCC, KRAS and TP53 genes have been associated with the progression of sporadic CRC, occurring at defined pathological stages of the tumor progression and consequently modulating several genes in the corresponding signaling pathways. Therefore, the identification of gene signatures that occur at each stage during the CRC progression is critical and can present an impact on the diagnosis and prognosis of the patient. In this study, our main goal was to determine these signatures, by evaluating the gene expression of paired colorectal adenoma and adenocarcinoma samples to identify novel genetic markers in association to the adenoma-adenocarcinoma stage transition. METHODS: Ten paired adenoma and adenocarcinoma colorectal samples were subjected to microarray gene expression analysis. In addition, mutations in APC, KRAS and TP53 genes were investigated by DNA sequencing in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. RESULTS: Gene expression analysis revealed a signature of 689 differentially expressed genes (DEG) (fold-change> 2, p< 0.05), between the adenoma and adenocarcinoma paired samples analyzed. Gene pathway analysis using the 689 DEG identified important cancer pathways such as remodeling of the extracellular matrix and epithelial-mesenchymal transition. Among these DEG, the ETV4 stood out as one of the most expressed in the adenocarcinoma samples, further confirmed in the adenocarcinoma set of samples from the TCGA database. Subsequent in vitro siRNA assays against ETV4 resulted in the decrease of cell proliferation, colony formation and cell migration in the HT29 and SW480 colorectal cell lines. DNA sequencing analysis revealed KRAS and TP53 gene pathogenic mutations, exclusively in the adenocarcinomas samples. CONCLUSION: Our study identified a set of genes with high potential to be used as biomarkers in CRC, with a special emphasis on the ETV4 gene, which demonstrated involvement in proliferation and migration.
Assuntos
Adenocarcinoma/genética , Adenoma/genética , Neoplasias Colorretais/genética , Genes Neoplásicos , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-ets/fisiologia , Adenocarcinoma/química , Adenocarcinoma/patologia , Adenoma/química , Adenoma/patologia , Idoso , Biomarcadores Tumorais/genética , Brasil , Divisão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/química , Neoplasias Colorretais/patologia , DNA de Neoplasias/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-ets/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ets/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Análise Serial de Tecidos , Transcriptoma , Ensaio Tumoral de Célula-TroncoRESUMO
The sporulating, filamentous soil bacterium Streptomyces venezuelae ATCC 10712 differentiates under submerged and surface growth conditions. In order to lay a solid foundation for the study of development-associated division for this organism, a congenic set of mutants was isolated, individually deleted for a gene encoding either a cytoplasmic (i.e. ftsZ) or core inner membrane (i.e. divIC, ftsL, ftsI, ftsQ, ftsW) component of the divisome. While ftsZ mutants are completely blocked for division, single mutants in the other core divisome genes resulted in partial, yet similar, blocks in sporulation septum formation. Double and triple mutants for core divisome membrane components displayed phenotypes that were similar to those of the single mutants, demonstrating that the phenotypes were not synergistic. Division in this organism is still partially functional without multiple core divisome proteins, suggesting that perhaps other unknown lineage-specific proteins perform redundant functions. In addition, by isolating an ftsZ2p mutant with an altered -10 region, the conserved developmentally controlled promoter was also shown to be required for sporulation-associated division. Finally, microscopic observation of FtsZ-YFP dynamics in the different mutant backgrounds led to the conclusion that the initial assembly of regular Z rings does not per se require the tested divisome membrane proteins, but the stability of Z rings is dependent on the divisome membrane components tested. The observation is consistent with the interpretation that Z ring instability likely results from and further contributes to the observed defects in sporulation septation in mutants lacking core divisome proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Divisão Celular , Streptomyces/citologia , Proteínas de Bactérias/genética , Divisão Celular/genética , Segregação de Cromossomos , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Fenótipo , Regiões Promotoras Genéticas , Esporos Bacterianos/citologia , Esporos Bacterianos/genética , Esporos Bacterianos/fisiologia , Streptomyces/genética , Streptomyces/fisiologiaRESUMO
Intracellular calcium (Ca2+) is crucial for signal transduction in Cryptococcus neoformans, the major cause of fatal fungal meningitis. The calcineurin pathway is the only Ca2+-requiring signaling cascade implicated in cryptococcal stress adaptation and virulence, with Ca2+ binding mediated by the EF-hand domains of the Ca2+ sensor protein calmodulin. In this study, we identified the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) as a member of the EF-hand superfamily. We demonstrated that Ncs1 has a role in Ca2+ homeostasis under stress and nonstress conditions, as the ncs1Δ mutant is sensitive to a high Ca2+ concentration and has an elevated basal Ca2+ level. Furthermore, NCS1 expression is induced by Ca2+, with the Ncs1 protein adopting a punctate subcellular distribution. We also demonstrate that, in contrast to the case with Saccharomyces cerevisiae, NCS1 expression in C. neoformans is regulated by the calcineurin pathway via the transcription factor Crz1, as NCS1 expression is reduced by FK506 treatment and CRZ1 deletion. Moreover, the ncs1Δ mutant shares a high temperature and high Ca2+ sensitivity phenotype with the calcineurin and calmodulin mutants (cna1Δ and cam1Δ), and the NCS1 promoter contains two calcineurin/Crz1-dependent response elements (CDRE1). Ncs1 deficiency coincided with reduced growth, characterized by delayed bud emergence and aberrant cell division, and hypovirulence in a mouse infection model. In summary, our data show that Ncs1 has a significant role as a Ca2+ sensor in C. neoformans, working with calcineurin to regulate Ca2+ homeostasis and, consequently, promote fungal growth and virulence.IMPORTANCECryptococcus neoformans is the major cause of fungal meningitis in HIV-infected patients. Several studies have highlighted the important contributions of Ca2+ signaling and homeostasis to the virulence of C. neoformans Here, we identify the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) and demonstrate its role in Ca2+ homeostasis, bud emergence, cell cycle progression, and virulence. We also show that Ncs1 function is regulated by the calcineurin/Crz1 signaling cascade. Our work provides evidence of a link between Ca2+ homeostasis and cell cycle progression in C. neoformans.
Assuntos
Calcineurina/genética , Proteínas de Ligação ao Cálcio/genética , Divisão Celular/genética , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Proteínas Sensoras de Cálcio Neuronal/genética , Neuropeptídeos/genética , Animais , Cryptococcus neoformans/química , Feminino , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Virulência/genéticaRESUMO
Prostate cancer (PCa) has different molecular features along progression, including androgen profile, which is associated to therapy inefficiency leading to more aggressive phenotype. Docosahexaenoic acid (DHA) has antiproliferative and pro-apoptotic properties in different cancers associated to cell metabolism modulation. The latter is of particular interest since metabolic reprogramming is one of PCa hallmarks, but is not clear how this occurs among disease progression. Therefore, we evaluated DHA antiproliferative potential in distinct androgenic backgrounds associated to metabolism modulation and androgen-regulated genes. For this purpose, pre-malignant PNT1A and tumor AR-positive 22rv1, and AR-negative PC3 cells were incubated with DHA at 100 µM-48 h. DHA reduced at least 26% cell number for all lineages due to S-phase decrease in AR-positive and G2/M arrest in AR-negative. Mitochondrial metabolic rate decreased in PNT1A (~38%) and increased in tumor cells (at least 40%). This was associated with ROS overproduction (1.6-fold PNT1A; 2.1 22rv1; 2.2 PC3), lipid accumulation (3-fold PNT1A; 1.8 22rv1; 3.6 PC3) and mitochondria damage in all cell lines. AKT, AMPK and PTEN were not activated in any cell line, but p-ERK1/2 increased (1.5-fold) in PNT1A. Expression of androgen-regulated and nuclear receptors genes showed that DHA affected them in a distinct pattern in each cell line, but most converged to metabolism regulation, response to hormones, lipids and stress. In conclusion, regardless of androgenic or PTEN background DHA exerted antiproliferative effect associated to cell cycle impairment, lipid deregulation and oxidative stress, but differentially regulated gene expression probably due to distinct molecular features of each pathologic stage.
Assuntos
Ciclo Celular/genética , Ácidos Docosa-Hexaenoicos/metabolismo , Redes e Vias Metabólicas/genética , Próstata/metabolismo , Neoplasias da Próstata/genética , Androgênios/genética , Androgênios/metabolismo , Apoptose/genética , Divisão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Redes Reguladoras de Genes/genética , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
A growing body of evidence suggests a key role of tumor microenvironment, especially for bone marrow mesenchymal stem cells (MSC), in the maintenance and progression of multiple myeloma (MM), through direct and indirect interactions with tumor plasma cells. Thus, this study aimed to investigate the gene expression and functional alterations of MSC from MM patients (MM-MSC) in comparison with their normal counterparts from normal donors (ND-MSC). Gene expression analysis (Affymetrix) was performed in MM-MSC and ND-MSC after in vitro expansion. To validate these findings, some genes were selected to be evaluated by quantitative real time PCR (RT-qPCR), and also functional in vitro analyses were performed. We demonstrated that MM-MSC have a distinct gene expression profile than ND-MSC, with 485 differentially expressed genes (DEG) - 280 upregulated and 205 downregulated. Bioinformatics analyses revealed that the main enriched functions among downregulated DEG were related to cell cycle progression, immune response activation and bone metabolism. Four genes were validated by qPCR - ZNF521 and SEMA3A, which are involved in bone metabolism, and HLA-DRA and CHIRL1, which are implicated in the activation of immune response. Taken together, our results suggest that MM-MSC have constitutive abnormalities that remain present even in the absence of tumors cells. The alterations found in cell cycle progression, immune system activation, and osteoblastogenesis suggest, respectively, that MM-MSC are permanently dependent of tumor cells, might contribute to immune evasion and play an essential role in bone lesions frequently found in MM patients.
Assuntos
Osso e Ossos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mieloma Múltiplo/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/metabolismo , Divisão Celular/genética , Divisão Celular/fisiologia , Feminino , Perfilação da Expressão Gênica/métodos , Cadeias alfa de HLA-DR/genética , Cadeias alfa de HLA-DR/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologiaRESUMO
In the root meristem, the quiescent center (QC) is surrounded by stem cells, which in turn generate the different cell types of the root. QC cells rarely divide under normal conditions but can replenish damaged stem cells. In the proximal meristem, the daughters of stem cells, which are referred to as transit-amplifying cells, undergo additional rounds of cell division prior to differentiation. Here, we describe the functions of GRF-INTERACTING FACTORs (GIFs), including ANGUSTIFOLIA3 (AN3), in Arabidopsis thaliana roots. GIFs have been shown to interact with GRF transcription factors and SWI/SNF chromatin remodeling complexes. We found that combinations of GIF mutants cause the loss of QC identity. However, despite their QC impairment, GIF mutants have a significantly enlarged root meristem with additional lateral root cap layers. We show that the increased expression of PLETHORA1 (PLT1) is at least partially responsible for the large root meristems of an3 mutants. Furthermore, we found that GIFs are necessary for maintaining the precise expression patterns of key developmental regulators and that AN3 complexes bind directly to the promoter regions of PLT1 as well as SCARECROW We propose that AN3/GIFs participate in different pathways that control QC organization and the size of the meristem.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Diferenciação Celular/genética , Divisão Celular/genética , Montagem e Desmontagem da Cromatina/genética , Homeostase/genética , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/fisiologia , Mutação , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The flagellated protist Tritrichomonas foetus is a parasite that causes bovine trichomonosis, a major sexually transmitted disease in cattle. Cell division has been described as a key player in controlling cell survival in other cells, including parasites but there is no information on the regulation of this process in T. foetus. The regulation of cytokinetic abscission, the final stage of cell division, is mediated by members of the ESCRT (endosomal sorting complex required for transport) machinery. VPS32 is a subunit within the ESCRTIII complex and here, we report that TfVPS32 is localized on cytoplasmic vesicles and a redistribution of the protein to the midbody is observed during the cellular division. In concordance with its localization, deletion of TfVPS32 C-terminal alpha helices (α5 helix and/or α4-5 helix) leads to abnormal T. foetus growth, an increase in the percentage of multinucleated parasites and cell cycle arrest at G2/M phase. Together, these results indicate a role of this protein in controlling normal cell division.
Assuntos
Divisão Celular/genética , Proteínas de Protozoários/genética , Tritrichomonas foetus/fisiologia , Citocinese/genética , Proteínas de Protozoários/metabolismo , Tritrichomonas foetus/genéticaRESUMO
KEY MESSAGE: Arabidopsis med12 and med13 mutants exhibit shoot and root phenotypes related to an altered auxin homeostasis. Sucrose supplementation reactivates both cell division and elongation in primary roots as well as auxin-responsive and stem cell niche gene expression in these mutants. An analysis of primary root growth of WT, med12, aux1-7 and med12 aux1 single and double mutants in response to sucrose and/or N-1-naphthylphthalamic acid (NPA) placed MED12 upstream of auxin transport for the sugar modulation of root growth. The MEDIATOR (MED) complex plays diverse functions in plant development, hormone signaling and biotic and abiotic stress tolerance through coordination of transcription. Here, we performed genetic, developmental, molecular and pharmacological analyses to characterize the role of MED12 and MED13 on the configuration of root architecture and its relationship with auxin and sugar responses. Arabidopsis med12 and med13 single mutants exhibit shoot and root phenotypes consistent with altered auxin homeostasis including altered primary root growth, lateral root development, and root hair elongation. MED12 and MED13 were required for activation of cell division and elongation in primary roots, as well as auxin-responsive and stem cell niche gene expression. Remarkably, most of these mutant phenotypes were rescued by supplying sucrose to the growth medium. The growth response of primary roots of WT, med12, aux1-7 and med12 aux1 single and double mutants to sucrose and application of auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) revealed the correlation of med12 phenotype with the activity of the auxin intake permease and suggests that MED12 acts upstream of AUX1 in the root growth response to sugar. These data provide compelling evidence that MEDIATOR links sugar sensing to auxin transport and distribution during root morphogenesis.