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1.
Int J Mol Sci ; 23(9)2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35563018

RESUMO

DYT1 dystonia is a debilitating neurological movement disorder that arises upon Torsin ATPase deficiency. Nuclear envelope (NE) blebs that contain FG-nucleoporins (FG-Nups) and K48-linked ubiquitin are the hallmark phenotype of Torsin manipulation across disease models of DYT1 dystonia. While the aberrant deposition of FG-Nups is caused by defective nuclear pore complex assembly, the source of K48-ubiquitylated proteins inside NE blebs is not known. Here, we demonstrate that the characteristic K48-ubiquitin accumulation inside blebs requires p97 activity. This activity is highly dependent on the p97 adaptor UBXD1. We show that p97 does not significantly depend on the Ufd1/Npl4 heterodimer to generate the K48-ubiquitylated proteins inside blebs, nor does inhibiting translation affect the ubiquitin sequestration in blebs. However, stimulating global ubiquitylation by heat shock greatly increases the amount of K48-ubiquitin sequestered inside blebs. These results suggest that blebs have an extraordinarily high capacity for sequestering ubiquitylated protein generated in a p97-dependent manner. The p97/UBXD1 axis is thus a major factor contributing to cellular DYT1 dystonia pathology and its modulation represents an unexplored potential for therapeutic development.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Adenosina Trifosfatases , Proteínas Relacionadas à Autofagia , Distonia , Membrana Nuclear , Proteínas Nucleares , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Estruturas da Membrana Celular/metabolismo , Distonia/genética , Distonia/metabolismo , Distonia Muscular Deformante , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ubiquitina/metabolismo
2.
Oxid Med Cell Longev ; 2022: 3613319, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35281461

RESUMO

Stem cell-based therapy has been indicated to be beneficial for intervertebral disc regeneration. However, the underlying mechanisms have not been fully identified. The present study showed that bone marrow mesenchymal stem cells (BMSCs) donated mitochondria to adjacent nucleus pulposus cells (NPCs) in a coculture system. The mode of mitochondrial transfer between these cells was intercellular tunneling nanotube (TNT), which acted as a transportation expressway for mitochondria. NPCs acquired additional mitochondria from BMSCs in a concentration-dependent manner after rotenone-induced mitochondrial dysfunction in NPCs. Further research demonstrated that TNT-mediated mitochondrial transfer rescued NPCs from mitochondrial dysfunction and apoptosis, which was indicated by the recovery of the mitochondrial respiratory chain, the increase in mitochondrial membrane potential, and the decreases in reactive oxygen species (ROS) levels and apoptosis rates. Furthermore, Miro1, a critical protein that regulates mitochondrial movement, was knocked down in BMSCs and significantly reduced mitochondrial transfer from BMSCs to NPCs. These results suggested that Miro1 depletion inhibited the rescue of NPCs with mitochondrial dysfunction. Taken together, our data shed light on a novel mechanism by which BMSCs rescue impaired NPCs, providing a concrete foundation to study the critical role of intercellular interactions in disc regeneration.


Assuntos
Estruturas da Membrana Celular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Núcleo Pulposo/metabolismo , Apoptose , Células Cultivadas , Nanotubos
3.
Int J Mol Sci ; 23(4)2022 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-35216348

RESUMO

Tunneling nanotubes (TNTs), discovered in 2004, are thin, long protrusions between cells utilized for intercellular transfer and communication. These newly discovered structures have been demonstrated to play a crucial role in homeostasis, but also in the spreading of diseases, infections, and metastases. Gaining much interest in the medical research field, TNTs have been shown to transport nanomedicines (NMeds) between cells. NMeds have been studied thanks to their advantageous features in terms of reduced toxicity of drugs, enhanced solubility, protection of the payload, prolonged release, and more interestingly, cell-targeted delivery. Nevertheless, their transfer between cells via TNTs makes their true fate unknown. If better understood, TNTs could help control NMed delivery. In fact, TNTs can represent the possibility both to improve the biodistribution of NMeds throughout a diseased tissue by increasing their formation, or to minimize their formation to block the transfer of dangerous material. To date, few studies have investigated the interaction between NMeds and TNTs. In this work, we will explain what TNTs are and how they form and then review what has been published regarding their potential use in nanomedicine research. We will highlight possible future approaches to better exploit TNT intercellular communication in the field of nanomedicine.


Assuntos
Estruturas da Membrana Celular/metabolismo , Animais , Transporte Biológico/fisiologia , Humanos , Nanomedicina/métodos , Nanotubos , Distribuição Tecidual/fisiologia
4.
Proc Natl Acad Sci U S A ; 119(7)2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35135877

RESUMO

Reduced blood flow and impaired neurovascular coupling are recognized features of glaucoma, the leading cause of irreversible blindness worldwide, but the mechanisms underlying these defects are unknown. Retinal pericytes regulate microcirculatory blood flow and coordinate neurovascular coupling through interpericyte tunneling nanotubes (IP-TNTs). Using two-photon microscope live imaging of the mouse retina, we found reduced capillary diameter and impaired blood flow at pericyte locations in eyes with high intraocular pressure, the most important risk factor to develop glaucoma. We show that IP-TNTs are structurally and functionally damaged by ocular hypertension, a response that disrupted light-evoked neurovascular coupling. Pericyte-specific inhibition of excessive Ca2+ influx rescued hemodynamic responses, protected IP-TNTs and neurovascular coupling, and enhanced retinal neuronal function as well as survival in glaucomatous retinas. Our study identifies pericytes and IP-TNTs as potential therapeutic targets to counter ocular pressure-related microvascular deficits, and provides preclinical proof of concept that strategies aimed to restore intrapericyte calcium homeostasis rescue autoregulatory blood flow and prevent neuronal dysfunction.


Assuntos
Estruturas da Membrana Celular/fisiologia , Glaucoma/patologia , Pericitos/fisiologia , Retina/citologia , Retina/patologia , Animais , Antígenos , Cálcio/metabolismo , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Glaucoma/etiologia , Fenômenos Magnéticos , Masculino , Camundongos , Microesferas , Nanotubos , Regiões Promotoras Genéticas , Proteoglicanas , Vasos Retinianos/patologia , Técnicas de Cultura de Tecidos
5.
FEBS Open Bio ; 12(1): 203-210, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34738322

RESUMO

Tunneling nanotubes (TNTs) are F-actin-based open-ended tubular extensions that form following stresses, such as nutritional deprivation and oxidative stress. The chemotherapy agent 5-fluorouracil (5-FU) represents a significant stressor to cancer cells and induces thymidine deficiency, a state similar to nutritional deprivation. However, the ability of 5-FU to induce TNT formation in cancer cells and potentially enhance survival has not been explored. In this study, we examined whether 5-FU can induce TNT formation in MCF-7 breast cancer cells. Cytotoxic doses of 5-FU (150-350 µm) were observed to significantly induce TNT formation beginning at 24 h after exposure. TNTs formed following 5-FU treatment probably originated as extensions of gap junctions as MCF-7 cells detach from cell clusters. TNTs act as conduits for exchange of cellular components and we observed mitochondrial exchange through TNTs following 5-FU treatment. 5-FU-induced TNT formation was inhibited by over 80% following treatment with the F-actin-depolymerizing agent, cytochalasin B (cytoB). The inhibition of TNTs by cytoB corresponded with increased 5-FU-induced cytotoxicity by 30-62% starting at 48 h, suggesting TNT formation aides in MCF-7 cell survival against 5-FU. Two other widely used chemotherapy agents, docetaxel and doxorubicin induced TNT formation at much lower levels than 5-FU. Our work suggests that the therapeutic targeting of TNTs may increase 5-FU chemotherapy efficacy and decrease drug resistance in cancer cells, and these findings merits further investigation.


Assuntos
Neoplasias da Mama , Neoplasias da Mama/tratamento farmacológico , Comunicação Celular , Estruturas da Membrana Celular , Feminino , Fluoruracila/farmacologia , Humanos , Células MCF-7 , Nanotubos
6.
Methods Mol Biol ; 2424: 199-216, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34918297

RESUMO

In vivo and ex vivo analyses of omental adhesion in ovarian cancer (OvCa) are necessary to understand the dynamics of OvCa metastasis. Here we describe methods to analyze OvCa omental adhesion, including in vivo and ex vivo fluorescent imaging, advanced microscopy, and histological techniques. The use of fluorescently tagged OvCa cells allows for omental tumor visualization and quantification in adhesion and tumor studies. Additionally, advanced microscopy modalities allow for visualization and multiplexed analysis of OvCa omental adhesion. Second harmonic generation microscopy permits the visualization and analysis of omental collagen, specifically the tumor-associated collagen signature that forms as the tumor progresses. Scanning electron microscopy is used for the observation of microscopic details between OvCa cells and the omentum, such as tunneling nanotubes or microvilli. Histological methods are used to investigate several intratumoral properties including visualizing tumor structure using hematoxylin and eosin stain; quantifying collagen with Masson's trichrome stain; analyzing collagen structure with a collagen hybridizing peptide; and identifying a number of markers including, but not limited to proliferation, immune cell types, adhesion molecules, and fibroblasts with immunohistochemistry. Both the in vivo and ex vivo imaging modalities and subsequent analysis can provide insight into the interaction of metastasizing OvCa cells and the omentum.


Assuntos
Omento , Neoplasias Ovarianas , Carcinoma Epitelial do Ovário , Estruturas da Membrana Celular , Colágeno , Feminino , Humanos , Nanotubos
7.
Neurosci Lett ; 769: 136392, 2022 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-34902517

RESUMO

Stem cells from human exfoliated deciduous teeth (SHED) have stromal-derived inducing activity (SDIA): which means these stromal cells induce neural differentiation where they are used as a substratum for embryonic stem cell (ESCs) culture. Recent studies show that mitochondria or mitochondrial products, as paracrine factors, can be released and transferred from one cell to another. With this information, we were curious to know whether in the SDIA co-culture system, SHED release or donate their mitochondria to ESCs. For this purpose, before co-culture, SHED s' mitochondria and ESCs s' cell membranes were separately labeled with specific fluorescent probes. After co-culture, SHED s' mitochondria were tracked by fluorescent microscope and flow cytometry analysis. Co-culture also performed in the presence of inhibitors that block probable transfer pathways suchlike tunneling nanotubes, gap junctions or vesicles. Results showed that mitochondrial transfer takes place from SHED to ESCs. This transfer partly occurs by tunneling nanotubes and not through gap junctions or vesicles; also was not dependent on intracellular calcium level. This kind of horizontal gene transfer may open a new prospect for further research on probable role of mitochondria on fate choice and neural induction processes.


Assuntos
Comunicação Celular , Estruturas da Membrana Celular/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/fisiologia , Cálcio/metabolismo , Linhagem Celular , Técnicas de Cocultura/métodos , Matriz Extracelular/metabolismo , Junções Comunicantes/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Mitocôndrias/metabolismo , Nanotubos , Dente Decíduo/citologia
8.
Biochem J ; 478(22): 3977-3998, 2021 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-34813650

RESUMO

Tunneling nanotubes (TNTs) are F-actin-based, membrane-enclosed tubular connections between animal cells that transport a variety of cellular cargo. Over the last 15 years since their discovery, TNTs have come to be recognized as key players in normal cell communication and organism development, and are also exploited for the spread of various microbial pathogens and major diseases like cancer and neurodegenerative disorders. TNTs have also been proposed as modalities for disseminating therapeutic drugs between cells. Despite the rapidly expanding and wide-ranging relevance of these structures in both health and disease, there is a glaring dearth of molecular mechanistic knowledge regarding the formation and function of these important but enigmatic structures. A series of fundamental steps are essential for the formation of functional nanotubes. The spatiotemporally controlled and directed modulation of cortical actin dynamics would be required to ensure outward F-actin polymerization. Local plasma membrane deformation to impart negative curvature and membrane addition at a rate commensurate with F-actin polymerization would enable outward TNT elongation. Extrinsic tactic cues, along with cognate intrinsic signaling, would be required to guide and stabilize the elongating TNT towards its intended target, followed by membrane fusion to create a functional TNT. Selected cargoes must be transported between connected cells through the action of molecular motors, before the TNT is retracted or destroyed. This review summarizes the current understanding of the molecular mechanisms regulating these steps, also highlighting areas that deserve future attention.


Assuntos
Comunicação Celular , Animais , Transporte Biológico , Linhagem Celular , Membrana Celular , Estruturas da Membrana Celular/imunologia , Estruturas da Membrana Celular/metabolismo , Estruturas da Membrana Celular/ultraestrutura , Humanos , Fusão de Membrana , Nanotubos/ultraestrutura
9.
Faraday Discuss ; 232(0): 358-374, 2021 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-34647559

RESUMO

Heterogeneity in cell membrane structure, typified by microdomains with different biophysical and biochemical properties, is thought to impact on a variety of cell functions. Integral membrane proteins act as nanometre-sized probes of the lipid environment and their thermally-driven movements can be used to report local variations in membrane properties. In the current study, we have used total internal reflection fluorescence microscopy (TIRFM) combined with super-resolution tracking of multiple individual molecules, in order to create high-resolution maps of local membrane viscosity. We used a quadrat sampling method and show how statistical tests for membrane heterogeneity can be conducted by analysing the paths of many molecules that pass through the same unit area of membrane. We describe experiments performed on cultured primary cells, stable cell lines and ex vivo tissue slices using a variety of membrane proteins, under different imaging conditions. In some cell types, we find no evidence for heterogeneity in mobility across the plasma membrane, but in others we find statistically significant differences with some regions of membrane showing significantly higher viscosity than others.


Assuntos
Proteínas de Membrana , Imagem Individual de Molécula , Membrana Celular , Estruturas da Membrana Celular , Microscopia de Fluorescência
10.
BMC Mol Cell Biol ; 22(1): 52, 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34615464

RESUMO

BACKGROUND: Mitochondrial DNA (mtDNA) carrying certain pathogenic mutations or single nucleotide variants (SNVs) enhances the invasion and metastasis of tumor cells, and some of these mutations are homoplasmic in tumor cells and even in tumor tissues. On the other hand, intercellular transfer of mitochondria and cellular components via extracellular vesicles (EVs) and tunneling nanotubes (TNTs) has recently attracted intense attention in terms of cell-to-cell communication in the tumor microenvironment. It remains unclear whether metastasis-enhancing pathogenic mutant mtDNA in tumor cells is intercellularly transferred between tumor cells and stromal cells. In this study, we investigated whether mtDNA with the NADH dehydrogenase subunit 6 (ND6) G13997A pathogenic mutation in highly metastatic cells can be horizontally transferred to low-metastatic cells and stromal cells in the tumor microenvironment. RESULTS: When MitoTracker Deep Red-labeled high-metastatic Lewis lung carcinoma A11 cells carrying the ND6 G13997A mtDNA mutation were cocultured with CellLight mitochondria-GFP-labeled low-metastatic P29 cells harboring wild-type mtDNA, bidirectional transfer of red- and green-colored vesicles, probably mitochondria-related EVs, was observed in a time-dependent manner. Similarly, intercellular transfer of mitochondria-related EVs occurred between A11 cells and α-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts (CAFs, WA-mFib), macrophages (RAW264.7) and cytotoxic T cells (CTLL-2). Intercellular transfer was suppressed by inhibitors of EV release. The large and small EV fractions (L-EV and S-EV, respectively) prepared from the conditioned medium by differential ultracentrifugation both were found to contain mtDNA, although only S-EVs were efficiently incorporated into the cells. Several subpopulations had evidence of LC3-II and contained degenerated mitochondrial components in the S-EV fraction, signaling to the existence of autophagy-related S-EVs. Interestingly, the S-EV fraction contained a MitoTracker-positive subpopulation, which was inhibited by the respiration inhibitor antimycin A, indicating the presence of mitochondria with membrane potential. It was also demonstrated that mtDNA was transferred into mtDNA-less ρ0 cells after coculture with the S-EV fraction. In syngeneic mouse subcutaneous tumors formed by a mixture of A11 and P29 cells, the mitochondria-related EVs released from A11 cells reached distantly positioned P29 cells and CAFs. CONCLUSIONS: These results suggest that metastasis-enhancing pathogenic mtDNA derived from metastatic tumor cells is transferred to low-metastatic tumor cells and stromal cells via S-EVs in vitro and in the tumor microenvironment, inferring a novel mechanism of enhancement of metastatic potential during tumor progression.


Assuntos
DNA Mitocondrial , Vesículas Extracelulares , Animais , Estruturas da Membrana Celular , DNA Mitocondrial/genética , Vesículas Extracelulares/metabolismo , Camundongos , Mitocôndrias/genética , Mutação , Nanotubos , Células Estromais
11.
Biol Futur ; 72(1): 25-36, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34554502

RESUMO

Nanotubular connections between mammalian cell types came into the focus only two decades ago, when "live cell super-resolution imaging" was introduced. Observations of these long-time overlooked structures led to understanding mechanisms of their growth/withdrawal and exploring some key genetic and signaling factors behind their formation. Unbelievable level of multiple supportive collaboration between tumor cells undergoing cytotoxic chemotherapy, cross-feeding" between independent bacterial strains or "cross-dressing" collaboration of immune cells promoting cellular immune response, all via nanotubes, have been explored recently. Key factors and "calling signals" determining the spatial directionality of their growth and their overall in vivo significance, however, still remained debated. Interestingly, prokaryotes, including even ancient archaebacteria, also seem to use such NT connections for intercellular communication. Herein, we will give a brief overview of current knowledge of membrane nanotubes and depict a simple model about their possible "historical role".


Assuntos
Comunicação Celular/fisiologia , Estruturas da Membrana Celular/fisiologia , Sistema Imunitário/fisiologia , Nanotubos/química , Animais , Transporte Biológico/fisiologia , Células Cultivadas , Humanos , Sistema Imunitário/citologia , Modelos Biológicos , Nanotubos/ultraestrutura , Células Procarióticas/fisiologia
12.
Cell ; 184(20): 5089-5106.e21, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34555357

RESUMO

Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of inflammatory pathways. Here, we studied how microglia handle and cope with α-synuclein (α-syn) fibrils and their clearance. We found that microglia exposed to α-syn establish a cellular network through the formation of F-actin-dependent intercellular connections, which transfer α-syn from overloaded microglia to neighboring naive microglia where the α-syn cargo got rapidly and effectively degraded. Lowering the α-syn burden attenuated the inflammatory profile of microglia and improved their survival. This degradation strategy was compromised in cells carrying the LRRK2 G2019S mutation. We confirmed the intercellular transfer of α-syn assemblies in microglia using organotypic slice cultures, 2-photon microscopy, and neuropathology of patients. Together, these data identify a mechanism by which microglia create an "on-demand" functional network in order to improve pathogenic α-syn clearance.


Assuntos
Estruturas da Membrana Celular/metabolismo , Microglia/metabolismo , Proteólise , alfa-Sinucleína/metabolismo , Actinas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Citoesqueleto/metabolismo , Regulação para Baixo , Feminino , Humanos , Inflamação/genética , Inflamação/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Microglia/patologia , Microglia/ultraestrutura , Mitocôndrias/metabolismo , Nanotubos , Agregados Proteicos , Espécies Reativas de Oxigênio/metabolismo , Transcriptoma/genética
13.
Lab Invest ; 101(12): 1571-1584, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34537825

RESUMO

Osteoclasts are multinucleated cells formed through specific recognition and fusion of mononuclear osteoclast precursors derived from hematopoietic stem cells. Detailed cellular events concerning cell fusion in osteoclast differentiation remain ambiguous. Tunneling nanotubes (TNTs), actin-based membrane structures, play an important role in intercellular communication between cells. We have previously reported the presence of TNTs in the fusion process of osteoclastogenesis. Here we analyzed morphological details of TNTs using scanning electron microscopy. The osteoclast precursor cell line RAW-D was stimulated to form osteoclast-like cells, and morphological details in the appearance of TNTs were extensively analyzed. Osteoclast-like cells could be classified into three types; early osteoclast precursors, late osteoclast precursors, and multinucleated osteoclast-like cells based on the morphological characteristics. TNTs were frequently observed among these three types of cells. TNTs could be classified into thin, medium, and thick TNTs based on the diameter and length. The shapes of TNTs were dynamically changed from thin to thick. Among them, medium TNTs were often observed between two remote cells, in which side branches attached to the culture substrates and beaded bulge-like structures were often observed. Cell-cell interaction through TNTs contributed to cell migration and rapid transport of information between cells. TNTs were shown to be involved in cell-cell fusion between osteoclast precursors and multinucleated osteoclast-like cells, in which movement of membrane vesicles and nuclei was observed. Formation of TNTs was also confirmed in primary cultures of osteoclasts. Furthermore, we have successfully detected TNTs formed between osteoclasts observed in the bone destruction sites of arthritic rats. Thus, formation of TNTs may be important for the differentiation of osteoclasts both in vitro and in vivo. TNTs could be one target cellular structure for the regulation of osteoclast differentiation and function in bone diseases.


Assuntos
Estruturas da Membrana Celular/ultraestrutura , Nanotubos/ultraestrutura , Osteogênese , Animais , Fusão Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos Endogâmicos Lew
14.
Biol Open ; 10(9)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34494646

RESUMO

Well-orchestrated intercellular communication networks are pivotal to maintaining cardiac homeostasis and to ensuring adaptative responses and repair after injury. Intracardiac communication is sustained by cell-cell crosstalk, directly via gap junctions (GJ) and tunneling nanotubes (TNT), indirectly through the exchange of soluble factors and extracellular vesicles (EV), and by cell-extracellular matrix (ECM) interactions. GJ-mediated communication between cardiomyocytes and with other cardiac cell types enables electrical impulse propagation, required to sustain synchronized heart beating. In addition, TNT-mediated organelle transfer has been associated with cardioprotection, whilst communication via EV plays diverse pathophysiological roles, being implicated in angiogenesis, inflammation and fibrosis. Connecting various cell populations, the ECM plays important functions not only in maintaining the heart structure, but also acting as a signal transducer for intercellular crosstalk. Although with distinct etiologies and clinical manifestations, intercellular communication derailment has been implicated in several cardiac disorders, including myocardial infarction and hypertrophy, highlighting the importance of a comprehensive and integrated view of complex cell communication networks. In this review, I intend to provide a critical perspective about the main mechanisms contributing to regulate cellular crosstalk in the heart, which may be considered in the development of future therapeutic strategies, using cell-based therapies as a paradigmatic example. This Review has an associated Future Leader to Watch interview with the author.


Assuntos
Comunicação Celular/fisiologia , Estruturas da Membrana Celular/fisiologia , Junções Comunicantes/fisiologia , Cardiopatias/fisiopatologia , Miocárdio/citologia , Matriz Extracelular/fisiologia , Humanos , Miócitos Cardíacos/fisiologia , Nanotubos
15.
Avian Dis ; 65(3): 335-339, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34427404

RESUMO

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that causes immunosuppression, T cell lymphomas, and neuropathic disease in infected chickens. To protect chickens from MDV infection, an avirulent live vaccine of turkey herpesvirus (HVT) has been successfully used in chickens worldwide. Many vaccine manufacturers have used chicken embryo fibroblast (CEF) cells to produce the HVT vaccine. Generally, it has been suggested that HVT is a highly cell-associated herpesvirus that spread via cell-to-cell contact, but it is unclear how HVT is transmitted from infected cells to uninfected target cells. Here, we show via immunofluorescence analysis that nanotubes containing the actin cytoskeleton and HVT antigens from infected CEF cells were observed to contact neighboring cells. When the infected cells were treated with inhibitors for actin polymerization or depolymerization, the formation and extension of the nanotubes from infected cells were greatly inhibited and the intercellular contact was abolished, leading to a drastic reduction in plaque formation and viral titers of the cell-associated virus. Our data indicate that cell-to-cell contacts via nanotubes composed of actin filaments are essential for efficient viral spreading and replication. This finding might contribute to the further improvement of efficient HVT vaccine production.


Nota de investigación­Transmisión de célula a célula del virus herpes del pavo en células embrionarias de pollo a través de tunelización por nanotubos. El virus de la enfermedad de Marek (MDV) es un alfaherpesvirus oncogénico que causa inmunosupresión, linfomas de células T y enfermedad neuropática en pollos infectados. Para proteger a los pollos de la infección por MDV, se ha utilizado con éxito una vacuna viva avirulenta del virus herpes del pavo (HVT) en pollos de todo el mundo. Muchos fabricantes de vacunas han utilizado células de fibroblasto de embrión de pollo (CEF) para producir la vacuna HVT. En general, se ha sugerido que el HVT es un virus herpes muy asociado a células que se propaga mediante el contacto entre células, pero no está claro cómo se transmite el virus HVT a partir de las células infectadas a las células blanco no infectadas. Aquí, se demuestra mediante análisis de inmunofluorescencia que nanotubos que contienen el citoesqueleto de actina y los antígenos del HVT dentro las células de fibroblasto de embrión de pollo infectadas son lo que contactan con las células vecinas. Cuando las células infectadas se trataron con inhibidores para la polimerización o despolimerización de actina, la formación y extensión de los nanotubos de las células infectadas se inhibió en gran medida y se abolió el contacto intercelular, lo que llevó a una reducción drástica en la formación de placa y de los títulos virales de virus asociados a células. Estos datos indican que los contactos entre células a través de nanotubos compuestos de filamentos de actina son esenciales para la propagación y replicación viral eficiente. Este hallazgo podría contribuir a la mejora adicional de la producción eficiente de vacunas HVT.


Assuntos
Herpesvirus Galináceo 2 , Doença de Marek , Nanotubos , Animais , Estruturas da Membrana Celular , Embrião de Galinha , Galinhas , Herpesvirus Meleagrídeo 1 , Doença de Marek/prevenção & controle
16.
Oxid Med Cell Longev ; 2021: 6697861, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34373767

RESUMO

Cellular senescence is a state of irreversible cell proliferation arrest induced by various stressors including telomere attrition, DNA damage, and oncogene induction. While beneficial as an acute response to stress, the accumulation of senescent cells with increasing age is thought to contribute adversely to the development of cancer and a number of other age-related diseases, including neurodegenerative diseases for which there are currently no effective disease-modifying therapies. Non-cell-autonomous effects of senescent cells have been suggested to arise through the SASP, a wide variety of proinflammatory cytokines, chemokines, and exosomes secreted by senescent cells. Here, we report an additional means of cell communication utilised by senescent cells via large numbers of membrane-bound intercellular bridges-or tunnelling nanotubes (TNTs)-containing the cytoskeletal components actin and tubulin, which form direct physical connections between cells. We observe the presence of mitochondria in these TNTs and show organelle transfer through the TNTs to adjacent cells. While transport of individual mitochondria along single TNTs appears by time-lapse studies to be unidirectional, we show by differentially labelled co-culture experiments that organelle transfer through TNTs can occur between different cells of equivalent cell age, but that senescent cells, rather than proliferating cells, appear to be predominant mitochondrial donors. Using small molecule inhibitors, we demonstrate that senescent cell TNTs are dependent on signalling through the mTOR pathway, which we further show is mediated at least in part through the downstream actin-cytoskeleton regulatory factor CDC42. These findings have significant implications for the development of senomodifying therapies, as they highlight the need to account for local direct cell-cell contacts as well as the SASP in order to treat cancer and diseases of ageing in which senescence is a key factor.


Assuntos
Estruturas da Membrana Celular/metabolismo , Senescência Celular , Citoesqueleto/metabolismo , Mitocôndrias/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Nanotubos
17.
J Neuroinflammation ; 18(1): 124, 2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34082772

RESUMO

BACKGROUND: Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by brain accumulation of aggregated amyloid-beta (Aß) and alpha-synuclein (αSYN), respectively. In order to develop effective therapies, it is crucial to understand how the Aß/αSYN aggregates can be cleared. Compelling data indicate that neuroinflammatory cells, including astrocytes and microglia, play a central role in the pathogenesis of AD and PD. However, how the interplay between the two cell types affects their clearing capacity and consequently the disease progression remains unclear. METHODS: The aim of the present study was to investigate in which way glial crosstalk influences αSYN and Aß pathology, focusing on accumulation and degradation. For this purpose, human-induced pluripotent cell (hiPSC)-derived astrocytes and microglia were exposed to sonicated fibrils of αSYN or Aß and analyzed over time. The capacity of the two cell types to clear extracellular and intracellular protein aggregates when either cultured separately or in co-culture was studied using immunocytochemistry and ELISA. Moreover, the capacity of cells to interact with and process protein aggregates was tracked using time-lapse microscopy and a customized "close-culture" chamber, in which the apical surfaces of astrocyte and microglia monocultures were separated by a <1 mm space. RESULTS: Our data show that intracellular deposits of αSYN and Aß are significantly reduced in co-cultures of astrocytes and microglia, compared to monocultures of either cell type. Analysis of conditioned medium and imaging data from the "close-culture" chamber experiments indicate that astrocytes secrete a high proportion of their internalized protein aggregates, while microglia do not. Moreover, co-cultured astrocytes and microglia are in constant contact with each other via tunneling nanotubes and other membrane structures. Notably, our live cell imaging data demonstrate that microglia, when attached to the cell membrane of an astrocyte, can attract and clear intracellular protein deposits from the astrocyte. CONCLUSIONS: Taken together, our data demonstrate the importance of astrocyte and microglia interactions in Aß/αSYN clearance, highlighting the relevance of glial cellular crosstalk in the progression of AD- and PD-related brain pathology.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Astrócitos/metabolismo , Astrócitos/patologia , Microglia/metabolismo , Microglia/patologia , Agregados Proteicos , Agregação Patológica de Proteínas , alfa-Sinucleína/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Estruturas da Membrana Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Humanos , Células-Tronco Pluripotentes Induzidas , Microscopia Confocal , Nanotubos , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteólise
18.
J Pineal Res ; 71(1): e12747, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34085316

RESUMO

Mitochondrial dysfunction is considered one of the hallmarks of ischemia/reperfusion injury. Mitochondria are plastic organelles that undergo continuous biogenesis, fusion, and fission. They can be transferred between cells through tunneling nanotubes (TNTs), dynamic structures that allow the exchange of proteins, soluble molecules, and organelles. Maintaining mitochondrial dynamics is crucial to cell function and survival. The present study aimed to assess the effects of melatonin on mitochondrial dynamics, TNT formation, and mitochondria transfer in HT22 cells exposed to oxygen/glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin treatment during the reoxygenation phase reduced mitochondrial reactive oxygen species (ROS) production, improved cell viability, and increased the expression of PGC1α and SIRT3. Melatonin also preserved the expression of the membrane translocase proteins TOM20 and TIM23, and of the matrix protein HSP60, which are involved in mitochondrial biogenesis. Moreover, it promoted mitochondrial fusion and enhanced the expression of MFN2 and OPA1. Remarkably, melatonin also fostered mitochondrial transfer between injured HT22 cells through TNT connections. These results provide new insights into the effect of melatonin on mitochondrial network reshaping and cell survival. Fostering TNTs formation represents a novel mechanism mediating the protective effect of melatonin in ischemia/reperfusion injury.


Assuntos
Isquemia Encefálica/patologia , Estruturas da Membrana Celular/efeitos dos fármacos , Melatonina/farmacologia , Mitocôndrias/efeitos dos fármacos , Neurônios/ultraestrutura , Animais , Linhagem Celular , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/ultraestrutura , Camundongos , Mitocôndrias/metabolismo , Nanotubos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Traumatismo por Reperfusão/patologia
19.
Appl Opt ; 60(17): 5081-5086, 2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34143073

RESUMO

A new, to the best of our knowledge, method for Stokes vector imaging is proposed to achieve imaging and dynamic monitoring of a non-labeled cytomembrane. In this work, a polarization state vector is described by a Stokes vector and expressed in chrominance space. A physical quantity called polarization chromaticity value (PCV) corresponding to a Stokes vector is used as the imaging parameter to perform Stokes vector imaging. By using the PCV imaging technique, the Stokes vector can be expressed in three-dimensional real space rather than in a Poincare sphere. Furthermore, a four-way Stokes parameter confocal microscopy system is designed to measure four Stokes parameters simultaneously and obtain micro-imaging. Label-free living onion cell membranes and their plasmolysis process are selected as the representative micro-anisotropy experimental analysis. It is proved that PCV imaging can perform visualization of cytomembranes, and further, microscopic orientation is demonstrated. The prospect of universal measurement of anisotropy details for analysis and diagnosis is provided.


Assuntos
Estruturas da Membrana Celular/fisiologia , Microscopia Confocal/métodos , Microscopia de Polarização/métodos , Cebolas/citologia , Imagem Óptica/métodos , Células Vegetais/fisiologia , Anisotropia , Interpretação de Imagem Assistida por Computador
20.
J Histochem Cytochem ; 69(6): 407-414, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33884901

RESUMO

During autophagy, autophagosomes are formed to engulf cytoplasmic contents. p62/SQSTM-1 is an autophagic adaptor protein that forms p62 bodies. A unique feature of p62 bodies is that they seem to directly associate with membranous structures. We first investigated the co-localization of mKate2-p62 bodies with phospholipids using click chemistry with propargyl-choline. Propargyl-choline-labeled phospholipids were detected inside the mKate2-p62 bodies, suggesting that phospholipids were present inside the bodies. To clarify whether or not p62 bodies come in contact with membranous structures directly, we investigated the ultrastructures of p62 bodies using in-resin correlative light and electron microscopy of the Epon-embedded cells expressing mKate2-p62. Fluorescent-positive p62 bodies were detected as uniformly lightly osmificated structures by electron microscopy. Membranous structures were detected on and inside the p62 bodies. In addition, multimembranous structures with rough endoplasmic reticulum-like structures that resembled autophagosomes directly came in contact with amorphous-shaped p62 bodies. These results suggested that p62 bodies are unique structures that can come in contact with membranous structures directly.


Assuntos
Autofagia , Estruturas da Membrana Celular/metabolismo , Proteína Sequestossoma-1/metabolismo , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Estruturas da Membrana Celular/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Células HeLa , Humanos , Fosfolipídeos/metabolismo , Proteína Sequestossoma-1/análise
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