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1.
Nat Commun ; 11(1): 5137, 2020 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-33046691

RESUMO

Periodic organization of cells is required for the function of many organs and tissues. The development of such periodic patterns is typically associated with mechanisms based on intercellular signaling such as lateral inhibition and Turing patterning. Here we show that the transition from disordered to ordered checkerboard-like pattern of hair cells and supporting cells in the mammalian hearing organ, the organ of Corti, is likely based on mechanical forces rather than signaling events. Using time-lapse imaging of mouse cochlear explants, we show that hair cells rearrange gradually into a checkerboard-like pattern through a tissue-wide shear motion that coordinates intercalation and delamination events. Using mechanical models of the tissue, we show that global shear and local repulsion forces on hair cells are sufficient to drive the transition from disordered to ordered cellular pattern. Our findings suggest that mechanical forces drive ordered hair cell patterning in a process strikingly analogous to the process of shear-induced crystallization in polymer and granular physics.


Assuntos
Células Ciliadas Auditivas/química , Órgão Espiral/crescimento & desenvolvimento , Animais , Fenômenos Biomecânicos , Células Ciliadas Auditivas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Órgão Espiral/química , Resistência ao Cisalhamento , Imagem com Lapso de Tempo
2.
Nat Commun ; 11(1): 5083, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033259

RESUMO

In hippocampal pyramidal cells, a small subset of dendritic spines contain endoplasmic reticulum (ER). In large spines, ER frequently forms a spine apparatus, while smaller spines contain just a single tubule of smooth ER. Here we show that the ER visits dendritic spines in a non-random manner, targeting spines during periods of high synaptic activity. When we blocked ER motility using a dominant negative approach against myosin V, spine synapses became stronger compared to controls. We were not able to further potentiate these maxed-out synapses, but long-term depression (LTD) was readily induced by low-frequency stimulation. We conclude that the brief ER visits to active spines have the important function of preventing runaway potentiation of individual spine synapses, keeping most of them at an intermediate strength level from which both long-term potentiation (LTP) and LTD are possible.


Assuntos
Espinhas Dendríticas/metabolismo , Retículo Endoplasmático/metabolismo , Sinapses/metabolismo , Animais , Hipocampo/metabolismo , Potenciação de Longa Duração , Miosina Tipo V/metabolismo , Ratos Wistar , Imagem com Lapso de Tempo
3.
Phys Rev Lett ; 125(12): 128101, 2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-33016741

RESUMO

The efficiency of a virus to establish its infection in host cells varies broadly among viruses. It remains unclear if there is a key step in this process that controls viral infectivity. To address this question, we use single-particle tracking and Brownian dynamics simulation to examine human immunodeficiency virus type 1 (HIV-1) infection in cell culture. We find that the frequency of viral-cell encounters is consistent with diffusion-limited interactions. However, even under the most favorable conditions, only 1% of the viruses can become immobilized on cell surface and subsequently enter the cell. This is a result of weak interaction between viral surface gp120 and CD4 receptor, which is insufficient to form a stable complex the majority of the time. We provide the first direct quantitation for efficiencies of these events relevant to measured HIV-1 infectivity and demonstrate that immobilization on host cell surface post-virion-diffusion is the key step in viral infection. Variation of its probability controls the efficiency of a virus to infect its host cells. These results explain the low infectivity of cell-free HIV-1 in vitro and offer a potential rationale for the pervasive high efficiency of cell-to-cell transmission of animal viruses.


Assuntos
HIV-1/patogenicidade , Antígenos CD4/metabolismo , Linhagem Celular , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Humanos , Imagem Óptica , Imagem com Lapso de Tempo , Vírion/metabolismo , Vírion/patogenicidade
4.
PLoS Biol ; 18(8): e3000826, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776935

RESUMO

Ca2+/calmodulin-dependent kinase II (CaMKII) regulates synaptic plasticity in multiple ways, supposedly including the secretion of neuromodulators like brain-derived neurotrophic factor (BDNF). Here, we show that neuromodulator secretion is indeed reduced in mouse α- and ßCaMKII-deficient (αßCaMKII double-knockout [DKO]) hippocampal neurons. However, this was not due to reduced secretion efficiency or neuromodulator vesicle transport but to 40% reduced neuromodulator levels at synapses and 50% reduced delivery of new neuromodulator vesicles to axons. αßCaMKII depletion drastically reduced neuromodulator expression. Blocking BDNF secretion or BDNF scavenging in wild-type neurons produced a similar reduction. Reduced neuromodulator expression in αßCaMKII DKO neurons was restored by active ßCaMKII but not inactive ßCaMKII or αCaMKII, and by CaMKII downstream effectors that promote cAMP-response element binding protein (CREB) phosphorylation. These data indicate that CaMKII regulates neuromodulation in a feedback loop coupling neuromodulator secretion to ßCaMKII- and CREB-dependent neuromodulator expression and axonal targeting, but CaMKIIs are dispensable for the secretion process itself.


Assuntos
Astrócitos/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Cálcio/metabolismo , Neurônios/metabolismo , Subunidades Proteicas/genética , Animais , Astrócitos/citologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Fosforilação , Cultura Primária de Células , Subunidades Proteicas/deficiência , Sinapses/fisiologia , Transmissão Sináptica , Imagem com Lapso de Tempo
5.
Nat Commun ; 11(1): 4271, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848153

RESUMO

Performing multi-color nanoscopy for extended times is challenging due to the rapid photobleaching rate of most fluorophores. Here we describe a new fluorophore (Yale-595) and a bio-orthogonal labeling strategy that enables two-color super-resolution (STED) and 3D confocal imaging of two organelles simultaneously for extended times using high-density environmentally sensitive (HIDE) probes. Because HIDE probes are small, cell-permeant molecules, they can visualize dual organelle dynamics in hard-to-transfect cell lines by super-resolution for over an order of magnitude longer than with tagged proteins. The extended time domain possible using these tools reveals dynamic nanoscale targeting between different organelles.


Assuntos
Corantes Fluorescentes , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Organelas/metabolismo , Linhagem Celular , Corantes Fluorescentes/química , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Imageamento Tridimensional , Microscopia Confocal , Fotodegradação , Imagem com Lapso de Tempo
6.
Nat Commun ; 11(1): 4097, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796861

RESUMO

Staphylococcus aureus is generally thought to divide in three alternating orthogonal planes over three consecutive division cycles. Although this mode of division was proposed over four decades ago, the molecular mechanism that ensures this geometry of division has remained elusive. Here we show, for three different strains, that S. aureus cells do not regularly divide in three alternating perpendicular planes as previously thought. Imaging of the divisome shows that a plane of division is always perpendicular to the previous one, avoiding bisection of the nucleoid, which segregates along an axis parallel to the closing septum. However, one out of the multiple planes perpendicular to the septum which divide the cell in two identical halves can be used in daughter cells, irrespective of its orientation in relation to the penultimate division plane. Therefore, division in three orthogonal planes is not the rule in S. aureus.


Assuntos
Proteínas de Bactérias/metabolismo , Staphylococcus aureus/citologia , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/genética , Microbiologia , Imagem com Lapso de Tempo
7.
Toxicol Lett ; 333: 105-114, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32736005

RESUMO

Maduramicin frequently induces severe cardiotoxicity in broiler chickens as well as in humans who consume maduramicin accidentally. Apoptosis and non-apoptotic cell death occur concurrently in the process of maduramicin-induced cardiotoxicity; however, the underlying mechanism of non-apoptotic cell death is largely unknown. Here, we report the relationship between maduramicin-caused cytoplasmic vacuolization and methuosis-like cell death as well as the underlying mechanism in primary chicken myocardial cells. Maduramicin induced a significant increase of cytoplasmic vacuoles with a degree of cell specificity in primary chicken embryo fibroblasts and chicken hepatoma cells (LMH), along with a decrease of ATP and an increase of LDH. The accumulated vacuoles were partly derived from cellular endocytosis rather than the swelling of endoplasm reticulum, lysosomes, and mitochondria. Moreover, the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) did not prevent maduramicin-induced cytoplasmic vacuolization. DNA ladder and cleavage of PARP were not observed in chicken myocardial cells during maduramicin exposure. Pretreatment with 3-methyladenine (3-MA) and cholorquine (CQ) of chicken myocardial cells did not attenuate cytoplasmic vacuolization and cytotoxicity, although LC3 and p62 were activated. Bafilomycin A1 almost completely prevented the generation of cytoplasmic vacuoles and significantly attenuated cytotoxicity induced by maduramicin, along with downregulation of K-Ras and upregulation of Rac1. Taken together, "methuosis" due to excessive cytoplasmic vacuolization mediates the cardiotoxicity of maduramicin. This provides new insights for understanding a nonclassical form of cell death in the field of drug-induced cytotoxicity.


Assuntos
Morte Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Lactonas/toxicidade , Miócitos Cardíacos/efeitos dos fármacos , Drogas Veterinárias/toxicidade , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Galinhas , Citoplasma , Fragmentação do DNA/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Microscopia Eletrônica de Transmissão , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Cultura Primária de Células , Imagem com Lapso de Tempo , Vacúolos/efeitos dos fármacos , Vacúolos/ultraestrutura
8.
PLoS One ; 15(8): e0236164, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760085

RESUMO

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has been widely used for biomedical applications. Here, we have analyzed the effect of HA on the rescue of primary cells under stress as well as its potential to recover muscle atrophy and validated the developed model in vitro using primary muscle cells derived from rats. The potentials of different HAs were elucidated through comparative analyses using pharmaceutical grade a) high (HHA) and b) low molecular weight (LHA) hyaluronans, c) hybrid cooperative complexes (HCC) of HA in three experimental set-ups. The cells were characterized based on the expression of myogenin, a muscle-specific biomarker, and the proliferation was analyzed using Time-Lapse Video Microscopy (TLVM). Cell viability in response to H2O2 challenge was evaluated by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the expression of the superoxide dismutase enzyme (SOD-2) was assessed by western blotting. Additionally, in order to establish an in vitro model of atrophy, muscle cells were treated with tumor necrosis factor-alpha (TNF-α), along with hyaluronans. The expression of Atrogin, MuRF-1, nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB), and Forkhead-box-(Fox)-O-3 (FoxO3a) was evaluated by western blotting to elucidate the molecular mechanism of atrophy. The results showed that HCC and HHA increased cell proliferation by 1.15 and 2.3 folds in comparison to un-treated cells (control), respectively. Moreover, both pre- and post-treatments of HAs restored the cell viability, and the SOD-2 expression was found to be reduced by 1.5 fold in HA-treated cells as compared to the stressed condition. Specifically in atrophic stressed cells, HCC revealed a noteworthy beneficial effect on the myogenic biomarkers indicating that it could be used as a promising platform for tissue regeneration with specific attention to muscle cell protection against stressful agents.


Assuntos
Ácido Hialurônico/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Atrofia Muscular/terapia , Medicina Regenerativa/métodos , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/metabolismo , Géis , Humanos , Ácido Hialurônico/química , Peróxido de Hidrogênio/toxicidade , Microscopia Intravital , Peso Molecular , Fibras Musculares Esqueléticas/patologia , Atrofia Muscular/patologia , Miogenina/análise , Miogenina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Cultura Primária de Células , Ratos , Superóxido Dismutase/análise , Superóxido Dismutase/metabolismo , Imagem com Lapso de Tempo , Fator de Necrose Tumoral alfa/metabolismo
9.
Curr Protoc Stem Cell Biol ; 54(1): e118, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640120

RESUMO

The normal development of the pulmonary system is critical to transitioning from placental-dependent fetal life to alveolar-dependent newborn life. Human lung development and disease have been difficult to study due to the lack of an in vitro model system containing cells from the large airways and distal alveolus. This article describes a system that allows human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to differentiate and form three-dimensional (3D) structures that emulate the development, cytoarchitecture, and function of the lung ("organoids"), containing epithelial and mesenchymal cell populations, and including the production of surfactant and presence of ciliated cells. The organoids can also be invested with mesoderm derivatives, differentiated from the same human pluripotent stem cells, such as alveolar macrophages and vasculature. Such lung organoids may be used to study the impact of environmental modifiers and perturbagens (toxins, microbial or viral pathogens, alterations in microbiome) or the efficacy and safety of drugs, biologics, and gene transfer. © 2020 Wiley Periodicals LLC. Basic Protocol: hESC/hiPSC dissection, definitive endoderm formation, and lung progenitor cell induction.


Assuntos
Infecções por Coronavirus/patologia , Pulmão/citologia , Organoides/citologia , Pneumonia Viral/patologia , Infecções Respiratórias/patologia , Betacoronavirus , Técnicas de Cultura de Células , Diferenciação Celular , Infecções por Coronavirus/terapia , Endoderma/citologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Pulmão/crescimento & desenvolvimento , Pulmão/fisiologia , Modelos Biológicos , Pandemias , Modelagem Computacional Específica para o Paciente , Pneumonia Viral/terapia , Infecções Respiratórias/terapia , Imagem com Lapso de Tempo
10.
PLoS One ; 15(6): e0234180, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32511278

RESUMO

The autophagy-endolysosomal pathway is an evolutionally conserved degradation system that is tightly linked to a wide variety of physiological processes. Dysfunction of this system is associated with many pathological conditions such as cancer, inflammation and neurodegenerative diseases. Therefore, monitoring the cellular autophagy-endolysosomal activity is crucial for studies on the pathogenesis as well as therapeutics of such disorders. To this end, we here sought to create a novel means exploiting Keima, an acid-stable fluorescent protein possessing pH-dependent fluorescence excitation spectra, for precisely monitoring the autophagy-endolysosomal system. First, we generated three lines of transgenic (tg) mouse expressing monomeric Keima-fused MAP1LC3B (mKeima-LC3B). Then, these tg mice were subjected to starvation by food-restriction, and also challenged to neurodegeneration by genetically crossing with a mouse model of amyotrophic lateral sclerosis; i.e., SOD1H46R transgenic mouse. Unexpectedly, despite that a lipidated-form of endogenous LC3 (LC3-II) was significantly increased, those of mKeima-LC3B (mKeima-LC3B-II) were not changed under both stressed conditions. It was also noted that mKeima-LC3B-positive aggregates were progressively accumulated in the spinal cord of SOD1H46R;mKeima-LC3B double-tg mice, suggestive of acid-resistance and aggregate-prone natures of long-term overexpressed mKeima-LC3B in vivo. Next, we characterized mouse embryonic fibroblasts (MEFs) derived from mKeima-LC3B-tg mice. In contrast with in vivo, levels of mKeima-LC3B-I were decreased under starved conditions. Furthermore, when starved MEFs were treated with chloroquine (CQ), the abundance of mKeima-LC3B-II was significantly increased. Remarkably, when cultured medium was repeatedly changed between DMEM (nutrient-rich) and EBSS (starvation), acidic/neutral signal ratios of mKeima-LC3B-positive compartments were rapidly and reversibly shifted, which were suppressed by the CQ treatment, indicating that intraluminal pH of mKeima-LC3B-positive vesicles was changeable upon nutritional conditions of culture media. Taken together, although mKeima-LC3B-tg mice may not be an appropriate tool to monitor the autophagy-endolysosomal system in vivo, mKeima-LC3B must be one of the most sensitive reporter molecules for monitoring this system under in vitro cultured conditions.


Assuntos
Autofagia/fisiologia , Endossomos/metabolismo , Proteínas Luminescentes/genética , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Animais , Células Cultivadas , Meios de Cultura/farmacologia , Endossomos/genética , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/metabolismo , Lisossomos/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Inanição , Superóxido Dismutase-1/genética , Imagem com Lapso de Tempo
11.
J Vis Exp ; (159)2020 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-32510504

RESUMO

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations of bacteria move over large distances in characteristic tendril-shaped communities over the course of hours. Swarming is sensitive to several factors including medium moisture, humidity, and nutrient content. In addition, the collective stress response, which is observed in P. aeruginosa that are stressed by antibiotics or bacteriophage (phage), repels swarms from approaching the area containing the stress. The methods described here address how to control the critical factors that affect swarming. We introduce a simple method to monitor swarming dynamics and the collective stress response with high temporal resolution using a flatbed document scanner, and describe how to compile and perform a quantitative analysis of swarms. This simple and cost-effective method provides precise and well-controlled quantification of swarming and may be extended to other types of plate-based growth assays and bacterial species.


Assuntos
Pseudomonas aeruginosa/fisiologia , Estresse Fisiológico , Imagem com Lapso de Tempo , Antibacterianos/farmacologia , Bacteriófagos/fisiologia , Análise Custo-Benefício , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/virologia , Imagem com Lapso de Tempo/economia
12.
J Vis Exp ; (160)2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32597868

RESUMO

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.


Assuntos
Separação Celular/métodos , Implantação do Embrião , Embrião de Mamíferos/citologia , Trofoblastos/citologia , Animais , Biomarcadores/metabolismo , Blastocisto/citologia , Forma Celular , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Feminino , Humanos , Gravidez , Imagem com Lapso de Tempo , Fixação de Tecidos , Tripsina/metabolismo , Vitrificação
13.
PLoS One ; 15(5): e0231944, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365105

RESUMO

Intrauterine bleeding during pregnancy is a major risk factor for preterm birth. Thrombin, the most abundant coagulation factor in blood, is associated with uterine myometrial contraction. Here, we investigated the molecular mechanism and signaling of thrombin-induced myometrial contraction. First, histologic studies of placental abruption, as a representative intrauterine bleeding, revealed that thrombin was expressed within the infiltrating hemorrhage and that thrombin receptor (protease-activated receptor 1, PAR1) was highly expressed in myometrial cells surrounding the hemorrhage. Treatment of human myometrial cells with thrombin resulted in augmented contraction via PAR1. Thrombin-induced signaling to myosin was then mediated by activation of myosin light chain kinase- and Rho-induced phosphorylation of myosin light chain-2. In addition, thrombin increased prostaglandin-endoperoxidase synthase-2 (PTGS2 or COX2) mRNA and prostaglandin E2 and F2α synthesis in human myometrial cells. Thrombin significantly increased the mRNA level of interleukine-1ß, whereas it decreased the expressions of prostaglandin EP3 and F2α receptors. Progesterone partially blocked thrombin-induced myometrial contractions, which was accompanied by suppression of the thrombin-induced increase of PTGS2 and IL1B mRNA expressions as well as suppression of PAR1 expression. Collectively, thrombin induces myometrial contractions by two mechanisms, including direct activation of myosin and indirect increases in prostaglandin synthesis. The results suggest a therapeutic potential of progesterone for preterm labor complicated by intrauterine bleeding.


Assuntos
Miométrio/efeitos dos fármacos , Trombina/farmacologia , Contração Uterina/efeitos dos fármacos , Miosinas Cardíacas/metabolismo , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Dinoprosta/metabolismo , Feminino , Humanos , Contração Muscular/efeitos dos fármacos , Miométrio/fisiologia , Cadeias Leves de Miosina/metabolismo , Fosforilação/efeitos dos fármacos , Gravidez , Progesterona/metabolismo , Progesterona/farmacologia , Receptor PAR-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trombina/metabolismo , Imagem com Lapso de Tempo , Contração Uterina/fisiologia
14.
PLoS One ; 15(5): e0233059, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32433687

RESUMO

Complex extracellular structures exist throughout phylogeny, but the dynamics of their formation and dissolution are often opaque. One example is the pharyngeal grinder of the nematode Caenorhabditis elegans, an extracellular structure that ruptures bacteria during feeding. During each larval transition stage, called lethargus, the grinder is replaced with one of a larger size. Here, we characterize at the ultrastructural level the deconstruction of the larval grinder and the construction of the adult grinder during the fourth larval stage (L4)-to-adult transition. Early in L4 lethargus, pharyngeal muscle cells trans-differentiate from contractile to secretory cells, as evidenced by the appearance of clear and dense core vesicles and disruptions in sarcomere organization. This is followed, within minutes, by the dissolution of the L4 grinder and the formation and maturation of the adult grinder. Components of the nascent adult grinder are deposited basally, and are separated from the dissolving larval grinder by a visible apical layer. The complete grinder is a lamellated extracellular matrix comprised of five layers. Following grinder formation, pharyngeal muscle cells regain ultrastructural contractile properties, and muscle contractions resume. Our findings add to our understanding of how complex extracellular structures assemble and dissemble.


Assuntos
Caenorhabditis elegans/fisiologia , Muda , Erupção Dentária , Animais , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/metabolismo , Larva , Metaloendopeptidases/metabolismo , Microscopia Eletrônica de Transmissão , Músculos Faríngeos/ultraestrutura , Sono , Imagem com Lapso de Tempo
15.
PLoS One ; 15(5): e0233012, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469878

RESUMO

Leukocyte migration is controlled by a membrane-based chemosensory pathway on the leading edge pseudopod that guides cell movement up attractant gradients during the innate immune and inflammatory responses. This study employed single cell and population imaging to investigate drug-induced perturbations of leading edge pseudopod morphology in cultured, polarized RAW macrophages. The drugs tested included representative therapeutics (acetylsalicylic acid, diclofenac, ibuprofen, acetaminophen) as well as control drugs (PDGF, Gö6976, wortmannin). Notably, slow addition of any of the four therapeutics to cultured macrophages, mimicking the slowly increasing plasma concentration reported for standard oral dosage in patients, yielded no detectable change in pseudopod morphology. This finding is consistent with the well established clinical safety of these drugs. However, rapid drug addition to cultured macrophages revealed four distinct classes of effects on the leading edge pseudopod: (i) non-perturbing drug exposures yielded no detectable change in pseudopod morphology (acetylsalicylic acid, diclofenac); (ii) adaptive exposures yielded temporary collapse of the extended pseudopod and its signature PI(3,4,5)P3 lipid signal followed by slow recovery of extended pseudopod morphology (ibuprofen, acetaminophen); (iii) disruptive exposures yielded long-term pseudopod collapse (Gö6976, wortmannin); and (iv) activating exposures yielded pseudopod expansion (PDGF). The novel observation of adaptive exposures leads us to hypothesize that rapid addition of an adaptive drug overwhelms an intrinsic or extrinsic adaptation system yielding temporary collapse followed by adaptive recovery, while slow addition enables gradual adaptation to counteract the drug perturbation in real time. Overall, the results illustrate an approach that may help identify therapeutic drugs that temporarily inhibit the leading edge pseudopod during extreme inflammation events, and toxic drugs that yield long term inhibition of the pseudopod with negative consequences for innate immunity. Future studies are needed to elucidate the mechanisms of drug-induced pseudopod collapse, as well as the mechanisms of adaptation and recovery following some inhibitory drug exposures.


Assuntos
Macrófagos/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Acetaminofen/farmacologia , Adaptação Fisiológica , Animais , Aspirina/farmacologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/fisiologia , Diclofenaco/farmacologia , Humanos , Ibuprofeno/farmacologia , Imunidade Inata/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/fisiologia , Camundongos , Pseudópodes/fisiologia , Pseudópodes/ultraestrutura , Células RAW 264.7 , Imagem com Lapso de Tempo
16.
Proc Natl Acad Sci U S A ; 117(21): 11265-11273, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32439711

RESUMO

The nucleation of Alzheimer-associated Aß peptide monomers can be catalyzed by preexisting Aß fibrils. This leads to autocatalytic amplification of aggregate mass and underlies self-replication and generation of toxic oligomers associated with several neurodegenerative diseases. However, the nature of the interactions between the monomeric species and the fibrils during this key process, and indeed the ultrastructural localization of the interaction sites have remained elusive. Here we used NMR and optical spectroscopy to identify conditions that enable the capture of transient species during the aggregation and secondary nucleation of the Aß42 peptide. Cryo-electron microscopy (cryo-EM) images show that new aggregates protrude from the entire length of the progenitor fibril. These protrusions are morphologically distinct from the well-ordered fibrils dominating at the end of the aggregation process. The data provide direct evidence that self-replication through secondary nucleation occurs along the sides of fibrils, which become heavily decorated under the current solution conditions (14 µM Aß42, 20 mM sodium phosphate, 200 µM EDTA, pH 6.8).


Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/ultraestrutura , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Doença de Alzheimer/patologia , Amiloide/metabolismo , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/química , Benzotiazóis/química , Benzotiazóis/metabolismo , Microscopia Crioeletrônica , Humanos , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Fragmentos de Peptídeos/química , Imagem com Lapso de Tempo
17.
Proc Natl Acad Sci U S A ; 117(23): 12961-12968, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32444487

RESUMO

Viral immune evasion is currently understood to focus on deflecting CD8 T cell recognition of infected cells by disrupting antigen presentation pathways. We evaluated viral interference with the ultimate step in cytotoxic T cell function, the death of infected cells. The viral inhibitor of caspase-8 activation (vICA) conserved in human cytomegalovirus (HCMV) and murine CMV (MCMV) prevents the activation of caspase-8 and proapoptotic signaling. We demonstrate the key role of vICA from either virus, in deflecting antigen-specific CD8 T cell-killing of infected cells. vICA-deficient mutants, lacking either UL36 or M36, exhibit greater susceptibility to CD8 T cell control than mutants lacking the set of immunoevasins known to disrupt antigen presentation via MHC class I. This difference is evident during infection in the natural mouse host infected with MCMV, in settings where virus-specific CD8 T cells are adoptively transferred. Finally, we identify the molecular mechanism through which vICA acts, demonstrating the central contribution of caspase-8 signaling at a point of convergence of death receptor-induced apoptosis and perforin/granzyme-dependent cytotoxicity.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Evasão da Resposta Imune , Linfócitos T Citotóxicos/imunologia , Animais , Apoptose/imunologia , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular , Técnicas de Cocultura , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/virologia , Modelos Animais de Doenças , Fibroblastos , Granzimas/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Camundongos Knockout , Muromegalovirus/genética , Muromegalovirus/imunologia , Muromegalovirus/metabolismo , Mutagênese , Perforina/genética , Perforina/metabolismo , Receptores de Morte Celular/metabolismo , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/metabolismo , Imagem com Lapso de Tempo , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/metabolismo
18.
Proc Natl Acad Sci U S A ; 117(23): 12847-12855, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32457163

RESUMO

Microtubule network remodeling is essential for fundamental cellular processes including cell division, differentiation, and motility. Microtubules are active biological polymers whose ends stochastically and independently switch between phases of growth and shrinkage. Microtubule treadmilling, in which the microtubule plus end grows while the minus end shrinks, is observed in cells; however, the underlying mechanisms are not known. Here, we use a combination of computational and in vitro reconstitution approaches to determine the conditions leading to robust microtubule treadmilling. We find that microtubules polymerized from tubulin alone can treadmill, albeit with opposite directionality and order-of-magnitude slower rates than observed in cells. We then employ computational simulations to predict that the combinatory effects of four microtubule-associated proteins (MAPs), namely EB1, XMAP215, CLASP2, and MCAK, can promote fast and sustained plus-end-leading treadmilling. Finally, we experimentally confirm the predictions of our computational model using a multi-MAP, in vitro microtubule dynamics assay to reconstitute robust plus-end-leading treadmilling, consistent with observations in cells. Our results demonstrate how microtubule dynamics can be modulated to achieve a dynamic balance between assembly and disassembly at opposite polymer ends, resulting in treadmilling over long periods of time. Overall, we show how the collective effects of multiple components give rise to complex microtubule behavior that may be used for global network remodeling in cells.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Simulação de Dinâmica Molecular , Proteínas Recombinantes/metabolismo , Células Sf9 , Imagem com Lapso de Tempo
19.
Nat Commun ; 11(1): 2444, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415068

RESUMO

The essential role of ORAI1 channels in receptor-evoked Ca2+ signaling is well understood, yet little is known about the physiological activation of the ORAI channel trio natively expressed in all cells. The roles of ORAI2 and ORAI3 have remained obscure. We show that ORAI2 and ORAI3 channels play a critical role in mediating the regenerative Ca2+ oscillations induced by physiological receptor activation, yet ORAI1 is dispensable in generation of oscillations. We reveal that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca2+-dependent inactivation. This broadened bandwidth of Ca2+ influx is translated by cells into differential activation of NFAT1 and NFAT4 isoforms. Our results uncover a long-sought role for ORAI2 and ORAI3, revealing an intricate control mechanism whereby heteromerization of ORAI channels mediates graded Ca2+ signals that extend the agonist-sensitivity to fine-tune transcriptional control.


Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carbacol/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Fatores de Transcrição NFATC/metabolismo , Proteína ORAI1/metabolismo , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Molécula 1 de Interação Estromal/metabolismo , Imagem com Lapso de Tempo
20.
PLoS One ; 15(4): e0231774, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302356

RESUMO

Cancer is a complex disease caused by multiple types of interactions. To simplify and normalize the assessment of drug effects, spheroid microenvironments have been utilized. Research models that involve agent measurement with the examination of clonogenic survival by monitoring culture process with image analysis have been developed for spheroid-based screening. Meanwhile, computer simulations using various models have enabled better predictions for phenomena in cancer. However, user-based parameters that are specific to a researcher's own experimental conditions must be inputted. In order to bridge the gap between experimental and simulated conditions, we have developed an in silico analysis method with virtual three-dimensional embodiment computed using the researcher's own samples. The present work focused on HeLa spheroid growth in soft agar culture, with spheroids being modeled in silico based on time-lapse images capturing spheroid growth. The spheroids in silico were optimized by adjusting the growth curves to those obtained from time-lapse images of spheroids and were then assigned virtual inner proliferative activity by using generations assigned to each cellular particle. The ratio and distribution of the virtual inner proliferative activities were confirmed to be similar to the proliferation zone ratio and histochemical profiles of HeLa spheroids, which were also consistent with those identified in an earlier study. We validated that time-lapse images of HeLa spheroids provided virtual inner proliferative activity for spheroids in vitro. The present work has achieved the first step toward an in silico analysis method using computational simulation based on a researcher's own samples, helping to bridge the gap between experiment and simulation.


Assuntos
Ágar/farmacologia , Simulação por Computador , Esferoides Celulares/citologia , Imagem com Lapso de Tempo , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Células HeLa , Humanos , Esferoides Celulares/efeitos dos fármacos
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