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1.
Nat Commun ; 12(1): 4969, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404787

RESUMO

Multimeric cytoskeletal protein complexes orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed, differential detergent fractionation to simultaneously detect protein complexes in hundreds of individual cells. Fractionation occurs by 60 s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Proteins are measured with a ~5-hour immunoassay. Co-detection of cytoskeletal protein complexes in U2OS cells treated with filamentous actin (F-actin) destabilizing Latrunculin A detects a unique subpopulation (~2%) exhibiting downregulated F-actin, but upregulated microtubules. Thus, some cells may upregulate other cytoskeletal complexes to counteract the stress of Latrunculin A treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin. We find heat shock may dysregulate filamentous and globular actin correlation. In this work, our assay overcomes selectivity limitations to biochemically quantify single-cell protein complexes perturbed with diverse stimuli.


Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Heterogeneidade Genética , Actinas/genética , Actinas/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular , Linhagem Celular , Resposta ao Choque Térmico , Humanos , Microtúbulos/metabolismo , Modelos Biológicos , Análise de Célula Única/métodos , Tiazolidinas/farmacologia
2.
Nat Microbiol ; 6(9): 1175-1187, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373624

RESUMO

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance by daughter cells is poorly understood. Here, we identify Cell Cycle Regulator protein interacting with FtsZ (CcrZ) as a conserved and essential protein in pneumococci and related Firmicutes such as Bacillus subtilis and Staphylococcus aureus. CcrZ couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae interacts directly with the cytoskeleton protein FtsZ, which places CcrZ in the middle of the newborn cell where the DnaA-bound origin is positioned. This work uncovers a mechanism for control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.


Assuntos
Proteínas de Bactérias/metabolismo , Ciclo Celular , Proteínas do Citoesqueleto/metabolismo , Replicação do DNA , Streptococcus pneumoniae/citologia , Streptococcus pneumoniae/metabolismo , Bacillus subtilis/citologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , Streptococcus pneumoniae/genética
3.
FASEB J ; 35(9): e21811, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34369620

RESUMO

Actin cytoskeletal reorganization plays an important role in regulating smooth muscle contraction, which is essential for the modulation of various physiological functions including airway tone. The adapter protein Abi1 (Abelson interactor 1) participates in the control of smooth muscle contraction. The mechanisms by which Abi1 coordinates smooth muscle function are not fully understood. Here, we found that contractile stimulation elicited Abi1 acetylation in human airway smooth muscle (HASM) cells. Mutagenesis analysis identified lysine-416 (K416) as a major acetylation site. Replacement of K416 with Q (glutamine) enhanced the interaction of Abi1 with neuronal Wiskott-Aldrich syndrome protein (N-WASP), an important actin-regulatory protein. Moreover, the expression of K416Q Abi1 promoted actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19 and vimentin phosphorylation at Ser-56. Furthermore, p300 is a lysine acetyltransferase that catalyzes acetylation of histone and non-histone proteins in various cell types. Here, we discovered that a portion of p300 was localized in the cytoplasm of HASM cells. Knockdown of p300 reduced the agonist-induced Abi1 acetylation in HASM cells and in mouse airway smooth muscle tissues. Smooth muscle conditional knockout of p300 inhibited actin polymerization and the contraction of airway smooth muscle tissues without affecting myosin light chain phosphorylation and vimentin phosphorylation. Together, our results suggest that contractile stimulation induces Abi1 acetylation via p300 in smooth muscle. Acetylation at K416 promotes the coupling of Abi1 with N-WASP, which facilitates actin polymerization and smooth muscle contraction. This is a novel acetylation-dependent regulation of the actin cytoskeleton in smooth muscle.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Acetilação , Animais , Células Cultivadas , Proteína p300 Associada a E1A/metabolismo , Humanos , Lisina Acetiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação/fisiologia , Transdução de Sinais/fisiologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo
4.
Nat Commun ; 12(1): 4926, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389728

RESUMO

The sperm head-to-tail coupling apparatus (HTCA) ensures sperm head-tail integrity while defective HTCA causes acephalic spermatozoa, rendering males infertile. Here, we show that CENTLEIN is indispensable for HTCA integrity and function, and that inactivation of CENTLEIN in mice leads to sperm decapitation and male sterility. We demonstrate that CENTLEIN directly interacts with both SUN5 and PMFBP1, two proteins localized in the HTCA and related with acephalic spermatozoa syndrome. We find that the absence of Centlein sets SUN5 and PMFBP1 apart, the former close to the sperm head and the latter in the decapitated tail. We show that lack of Sun5 results in CENTLEIN and PMFBP1 left in the decapitated tail, while disruption of Pmfbp1 results in SUN5 and CENTLEIN left on the detached sperm head. These results demonstrate that CENTLEIN cooperating with SUN5 and PMFBP1 participates in the HTCA assembly and integration of sperm head to the tail, indicating that impairments of CENTLEIN might be associated with acephalic spermatozoa syndrome in humans.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Cabeça do Espermatozoide/metabolismo , Cauda do Espermatozoide/metabolismo , Espermatozoides/metabolismo , Animais , Proteínas de Ciclo Celular , Células Cultivadas , Proteínas do Citoesqueleto/genética , Células HEK293 , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Mutação , Ligação Proteica , Espermatozoides/citologia , Teratozoospermia/genética , Teratozoospermia/metabolismo
5.
Nature ; 596(7871): 285-290, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34321666

RESUMO

Ageing is driven by a loss of cellular integrity1. Given the major role of ubiquitin modifications in cell function2, here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome3, a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament4 and the EPS-8 modulator of RAC signalling5. While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.


Assuntos
Envelhecimento/metabolismo , Caenorhabditis elegans/metabolismo , Proteoma/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Citoesqueleto de Actina/metabolismo , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas do Citoesqueleto/metabolismo , Intestinos/microbiologia , Longevidade , Músculos/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteoma/química , Proteínas rac de Ligação ao GTP/metabolismo
6.
Hum Genet ; 140(9): 1367-1377, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34255152

RESUMO

Spermatozoa are polarized cells with a head and a flagellum joined together by the connecting piece. Flagellum integrity is critical for normal sperm function, and flagellum defects consistently lead to male infertility. Multiple morphological abnormalities of the flagella (MMAF) is a distinct sperm phenotype consistently leading to male infertility due to a reduced or absent sperm motility associated with severe morphological and ultrastructural flagellum defects. Despite numerous genes recently described to be recurrently associated with MMAF, more than half of the cases analyzed remain unresolved, suggesting that many yet uncharacterized gene defects account for this phenotype. By performing a retrospective exome analysis of the unsolved cases from our initial cohort of 167 infertile men with a MMAF phenotype, we identified one individual carrying a homozygous frameshift variant in CFAP206, a gene encoding a microtubule-docking adapter for radial spoke and inner dynein arm. Immunostaining experiments in the patient's sperm cells demonstrated the absence of WDR66 and RSPH1 proteins suggesting severe radial spokes and calmodulin and spoke-associated complex defects. Using the CRISPR-Cas9 technique, we generated homozygous Cfap206 knockout (KO) mice which presented with male infertility due to functional, structural and ultrastructural sperm flagellum defects associated with a very low rate of embryo development using ICSI. Overall, we showed that CFAP206 is essential for normal sperm flagellum structure and function in human and mouse and that bi-allelic mutations in CFAP206 cause male infertility in man and mouse by inducing morphological and functional defects of the sperm flagellum that may also cause ICSI failures.


Assuntos
Proteínas do Citoesqueleto , Mutação da Fase de Leitura , Homozigoto , Infertilidade Masculina , Cauda do Espermatozoide/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Camundongos
7.
J Biol Chem ; 296: 100640, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34237899

RESUMO

Myosins are actin-based motor proteins known to perform a variety of different mechanical tasks in cells including transporting cargo, generating tension, and linking the cytoskeleton and membrane. Myosins that function as transporters often form complexes with adaptor proteins and vesicular membranes, making it unclear how they transport their cargo through the actin cytoskeletal network. Rai et al. now use single-molecule kinetics, FRET, and DNA origami scaffolds that mimic motor-adaptor complexes to reveal that the myosin VI-Dab2 complex, which is held together weakly and turns over rapidly, can facilitate processive transport without disruption of the cytoskeleton.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Citoesqueleto de Actina/metabolismo , Transporte Biológico , Proteínas do Citoesqueleto/metabolismo , Humanos , Ligação Proteica , Frações Subcelulares/metabolismo
8.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200891

RESUMO

Preterm labor (PTL) is one of the obstetric complications, and is known to be associated with abnormal maternal inflammatory response and intrauterine inflammation and/or infection. However, the expression of specific miRNAs associated with PTL is not clear. In this study, we performed combination analysis of miRNA array and gene array, and then selected one miRNA (miR-373-3p) and its putative target genes (CD44 and RDX) that exhibited large expression differences in term and PTL placentas with or without inflammation. Using qRT-PCR and luciferase assays, we confirmed that miR-373-3p directly targeted CD44 and RDX. Overexpression of miR-373-3p reduced the migration and invasion of trophoblast cells, while inhibition of miR-373-3p restored the migration and invasion abilities of trophoblast cells. Finally, we validated the expression of miR-373-3p and its target genes in clinical patients' blood. miR-373-3p was increased in PTL patients' blood, and was the most expressed in PTL patients' blood with inflammation. In addition, by targeting the miR-373-3p, CD44 and RDX was decreased in PTL patients' blood, and their expression were the lowest in PTL patients' blood with inflammation. Taken together, these findings suggest that miR-373-3p and its target genes can be potential biomarkers for diagnosis of PTL.


Assuntos
Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Receptores de Hialuronatos/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Placenta/patologia , Trofoblastos/patologia , Proliferação de Células , Proteínas do Citoesqueleto/genética , Feminino , Humanos , Receptores de Hialuronatos/genética , Proteínas de Membrana/genética , Placenta/metabolismo , Gravidez , Trofoblastos/metabolismo
9.
Nat Commun ; 12(1): 4096, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215746

RESUMO

Non-centrosomal microtubule arrays serve crucial functions in cells, yet the mechanisms of their generation are poorly understood. During budding of the epithelial tubes of the salivary glands in the Drosophila embryo, we previously demonstrated that the activity of pulsatile apical-medial actomyosin depends on a longitudinal non-centrosomal microtubule array. Here we uncover that the exit from the last embryonic division cycle of the epidermal cells of the salivary gland placode leads to one centrosome in the cells losing all microtubule-nucleation capacity. This restriction of nucleation activity to the second, Centrobin-enriched, centrosome is key for proper morphogenesis. Furthermore, the microtubule-severing protein Katanin and the minus-end-binding protein Patronin accumulate in an apical-medial position only in placodal cells. Loss of either in the placode prevents formation of the longitudinal microtubule array and leads to loss of apical-medial actomyosin and impaired apical constriction. We thus propose a mechanism whereby Katanin-severing at the single active centrosome releases microtubule minus-ends that are then anchored by apical-medial Patronin to promote formation of the longitudinal microtubule array crucial for apical constriction and tube formation.


Assuntos
Divisão Celular/fisiologia , Centrossomo/metabolismo , Microtúbulos/metabolismo , Actinas , Actomiosina/metabolismo , Animais , Centrossomo/ultraestrutura , Proteínas do Citoesqueleto/metabolismo , Drosophila , Katanina , Masculino , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Morfogênese , Glândulas Salivares , Tubulina (Proteína)/metabolismo
10.
Commun Biol ; 4(1): 850, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34239035

RESUMO

The retinal pigmented epithelium (RPE) is a monolayer of multifunctional cells located at the back of the eye. High membrane turnover and polarization, including formation of actin-based apical microvilli, are essential for RPE function and retinal health. Herein, we demonstrate an important role for ßA3/A1-crystallin in RPE. ßA3/A1-crystallin deficiency leads to clathrin-mediated epidermal growth factor receptor (EGFR) endocytosis abnormalities and actin network disruption at the apical side that result in RPE polarity disruption and degeneration. We found that ßA3/A1-crystallin binds to phosphatidylinositol transfer protein (PITPß) and that ßA3/A1-crystallin deficiency diminishes phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), thus probably decreasing ezrin phosphorylation, EGFR activation, internalization, and degradation. We propose that ßA3/A1-crystallin acquired its RPE function before evolving as a structural element in the lens, and that in the RPE, it modulates the PI(4,5)P2 pool through PITPß/PLC signaling axis, coordinates EGFR activation, regulates ezrin phosphorylation and ultimately the cell polarity.


Assuntos
Polaridade Celular/fisiologia , Endocitose , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Cadeia A de beta-Cristalina/metabolismo , Animais , Polaridade Celular/genética , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Camundongos Knockout , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosforilação , Ligação Proteica , Epitélio Pigmentado da Retina/citologia , Cadeia A de beta-Cristalina/genética
11.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299211

RESUMO

Glaucoma is a leading cause of irreversible blindness worldwide, and increased intraocular pressure (IOP) is a major risk factor. We aimed to determine if early functional and molecular differences in the glaucomatous retina manifest before significant retinal ganglion cell (RGC) loss is apparent. Adenoviral vectors expressing a pathogenic form of myocilin (Ad5.MYOC) were used to induce IOP elevation in C57BL/6 mice. IOP and pattern electroretinograms (pERG) were recorded, and retinas were prepared for RNA sequencing, immunohistochemistry, or to determine RGC loss. Ocular injection of Ad5.MYOC leads to reliable IOP elevation, resulting in significant loss of RGC after nine weeks. A significant decrease in the pERG amplitude was evident in eyes three weeks after IOP elevation. Retinal gene expression analysis revealed increased expression for 291 genes related to complement cascade, inflammation, and antigen presentation in hypertensive eyes. Decreased expression was found for 378 genes associated with the γ-aminobutyric acid (GABA)ergic and glutamatergic systems and axon guidance. These data suggest that early functional changes in RGC might be due to reduced GABAA receptor signaling and neuroinflammation that precedes RGC loss in this glaucoma model. These initial changes may offer new targets for early detection of glaucoma and the development of new interventions.


Assuntos
Neurônios GABAérgicos/metabolismo , Glaucoma/patologia , Células Ganglionares da Retina/patologia , Ácido gama-Aminobutírico/metabolismo , Animais , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Neurônios GABAérgicos/patologia , Regulação da Expressão Gênica , Glaucoma/etiologia , Glaucoma/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Pressão Intraocular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Ganglionares da Retina/metabolismo
12.
Nat Commun ; 12(1): 3624, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131132

RESUMO

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/ß-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Junções Aderentes/metabolismo , Artrite/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Artrite/patologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Caderinas/metabolismo , Proteínas do Citoesqueleto/genética , Feminino , Proteínas de Homeodomínio , Proteínas com Domínio LIM/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos , beta Catenina/metabolismo
13.
Redox Biol ; 44: 102014, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34062408

RESUMO

The cytoskeleton is a supramolecular structure consisting of interacting protein networks that support cell dynamics in essential processes such as migration and division, as well as in responses to stress. Fast cytoskeletal remodeling is achieved with the participation of regulatory proteins and posttranslational modifications (PTMs). Redox-related PTMs are emerging as critical players in cytoskeletal regulation. Here we used a cellular model of mild nitroxidative stress in which a peroxynitrite donor induced transient changes in the organization of three key cytoskeletal proteins, i.e., vimentin, actin and tubulin. Nitroxidative stress-induced reconfiguration of intermediate filaments, microtubules and actin structures were further correlated with their PTM profiles and dynamics of the PTM landscape. Using high-resolution mass spectrometry, 62 different PTMs were identified and relatively quantified in vimentin, actin and tubulin, including 12 enzymatic, 13 oxidative and 2 nitric oxide-derived modifications as well as 35 modifications by carbonylated lipid peroxidation products, thus evidencing the occurrence of a chain reaction with formation of numerous reactive species and activation of multiple signaling pathways. Our results unveil the presence of certain modifications under basal conditions and their modulation in response to stress in a target-, residue- and reactive species-dependent manner. Thus, some modifications accumulated during the experiment whereas others varied transiently. Moreover, we identified protein PTM "hot spots", such as the single cysteine residue of vimentin, which was detected in seven modified forms, thus, supporting its role in PTM crosstalk and redox sensing. Finally, identification of novel PTMs in these proteins paves the way for unveiling new cytoskeleton regulatory mechanisms.


Assuntos
Proteínas do Citoesqueleto , Processamento de Proteína Pós-Traducional , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Filamentos Intermediários/metabolismo , Vimentina/metabolismo
14.
Toxicol Appl Pharmacol ; 426: 115607, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34089742

RESUMO

Sertoli cells (SC) structurally support and transport nutrients to germ cells during spermatogenesis facilitated by an active cytoskeleton. Chemical perturbation of SC microtubule (MT) dynamics instability leads to premature germ cell exfoliation demonstrating that this process is essential for male fertility, yet the effects of MT damaging drugs on SC lipid metabolism have been less explored. The aim of this study was to advance our understanding of how adequate SC MT dynamicity is needed to finely tune lipid homeostasis. To elucidate the role of MT dynamics instability on the latter, we suppressed MT dynamicity by long-term exposures to 10 nM of nocodazole (NCZ) on TM4-SC cultures. Inhibition of MT dynamics instability affected the distribution of [3H] arachidonate on TM4-SC. Triacylglycerols (TAG) exhibited a higher proportion of the [3H] label, with significantly lower percentages in the mitochondrial phospholipid cardiolipin, and notably, also in phosphatidylethanolamine. A noteworthy and progressive accumulation of lipid droplets during the period of exposure to NCZ was accompanied by increased TAG levels but not cholesterol levels in TM4-SC. NCZ-exposed cells reduced their mitochondrial membrane potential and increased ROS production without triggering apoptosis, had a compromised autophagic flux, and lost their transferrin expression. Although SC morphology was preserved, the NCZ-exposed cells displayed alteration of the normal organization of microfilaments (f-actin) and intermediate filaments (vimentin). Our findings suggest that a preserved MT dynamicity is essential in the maintenance of lipid and fatty acids homeostasis in SC, and thus highlights a novel target in these cells for drugs that impair MT dynamicity.


Assuntos
Metabolismo dos Lipídeos , Microtúbulos/metabolismo , Células de Sertoli/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Homeostase/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Microtúbulos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Nocodazol/farmacologia , Células de Sertoli/efeitos dos fármacos , Moduladores de Tubulina/farmacologia
15.
Nat Commun ; 12(1): 3803, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155197

RESUMO

The adenomatous polyposis coli (APC) is a frequently mutated tumour suppressor gene in cancers. However, whether APC is regulated at the epitranscriptomic level remains elusive. In this study, we analysed TCGA data and separated 200 paired oesophageal squamous cell carcinoma (ESCC) specimens and their adjacent normal tissues and demonstrated that methyltransferase-like 3 (METTL3) is highly expressed in tumour tissues. m6A-RNA immunoprecipitation sequencing revealed that METTL3 upregulates the m6A modification of APC, which recruits YTHDF for APC mRNA degradation. Reduced APC expression increases the expression of ß-catenin and ß-catenin-mediated cyclin D1, c-Myc, and PKM2 expression, thereby leading to enhanced aerobic glycolysis, ESCC cell proliferation, and tumour formation in mice. In addition, downregulated APC expression correlates with upregulated METTL3 expression in human ESCC specimens and poor prognosis in ESCC patients. Our findings reveal a mechanism by which the Wnt/ß-catenin pathway is upregulated in ESCC via METTL3/YTHDF-coupled epitranscriptomal downregulation of APC.


Assuntos
Adenosina/análogos & derivados , Proteínas do Citoesqueleto/genética , Metiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina/metabolismo , Animais , Carcinogênese , Proliferação de Células , Proteínas do Citoesqueleto/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Metiltransferases/genética , Camundongos , Prognóstico , RNA Mensageiro/metabolismo , Efeito Warburg em Oncologia , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
16.
Aging (Albany NY) ; 13(11): 15569-15579, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34106879

RESUMO

OBJECTIVE: This study aimed to investigate the regulatory effects of repressor element-1 silencing transcription factor (REST) on the glutamate receptors and immediate early genes (IEGs) in the SH-SY5Y cells. METHODS: The genes regulated by REST were screened by bioinformatics between AD patients and the control group. Then, SH-SY5Y cells were treated with 10 µM Aß or REST siRNA/cDNA, and the expressions of synaptic genes and IEGs were detected. Moreover, the protein expression of synaptophysin and PSD-95 was detected by Western blotting in the primary mouse hippocampal neurons. RESULTS: Firstly, 464 differentially expressed genes regulated by REST were identified between Alzheimer's disease (AD) patients and controls, and REST was closely related to the glutamatergic synapses and long-term potentiation. GRIA1, GRIN2A, GRIN1, and ARC showed significant variations with the changes of REST. Moreover, the loss of REST reduced the expression of synaptophysin and PSD-95, which was related to synaptic plasticity. CONCLUSION: REST maintains synaptic plasticity by affecting both glutamate receptors and IEGs, and the imbalance between neural excitation and inhibition mediated by REST compromises neural function, contributing to cognitive impairment.


Assuntos
Genes Precoces , Plasticidade Neuronal/genética , Receptores de Glutamato/metabolismo , Proteínas Repressoras/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Técnicas de Silenciamento de Genes , Hipocampo/citologia , Humanos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sinapses/metabolismo , Sinaptofisina/metabolismo
17.
Nat Commun ; 12(1): 3310, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083531

RESUMO

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far, however, not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we design an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ filaments actively transform these tubes into spring-like structures, where GTPase activity promotes spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, the directional forces that FtsZ-YFP-mts rings exert upon GTP hydrolysis can be estimated to be in the pN range. They are sufficient to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. We hypothesize that these forces result from torsional stress in a GTPase activity dependent manner.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo , Fenômenos Biomecânicos , Divisão Celular/fisiologia , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Hidrólise , Lipossomos/metabolismo , Proteínas Luminescentes/metabolismo , Membranas/metabolismo , Modelos Biológicos , Pinças Ópticas , Proteínas Recombinantes de Fusão/metabolismo , Torção Mecânica
18.
Development ; 148(13)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34128978

RESUMO

Intramembranous ossification, which consists of direct conversion of mesenchymal cells to osteoblasts, is a characteristic process in skull development. One crucial role of these osteoblasts is to secrete collagen-containing bone matrix. However, it remains unclear how the dynamics of collagen trafficking is regulated during skull development. Here, we reveal the regulatory mechanisms of ciliary and golgin proteins required for intramembranous ossification. During normal skull formation, osteoblasts residing on the osteogenic front actively secreted collagen. Mass spectrometry and proteomic analysis determined endogenous binding between ciliary protein IFT20 and golgin protein GMAP210 in these osteoblasts. As seen in Ift20 mutant mice, disruption of neural crest-specific GMAP210 in mice caused osteopenia-like phenotypes due to dysfunctional collagen trafficking. Mice lacking both IFT20 and GMAP210 displayed more severe skull defects compared with either IFT20 or GMAP210 mutants. These results demonstrate that the molecular complex of IFT20 and GMAP210 is essential for the intramembranous ossification during skull development.


Assuntos
Proteínas da Matriz do Complexo de Golgi/metabolismo , Crânio/crescimento & desenvolvimento , Crânio/metabolismo , Animais , Calcificação Fisiológica , Proteínas de Transporte/metabolismo , Diferenciação Celular , Proliferação de Células , Colágeno/metabolismo , Proteínas do Citoesqueleto/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/genética , Camundongos , Camundongos Knockout , Crista Neural/metabolismo , Osteoblastos , Osteogênese , Proteômica
19.
J Bacteriol ; 203(16): e0020421, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34031040

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen of acute clinical importance. Combination treatment with an FtsZ inhibitor potentiates the activity of penicillin binding protein (PBP)-targeting ß-lactam antibiotics against MRSA. To explore the mechanism underlying this synergistic behavior, we examined the impact of treatment with the FtsZ inhibitor TXA707 on the spatial localization of the five PBP proteins expressed in MRSA. In the absence of drug treatment, PBP1, PBP2, PBP3, and PBP4 colocalize with FtsZ at the septum, contributing to new cell wall formation. In contrast, PBP2a localizes to distinct foci along the cell periphery. Upon treatment with TXA707, septum formation becomes disrupted, and FtsZ relocalizes away from midcell. PBP1 and PBP3 remain significantly colocalized with FtsZ, while PBP2, PBP4, and PBP2a localize away from FtsZ to specific sites along the periphery of the enlarged cells. We also examined the impact on PBP2a and PBP2 localization of treatment with ß-lactam antibiotic oxacillin alone and in synergistic combination with TXA707. Significantly, PBP2a localizes to the septum in approximately 15% of the oxacillin-treated cells, a behavior that likely contributes to the ß-lactam resistance of MRSA. Combination treatment with TXA707 causes both PBP2a and PBP2 to localize in malformed septum-like structures. Our collective results suggest that PBP2, PBP4, and PBP2a may function collaboratively in peripheral cell wall repair and maintenance in response to FtsZ inhibition by TXA707. Cotreatment with oxacillin appears to reduce the availability of PBP2a to assist in this repair, thereby rendering the MRSA cells more susceptible to the ß-lactam. IMPORTANCE MRSA is a multidrug-resistant bacterial pathogen of acute clinical importance, infecting many thousands of individuals globally each year. The essential cell division protein FtsZ has been identified as an appealing target for the development of new drugs to combat MRSA infections. Through synergistic actions, FtsZ-targeting agents can sensitize MRSA to antibiotics like the ß-lactams that would otherwise be ineffective. This study provides key insights into the mechanism underlying this synergistic behavior as well as MRSA resistance to ß-lactam drugs. The results of this work will help guide the identification and optimization of combination drug regimens that can effectively treat MRSA infections and reduce the potential for future resistance.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas do Citoesqueleto/antagonistas & inibidores , Staphylococcus aureus Resistente à Meticilina/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Sinergismo Farmacológico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas/genética , Transporte Proteico/efeitos dos fármacos , beta-Lactamas/farmacologia
20.
Neuron ; 109(12): 1963-1978.e5, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34033754

RESUMO

Our daily life depends on muscle contraction, a process that is controlled by the neuromuscular junction (NMJ). However, the mechanisms of NMJ assembly remain unclear. Here we show that Rapsn, a protein critical for NMJ formation, undergoes liquid-liquid phase separation (LLPS) and condensates into liquid-like assemblies. Such assemblies can recruit acetylcholine receptors (AChRs), cytoskeletal proteins, and signaling proteins for postsynaptic differentiation. Rapsn LLPS requires multivalent binding of tetratricopeptide repeats (TPRs) and is increased by Musk signaling. The capacity of Rapsn to condensate and co-condensate with interaction proteins is compromised by mutations of congenital myasthenic syndromes (CMSs). NMJ formation is impaired in mutant mice carrying a CMS-associated, LLPS-deficient mutation. These results reveal a critical role of Rapsn LLPS in forming a synaptic semi-membraneless compartment for NMJ formation.


Assuntos
Proteínas Musculares/genética , Síndromes Miastênicas Congênitas/genética , Mioblastos/metabolismo , Junção Neuromuscular/metabolismo , Receptores Colinérgicos/metabolismo , Membranas Sinápticas/metabolismo , Animais , Proteínas do Citoesqueleto/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Placa Motora/embriologia , Placa Motora/metabolismo , Proteínas Musculares/metabolismo , Síndromes Miastênicas Congênitas/embriologia , Síndromes Miastênicas Congênitas/metabolismo , Junção Neuromuscular/embriologia , Transporte Proteico , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Repetições de Tetratricopeptídeos
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