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1.
PLoS Genet ; 17(10): e1009334, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34710087

RESUMO

Homozygous nonsense mutations in CEP55 are associated with several congenital malformations that lead to perinatal lethality suggesting that it plays a critical role in regulation of embryonic development. CEP55 has previously been studied as a crucial regulator of cytokinesis, predominantly in transformed cells, and its dysregulation is linked to carcinogenesis. However, its molecular functions during embryonic development in mammals require further investigation. We have generated a Cep55 knockout (Cep55-/-) mouse model which demonstrated preweaning lethality associated with a wide range of neural defects. Focusing our analysis on the neocortex, we show that Cep55-/- embryos exhibited depleted neural stem/progenitor cells in the ventricular zone as a result of significantly increased cellular apoptosis. Mechanistically, we demonstrated that Cep55-loss downregulates the pGsk3ß/ß-Catenin/Myc axis in an Akt-dependent manner. The elevated apoptosis of neural stem/progenitors was recapitulated using Cep55-deficient human cerebral organoids and we could rescue the phenotype by inhibiting active Gsk3ß. Additionally, we show that Cep55-loss leads to a significant reduction of ciliated cells, highlighting a novel role in regulating ciliogenesis. Collectively, our findings demonstrate a critical role of Cep55 during brain development and provide mechanistic insights that may have important implications for genetic syndromes associated with Cep55-loss.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Neocórtex/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Animais , Apoptose/fisiologia , Carcinogênese/metabolismo , Células Cultivadas , Citocinese/fisiologia , Homozigoto , Humanos , Camundongos , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Fenótipo
2.
Nat Commun ; 12(1): 6065, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34663803

RESUMO

Different types of cellular membranes have unique lipid compositions that are important for their functional identity. PI(4,5)P2 is enriched in the plasma membrane where it contributes to local activation of key cellular events, including actomyosin contraction and cytokinesis. However, how cells prevent PI(4,5)P2 from accumulating in intracellular membrane compartments, despite constant intermixing and exchange of lipid membranes, is poorly understood. Using the C. elegans early embryo as our model system, we show that the evolutionarily conserved lipid transfer proteins, PDZD-8 and TEX-2, act together with the PI(4,5)P2 phosphatases, OCRL-1 and UNC-26/synaptojanin, to prevent the build-up of PI(4,5)P2 on endosomal membranes. In the absence of these four proteins, large amounts of PI(4,5)P2 accumulate on endosomes, leading to embryonic lethality due to ectopic recruitment of proteins involved in actomyosin contractility. PDZD-8 localizes to the endoplasmic reticulum and regulates endosomal PI(4,5)P2 levels via its lipid harboring SMP domain. Accumulation of PI(4,5)P2 on endosomes is accompanied by impairment of their degradative capacity. Thus, cells use multiple redundant systems to maintain endosomal PI(4,5)P2 homeostasis.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Actomiosina/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Membrana Celular/metabolismo , Citocinese , Desenvolvimento Embrionário , Retículo Endoplasmático/metabolismo , Homeostase , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso , Fosfatidilinositóis , Monoéster Fosfórico Hidrolases
3.
Elife ; 102021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34596529

RESUMO

In multiple cell lineages, Delta-Notch signalling regulates cell fate decisions owing to unidirectional signalling between daughter cells. In Drosophila pupal sensory organ lineage, Notch regulates the intra-lineage pIIa/pIIb fate decision at cytokinesis. Notch and Delta that localise apically and basally at the pIIa-pIIb interface are expressed at low levels and their residence time at the plasma membrane is in the order of minutes. How Delta can effectively interact with Notch to trigger signalling from a large plasma membrane area remains poorly understood. Here, we report that the signalling interface possesses a unique apico-basal polarity with Par3/Bazooka localising in the form of nano-clusters at the apical and basal level. Notch is preferentially targeted to the pIIa-pIIb interface, where it co-clusters with Bazooka and its cofactor Sanpodo. Clusters whose assembly relies on Bazooka and Sanpodo activities are also positive for Neuralized, the E3 ligase required for Delta activity. We propose that the nano-clusters act as snap buttons at the new pIIa-pIIb interface to allow efficient intra-lineage signalling.


Assuntos
Divisão Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Órgãos dos Sentidos/metabolismo , Células-Tronco/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem da Célula , Polaridade Celular , Citocinese , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Receptores Notch/genética , Órgãos dos Sentidos/citologia , Transdução de Sinais , Fatores de Tempo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
4.
Nat Commun ; 12(1): 5429, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521822

RESUMO

Bacillus subtilis is a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, here we report broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.


Assuntos
Aminoácidos/genética , Aminoacil-tRNA Sintetases/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Código Genético , Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/classificação , Aminoacil-tRNA Sintetases/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Códon , Citocinese/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Ligação Proteica , Biossíntese de Proteínas , Mapeamento de Interação de Proteínas , RNA de Transferência/genética , RNA de Transferência/metabolismo
5.
J Cell Sci ; 134(19)2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34523683

RESUMO

In fission yeast, polarized cell growth stops during division and resumes after cytokinesis completes and cells separate. It is unclear how growth reactivation is timed to occur immediately after cell separation. We uncoupled these sequential events by delaying cytokinesis with a temporary Latrunculin A treatment. Mitotic cells recovering from treatment initiate end growth during septation, displaying a polar elongation simultaneous with septation (PrESS) phenotype. PrESS cell ends reactivate Cdc42, a major regulator of polarized growth, during septation, but at a fixed time after anaphase B. A candidate screen implicates Rga4, a negative regulator of Cdc42, in this process. We show that Rga4 appears punctate at the cell sides during G2, but is diffuse during mitosis, extending to the ends. Although the Morphogenesis Orb6 (MOR) pathway is known to promote cell separation and growth by activating protein synthesis, we find that, for polarized growth, removal of Rga4 from the ends is also necessary. Therefore, we propose that growth resumes after division once the MOR pathway is activated and the ends lose Rga4 in a cell-cycle-dependent manner.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Anáfase , Proteínas de Ciclo Celular/genética , Citocinese , Proteínas Ativadoras de GTPase/genética , Proteínas Serina-Treonina Quinases , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteína cdc42 de Ligação ao GTP
6.
Int J Mol Sci ; 22(18)2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34576232

RESUMO

Neuroblastoma, the most common extra-cranial solid tumor of early childhood, is one of the major therapeutic challenges in child oncology: it is highly heterogenic at a genetic, biological, and clinical level. The high-risk cases have one of the least favorable outcomes amongst pediatric tumors, and the mortality rate is still high, regardless of the use of intensive multimodality therapies. Here, we observed that neuroblastoma cells display an increased expression of Cockayne Syndrome group B (CSB), a pleiotropic protein involved in multiple functions such as DNA repair, transcription, mitochondrial homeostasis, and cell division, and were recently found to confer cell robustness when they are up-regulated. In this study, we demonstrated that RNAi-mediated suppression of CSB drastically impairs tumorigenicity of neuroblastoma cells by hampering their proliferative, clonogenic, and invasive capabilities. In particular, we observed that CSB ablation induces cytokinesis failure, leading to caspases 9 and 3 activation and, subsequently, to massive apoptotic cell death. Worthy of note, a new frontier in cancer treatment, already proved to be successful, is cytokinesis-failure-induced cell death. In this context, CSB ablation seems to be a new and promising anticancer strategy for neuroblastoma therapy.


Assuntos
Citocinese/fisiologia , DNA Helicases/fisiologia , Enzimas Reparadoras do DNA/fisiologia , Neuroblastoma/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , Interferência de RNA , Apoptose , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Centrossomo , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Fuso Acromático
7.
Cells ; 10(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34571985

RESUMO

Golgi phosphoprotein 3 (GOLPH3) is a highly conserved peripheral membrane protein localized to the Golgi apparatus and the cytosol. GOLPH3 binding to Golgi membranes depends on phosphatidylinositol 4-phosphate [PI(4)P] and regulates Golgi architecture and vesicle trafficking. GOLPH3 overexpression has been correlated with poor prognosis in several cancers, but the molecular mechanisms that link GOLPH3 to malignant transformation are poorly understood. We recently showed that PI(4)P-GOLPH3 couples membrane trafficking with contractile ring assembly during cytokinesis in dividing Drosophila spermatocytes. Here, we use affinity purification coupled with mass spectrometry (AP-MS) to identify the protein-protein interaction network (interactome) of Drosophila GOLPH3 in testes. Analysis of the GOLPH3 interactome revealed enrichment for proteins involved in vesicle-mediated trafficking, cell proliferation and cytoskeleton dynamics. In particular, we found that dGOLPH3 interacts with the Drosophila orthologs of Fragile X mental retardation protein and Ataxin-2, suggesting a potential role in the pathophysiology of disorders of the nervous system. Our findings suggest novel molecular targets associated with GOLPH3 that might be relevant for therapeutic intervention in cancers and other human diseases.


Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Doenças do Sistema Nervoso/metabolismo , Sistema Nervoso/metabolismo , Proteínas Oncogênicas/metabolismo , Animais , Proliferação de Células/fisiologia , Citocinese/fisiologia , Citoesqueleto/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Mapas de Interação de Proteínas/fisiologia , Transporte Proteico/fisiologia
8.
J Cell Sci ; 134(19)2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34518877

RESUMO

At mitotic exit the cell cycle engine is reset to allow crucial processes, such as cytokinesis and replication origin licensing, to take place before a new cell cycle begins. In budding yeast, the cell cycle clock is reset by a Hippo-like kinase cascade called the mitotic exit network (MEN), whose activation is triggered at spindle pole bodies (SPBs) by the Tem1 GTPase. Yet, MEN activity must be extinguished once MEN-dependent processes have been accomplished. One factor contributing to switching off the MEN is the Amn1 protein, which binds Tem1 and inhibits it through an unknown mechanism. Here, we show that Amn1 downregulates Tem1 through a dual mode of action. On one side, it evicts Tem1 from SPBs and escorts it into the nucleus. On the other, it promotes Tem1 degradation as part of a Skp, Cullin and F-box-containing (SCF) ubiquitin ligase. Tem1 inhibition by Amn1 takes place after cytokinesis in the bud-derived daughter cell, consistent with its asymmetric appearance in the daughter cell versus the mother cell. This dual mechanism of Tem1 inhibition by Amn1 may contribute to the rapid extinguishing of MEN activity once it has fulfilled its functions.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteínas de Saccharomyces cerevisiae , Transporte Ativo do Núcleo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinese , Regulação para Baixo/genética , Humanos , Mitose , Proteínas Monoméricas de Ligação ao GTP/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo
9.
Dev Cell ; 56(17): 2486-2500.e6, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34480876

RESUMO

During cytokinesis, animal cells rapidly remodel the equatorial cortex to build an aligned array of actin filaments called the contractile ring. Local reorientation of filaments by active equatorial compression is thought to underlie the emergence of filament alignment during ring assembly. Here, combining single molecule analysis and modeling in one-cell C. elegans embryos, we show that filaments turnover is far too fast for reorientation of individual filaments by equatorial compression to explain the observed alignment, even if favorably oriented filaments are selectively stabilized. By tracking single formin/CYK-1::GFP particles to monitor local filament assembly, we identify a mechanism that we call filament-guided filament assembly (FGFA), in which existing filaments serve as templates to orient the growth of new filaments. FGFA sharply increases the effective lifetime of filament orientation, providing structural memory that allows cells to build highly aligned filament arrays in response to equatorial compression, despite rapid turnover of individual filaments.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citocinese/fisiologia , Citoesqueleto/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo
10.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445265

RESUMO

Standard toxicity tests might not be fully adequate for evaluating nanomaterials since their unique features are also responsible for unexpected interactions. The in vitro cytokinesis-block micronucleus (CBMN) test is recommended for genotoxicity testing, but cytochalasin-B (Cyt-B) may interfere with nanoparticles (NP), leading to inaccurate results. Our objective was to determine whether Cyt-B could interfere with MN induction by TiO2 NP in human SH-SY5Y cells, as assessed by CBMN test. Cells were treated for 6 or 24 h, according to three treatment options: co-treatment with Cyt-B, post-treatment, and delayed co-treatment. Influence of Cyt-B on TiO2 NP cellular uptake and MN induction as evaluated by flow cytometry (FCMN) were also assessed. TiO2 NP were significantly internalized by cells, both in the absence and presence of Cyt-B, indicating that this chemical does not interfere with NP uptake. Dose-dependent increases in MN rates were observed in CBMN test after co-treatment. However, FCMN assay only showed a positive response when Cyt-B was added simultaneously with TiO2 NP, suggesting that Cyt-B might alter CBMN assay results. No differences were observed in the comparisons between the treatment options assessed, suggesting they are not adequate alternatives to avoid Cyt-B interference in the specific conditions tested.


Assuntos
Citocinese/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico , Nanopartículas/efeitos adversos , Titânio/efeitos adversos , Linhagem Celular Tumoral , Citocalasina B/farmacologia , Relação Dose-Resposta a Droga , Humanos , Titânio/farmacologia
11.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445214

RESUMO

Deubiquitinating enzymes play key roles in the precise modulation of Aurora B-an essential cell cycle regulator. The expression of Aurora B increases before the onset of mitosis and decreases during mitotic exit; an imbalance in these levels has a severe impact on the fate of the cell cycle. Dysregulation of Aurora B can lead to aberrant chromosomal segregation and accumulation of errors during mitosis, eventually resulting in cytokinesis failure. Thus, it is essential to identify the precise regulatory mechanisms that modulate Aurora B levels during the cell division cycle. Using a deubiquitinase knockout strategy, we identified USP48 as an important candidate that can regulate Aurora B protein levels during the normal cell cycle. Here, we report that USP48 interacts with and stabilizes the Aurora B protein. Furthermore, we showed that the deubiquitinating activity of USP48 helps to maintain the steady-state levels of Aurora B protein by regulating its half-life. Finally, USP48 knockout resulted in delayed progression of cell cycle due to accumulation of mitotic defects and ultimately cytokinesis failure, suggesting the role of USP48 in cell cycle regulation.


Assuntos
Aurora Quinase B/metabolismo , Citocinese , Mitose , Proteases Específicas de Ubiquitina/metabolismo , Aurora Quinase B/genética , Estabilidade Enzimática , Células HEK293 , Células HeLa , Humanos , Proteases Específicas de Ubiquitina/genética
12.
Artigo em Inglês | MEDLINE | ID: mdl-34454691

RESUMO

BACKGROUND: 5-aza-2'-deoxycytidine (5azadC, decitabine) is a DNA hypomethylating agent used in the treatment of myelodysplastic syndromes. Due to cytotoxic side effects dose optimization is essential. The aim of this study was to define and quantify the effects of 5azadC on biomarkers of chromosomal stability, and telomere length, in human lymphoblastoid cell line, WIL2-NS, at clinically relevant dosages. METHODS: Human WIL2-NS cells were maintained in complete medium containing 0, 0.2 or 1.0 µM 5azadC for four days, and analysed daily for telomere length (flow cytometry), chromosomal stability (cytokinesis-block micronucleus cytome (CBMN-cyt) assay), and global methylation (%5me-C). RESULTS: DNA methylation decreased significantly in 1.0 µM 5azadC, relative to control (p < 0.0001). Exposure to 1.0 µM 5azadC resulted in 1.7-fold increase in telomere length (p < 0.0001), in parallel with rapid increase in biomarkers of DNA damage; (micronuclei (MN, 6-fold increase), nucleoplasmic bridges (NPB, a 12-fold increase), and nuclear buds (NBud, a 13-fold increase) (all p < 0.0001). Fused nuclei (FUS), indicative of mitotic dysfunction, showed a 5- and 13-fold increase in the 0.2 µM and 1.0 µM conditions, respectively (p = 0.001) after 4 days. CONCLUSIONS: These data show that (i) clinically relevant concentrations of 5azadC are highly genotoxic; (ii) hypomethylation was associated with increased TL and DNA damage; and (iii) longer TL was associated with chromosomal instability. These findings suggest that lower doses of 5azdC may be effective as a hypomethylating agent, while potentially reducing DNA damage and risk for secondary disease.


Assuntos
Dano ao DNA/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Decitabina/farmacologia , Telômero/efeitos dos fármacos , Biomarcadores/metabolismo , Linhagem Celular , Instabilidade Cromossômica/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Humanos , Linfócitos/efeitos dos fármacos , Testes para Micronúcleos/métodos , Mitose/efeitos dos fármacos
13.
J Cell Sci ; 134(16)2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34402513

RESUMO

The F-BAR protein Imp2 is an important contributor to cytokinesis in the fission yeast Schizosaccharomyces pombe. Because cell cycle-regulated phosphorylation of the central intrinsically disordered region (IDR) of the Imp2 paralog Cdc15 controls Cdc15 oligomerization state, localization and ability to bind protein partners, we investigated whether Imp2 is similarly phosphoregulated. We found that Imp2 is endogenously phosphorylated on 28 sites within its IDR, with the bulk of phosphorylation being constitutive. In vitro, the casein kinase 1 (CK1) isoforms Hhp1 and Hhp2 can phosphorylate 17 sites, and Cdk1 (also known as Cdc2) can phosphorylate the remaining 11 sites. Mutations that prevent Cdk1 phosphorylation result in precocious Imp2 recruitment to the cell division site, and mutations designed to mimic these phosphorylation events delay Imp2 accumulation at the contractile ring (CR). Mutations that eliminate CK1 phosphorylation sites allow CR sliding, and phosphomimetic substitutions at these sites reduce Imp2 protein levels and slow CR constriction. Thus, like Cdc15, the Imp2 IDR is phosphorylated at many sites by multiple kinases. In contrast to Cdc15, for which phosphorylation plays a major cell cycle regulatory role, Imp2 phosphorylation is primarily constitutive, with milder effects on localization and function. This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinese/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
14.
Elife ; 102021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34423780

RESUMO

Structures and machines require smoothening of raw materials. Self-organized smoothening guides cell and tissue morphogenesis and is relevant to advanced manufacturing. Across the syncytial Drosophila embryo surface, smooth interfaces form between expanding Arp2/3-based actin caps and surrounding actomyosin networks, demarcating the circumferences of nascent dome-like compartments used for pseudocleavage. We found that forming a smooth and circular boundary of the surrounding actomyosin domain requires Arp2/3 in vivo. To dissect the physical basis of this requirement, we reconstituted the interacting networks using node-based models. In simulations of actomyosin networks with local clearances in place of Arp2/3 domains, rough boundaries persisted when myosin contractility was low. With addition of expanding Arp2/3 network domains, myosin domain boundaries failed to smoothen, but accumulated myosin nodes and tension. After incorporating actomyosin mechanosensitivity, Arp2/3 network growth locally induced a surrounding contractile actomyosin ring that smoothened the interface between the cytoskeletal domains, an effect also evident in vivo. In this way, a smooth structure can emerge from the lateral interaction of irregular active materials.


Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Embrião não Mamífero/fisiologia , Actinas/genética , Actomiosina/metabolismo , Animais , Citocinese/fisiologia , Citoesqueleto/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento
15.
Development ; 148(18)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34397091

RESUMO

Zebrafish are excellent at regenerating their heart by reinitiating proliferation in pre-existing cardiomyocytes. Studying how zebrafish achieve this holds great potential in developing new strategies to boost mammalian heart regeneration. Nevertheless, the lack of appropriate live-imaging tools for the adult zebrafish heart has limited detailed studies into the dynamics underlying cardiomyocyte proliferation. Here, we address this by developing a system in which cardiac slices of the injured zebrafish heart are cultured ex vivo for several days while retaining key regenerative characteristics, including cardiomyocyte proliferation. In addition, we show that the cardiac slice culture system is compatible with live timelapse imaging and allows manipulation of regenerating cardiomyocytes with drugs that normally would have toxic effects that prevent their use. Finally, we use the cardiac slices to demonstrate that adult cardiomyocytes with fully assembled sarcomeres can partially disassemble their sarcomeres in a calpain- and proteasome-dependent manner to progress through nuclear division and cytokinesis. In conclusion, we have developed a cardiac slice culture system, which allows imaging of native cardiomyocyte dynamics in real time to discover cellular mechanisms during heart regeneration.


Assuntos
Proliferação de Células/fisiologia , Miócitos Cardíacos/fisiologia , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados/metabolismo , Animais Geneticamente Modificados/fisiologia , Calpaína/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Células Cultivadas , Citocinese/fisiologia , Feminino , Coração/fisiologia , Masculino , Mamíferos/metabolismo , Mamíferos/fisiologia , Miócitos Cardíacos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Regeneração/fisiologia , Sarcômeros/metabolismo , Sarcômeros/fisiologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
16.
PLoS Pathog ; 17(8): e1009329, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34339455

RESUMO

The flagellar pocket (FP) is the only endo- and exocytic organelle in most trypanosomes and, as such, is essential throughout the life cycle of the parasite. The neck of the FP is maintained enclosed around the flagellum via the flagellar pocket collar (FPC). The FPC is a macromolecular cytoskeletal structure and is essential for the formation of the FP and cytokinesis. FPC biogenesis and structure are poorly understood, mainly due to the lack of information on FPC composition. To date, only two FPC proteins, BILBO1 and FPC4, have been characterized. BILBO1 forms a molecular skeleton upon which other FPC proteins can, theoretically, dock onto. We previously identified FPC4 as the first BILBO1 interacting partner and demonstrated that its C-terminal domain interacts with the BILBO1 N-terminal domain (NTD). Here, we report by yeast two-hybrid, bioinformatics, functional and structural studies the characterization of a new FPC component and BILBO1 partner protein, BILBO2 (Tb927.6.3240). Further, we demonstrate that BILBO1 and BILBO2 share a homologous NTD and that both domains interact with FPC4. We have determined a 1.9 Å resolution crystal structure of the BILBO2 NTD in complex with the FPC4 BILBO1-binding domain. Together with mutational analyses, our studies reveal key residues for the function of the BILBO2 NTD and its interaction with FPC4 and evidenced a tripartite interaction between BILBO1, BILBO2, and FPC4. Our work sheds light on the first atomic structure of an FPC protein complex and represents a significant step in deciphering the FPC function in Trypanosoma brucei and other pathogenic kinetoplastids.


Assuntos
Citocinese , Citoesqueleto/metabolismo , Flagelos/metabolismo , Organelas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Homologia de Sequência , Técnicas do Sistema de Duplo-Híbrido
17.
EMBO Rep ; 22(10): e52387, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34431205

RESUMO

The isotropic metaphase actin cortex progressively polarizes as the anaphase spindle elongates during mitotic exit. This involves the loss of actomyosin cortex from opposing cell poles and the accumulation of an actomyosin belt at the cell centre. Although these spatially distinct cortical remodelling events are coordinated in time, here we show that they are independent of each other. Thus, actomyosin is lost from opposing poles in anaphase cells that lack an actomyosin ring owing to centralspindlin depletion. In examining potential regulators of this process, we identify a role for Aurora B kinase in actin clearance at cell poles. Upon combining Aurora B inhibition with centralspindlin depletion, cells exiting mitosis fail to change shape and remain completely spherical. Additionally, we demonstrate a requirement for Aurora B in the clearance of cortical actin close to anaphase chromatin in cells exiting mitosis with a bipolar spindle and in monopolar cells forced to divide while flat. Altogether, these data suggest a novel role for Aurora B activity in facilitating DNA-mediated polar relaxation at anaphase, polarization of the actomyosin cortex, and cell division.


Assuntos
Actomiosina , Citocinese , Anáfase , Aurora Quinase B/genética , Mitose , Fuso Acromático
18.
J Cell Sci ; 134(18)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34435638

RESUMO

Rho5 is the yeast homolog of the human small GTPase Rac1. We characterized the genes encoding Rho5 and the subunits of its dimeric activating guanine-nucleotide-exchange factor (GEF), Dck1 and Lmo1, in the yeast Kluyveromyces lactis. Rapid translocation of the three GFP-tagged components to mitochondria upon oxidative stress and carbon starvation indicate a similar function of KlRho5 in energy metabolism and mitochondrial dynamics as described for its Saccharomyces cerevisiae homolog. Accordingly, Klrho5 deletion mutants are hyper-resistant towards hydrogen peroxide. Moreover, synthetic lethalities of rho5 deletions with key components in nutrient sensing, such as sch9 and gpr1, are not conserved in K. lactis. Instead, Klrho5 deletion mutants display morphological defects with strengthened lateral cell walls and protruding bud scars. The latter result from aberrant cytokinesis, as observed by following the budding process in vivo and by transmission electron microscopy of the bud neck region. This phenotype can be suppressed by KlCDC42G12V, which encodes a hyper-active variant. Data from live-cell fluorescence microscopy support the notion that KlRho5 interferes with the actin moiety of the contractile actomyosin ring, with consequences different from those previously reported for mutants lacking myosin.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteínas de Saccharomyces cerevisiae , Actomiosina/metabolismo , Citocinese/genética , Humanos , Kluyveromyces , Estresse Oxidativo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Antimicrob Agents Chemother ; 65(11): e0063221, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34424040

RESUMO

Trypanosoma brucei subspecies cause African sleeping sickness in humans, an infection that is commonly fatal if not treated, and available therapies are limited. Previous studies have shown that heat shock protein 90 (Hsp90) inhibitors have potent and vivid activity against bloodstream-form trypanosomes. Hsp90s are phylogenetically conserved and essential catalysts that function at the crux of cell biology, where they ensure the proper folding of proteins and their assembly into multicomponent complexes. To assess the specificity of Hsp90 inhibitors and further define the role of Hsp90s in African trypanosomes, we used RNA interference (RNAi) to knock down cytosolic and mitochondrial Hsp90s (HSP83 and HSP84, respectively). Loss of either protein led to cell death, but the phenotypes were distinctly different. Depletion of cytosolic HSP83 closely mimicked the consequences of chemically depleting Hsp90 activity with inhibitor 17-AAG. In these cells, cytokinesis was severely disrupted, and segregation of the kinetoplast (the massive mitochondrial DNA structure unique to this family of eukaryotic pathogens) was impaired, leading to cells with abnormal kinetoplast DNA (kDNA) structures. Quite differently, knockdown of mitochondrial HSP84 did not impair cytokinesis but halted the initiation of new kDNA synthesis, generating cells without kDNA. These findings highlight the central role of Hsp90s in chaperoning cell cycle regulators in trypanosomes, reveal their unique function in kinetoplast replication, and reinforce their specificity and value as drug targets.


Assuntos
Preparações Farmacêuticas , Trypanosoma brucei brucei , Citocinese/genética , Replicação do DNA/genética , DNA de Cinetoplasto/genética , DNA Mitocondrial , Humanos , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética
20.
J Biochem ; 170(3): 369-377, 2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34424293

RESUMO

Cardiolipin (CL) localizes to curved membranes such as cristae in mitochondria as well as cell poles and division sites in rod-shaped bacteria. CL is believed to stabilize the membrane curvature by localizing to sites of negative curvature. However, this hypothesis has not been tested because of a lack of appropriate tools to distinguish CL inside and outside lipid bilayers. In this study, we provided the first evidence that CL localized to regions of negative curvature in Escherichia coli using the novel CL probe erylysin A-EGFP (EryA-EGFP). Staining in E.coli illustrated that CL localized to the inner leaflets at cell poles and the outer leaflets at division sites. Furthermore, we revealed that EryA-EGFP inhibited cytokinesis. We propose that cytokinesis completes after CL in the outer leaflets transfers to the inner leaflets at division sites by inspecting the mechanism of inhibition of cytokinesis. Moreover, the cytoskeletal protein RodZ was abnormally distributed when cytokinesis was inhibited by EryA-EGFP, suggesting that RodZ participates in cytokinesis. In summary, we revealed the detailed distribution of CL and proposed a new model of cytokinesis.


Assuntos
Cardiolipinas/metabolismo , Citocinese , Proteínas de Escherichia coli/metabolismo , Pterocarpanos/metabolismo , Divisão Celular , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/metabolismo , Bicamadas Lipídicas/metabolismo , Mitocôndrias/metabolismo
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