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1.
Methods Mol Biol ; 2364: 3-24, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34542846

RESUMO

Super-resolution (SR) imaging techniques have advanced rapidly in recent years, but only a subset of these techniques is gentle enough to be used by cell biologists to study living cells with minimal photodamage. Our research is focused on studies of the dynamic remodeling of the actin cytoskeleton in living pancreatic beta cells during insulin secretion. These studies require super-resolution light microscopic techniques that are gentle enough to record rapid changes of the actin cytoskeleton in real time. In this chapter, we describe an SR technique that breaks the diffraction limit of the conventional light microscope called TIRF-SIM. Using this SR techniques, we have been able to show that (1) microvilli on pancreatic beta cells translocate in the plane of the plasma membrane and (2) the cortical actin network reorganizes when cells are stimulated to secrete insulin. We describe the FIJI plugins that were used to process and analyze the TIRF-SIM images to obtain quantitative data.


Assuntos
Citoesqueleto de Actina , Actinas , Membrana Celular , Microscopia de Fluorescência
2.
Methods Mol Biol ; 2364: 81-100, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34542849

RESUMO

Budding yeast, Saccharomyces cerevisiae, is an appealing model organism to study the organization and function of the actin cytoskeleton. With the advent of techniques to perform high-resolution, multidimensional analysis of the yeast cell, imaging of yeast has emerged as an important tool for research on the cytoskeleton. This chapter describes techniques and approaches for visualizing the actin cytoskeleton in fixed yeast cells with wide-field and super-resolution fluorescence microscopy.


Assuntos
Saccharomyces cerevisiae , Citoesqueleto de Actina , Actinas , Citoesqueleto , Microscopia de Fluorescência
3.
Methods Mol Biol ; 2364: 25-52, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34542847

RESUMO

The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of a cell. Fibrillar polymers-actin filaments, microtubules, and intermediate filaments-are major constituents of the cytoskeleton, which constantly change their organization during cellular activities. The actin cytoskeleton is especially polymorphic, as actin filaments can form multiple higher-order assemblies performing different functions. Structural information about cytoskeleton organization is critical for understanding its functions and mechanisms underlying various forms of cellular activity. Because of the nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a key tool to determine the structure of the cytoskeleton.This article describes application of rotary shadowing (or platinum replica ) EM (PREM) for visualization of the cytoskeleton . The procedure is applicable to thin cultured cells growing on glass coverslips and consists of detergent extraction (or mechanical "unroofing") of cells to expose their cytoskeleton , chemical fixation to provide stability, ethanol dehydration and critical point drying to preserve three-dimensionality, rotary shadowing with platinum to create contrast, and carbon coating to stabilize replicas. This technique provides easily interpretable three-dimensional images, in which individual cytoskeletal fibers are clearly resolved and individual proteins can be identified by immunogold labeling. More importantly, PREM is easily compatible with live cell imaging, so that one can correlate the dynamics of a cell or its components, e.g., expressed fluorescent proteins, with high-resolution structural organization of the cytoskeleton in the same cell.


Assuntos
Microscopia Eletrônica , Citoesqueleto de Actina , Actinas , Citoesqueleto , Detergentes , Microtúbulos , Platina
4.
Methods Mol Biol ; 2364: 53-80, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34542848

RESUMO

Although budding yeast, Saccharomyces cerevisiae, is widely used as a model organism in biological research, studying cell biology in yeast was hindered due to its small size, rounded morphology, and cell wall. However, with improved techniques, researchers can acquire high-resolution images and carry out rapid multidimensional analysis of a yeast cell. As a result, imaging in yeast has emerged as an important tool to study cytoskeletal organization, function, and dynamics. This chapter describes techniques and approaches for visualizing the actin cytoskeleton in live yeast cells.


Assuntos
Saccharomyces cerevisiae , Citoesqueleto de Actina , Actinas , Divisão Celular , Proteínas de Saccharomyces cerevisiae
5.
Methods Mol Biol ; 2364: 101-137, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34542850

RESUMO

The actin cytoskeleton plays a fundamental role in the regulation of multiple cellular pathways, including trafficking and locomotion. The functional integrity of the cytoskeleton is important during aging, as the decline of cytoskeletal integrity contributes to the physiological consequence of aging. Moreover, improving cytoskeletal form and function throughout aging is sufficient to drive life span extension and promote organismal health in multiple model systems. For these reasons, optimized protocols for visualization of the actin cytoskeleton and its downstream consequences on health span and life span are critical for understanding the aging process. In C. elegans, the actin cytoskeleton shows diverse morphologies across tissues, potentially due to the significantly different functions of each cell type. This chapter describes an imaging platform utilizing LifeAct to visualize the actin cytoskeleton in live, whole nematodes throughout the aging process and methods to perform follow-up studies on the life span and health span of these organisms.


Assuntos
Caenorhabditis elegans , Citoesqueleto de Actina , Actinas , Envelhecimento , Animais , Citoesqueleto
6.
Methods Mol Biol ; 2364: 177-198, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34542854

RESUMO

This chapter presents an overview of the most common F-actin influencing substances, used to study actin dynamics in living plant cells for studies on morphogenesis, motility, organelle movement, apoptosis, or abiotic stress. These substances can be divided into two major subclasses-F-actin-stabilizing and F-actin-polymerizing substances like jasplakinolide and chondramides and F-actin-severing compounds like cytochalasins and latrunculins. Jasplakinolide, which may have anti-cancer activities, was originally isolated from a marine sponge and can now be synthesized and has become commercially available, which is responsible for its wide distribution as membrane-permeable F-actin-stabilizing and F-actin-polymerizing agent. Recently an acyclic derivate of jasplakinolide was isolated. Cytochalasins, derived from fungi, show an F-actin-severing function, and many derivatives are commercially available (A, B, C, D, E, H, J), also making it a widely used compound for F-actin disruption. The same can be stated for latrunculins (A, B), derived from Red Sea sponges; however the mode of action is different by binding to G-actin and inhibiting incorporation into the filament. In the case of swinholide, isolated from red algae or the cyanobacterium Nostoc, a stable complex with actin dimers is formed, resulting in severing F-actin.For influencing F-actin dynamics in plant cells, only membrane permeable drugs are useful in a broad range. We, however, introduce also the phallotoxins and synthetic derivatives thereof, as they are widely used to visualize F-actin in fixed cells. A particular uptake mechanism has been shown for hepatocytes but has also been described in siphonal giant algae. The focus is set on F-actin dynamics in plant cells where alterations in cytoplasmic streaming can be particularly well studied; moreover fluorescence methods for phalloidin- and antibody staining as well as techniques for immunoelectron microscopy are explained.


Assuntos
Poríferos , Citoesqueleto de Actina , Actinas , Animais , Citocalasinas/farmacologia , Depsipeptídeos , Faloidina , Células Vegetais
7.
Methods Mol Biol ; 2364: 217-235, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34542856

RESUMO

The aim of this chapter is to present an innovative technique to visualize changes of the F-actin cytoskeleton in response to locally applied force. We developed an in vitro system that combines micromanipulation of force by magnetic tweezers with simultaneous live cell fluorescence microscopy. We applied pulling forces to magnetic beads coated with the Neisseria gonorrhoeae Type IV pili in the same order of magnitude than the forces generated by live bacteria. We saw quick and robust F-actin accumulation in individual cells at the sites where pulling forces were applied. Using the magnetic tweezers, we were able to mimic the local response of the F-actin cytoskeleton to bacteria-generated forces. In this chapter, we describe our magnetic tweezers system and show how to control it in order to study cellular responses to force.


Assuntos
Citoesqueleto de Actina , Actinas , Citoesqueleto , Micromanipulação , Neisseria gonorrhoeae
8.
Methods Mol Biol ; 2364: 319-326, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34542860

RESUMO

Several model systems have been developed to investigate mechanisms and regulation of intracellular organelle motility. The fish retinal pigment epithelial (RPE) cell represents an unusual but simple system for the study of actin-dependent organelle motility. Primary cultures of RPE dissociated from the eye are amenable to motility studies using a simple perfusion chamber and conventional phase contrast microscopy. In vivo, melanin-containing pigment granules (melanosomes) within fish RPE migrate distances up to 100 µm in response to light flux. When sheets of RPE are removed from the eye and dissociated, they attach to the substrate with apical projections extending radially from the central cell body. Melanosomes can be chemically triggered to aggregate or disperse throughout the projections. Melanosome migration in RPE apical projections is dependent on actin filaments and thus renders this model system useful for investigations of actin-dependent organelle motility.


Assuntos
Melanossomas , Perciformes , Actinas , Animais , Epitélio Pigmentado Ocular , Pigmentos da Retina
9.
J Cell Biol ; 221(1)2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34812842

RESUMO

Epithelial cells assemble specialized actomyosin structures at E-Cadherin-based cell-cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms that build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized, and highly organized actomyosin cytoskeleton at the zonula adherens, combining genetic and pharmacologic approaches with superresolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins, or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micron-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, overlying disorganized actin filaments. This suggested that myosin arrays might bundle actin at mature junctions. Consistent with this idea, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization and prevented actin bundling and polarization. We obtained similar results in Caco-2 cells. These results suggest a novel role for myosin self-assembly, helping drive actin organization to facilitate cell shape change.


Assuntos
Junções Aderentes/metabolismo , Citoesqueleto/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Células CACO-2 , Proteínas de Ligação a DNA , Cães , Forminas/metabolismo , Humanos , Células Madin Darby de Rim Canino , Modelos Biológicos , Quinase de Cadeia Leve de Miosina/metabolismo , Quinases Associadas a rho/metabolismo
10.
Environ Toxicol ; 37(1): 28-40, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34558770

RESUMO

Benzophenone-3 (BP-3), one of the most commonly utilized ultraviolet filters in personal care products, has aroused public concern in recent years for its high chances of human exposure. Previous studies have found that BP-3 can impair testes development and spermatogenesis, but the targets of BP-3 are still unknown. In this study, primary Sertoli cells from 20-day-old mice were treated in vitro with 0-100 µM BP-3 for 24 h to identify its toxicity on Sertoli cells and Sertoli cell barrier. Results demonstrated that BP-3 could induce a notable change in cell morphology and impair Sertoli cell viability. The analysis of transepithelial electrical resistance showed that the integrity of the Sertoli cell barrier was destroyed by BP-3 (100 µM). Some structural proteins of the barrier including ZO-1, Occludin, and Connexin43 were lower expressed and the localization of basal ectoplasmic specializations protein ß-catenin was altered because of BP-3 treatment. Further exploration suggested that BP-3 led to Sertoli cell F-actin disorganization by affecting the expression of Rictor, a key component of the mTORC2 complex. Moreover, although increased DNA damage marker γH2A.X was observed in the treatment group, the cell apoptosis rate was changeless which was further confirmed by increased BAX and stable Bcl-2 (two primary apoptosis regulating proteins). In conclusion, this study revealed that BP-3 had the potential to perturb the Sertoli cell barrier through altered junction proteins and disorganized F-actin, but it could hardly evoke Sertoli cell apoptosis.


Assuntos
Actinas , Células de Sertoli , Animais , Apoptose , Benzofenonas , Barreira Hematotesticular , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Espermatogênese , Junções Íntimas
11.
Phytomedicine ; 95: 153882, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34968897

RESUMO

BACKGROUND: YiQiFuMai lyophilized injection (YQFM) is derived from a traditional Chinese medicine prescription termed Shengmai San.YQFM is clinically applied to the treatment of cardiovascular and cerebrovascular diseases. It has been found that critical components of YQFM affect non-muscle myosin heavy chain IIA (NMMHC IIA), but its regulation in the excessive autophagy and the underlying mechanism has yet to be clarified. PURPOSE: To evaluate whether YQFM has neuroprotective effects on cerebral ischemia/reperfusion-induced injury by inhibiting NMMHC IIA-actin-ATG9A interaction for autophagosome formation. METHODS: The neuroprotective effects of YQFM were investigated in vivo in mice with middle cerebral artery occlusion/reperfusion (MCAO/R) (n = 6) by detecting neurological deficits, infarct volume, and histopathological changes. The NMMHC IIA-actin-ATG9A interaction was determined using immunofluorescence co-localization, co-immunoprecipitation, and proximity ligation assay. Rat pheochromocytoma (PC12) cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) were used to mimic neurons in in vitro experiments. RESULTS: In MCAO/R model mice, YQFM (1.342 g/kg) attenuated brain ischemia/reperfusion-induced injury by regulating NMMHC IIA-actin-mediated ATG9A trafficking. YQFM (400 µg/ml) also exerted similar effects on OGD/R-induced PC12 cells. Furthermore, RNAi of NMMHC IIA weakened the NMMHC IIA-F-actin-dependent ATG9A trafficking and, therefore, attenuated the neuroprotective activities of YQFM in vitro. CONCLUSION: These findings demonstrated that YQFM exerted neuroprotective effects by regulating the NMMHC IIA-actin-ATG9A interaction for autophagosome formation. This evidence sheds new light on the potential mechanism of YQFM in the treatment of cerebral ischemia/reperfusion.


Assuntos
Isquemia Encefálica , Medicamentos de Ervas Chinesas , Fármacos Neuroprotetores , Traumatismo por Reperfusão , Actinas , Animais , Autofagia , Proteínas Relacionadas à Autofagia , Isquemia Encefálica/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Proteínas de Membrana , Camundongos , Fármacos Neuroprotetores/farmacologia , Ratos , Traumatismo por Reperfusão/tratamento farmacológico , Proteínas de Transporte Vesicular
12.
Methods Mol Biol ; 2382: 1-16, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34705230

RESUMO

Cell division in plants consists of separating the mother cell in two daughter cells by the centrifugal growth of a new wall. This process involves the reorganization of the structural elements of the cell, namely the microtubules and actin cytoskeleton which allow the coordination, the orientation, and the progression of mitosis. In addition to its implication in those plant-specific structures, the actin cytoskeleton, in close association with the plasma membrane, exhibits specific patterning at the cortex of the dividing cells, and might act as a signaling component. This review proposes an overview of the techniques available to visualize the actin cytoskeleton in fixed tissues or living cells during division, including electron, fluorescent, and super-resolution microscopy techniques.


Assuntos
Células Vegetais , Citoesqueleto de Actina , Actinas , Citoesqueleto , Microtúbulos , Mitose , Plantas
13.
Methods Mol Biol ; 2382: 245-252, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34705244

RESUMO

Transgenic tobacco BY-2 cell lines stably expressing fluorescent protein-tagged marker proteins have been used to visualize the dynamic behaviors of cytoskeletons and organelles during plant cell division. Using time-lapse confocal imaging, we recently revealed that the pharmacological disruption of actin filaments results in the abnormal organization of phragmoplast microtubules during the early phase of cytokinesis in cell cycle-synchronized BY-2 cells. Additionally, disrupting the actin filaments shortens the time from cell plate emergence to the accumulation of green fluorescent protein-tagged NACK1 kinesin on the cell plate, suggesting that there are two functionally diverse types of microtubules in the phragmoplast. We herein describe a protocol for the cell cycle synchronization of BY-2 cells and the time-lapse confocal imaging of cytokinesis combined with a treatment with an actin polymerization inhibitor and the visualization of an emerging cell plate with a vital stain. This protocol is useful for examining the dynamic changes in protein localization or the intracellular architecture and the effects of actin disruption during plant cell division.


Assuntos
Citocinese , Tabaco , Actinas , Ciclo Celular , Divisão Celular , Microtúbulos , Imagem com Lapso de Tempo
14.
Invest Ophthalmol Vis Sci ; 62(15): 30, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34967855

RESUMO

Purpose: The development of myopia in guinea pigs can be inhibited by attenuating scleral hypoxia by increasing choroidal blood perfusion (ChBP). In this study, we reduced ChBP through surgical and pharmacological methods to determine the effect on myopia development. We also determined whether ChBP was reduced by quinpirole, a drug that enhances form-deprivation myopia (FDM). Methods: ChBP was reduced in the right eyes of guinea pigs via transection of the temporal ciliary arteries or daily injections of phenylephrine into the inferior peribulbar space for one week during normal ocular growth. Other guinea pigs were subjected to two weeks of monocular FDM-with facemasks, along with daily injections of quinpirole, a dopamine D2 receptor agonist, to enhance the FDM. Changes in refraction, axial length, ChBP, and choroidal thickness (ChT) were measured in both treated and fellow eyes of the treatment and control groups. Scleral hypoxia labeling with pimonidazole adducts and α-smooth muscle actin (α-SMA) protein were also measured. Results: Surgical and pharmacological reduction of ChBP induced myopia development in the treated eyes. These treatments rendered the scleral hypoxia and increased scleral α-SMA expression. Furthermore, quinpirole injections, which increased the magnitude of myopia, augmented the FDM-associated reductions in ChBP and ChT and increased the levels of scleral hypoxia and α-SMA protein. Conclusions: Decreased ChBP in guinea pigs leads to scleral hypoxia and scleral myofibroblast transdifferentiation with increased α-SMA expression, ultimately resulting in myopia development. In future clinical trials, ChBP reduction can serve as a potential biomarker for early detection of myopia development.


Assuntos
Corioide/irrigação sanguínea , Miopia/fisiopatologia , Fluxo Sanguíneo Regional/fisiologia , Actinas/metabolismo , Animais , Comprimento Axial do Olho , Velocidade do Fluxo Sanguíneo , Western Blotting , Corioide/efeitos dos fármacos , Corioide/patologia , Artérias Ciliares/cirurgia , Angiografia por Tomografia Computadorizada , Modelos Animais de Doenças , Agonistas de Dopamina/farmacologia , Eletrorretinografia , Cobaias , Hipóxia/metabolismo , Músculo Liso/metabolismo , Miopia/metabolismo , Fenilefrina/farmacologia , Quimpirol/farmacologia , Receptores de Dopamina D2/metabolismo , Refração Ocular/fisiologia , Esclera/metabolismo , Tomografia de Coerência Óptica
15.
Biomolecules ; 11(12)2021 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-34944497

RESUMO

BACKGROUND: Epithelial-mesenchymal transition (EMT), a phenotypic conversion of the epithelial to mesenchymal state, contributes to cancer progression. Currently, several microRNAs (miRNAs) are associated with EMT-mediated cancer progression, but the contribution of miR-34a to EMT in cancer cells remains controversial. The present study aimed to clarify the role of miR-34a in the EMT-related phenotypes of human non-small cell lung cancer (NSCLC) cell lines, A549 (p53 wild-type) and H1299 (p53-deficient). METHODS: The miR-34a mimic and p53 small interfering RNA (siRNA) were transfected into the cells using Lipofectamine, and the obtained total RNA and cell lysates were used for real-time polymerase chain reaction and Western blotting analysis, respectively. RESULTS: The introduction of the miR-34a mimic led to an increase in the mRNA and protein expression levels of α-smooth muscle actin (α-SMA), a mesenchymal marker gene, in A549, but not in H1299 cells. Additionally, miR-34a-induced the upregulation of p53 activity and migration was observed in A549, but not in H1299 cells. However, under the p53-knockdown condition, only α-SMA upregulation by miR-34a was abolished. CONCLUSION: These findings indicate a close relationship between p53 and miR-34a-induced EMT in p53-wild type NSCLC cells, which provides novel insights about the role of miR-34a in EMT-like phenotypic changes in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Proteína Supressora de Tumor p53/genética , Células A549 , Actinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Regulação para Cima
16.
PLoS One ; 16(12): e0260130, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34965258

RESUMO

The objective of the current study is to investigate the effect of rice bran oil (RBO) on hepatic fibrosis as a characteristic response to persistent liver injuries. Rats were randomly allocated into five groups: the negative control group, thioacetamide (TAA) group (thioacetamide 100 mg/kg thrice weekly for two successive weeks, ip), RBO 0.2 and 0.4 groups (RBO 0.2mL and 0.4 mL/rat/day, po) and standard group (silymarin 100 mg/kg/day, po) for two weeks after TAA injection. Blood and liver tissue samples were collected for biochemical, molecular, and histological analyses. Liver functions, oxidative stress, inflammation, liver fibrosis markers were assessed. The obtained results showed that RBO reduced TAA-induced liver fibrosis and suppressed the extracellular matrix formation. Compared to the positive control group, RBO dramatically reduced total bilirubin, AST, and ALT blood levels. Furthermore, RBO reduced MDA and increased GSH contents in the liver. Simultaneously RBO downregulated the NF-κß signaling pathway, which in turn inhibited the expression of some inflammatory mediators, including Cox-2, IL-1ß, and TNF-α. RBO attenuated liver fibrosis by suppressing the biological effects of TGF-ß1, α-SMA, collagen I, hydroxyproline, CTGF, and focal adhesion kinase (FAK). RBO reduced liver fibrosis by inhibiting hepatic stellate cell activation and modulating the interplay among the TGF-ß1 and FAK signal transduction. The greater dosage of 0.4 mL/kg has a more substantial impact. Hence, this investigation presents RBO as a promising antifibrotic agent in the TAA model through inhibition of TGF-ß1 /FAK/α-SMA.


Assuntos
Actinas/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Óleo de Farelo de Arroz/uso terapêutico , Fator de Crescimento Transformador beta1/metabolismo , Albuminas/metabolismo , Animais , Becaplermina/metabolismo , Biomarcadores/metabolismo , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Globulinas/metabolismo , Glutationa/metabolismo , Hidroxiprolina/metabolismo , Mediadores da Inflamação/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/patologia , Cirrose Hepática/sangue , Cirrose Hepática/induzido quimicamente , Masculino , Malondialdeído/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Wistar , Óleo de Farelo de Arroz/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tioacetamida , Transaminases/sangue , Transaminases/metabolismo
17.
Curr Microbiol ; 79(1): 23, 2021 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-34905091

RESUMO

Enhanced HSV-1 production is found in activated T-lymphocytes, but the mechanism is still unknown. In this paper, the HSV-1 entry step in CD3+CD4-CD8-Jurkat T lymphocytes was investigated. Observation under electron microscopy revealed the level of filopodia formation on the surface of activated Jurkat cells was significantly higher than that of non-activated Jurkat cells especially after adding HSV-1 for 15 min. A significant increase of actin protein was demonstrated in HSV-1 infected, activated Jurkat cells compared to HSV-1 infected, non-activated Jurkat cells. After the cells were treated with 2.5 and 5 µg/mL cytochalasin D, an inhibitor of actin polymerization that causes depolymerization of actin's filamentous form, the actin protein was decreased significantly, resulting in an absence of filopodia formation. In summary, this is the first study revealing that HSV-1 induced filopodia formation through actin polymerization in activated T cells similar to epithelial, mucosal and neuronal cells. This phenomenon supported the virus entry resulting to increased yield of HSV-1 production.


Assuntos
Actinas , Herpesvirus Humano 1 , Pseudópodes , Linfócitos T/virologia , Internalização do Vírus , Herpesvirus Humano 1/fisiologia , Humanos , Polimerização
18.
Cells ; 10(12)2021 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-34943931

RESUMO

Phosphatase and tensin homolog deleted on chromosome 10, or PTEN, is a well-characterized tumor suppressor with both lipid and protein phosphatase activities. PTEN is often downregulated by epigenetic mechanisms such as hypermethylation, which leads to constitutive activation of the PI3K-Akt pathway. Large datasets from next-generation sequencing, however, revealed that mutations in PTEN may not only hamper protein function but may also affect interactions with downstream effectors, leading to variable oncogenic readouts. Here, two novel PTEN mutations, Q171R and Y65S, identified in Filipino colorectal cancer patients, were phenotypically characterized in NIH3T3 and HCT116 cells, alongside the C124S canonical mutant and wild-type controls. The novel mutants increased cellular proliferation, resistance to apoptosis and migratory capacity. They induced gross morphological changes including cytoplasmic shrinkage, increased cellular protrusions and extensive cytoskeletal reorganization. The mutants also induced a modest increase in Akt phosphorylation. Further mechanistic studies will help determine the differential oncogenic potencies of these mutants, and resolve whether the structural constraints imposed by the mutations may have altered associations with downstream effectors.


Assuntos
Genes Supressores de Tumor , Mutação/genética , Oncogenes , PTEN Fosfo-Hidrolase/genética , Actinas/metabolismo , Animais , Apoptose , Caspase 3/metabolismo , Caspase 7/metabolismo , Movimento Celular/genética , Proliferação de Células , Forma Celular , Citoesqueleto/metabolismo , Células HCT116 , Humanos , Camundongos , Proteínas Mutantes/metabolismo , Células NIH 3T3 , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo
19.
Cells ; 10(12)2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34944081

RESUMO

The vitelline layer (VL) of a sea urchin egg is an intricate meshwork of glycoproteins that intimately ensheathes the plasma membrane. During fertilization, the VL plays important roles. Firstly, the receptors for sperm reside on the VL. Secondly, following cortical granule exocytosis, the VL is elevated and transformed into the fertilization envelope (FE), owing to the assembly and crosslinking of the extruded materials. As these two crucial stages involve the VL, its alteration was expected to affect the fertilization process. In the present study, we addressed this question by mildly treating the eggs with a reducing agent, dithiothreitol (DTT). A brief pretreatment with DTT resulted in partial disruption of the VL, as judged by electron microscopy and by a novel fluorescent polyamine probe that selectively labelled the VL. The DTT-pretreated eggs did not elevate the FE but were mostly monospermic at fertilization. These eggs also manifested certain anomalies at fertilization: (i) compromised Ca2+ signaling, (ii) blocked translocation of cortical actin filaments, and (iii) impaired cleavage. Some of these phenotypic changes were reversed by restoring the DTT-exposed eggs in normal seawater prior to fertilization. Our findings suggest that the FE is not the decisive factor preventing polyspermy and that the integrity of the VL is nonetheless crucial to the egg's fertilization response.


Assuntos
Ditiotreitol/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fertilização/fisiologia , Ouriços-do-Mar/fisiologia , Actinas/metabolismo , Animais , Cálcio/metabolismo , Fertilização/efeitos dos fármacos , Óvulo/efeitos dos fármacos , Óvulo/fisiologia , Óvulo/ultraestrutura , Ouriços-do-Mar/efeitos dos fármacos , Ouriços-do-Mar/ultraestrutura
20.
Dev Cell ; 56(24): 3349-3363.e6, 2021 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-34932950

RESUMO

Myoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We demonstrate that ERK1/2 inhibition (ERKi) induces robust differentiation and fusion of primary mouse myoblasts through a linear pathway involving RXR, ryanodine receptors, and calcium-dependent activation of CaMKII in nascent myotubes. CaMKII activation results in myotube growth via fusion with mononucleated myoblasts at a fusogenic synapse. Mechanistically, CaMKII interacts with and regulates MYMK and Rac1, and CaMKIIδ/γ knockout mice exhibit smaller regenerated myofibers following injury. In addition, the expression of a dominant negative CaMKII inhibits the formation of large multinucleated myotubes. Finally, we demonstrate the evolutionary conservation of the pathway in chicken myoblasts. We conclude that ERK1/2 represses a signaling cascade leading to CaMKII-mediated fusion of myoblasts to myotubes, providing an attractive target for the cultivated meat industry and regenerative medicine.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Fibras Musculares Esqueléticas/citologia , Mioblastos/citologia , Actinas/metabolismo , Animais , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo
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