Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 7.771
Filtrar
1.
Nat Commun ; 10(1): 4694, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619676

RESUMO

Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of bio-complexes. However, current techniques remain difficult to resolve the dipole assemblies on subcellular structures and their dynamics in living cells at super-resolution level. Here we report polarized structured illumination microscopy (pSIM), which achieves super-resolution imaging of dipoles by interpreting the dipoles in spatio-angular hyperspace. We demonstrate the application of pSIM on a series of biological filamentous systems, such as cytoskeleton networks and λ-DNA, and report the dynamics of short actin sliding across a myosin-coated surface. Further, pSIM reveals the side-by-side organization of the actin ring structures in the membrane-associated periodic skeleton of hippocampal neurons and images the dipole dynamics of green fluorescent protein-labeled microtubules in live U2OS cells. pSIM applies directly to a large variety of commercial and home-built SIM systems with various imaging modality.


Assuntos
Actinas/ultraestrutura , Citoesqueleto/ultraestrutura , DNA/ultraestrutura , Microscopia de Fluorescência/métodos , Microscopia de Polarização/métodos , Miosinas/ultraestrutura , Neurônios/ultraestrutura , Animais , Bacteriófago lambda/genética , Linhagem Celular Tumoral , Hipocampo/citologia , Humanos , Rim , Camundongos
2.
PLoS Biol ; 17(10): e3000475, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31584943

RESUMO

The Toxoplasma gondii inner membrane complex (IMC) is an important organelle involved in parasite motility and replication. The IMC resides beneath the parasite's plasma membrane and is composed of both membrane and cytoskeletal components. Although the protein composition of the IMC is becoming better understood, the protein-protein associations that enable proper functioning of the organelle remain largely unknown. Determining protein interactions in the IMC cytoskeletal network is particularly challenging, as disrupting the cytoskeleton requires conditions that disrupt protein complexes. To circumvent this problem, we demonstrate the application of a photoreactive unnatural amino acid (UAA) crosslinking system to capture protein interactions in the native intracellular environment. In addition to identifying binding partners, the UAA approach maps the binding interface of the bait protein used for crosslinking, providing structural information of the interacting proteins. We apply this technology to the essential IMC protein ILP1 and demonstrate that distinct regions of its C-terminal coiled-coil domain crosslink to the alveolins IMC3 and IMC6, as well as IMC27. We also show that the IMC3 C-terminal domain and the IMC6 N-terminal domain are necessary for binding to ILP1, further mapping interactions between ILP1 and the cytoskeleton. Together, this study develops a new approach to study protein-protein interactions in Toxoplasma and provides the first insight into the architecture of the cytoskeletal network of the apicomplexan IMC.


Assuntos
Azidas/química , Reagentes para Ligações Cruzadas/química , Proteínas do Citoesqueleto/química , Citoesqueleto/metabolismo , Membranas Intracelulares/metabolismo , Fenilalanina/análogos & derivados , Proteínas de Protozoários/química , Toxoplasma/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Expressão Gênica , Membranas Intracelulares/ultraestrutura , Fenilalanina/química , Processos Fotoquímicos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genética , Toxoplasma/ultraestrutura , Raios Ultravioleta
3.
Micron ; 126: 102750, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31522088

RESUMO

Globally, colon cancer is a predominant cause of increased morbidity and mortality annually; therefore, in addition to traditional treatments, new protocols are under continuous investigation. Nanotechnology-based cancer therapy is a new strategy and considered one of the most promising research directions for colon cancer. In this study, we used a silver nanoparticle (AgNP)-based methodology to treat colon cancer cells, and single cell approaches to examine how AgNPs exerted inhibiting effects on cells. We found that AgNPs could apparently destroy cytoskeleton and topography structures, alter cell membrane nanostructures, and thus increase membrane roughness, and depress cell membrane adhesion properties and cell stiffness. We also found that AgNPs caused mitochondrial dysfunctions including hyperpolarization of membrane potential and reactive oxygen species (ROS) accumulation. Notably, AgNPs altered all phenotypes or functions of cells in a dose-dependent manner. Therefore, our research provided a new paradigm for revealing killing mechanisms of AgNPs against colon cancer cells from single cell biophysical aspects, which could advance AgNP-based nanotechnology cancer therapy.


Assuntos
Membrana Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Citoesqueleto/efeitos dos fármacos , Nanopartículas Metálicas/química , Prata/farmacologia , Apoptose , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Humanos , Potencial da Membrana Mitocondrial , Microscopia de Força Atômica , Microtúbulos/efeitos dos fármacos , Prata/química , Análise de Célula Única
4.
Yonsei Med J ; 60(10): 890-897, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31538423

RESUMO

In 1993, I reported that Coxiella burnetii transforms human B cells into hairy cells (cbHCs), the first hairy cell reported outside of hairy cell leukemia (HCL). Over last few decades, advances in molecular biology have provided evidence supporting that C. burnetii induces hairiness and inhibits the apoptosis of host cells. The present review summarizes new information in support of cbHC. C. burnetii was shown to induce reorganization of the cytoskeleton and to inhibit apoptosis in host cells. Peritoneal B1a cells were found to be permissive for virulent C. burnetii Nine Mile phase I (NMI) strains in mice. C. burnetii severely impaired E-cad expression in circulating cells of Q fever patients. B-cell non-Hodgkin lymphoma was linked to C. burnetii. Mutation of BRAF V600E was pronounced in HCL, but "hairiness" was not linked to the mutation. Risk factors shared among coxiellosis and HCL in humans and animals were reported in patients with Q-fever. Accordingly, I propose that C. burnetii induces reorganization of the cytoskeleton and inhibits apoptosis as cytopathic effects that are not target cell specific. The observed hairiness in cbHC appears to be a fixed image of dynamic nature, and hairy cells in HCL are distinct among lymphoid cells in circulation. As the cytoskeleton plays key roles in maintaining cell structural integrity in health and disease, the pathophysiology of similar cytopathic effects should be addressed in other diseases, such as myopathies, B-cell dyscrasias, and autoimmune syndromes.


Assuntos
Coxiella burnetii/fisiologia , Citoesqueleto/metabolismo , Animais , Apoptose , Linfócitos B/microbiologia , Linfócitos B/ultraestrutura , Coxiella burnetii/ultraestrutura , Citoesqueleto/ultraestrutura , Humanos , Mutação/genética , Febre Q/microbiologia , Febre Q/patologia
5.
Protist ; 170(4): 385-396, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31493690

RESUMO

Ministeria vibrans (Filasterea) is a tiny amoeboid species described by Tong in 1997. It has been sporadically found in different habitats, and cultured strains were established. M. vibrans is well characterised by molecular phylogeny but until now was not ultrastructurally investigated in detail. Here, we provide the ultrastructure for this species based on a new strain isolated from oxygen-depleted water of the Baltic Sea. A thin vibrating flagellum could be observed but no vibrating movement of the cell body and no stalk. Our first ultrastructural study of a filasterean taxon revealed radial microvilli supported by bundles of microfilaments. Two centrioles located in the nuclear pit can migrate to the cell periphery and transform into the kinetid: the centriole orthogonal to the kinetosome with a fibrillar root and a basal foot that initiates microtubules. Microvilli in Ministeria suggest their presence in the common ancestor of Filasterea and Choanoflagellata. The kinetid structure of Ministeria is similar to that of the choanocytes of the most deep-branching sponges, differing essentially from the kinetid of choanoflagellates. Thus, kinetid and microvilli of Ministeria illustrate features of the common ancestor of three holozoan groups: Filasterea, Metazoa and Choanoflagellata.


Assuntos
Citoesqueleto/ultraestrutura , Eucariotos/ultraestrutura , Eucariotos/classificação , Microscopia Eletrônica de Transmissão , Filogenia , Especificidade da Espécie
6.
In Vitro Cell Dev Biol Anim ; 55(8): 641-655, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31297697

RESUMO

The equine hoof dermal-epidermal interface requires progenitor cells with distinct characteristics. This study was designed to provide accurate ultrastructural depictions of progenitor cells isolated from inflamed tissue and normal tissue before and after cryopreservation and following selection of cells expressing both keratin (K) 14 (ectodermal) and cluster of differentiation (CD) 105 (mesodermal). Passage 3 cell ultrastructure was assessed following 2D culture and after 3D culture on decellularized hoof tissue scaffolds. Outcome measures included light, transmission electron, and scanning electron microscopy, immunocytochemistry, and CD105+K14+ cell trilineage plasticity. Cells from normal tissue had typical progenitor cell characteristics. Those from inflamed tissue had organelles and morphology consistent with catabolic activities including lysosomes, irregular rough endoplasmic reticulum, and fewer vacuoles and early endosomes than those from normal tissue. Cryopreserved tissue cells appeared apoptotic with an irregular cell membrane covered by cytoplasmic protrusions closely associated with endocytic and exocytic vesicles, chromatin aggregated on the nuclear envelop, abundant, poorly organized rough endoplasmic reticulum, and plentiful lysosomes. Cells that were CD105+K14+ were distinguishable from heterogenous cells by infrequent microvilli on the cell surface, sparse endosomes and vesicles, and desmosomes between cells. Cells expressed ectodermal (K15) and mesodermal (CD105) proteins in 2D and 3D cultures. Inflamed and cryopreserved tissue isolates attached poorly to tissue scaffold while normal tissue cells attached well, but only CD105+K14+ cells produced extracellular matrix after 4 d. The CD105+K14+ cells exhibited osteoblastic, adipocytic, and neurocytic differentiation. Ultrastructural information provided by this study contributes to understanding of equine hoof progenitor cells to predict their potential contributions to tissue maintenance, healing, and damage as well post-implantation behavior.


Assuntos
Separação Celular , Criopreservação , Endoglina/metabolismo , Casco e Garras/patologia , Casco e Garras/ultraestrutura , Inflamação/patologia , Queratina-14/metabolismo , Células-Tronco/ultraestrutura , Animais , Diferenciação Celular , Linhagem da Célula , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Feminino , Cavalos , Masculino
7.
Nanoscale ; 11(27): 13089-13097, 2019 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-31268074

RESUMO

Atomic force microscopy allows for the nanomechanical surface characterization of a multitude of types of materials with highest spatial precision in various relevant environments. In recent years, researchers have refined this methodology to analyze living biological materials in vitro. The atomic force microscope thus has become an essential instrument for the (in many cases) non-destructive, high-resolution imaging of cells and visualization of their dynamic mechanical processes. Mapping force versus distance curves and the local evaluation of soft samples allow the operator to "see" beneath the sample surface and to capture the local mechanical properties. In this work, we combine atomic force microscopy with fluorescence microscopy to investigate cancerous epithelial breast cells in culture medium. With unprecedented spatial resolution, we provide tomographic images for the local elasticity of confluent layers of cells. For these particular samples, a layer of higher elastic modulus located directly beneath the cell membrane in comparison with the average elastic properties was observed. Strikingly, this layer appears to be perforated at unique locations of the sample surface of weakest mechanical properties where distinct features were visible permitting the tip to indent farthest into the cell's volume. We interpret this layer as the cell membrane mechanically supported by the components of the cytoskeleton that is populated with sites of integral membrane proteins. These proteins act as breaking points for the indenter thus explaining the mechanical weakness at these locations. In contrast, the highest mechanical strength of the cell was found at locations of the cell cores as cross-checked by fluorescence microscopy images of staining experiments, in particular at nucleoli sites as the cumulative elastic modulus there comprises cytoskeletal features and the tight packing ribosomal DNA of the cell.


Assuntos
Citoesqueleto , Módulo de Elasticidade , Proteínas de Membrana/metabolismo , Microscopia de Força Atômica , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Humanos , Microscopia de Fluorescência
8.
Parasitol Res ; 118(9): 2651-2667, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31270680

RESUMO

Representatives of Apicomplexa perform various kinds of movements that are linked to the different stages of their life cycle. Ancestral apicomplexan lineages, including gregarines, represent organisms suitable for research into the evolution and diversification of motility within the group. The vermiform trophozoites and gamonts of the archigregarine Selenidium pygospionis perform a very active type of bending motility. Experimental assays and subsequent light, electron, and confocal microscopic analyses demonstrated the fundamental role of the cytoskeletal proteins actin and tubulin in S. pygospionis motility and allowed us to compare the mechanism of its movement to the gliding machinery (the so-called glideosome concept) described in apicomplexan zoites. Actin-modifying drugs caused a reduction in the movement speed (cytochalasin D) or stopped the motility of archigregarines completely (jasplakinolide). Microtubule-disrupting drugs (oryzalin and colchicine) had an even more noticeable effect on archigregarine motility. The fading and disappearance of microtubules were documented in ultrathin sections, along with the formation of α-tubulin clusters visible after the immunofluorescent labelling of drug-treated archigregarines. The obtained data indicate that subpellicular microtubules most likely constitute the main motor structure involved in S. pygospionis bending motility, while actin has rather a supportive function.


Assuntos
Apicomplexa/crescimento & desenvolvimento , Apicomplexa/fisiologia , Citoesqueleto/metabolismo , Proteínas de Protozoários/metabolismo , Actinas/metabolismo , Animais , Apicomplexa/ultraestrutura , Citoesqueleto/ultraestrutura , Tomografia com Microscopia Eletrônica , Microtúbulos/metabolismo , Parasitos , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/metabolismo , Trofozoítos/ultraestrutura , Tubulina (Proteína)/metabolismo
9.
PLoS One ; 14(7): e0219353, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31356645

RESUMO

INTRODUCTION: Focal and Segmental GlomeruloSclerosis (FSGS) can cause nephrotic syndrome with a risk of progression to end-stage renal disease. The idiopathic form has a high rate of recurrence after transplantation, suggesting the presence of a systemic circulating factor that causes glomerular permeability and can be removed by plasmapheresis or protein-A immunoadsorption. RESULTS: To identify this circulating factor, the eluate proteins bound on therapeutic immunoadsorption with protein-A columns were analyzed by comparative electrophoresis and mass spectrometry. A soluble form of calcium/calmodulin-dependent serine protein kinase (CASK) was identified. CASK was immunoprecipitated only in the sera of patients with recurrent FSGS after transplantation and not in control patients. Recombinant-CASK (rCASK) induced the reorganization of the actin cytoskeleton in immortalized podocytes, a redistribution of synaptopodin, ZO-1,vinculin and ENA. rCASK also induced alterations in the permeability of a monolayer of podocytes and increased the motility of pdodocytes in vitro. The extracellular domain of CD98, a transmembrane receptor expressed on renal epithelial cells, has been found to co-immunoprecipitated with rCASK. The invalidation of CD98 with siRNA avoided the structural changes of rCask treated cells suggesting its involvement in physiopathology of the disease. In mice, recombinant CASK induced proteinuria and foot process effacement in podocytes. CONCLUSION: Our results suggest that CASK can induce the recurrence of FSGS after renal transplantation.


Assuntos
Glomerulosclerose Segmentar e Focal/sangue , Guanilato Quinases/sangue , Transplante de Rim , Adulto , Animais , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Feminino , Proteína-1 Reguladora de Fusão/metabolismo , Glomerulosclerose Segmentar e Focal/complicações , Humanos , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Masculino , Membranas/metabolismo , Membranas/ultraestrutura , Camundongos , Pessoa de Meia-Idade , Podócitos/metabolismo , Podócitos/patologia , Podócitos/ultraestrutura , Ligação Proteica , Proteinúria/complicações , Recidiva
10.
PLoS One ; 14(6): e0218197, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31188903

RESUMO

BACKGROUND: Endothelialization of small diameter synthetic vascular grafts is a potential solution to the thrombosis and intimal hyperplasia that plague current devices. Endothelial colony forming cells, which are blood-derived and similar to mature endothelial cells, are a potential cell source. Anisotropic spatial growth restriction micropatterning has been previously shown to affect the morphology and function of mature endothelial cells in a manner similar to unidirectional fluid shear stress. To date, endothelial colony forming cells have not been successfully micropatterned. This study addresses the hypothesis that micropatterning of endothelial colony forming cells will induce morphological elongation, cytoskeletal alignment, and changes in immunogenic and thrombogenic-related gene expression. METHODS: Spatially growth restrictive test surfaces with 25 µm-wide lanes alternating between collagen-I and a blocking polymer were created using microfluidics. Case-matched endothelial colony forming cells and control mature carotid endothelial cells were statically cultured on either micropatterned or non-patterned surfaces. Cell elongation was quantified using shape index. Using confocal microscopy, cytoskeletal alignment was visualized and density and apoptotic rate were determined. Gene expression was measured using quantitative PCR to measure KLF-2, eNOS, VCAM-1, and vWF. RESULTS: Endothelial colony forming cells were successfully micropatterned for up to 50 hours. Micropatterned cells displayed elongation and actin alignment. Micropatterning increased the packing densities of both cell types, but did not affect apoptotic rate, which was lower in endothelial colony forming cells. KLF-2 gene expression was increased in micropatterned relative to non-patterned endothelial colony forming cells after 50 hours. No significant differences were seen in the other genes tested. CONCLUSIONS: Endothelial colony forming cells can be durably micropatterned using spatial growth restriction. Micropatterning has a significant effect on the gross and subcellular morphologies of both cell types. Further study is required to fully understand the effect of micropatterning on endothelial colony forming cell gene expression.


Assuntos
Forma Celular , Citoesqueleto/ultraestrutura , Células Endoteliais/ultraestrutura , Células-Tronco Hematopoéticas/ultraestrutura , Mecanotransdução Celular , Animais , Artérias Carótidas/citologia , Artérias Carótidas/metabolismo , Adesão Celular , Proliferação de Células , Citoesqueleto/metabolismo , Dimetilpolisiloxanos/química , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Dispositivos Lab-On-A-Chip , Masculino , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Papio anubis , Cultura Primária de Células , Estresse Mecânico , Propriedades de Superfície , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo
11.
Methods Mol Biol ; 1992: 135-149, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148036

RESUMO

The cortical microtubule and actin meshworks play a central role in the shaping of plant cells. Transgenic plants expressing fluorescent protein markers specifically tagging the two main cytoskeletal systems are available, allowing noninvasive in vivo studies. Advanced microscopy techniques, in particular confocal laser scanning microscopy (CLSM), spinning disk confocal microscopy (SDCM), and variable angle epifluorescence microscopy (VAEM), can be nowadays used for imaging the cortical cytoskeleton of living cells with unprecedented spatial and temporal resolution. With the aid of free computing tools based on the publicly available ImageJ software package, quantitative information can be extracted from microscopic images and video sequences, providing insight into both architecture and dynamics of the cortical cytoskeleton.


Assuntos
Arabidopsis/ultraestrutura , Citoesqueleto/ultraestrutura , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Células Vegetais/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Microtúbulos/ultraestrutura
12.
Methods Mol Biol ; 1992: 151-171, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148037

RESUMO

The microtubule cytoskeleton plays important roles in cell morphogenesis. To investigate the mechanisms of cytoskeletal organization, for example, during growth or development, in genetic studies, or in response to environmental stimuli, image analysis tools for quantitative assessment are needed. Here, we present a method for texture measure-based quantification and comparative analysis of global microtubule cytoskeleton patterns and subsequent visualization of output data. In contrast to other approaches that focus on the extraction of individual cytoskeletal fibers and analysis of their orientation relative to the growth axis, CytoskeletonAnalyzer2D quantifies cytoskeletal organization based on the analysis of local binary patterns. CytoskeletonAnalyzer2D thus is particularly well suited to study cytoskeletal organization in cells where individual fibers are difficult to extract or which lack a clearly defined growth axis, such as leaf epidermal pavement cells. The tool is available as ImageJ plugin and can be combined with publicly available software and tools, such as R and Cytoscape, to visualize similarity networks of cytoskeletal patterns.


Assuntos
Citoesqueleto/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Microtúbulos/ultraestrutura , Imagem Óptica/métodos , Tabaco/citologia , Actinas/ultraestrutura , Microscopia Confocal/métodos , Software , Tabaco/ultraestrutura
13.
Nat Plants ; 5(5): 498-504, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31040442

RESUMO

Cotton (Gossypium hirsutum) fibres consist of single cells that grow in a highly polarized manner, assumed to be controlled by the cytoskeleton1-3. However, how the cytoskeletal organization and dynamics underpin fibre development remains unexplored. Moreover, it is unclear whether cotton fibres expand via tip growth or diffuse growth2-4. We generated stable transgenic cotton plants expressing fluorescent markers of the actin and microtubule cytoskeleton. Live-cell imaging revealed that elongating cotton fibres assemble a cortical filamentous actin network that extends along the cell axis to finally form actin strands with closed loops in the tapered fibre tip. Analyses of F-actin network properties indicate that cotton fibres have a unique actin organization that blends features of both diffuse and tip growth modes. Interestingly, typical actin organization and endosomal vesicle aggregation found in tip-growing cell apices were not observed in fibre tips. Instead, endomembrane compartments were evenly distributed along the elongating fibre cells and moved bi-directionally along the fibre shank to the fibre tip. Moreover, plus-end tracked microtubules transversely encircled elongating fibre shanks, reminiscent of diffusely growing cells. Collectively, our findings indicate that cotton fibres elongate via a unique tip-biased diffuse growth mode.


Assuntos
Fibra de Algodão , Citoesqueleto/ultraestrutura , Gossypium/ultraestrutura , Actinas/ultraestrutura , Proteínas de Fluorescência Verde , Imagem Tridimensional , Microscopia Intravital/métodos , Microtúbulos/ultraestrutura
14.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052288

RESUMO

A critical aim in neuroscience is to obtain a comprehensive view of how regulated neurotransmission is achieved. Our current understanding of synapses relies mainly on data from electrophysiological recordings, imaging, and molecular biology. Based on these methodologies, proteins involved in a synaptic vesicle (SV) formation, mobility, and fusion at the active zone (AZ) membrane have been identified. In the last decade, electron tomography (ET) combined with a rapid freezing immobilization of neuronal samples opened a window for understanding the structural machinery with the highest spatial resolution in situ. ET provides significant insights into the molecular architecture of the AZ and the organelles within the presynaptic nerve terminal. The specialized sensory ribbon synapses exhibit a distinct architecture from neuronal synapses due to the presence of the electron-dense synaptic ribbon. However, both synapse types share the filamentous structures, also commonly termed as tethers that are proposed to contribute to different steps of SV recruitment and exocytosis. In this review, we discuss the emerging views on the role of filamentous structures in SV exocytosis gained from ultrastructural studies of excitatory, mainly central neuronal compared to ribbon-type synapses with a focus on inner hair cell (IHC) ribbon synapses. Moreover, we will speculate on the molecular entities that may be involved in filament formation and hence play a crucial role in the SV cycle.


Assuntos
Citoesqueleto/metabolismo , Exocitose , Células Receptoras Sensoriais/metabolismo , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Citoesqueleto/ultraestrutura , Humanos , Células Receptoras Sensoriais/ultraestrutura , Membranas Sinápticas/ultraestrutura , Transmissão Sináptica , Vesículas Sinápticas/ultraestrutura
15.
Biomed Res ; 40(2): 79-85, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30982803

RESUMO

The vitreous of perinatal mice temporarily develops a unique vascular system, called the vasa hyaloidea propria (VHP). Observations showed the vessels possessed an extracellular matrix including the basement membrane in their entire length. Immunostaining of whole mount preparations of VHP with integrin ß1 antibody displayed a bush-like network consisting of long and straight fibers which were associated with the VHP but extended apart from the blood vessels. Electron microscopically, each fiber was composed of a bundle of thin filaments different from collagen fibrils. Macrophages associated with the VHP appeared to be arrested by the integrin bushes. The integrin bushes fragmented and disappeared by postnatal day 10, just before the regression of the VHP. Macrophages were involved in the digestion and clearance of integrin bushes. The vitreous integrin bushes appear to provide a scaffold for architectural maintenance of the hyaloid vessels and macrophages.


Assuntos
Membrana Basal/ultraestrutura , Vasos Sanguíneos/ultraestrutura , Citoesqueleto/ultraestrutura , Matriz Extracelular/ultraestrutura , Integrina beta1/ultraestrutura , Corpo Vítreo/ultraestrutura , Animais , Animais Recém-Nascidos , Membrana Basal/metabolismo , Vasos Sanguíneos/anatomia & histologia , Citoesqueleto/metabolismo , Embrião de Mamíferos , Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Imuno-Histoquímica , Integrina beta1/genética , Integrina beta1/metabolismo , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Microscopia Eletrônica , Neovascularização Fisiológica , Gravidez , Corpo Vítreo/anatomia & histologia , Corpo Vítreo/irrigação sanguínea
16.
Methods Cell Biol ; 151: 379-397, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948020

RESUMO

The rapid development, simplicity and optical clarity of the sea urchin embryo make it an excellent model system for studying the dynamic events of early development. An ever-growing palette of fluorescent proteins and biosensors can now be applied to studying sea urchin development, and there are now a wide variety of imaging modes that can be employed to image sea urchin embryogenesis. However, when performing live-cell imaging, one must take into consideration the sensitivity of embryos (and fluorescent probes) to the intense light associated with confocal microscopes. Here, we discuss general considerations for keeping embryos viable on the microscope stage, as well as probes for imaging cellular membranes and the cytoskeleton. We compare the relative merits of different confocal microscopes for live imaging of embryos and describe the potential for live-cell super-resolution microscopy.


Assuntos
Desenvolvimento Embrionário/genética , Microscopia Confocal/métodos , Imagem Óptica/métodos , Ouriços-do-Mar/ultraestrutura , Animais , Citoesqueleto/ultraestrutura , Embrião não Mamífero , Corantes Fluorescentes/química , Microtúbulos/ultraestrutura , Ouriços-do-Mar/crescimento & desenvolvimento
17.
Methods Cell Biol ; 151: 419-432, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948022

RESUMO

The cellular cortex-consisting of the plasma membrane and the adjacent outer few microns of the cytoplasm-is a critically important, dynamic and complex region in the sea urchin egg and embryo. Some 40 years ago it was discovered that isolated cortices could be obtained from eggs adhered to glass coverslips and since that time this preparation has been used in a wide range of studies, including seminal research on fertilization, exocytosis, the cytoskeleton, and cytokinesis. In this chapter, we discuss methods for isolating cortices from eggs and embryos, including those undergoing cell division. We also provide protocols for analyzing cortical architecture and dynamics using specific localization methods combined with super-resolution Structured Illumination and Stimulated Emission Depletion light microscopy and platinum replica transmission electron microscopy.


Assuntos
Citoplasma/ultraestrutura , Imagem Molecular/métodos , Óvulo/ultraestrutura , Ouriços-do-Mar/ultraestrutura , Animais , Membrana Celular/ultraestrutura , Citoesqueleto/ultraestrutura , Embrião não Mamífero , Exocitose/genética , Fertilização/genética , Ouriços-do-Mar/crescimento & desenvolvimento
18.
Nat Cell Biol ; 21(5): 603-613, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988424

RESUMO

Mitochondrial fission involves the preconstriction of an organelle followed by scission by dynamin-related protein Drp1. Preconstriction is facilitated by actin and non-muscle myosin II through a mechanism that remains unclear, largely due to the unknown cytoskeletal ultrastructure at mitochondrial constrictions. Here, using platinum replica electron microscopy, we show that mitochondria in cells are embedded in an interstitial cytoskeletal network that contains abundant unbranched actin filaments. Both spontaneous and induced mitochondrial constrictions typically associate with a criss-cross array of long actin filaments that comprise part of this interstitial network. Non-muscle myosin II is found adjacent to mitochondria but is not specifically enriched at the constriction sites. During ionomycin-induced mitochondrial fission, F-actin clouds colocalize with mitochondrial constriction sites, whereas dynamic myosin II clouds are present in the vicinity of constrictions. We propose that myosin II promotes mitochondrial constriction by inducing stochastic deformations of the interstitial actin network, which applies pressure on the mitochondrial surface and thus initiates curvature-sensing mechanisms that complete mitochondrial constriction.


Assuntos
Actinas/genética , Citoesqueleto/ultraestrutura , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/genética , Miosina Tipo II/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Animais , Células COS , Constrição , Citoesqueleto/metabolismo , Ionomicina/farmacologia , Mitocôndrias/genética , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miosina Tipo II/química , Miosina Tipo II/metabolismo
19.
Nat Commun ; 10(1): 1249, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890704

RESUMO

Phagocytosis of invading pathogens or cellular debris requires a dramatic change in cell shape driven by actin polymerization. For antibody-covered targets, phagocytosis is thought to proceed through the sequential engagement of Fc-receptors on the phagocyte with antibodies on the target surface, leading to the extension and closure of the phagocytic cup around the target. We find that two actin-dependent molecular motors, class 1 myosins myosin 1e and myosin 1f, are specifically localized to Fc-receptor adhesions and required for efficient phagocytosis of antibody-opsonized targets. Using primary macrophages lacking both myosin 1e and myosin 1f, we find that without the actin-membrane linkage mediated by these myosins, the organization of individual adhesions is compromised, leading to excessive actin polymerization, slower adhesion turnover, and deficient phagocytic internalization. This work identifies a role for class 1 myosins in coordinated adhesion turnover during phagocytosis and supports a mechanism involving membrane-cytoskeletal crosstalk for phagocytic cup closure.


Assuntos
Actinas/metabolismo , Adesão Celular/fisiologia , Miosina Tipo I/metabolismo , Miosinas/metabolismo , Fagocitose/fisiologia , Actinas/ultraestrutura , Animais , Células da Medula Óssea , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Feminino , Microscopia Intravital , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Microscopia de Fluorescência , Miosina Tipo I/genética , Miosinas/genética , Cultura Primária de Células , Células RAW 264.7 , Receptores Fc/metabolismo , Receptores Fc/ultraestrutura , Imagem com Lapso de Tempo
20.
Nanoscale ; 11(14): 6561-6565, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30916110

RESUMO

Due to their high biocompatibility and nontoxic nature, carbogenic fluorescent nanodots (FNDs) have already shown their application in bioimaging. However, their non-specific labeling has restricted their application in live cell super resolution microscopy (SRM). Here we introduce, for the first time, an orange emissive FND, specifically conjugated to the HeLa cell actin filament, for successful single molecule stochastic optical reconstruction microscopy (STORM) and super resolution radial fluctuation (SRRF) microscopy. The resolution obtained in SRRF (∼35 nm) was almost an order of magnitude less than the diffraction limited spot. Interestingly, in addition, the FND also showed electron microscope (EM) contrast inside the cell. We hope that this FND will not only replace some of the common dyes used for SRM, but will also be used as a dual responsive marker in correlative super resolution microscopy (CLEM).


Assuntos
Corantes Fluorescentes/química , Microscopia Eletrônica , Nanoestruturas/química , Citoesqueleto/ultraestrutura , Células HeLa , Humanos , Microscopia Confocal , Faloidina/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA