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1.
Braz. j. biol ; 84: e250151, 2024. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1350306

RESUMO

Abstract Mammals have a limited capacity to regenerate their tissues and organs. One of the mechanisms associated with natural regeneration is dedifferentiation. Several small molecules such as vitamin C and growth factors could improve reprogramming efficiency. In this study, the NTERA2-D1 (NT2) cells were induced towards differentiation (NT2-RA) with 10-5 M retinoic acid (RA) for three days and then subjected to various amounts of vitreous humor (VH). Results show that the growth rate of these cells was reduced, while this rate was partly restored upon treatment with VH (NT2-RA-VH). Cell cycle analysis with PI method also showed that the numbers of cells at the S phase of the cell cycle in these cells were increased. The levels of SSEA3 and TRA-1-81 antigens in NT2-RA were dropped but they increased in NT2- RA-VH to a level similar to the NT2 cells. The level of SSEA1 had an opposite pattern. Expression of OCT4 gene dropped after RA treatment, but it was recovered in NT2-RA-VH cells. In conclusion, we suggest VH as a potent mixture for improving the cellular reprogramming leading to dedifferentiation.


Resumo Os mamíferos têm uma capacidade limitada de regenerar seus tecidos e órgãos. Um dos mecanismos associados à regeneração natural é a desdiferenciação. Várias moléculas pequenas, como vitamina C e fatores de crescimento, podem melhorar a eficiência da reprogramação. Neste estudo, as células NTERA2-D1 (NT2) foram induzidas à diferenciação (NT2-RA) com ácido retinóico (RA) 10-5 M por três dias e depois submetidas a várias quantidades de humor vítreo (VH). Os resultados mostram que a taxa de crescimento dessas células foi reduzida, enquanto essa taxa foi parcialmente restaurada após o tratamento com VH (NT2-RA-VH). A análise do ciclo celular com o método PI também mostrou que o número de células na fase S do ciclo celular nessas células estava aumentado. Os níveis de antígenos SSEA3 e TRA-1-81 em NT2-RA diminuíram, mas aumentaram em NT2-RA-VH a um nível semelhante ao das células NT2. O nível de SSEA1 teve um padrão oposto. A expressão do gene OCT4 diminuiu após o tratamento com AR, mas foi recuperado em células NT2-RA-VH. Em conclusão, sugerimos o VH como uma mistura potente para melhorar a reprogramação celular levando à desdiferenciação.


Assuntos
Humanos , Corpo Vítreo , Proliferação de Células , Desdiferenciação Celular , Tretinoína , Células Tumorais Cultivadas , Diferenciação Celular , Divisão Celular , Linhagem Celular
2.
Environ Pollut ; 319: 121013, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36608730

RESUMO

Growing evidence suggested that microorganisms exhibited heterogeneous sensitivity to toxicants, but their underlying mechanisms remain largely unknown. The asynchronous cell cycle progression in natural population implies the connection between cell cycle and heterogeneity. Here, the heterogenous responses of Chlamydomonas reinhardtii upon Cu stress were confirmed with the aid of a fluorometric probe for imaging Cu(I), implying the connection with cell cycle. Our results further indicated that the increase of labile Cu(I) was related to the cell division, leading to the fluctuation of labile Cu(I) with diurnal cycle and cell cycle, respectively. However, lack of Cu mainly influenced the cell division. We demonstrated that G2/M phase was the critical stage requiring high Cu quota during cell division. Specifically, algae at G2/M phase required 10-fold of Cu quota compared with that at G1 phase, which was related to the mitochondrial replication. Eventually, the heterogeneous Cu uptake ability of algae at different cell phases led to the heterogeneous responses to Cu exposure. Overall, Cu could influence the cell cycle through mediating the cell division, and in turn algae at different cell phases exhibited different Cu sensitivities. This study firstly uncovered the underlying mechanisms of heterogeneous Cu sensitivity for phytoplankton, which could help to evaluate the potential ecological risks of Cu.


Assuntos
Chlamydomonas reinhardtii , Chlamydomonas , Ciclo Celular , Divisão Celular , Transporte Biológico , Chlamydomonas reinhardtii/metabolismo
3.
Biol Res ; 56(1): 1, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36597150

RESUMO

Cell cycle is one of the main cellular mechanisms involved in tumor progression. Almost all of the active molecular pathways in tumor cells directly or indirectly target the cell cycle progression. Therefore, it is necessary to assess the molecular mechanisms involved in cell cycle regulation in tumor cells. Since, early diagnosis has pivotal role in better cancer management and treatment, it is required to introduce the non-invasive diagnostic markers. Long non-coding RNAs (LncRNAs) have higher stability in body fluids in comparison with mRNAs. Therefore, they can be used as efficient non-invasive markers for the early detection of breast cancer (BCa). In the present review we have summarized all of the reported lncRNAs involved in cell cycle regulation in BCa. It has been reported that lncRNAs mainly affect the cell cycle in G1/S transition through the CCND1/CDK4-6 complex. Present review paves the way of introducing the cell cycle related lncRNAs as efficient markers for the early detection of BCa.


Assuntos
Neoplasias da Mama , RNA Longo não Codificante , Humanos , Feminino , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ciclo Celular/genética , Divisão Celular , Pontos de Checagem do Ciclo Celular
4.
Int J Mol Sci ; 24(1)2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36614249

RESUMO

Hepatocellular carcinoma (HCC) is the most common type of primary liver malignancy, with increased mortality and morbidity. Accumulating evidence suggested that 40S ribosomal protein S24 (RPS24) is related to malignant outcomes and progression. However, the role of RPS24 remains unclear in HCC. The mRNA and protein expression pattern of RPS24 in HCC was explored and confirmed based on the bioinformatics analysis and histological examination. The correlation between RPS24 expression and clinicopathological features, diagnostic value, prognosis, methylation status, and survival were evaluated. Then, we divided the HCC cohort into two groups based on the expression of RPS24, and performed the functional enrichment and immune cells infiltration analysis of RPS24. Furthermore, in vivo and in vitro experiments were performed to investigate the effect of RPS24 on HCC cells. RPS24 was observed to be elevated in HCC samples. RPS24 overexpression or RPS24 promoter methylation contributed to an unfavorable prognosis for HCC patients. The genes in the high RPS24 expression group were mainly enriched in DNA replication, cell cycle E2F targets, and the G2M checkpoint pathway. Moreover, the expression level of RPS24 was significantly related to immune infiltration and immunotherapy response. Our experiments also demonstrated that RPS24 knockdown suppressed the growth of HCC cells and tumor proliferation of the xenograft model. Therefore, RPS24 can be a potential adverse biomarker of HCC prognosis acting through facilitating cell proliferation and the formation of an immunosuppressive microenvironment in HCC. Targeting RPS24 may offer a promising therapeutic option for HCC management.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas Ribossômicas/genética , Ciclo Celular , Divisão Celular , Microambiente Tumoral/genética
5.
Proc Natl Acad Sci U S A ; 120(2): e2214829120, 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36595671

RESUMO

Hepatocellular carcinoma (HCC) remains a global health challenge whose incidence is growing worldwide. Previous evidence strongly supported the notion that the circadian clock controls physiological homeostasis of the liver and plays a key role in hepatocarcinogenesis. Despite the progress, cellular and molecular mechanisms underpinning this HCC-clock crosstalk remain unknown. Addressing this knowledge gap, we show here that although the human HCC cells Hep3B, HepG2, and Huh7 displayed variations in circadian rhythm profiles, all cells relied on the master circadian clock transcription factors, BMAL1 and CLOCK, for sustained cell growth. Down-regulating Bmal1 or Clock in the HCC cells induced apoptosis and arrested cell cycle at the G2/M phase. Mechanistically, we found that inhibiting Bmal1/Clock induced dysregulation of the cell cycle regulators Wee1 and p21 which cooperatively contribute to tumor cell death. Bmal1/Clock knockdown caused downregulation of Wee1 that led to apoptosis activation and upregulation of p21 which arrested the cell cycle at the G2/M phase. Collectively, our results suggest that the circadian clock regulators BMAL1 and CLOCK promote HCC cell proliferation by controlling Wee1 and p21 levels, thereby preventing apoptosis and cell cycle arrest. Our findings shed light on cellular impact of the clock proteins for maintaining HCC oncogenesis and provide proof-of-principle for developing cancer therapy based on modulation of the circadian clock.


Assuntos
Carcinoma Hepatocelular , Relógios Circadianos , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas CLOCK/genética , Ritmo Circadiano/genética , Relógios Circadianos/genética , Proliferação de Células , Ciclo Celular , Divisão Celular , Apoptose
6.
Nat Commun ; 14(1): 57, 2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36599833

RESUMO

Insulin acts through the insulin receptor (IR) tyrosine kinase to exert its classical metabolic and mitogenic actions. Here, using receptors with either short or long deletion of the ß-subunit or mutation of the kinase active site (K1030R), we have uncovered a second, previously unrecognized IR signaling pathway that is intracellular domain-dependent, but ligand and tyrosine kinase-independent (LYK-I). These LYK-I actions of the IR are linked to changes in phosphorylation of a network of proteins involved in the regulation of extracellular matrix organization, cell cycle, ATM signaling and cellular senescence; and result in upregulation of expression of multiple extracellular matrix-related genes and proteins, down-regulation of immune/interferon-related genes and proteins, and increased sensitivity to apoptosis. Thus, in addition to classical ligand and tyrosine kinase-dependent (LYK-D) signaling, the IR regulates a second, ligand and tyrosine kinase-independent (LYK-I) pathway, which regulates the cellular machinery involved in senescence, matrix interaction and response to extrinsic challenges.


Assuntos
Apoptose , Divisão Celular , Senescência Celular , Proteínas Tirosina Quinases , Receptor de Insulina , Apoptose/genética , Divisão Celular/genética , Insulina/metabolismo , Ligantes , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Senescência Celular/genética , Humanos , Animais , Camundongos
7.
Cell Mol Life Sci ; 80(1): 36, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36627412

RESUMO

Cell differentiation involves profound changes in global gene expression that often has to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibility data, we find that p27 is coincidentally necessary to repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2 and ASCL1. Our data reveal both a direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of Sox2 leads to reduced levels of its downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.


Assuntos
Inibidor de Quinase Dependente de Ciclina p27 , Neurogênese , Fatores de Transcrição SOXB1 , Animais , Camundongos , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Expressão Gênica , Neurogênese/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
8.
Development ; 150(2)2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36692218

RESUMO

The first characterised FUSE Binding Protein family member, FUBP1, binds single-stranded DNA to activate MYC transcription. Psi, the sole FUBP protein in Drosophila, binds RNA to regulate P-element and mRNA splicing. Our previous work revealed pro-growth functions for Psi, which depend, in part, on transcriptional activation of Myc. Genome-wide functions for FUBP family proteins in transcriptional control remain obscure. Here, through the first genome-wide binding and expression profiles obtained for a FUBP family protein, we demonstrate that, in addition to being required to activate Myc to promote cell growth, Psi also directly binds and activates stg to couple growth and cell division. Thus, Psi knockdown results in reduced cell division in the wing imaginal disc. In addition to activating these pro-proliferative targets, Psi directly represses transcription of the growth inhibitor tolkin (tok, a metallopeptidase implicated in TGFß signalling). We further demonstrate tok overexpression inhibits proliferation, while tok loss of function increases mitosis alone and suppresses impaired cell division caused by Psi knockdown. Thus, Psi orchestrates growth through concurrent transcriptional activation of the pro-proliferative genes Myc and stg, in combination with repression of the growth inhibitor tok.


Assuntos
Proteínas de Drosophila , Drosophila , Proteínas de Ligação a RNA , Animais , Divisão Celular , Proliferação de Células , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ativação Transcricional
9.
J Cell Sci ; 136(2)2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36661138

RESUMO

The fate of the two daughter cells is intimately connected to their positioning, which is in turn regulated by cell junction remodelling and orientation of the mitotic spindle. How multiple cues are integrated to dictate the ultimate positioning of daughters is not clear. Here, we identify novel mechanisms of regulation of daughter positioning in single MCF10A cells. The polarity protein, Scribble cooperates with E-cadherin for sequential roles in daughter positioning. First Scribble stabilises E-cadherin at the mitotic cortex as well as the retraction fibres, to mediate spindle orientation. Second, Scribble re-locates to the junction between the two daughters to allow a new E-cadherin-based-interface to form between them, influencing the width of the nascent daughter-daughter junction and subsequent cell positioning. Thus, E-cadherin and Scribble dynamically relocate to different intracellular sites during cell division to orient the mitotic spindle and control placement of the daughter cells after cell division. This article has an associated First Person interview with the first author of the paper.


Assuntos
Caderinas , Fuso Acromático , Humanos , Caderinas/genética , Caderinas/metabolismo , Divisão Celular/genética , Polaridade Celular/fisiologia , Junções Intercelulares/metabolismo , Fuso Acromático/metabolismo
10.
PLoS One ; 18(1): e0273136, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36662698

RESUMO

DivIVA, GpsB, FilP, and Scy are all involved in bacterial cell division. They have been reported to interact with each other, and although they have been the subject of considerable research interest, not much is known about the molecular basis for their biological activity. Although they show great variability in taxonomic occurrence, phenotypic profile, and molecular properties, we find that they nevertheless share a conserved N-terminal sequence motif, which points to a common evolutionary origin. The motif always occurs N-terminally to a coiled-coil helix that mediates dimerization. We define the motif and coiled coil jointly as a new domain, which we name DivIVA-like. In a large-scale survey of this domain in the protein sequence database, we identify a new family of proteins potentially involved in cell division, whose members, unlike all other DivIVA-like proteins, have between 2 and 8 copies of the domain in tandem. AlphaFold models indicate that the domains in these proteins assemble within a single chain, therefore not mediating dimerization.


Assuntos
Proteínas de Bactérias , Proteínas de Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas de Bactérias/metabolismo , Divisão Celular/genética , Domínios Proteicos , Bactérias Gram-Positivas/metabolismo
11.
PLoS One ; 18(1): e0280621, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36662844

RESUMO

In this paper, we perform a mathematical analysis of our proposed nonlinear, multiscale mathematical model of physiologically structured quiescent and proliferating cell populations at the macroscale and cell-cycle proteins at the microscale. Cell cycle dynamics (microscale) are driven by growth factors derived from the total cell population of quiescent and proliferating cells. Cell-cycle protein concentrations, on the other hand, determine the rates of transition between the two subpopulations. Our model demonstrates the underlying impact of cell cycle dynamics on the evolution of cell population in a tissue. We study the model's well-posedness, derive steady-state solutions, and find sufficient conditions for the stability of steady-state solutions using semigroup and spectral theory. Finally, we performed numerical simulations to see how the parameters affect the model's nonlinear dynamics.


Assuntos
Modelos Biológicos , Ciclo Celular/fisiologia , Divisão Celular
12.
Proc Natl Acad Sci U S A ; 120(4): e2210632120, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36669117

RESUMO

Plant cells are surrounded by a cell wall and do not migrate, which makes the regulation of cell division orientation crucial for development. Regulatory mechanisms controlling cell division orientation may have contributed to the evolution of body organization in land plants. The GRAS family of transcription factors was transferred horizontally from soil bacteria to an algal common ancestor of land plants. SHORTROOT (SHR) and SCARECROW (SCR) genes in this family regulate formative periclinal cell divisions in the roots of flowering plants, but their roles in nonflowering plants and their evolution have not been studied in relation to body organization. Here, we show that SHR cell autonomously inhibits formative periclinal cell divisions indispensable for leaf vein formation in the moss Physcomitrium patens, and SHR expression is positively and negatively regulated by SCR and the GRAS member LATERAL SUPPRESSOR, respectively. While precursor cells of a leaf vein lacking SHR usually follow the geometry rule of dividing along the division plane with the minimum surface area, SHR overrides this rule and forces cells to divide nonpericlinally. Together, these results imply that these bacterially derived GRAS transcription factors were involved in the establishment of the genetic regulatory networks modulating cell division orientation in the common ancestor of land plants and were later adapted to function in flowering plant and moss lineages for their specific body organizations.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Divisão Celular/genética , Raízes de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas
13.
Biomolecules ; 13(1)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36671489

RESUMO

BACKGROUND: The tumor immune microenvironment (TIME) of adrenocortical carcinoma (ACC) is heterogeneous. However, a classification of ACC based on the TIME remains unexplored. METHODS: We hierarchically clustered ACC based on the enrichment levels of twenty-three immune signatures to identify its immune-specific subtypes. Furthermore, we comprehensively compared the clinical and molecular profiles between the subtypes. RESULTS: We identified two immune-specific subtypes of ACC: Immunity-H and Immunity-L, which had high and low immune signature scores, respectively. We demonstrated that this subtyping method was stable and reproducible by analyzing five different ACC cohorts. Compared with Immunity-H, Immunity-L had lower levels of immune cell infiltration, worse overall and disease-free survival prognosis, and higher tumor stemness, genomic instability, proliferation potential, and intratumor heterogeneity. Furthermore, the ACC driver gene CTNNB1 was more frequently mutated in Immunity-L than in Immunity-H. Several proteins, such as mTOR, ERCC1, Akt, ACC1, Cyclin_E1, ß-catenin, FASN, and GAPDH, were more highly expressed in Immunity-L than in Immunity-H. In contrast, p53, Syk, Lck, PREX1, and MAPK were more highly expressed in Immunity-H. Pathway and gene ontology analysis showed that the immune, stromal, and apoptosis pathways were highly enriched in Immunity-H, while the cell cycle, steroid biosynthesis, and DNA damage repair pathways were highly enriched in Immunity-L. CONCLUSIONS: ACC can be classified into two stable immune-related subtypes, which have significantly different antitumor responses, molecular characteristics, and clinical outcomes. This subtyping may provide clinical implications for prognostic and immunotherapeutic stratification of ACC.


Assuntos
Neoplasias do Córtex Suprarrenal , Carcinoma Adrenocortical , Humanos , Carcinoma Adrenocortical/genética , Ciclo Celular , Divisão Celular , Intervalo Livre de Doença , Neoplasias do Córtex Suprarrenal/genética , Microambiente Tumoral/genética
14.
Cells ; 12(2)2023 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-36672251

RESUMO

Cell-cycle progression is regulated by numerous intricate endogenous mechanisms, among which intracellular forces and protein motors are central players. Although it seems unlikely that it is possible to speed up this molecular machinery by applying tiny external forces to the cell, we show that magnetic forcing of magnetosensitive bacteria reduces the duration of the mitotic phase. In such bacteria, the coupling of the cell cycle to the splitting of chains of biogenic magnetic nanoparticles (BMNs) provides a biological realization of such forcing. Using a static gradient magnetic field of a special spatial configuration, in probiotic bacteria E. coli Nissle 1917, we shortened the duration of the mitotic phase and thereby accelerated cell division. Thus, focused magnetic gradient forces exerted on the BMN chains allowed us to intervene in the processes of division and growth of bacteria. The proposed magnetic-based cell division regulation strategy can improve the efficiency of microbial cell factories and medical applications of magnetosensitive bacteria.


Assuntos
Escherichia coli , Campos Magnéticos , Escherichia coli/metabolismo , Divisão Celular , Ciclo Celular
15.
J Bacteriol ; 205(1): e0037322, 2023 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-36622232

RESUMO

FtsA, a homolog of actin, is essential for cell division of Escherichia coli and is widely conserved among many bacteria. FtsA helps to tether polymers of the bacterial tubulin homolog FtsZ to the cytoplasmic membrane as part of the cytokinetic Z ring. GFP fusions to FtsA have illuminated FtsA's localization in live E. coli, but these fusions have not been fully functional and required the presence of the native FtsA. Here, we characterize "sandwich" fusions of E. coli FtsA to either mCherry or msfGFP that are functional for cell division and exhibit fluorescent rings at midcell that persist throughout constriction until cell separation. FtsA within the Z ring moved circumferentially like FtsZ, and FtsA outside the rings formed highly dynamic patches at the membrane. Notably, both FtsA-mCherrysw and FtsA-msfGFPsw acted as mild hypermorphs, as they were not toxic when overproduced, bypassed the essential cell division protein ZipA, and suppressed several thermosensitive fts alleles, although not as effectively as the prototypical hypermorph FtsA*. Overall, our results indicate that fluorescent FtsA sandwich fusions can be used as the sole FtsA in E. coli and thus should shed new light on FtsA dynamics during the cell division cycle in this model system. IMPORTANCE FtsA is a key conserved cell division protein, and E. coli is the most well studied model system for bacterial cell division. One obstacle to full understanding of this process is the lack of a fully functional fluorescent reporter for FtsA in vivo. Here, we describe a fluorescent fusion to E. coli FtsA that promotes efficient cell division in the absence of the native FtsA and can be used to monitor FtsA dynamics during cell division.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Transporte/genética , Divisão Celular , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
16.
J Cell Biol ; 222(3)2023 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-36695784

RESUMO

The γ-tubulin ring complex (γ-TuRC) has essential roles in centrosomal and non-centrosomal microtubule organization during vertebrate mitosis. While there have been important advances in understanding γ-TuRC-dependent microtubule nucleation, γ-TuRC capping of microtubule minus-ends remains poorly characterized. Here, we utilized biochemical reconstitutions and cellular assays to characterize the human γ-TuRC's capping activity. Single filament assays showed that the γ-TuRC remained associated with a nucleated microtubule for tens of minutes. In contrast, caps at dynamic microtubule minus-ends displayed lifetimes of ∼1 min. Reconstituted γ-TuRCs with nucleotide-binding deficient γ-tubulin (γ-tubulinΔGTP) formed ring-shaped complexes that did not nucleate microtubules but capped microtubule minus-ends with lifetimes similar to those measured for wild-type complexes. In dividing cells, microtubule regrowth assays revealed that while knockdown of γ-tubulin suppressed non-centrosomal microtubule formation, add-back of γ-tubulinΔGTP could substantially restore this process. Our results suggest that γ-TuRC capping is a nucleotide-binding-independent activity that plays a role in non-centrosomal microtubule organization during cell division.


Assuntos
Proteínas Associadas aos Microtúbulos , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/química , Centro Organizador dos Microtúbulos , Divisão Celular
17.
Anal Chem ; 95(4): 2312-2320, 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36651064

RESUMO

Cell cycle is a significant factor toward cellular heterogeneity, so cell cycle discrimination is a precise measurement on the top of single-cell analysis. Single-cell analysis based on organic mass spectrometry has received great attention for its unique ability to profile single-cell metabolome, but the influence of cell cycle on cellular metabolome heterogeneity has been overlooked until now due to the lack of a compatible cell cycle discrimination method. Here, we report a robust protocol based on the combination of three small molecular indicators, consisting of two small molecular labels (Hoechst and docetaxel) and one cellular endogenous compound [phosphocholine (34:1)], to discriminate single cells at different cycle stages in real time by organic mass cytometry. More than 6000 HeLa cells were acquired by an improved organic mass cytometry system to build a cell cycle differentiation model. The model successfully discriminated single HeLa cells, SCC7, and Hep G2 cells, at G0/G1, S, and G2/M stages with larger than 85% sensitivity and larger than 89% specificity. Along with cell cycle discrimination, obvious heterogeneity of amino acids, nucleotides, energy metabolic intermediates, and phospholipids was observed among single cells at different cycle stages by this protocol, further demonstrating the necessity of cell cycle discrimination for cellular metabolome heterogeneity research and the potential of more endogenous small molecular compounds for cell cycle discrimination.


Assuntos
Metaboloma , Humanos , Células HeLa , Ciclo Celular , Divisão Celular , Espectrometria de Massas , Citometria de Fluxo
18.
PLoS Genet ; 19(1): e1010505, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36602967

RESUMO

Our understanding of the bacterial cell cycle is framed largely by population-based experiments that focus on the behavior of idealized average cells. Most famously, the contributions of Cooper and Helmstetter help to contextualize the phenomenon of overlapping replication cycles observed in rapidly growing bacteria. Despite the undeniable value of these approaches, their necessary reliance on the behavior of idealized average cells masks the stochasticity inherent in single-cell growth and physiology and limits their mechanistic value. To bridge this gap, we propose an updated and agnostic framework, informed by extant single-cell data, that quantitatively accounts for stochastic variations in single-cell dynamics and the impact of medium composition on cell growth and cell cycle progression. In this framework, stochastic timers sensitive to medium composition impact the relationship between cell cycle events, accounting for observed differences in the relationship between cell cycle events in slow- and fast-growing cells. We conclude with a roadmap for potential application of this framework to longstanding open questions in the bacterial cell cycle field.


Assuntos
Bactérias , Replicação do DNA , Replicação do DNA/genética , Ciclo Celular/genética , Divisão Celular/genética , Bactérias/genética , Cromossomos Bacterianos , DNA Bacteriano/genética
19.
Nat Commun ; 14(1): 151, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36631478

RESUMO

Oriented cell divisions are critical for the formation and maintenance of structured epithelia. Proper mitotic spindle orientation relies on polarised anchoring of force generators to the cell cortex by the evolutionarily conserved protein complex formed by the Gαi subunit of heterotrimeric G proteins, the Leucine-Glycine-Asparagine repeat protein (LGN) and the nuclear mitotic apparatus protein. However, the polarity cues that control cortical patterning of this ternary complex remain largely unknown in mammalian epithelia. Here we identify the membrane-associated protein Annexin A1 (ANXA1) as an interactor of LGN in mammary epithelial cells. Annexin A1 acts independently of Gαi to instruct the accumulation of LGN and nuclear mitotic apparatus protein at the lateral cortex to ensure cortical anchoring of Dynein-Dynactin and astral microtubules and thereby planar alignment of the mitotic spindle. Loss of Annexin A1 randomises mitotic spindle orientation, which in turn disrupts epithelial architecture and luminogenesis in three-dimensional cultures of primary mammary epithelial cells. Our findings establish Annexin A1 as an upstream cortical cue that regulates LGN to direct planar cell divisions during mammalian epithelial morphogenesis.


Assuntos
Anexina A1 , Animais , Anexina A1/metabolismo , Sinais (Psicologia) , Divisão Celular , Fuso Acromático/metabolismo , Morfogênese , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular/fisiologia , Mamíferos/metabolismo
20.
J Cell Sci ; 136(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36633091

RESUMO

Association with microtubules inhibits the fission of mitochondria in Schizosaccharomyces pombe. Here, we show that this attachment of mitochondria to microtubules is an important cell-intrinsic factor in determining cell division symmetry. By comparing mutant cells that exhibited enhanced attachment and no attachment of mitochondria to microtubules (Dnm1Δ and Mmb1Δ, respectively), we show that microtubules in these mutants displayed aberrant dynamics compared to wild-type cells, which resulted in errors in nuclear positioning. This translated to cell division asymmetry in a significant proportion of both Dnm1Δ and Mmb1Δ cells. Asymmetric division in Dnm1Δ and Mmb1Δ cells resulted in unequal distribution of mitochondria, with the daughter cell that received more mitochondria growing faster than the other daughter cell. Taken together, we show the existence of homeostatic feedback controls between mitochondria and microtubules in fission yeast, which directly influence mitochondrial partitioning and, thereby, cell growth. This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Humanos , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Divisão Celular/genética , Mitocôndrias/genética
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