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1.
Methods Mol Biol ; 2276: 397-407, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060057

RESUMO

Caenorhabditis elegans is a highly versatile model system, intensively used for functional, genetic, cytometric, and integrative studies. Due to its simplicity and large muscle cell number, C. elegans has frequently been used to study mitochondrial deficiencies caused by disease or drug toxicity. Here we describe a robust and efficient method to visualize and quantify mitochondrial morphology in vivo. This method has many practical and technical advantages above traditional (manual) methods and provides a comprehensive analysis of mitochondrial morphology.


Assuntos
Caenorhabditis elegans/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia Intravital/métodos , Microscopia Confocal/métodos , Mitocôndrias/ultraestrutura , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Mitocôndrias/metabolismo
2.
Methods Mol Biol ; 2268: 1-20, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085258

RESUMO

The understanding of how biological membranes are organized and how they function has constantly been evolving over the past decades. Instead of just serving as a medium in which specific proteins are located, certain parts of the lipid bilayer contribute to platforms that assemble signaling complexes by providing a microenvironment that facilitates effective protein-protein interactions. G protein-coupled receptors (GPCRs) and relevant signaling molecules, including the heterotrimeric G proteins, key enzymes such as kinases and phosphatases, trafficking proteins, and secondary messengers, preferentially partition to these highly organized cell membrane microdomains, called lipid rafts. Lipid rafts are essential for the trafficking and signaling of GPCRs. The study of GPCR biology in the context of lipid rafts involves the localization of the GPCR of interest in lipid rafts, at the basal state and upon receptor agonism, and the evaluation of the biological functions of the GPCR in appropriate cell lines. The lack of standardized methodologies to study lipid rafts, in general, and of the workings of GPCRs in lipid rafts, in particular, and the inescapable drawbacks of current methods have hampered the complete understanding of the underlying molecular mechanisms. Newer methodologies that allow the study of GPCRs in their native form are needed. The use of complementary approaches that produce mutually supportive results appears to be the best way for drawing conclusions with regard to the distribution and activity of GPCRs in lipid rafts.


Assuntos
Detergentes/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Immunoblotting/métodos , Microdomínios da Membrana/química , Microscopia Confocal/métodos , Receptores Acoplados a Proteínas G/metabolismo , Linhagem Celular , Proteínas Heterotriméricas de Ligação ao GTP/isolamento & purificação , Humanos , Microdomínios da Membrana/metabolismo , Receptores Acoplados a Proteínas G/isolamento & purificação , Transdução de Sinais
3.
Methods Mol Biol ; 2277: 157-173, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080151

RESUMO

Mitochondria have complex ultrastructure which includes continuous subcompartments, such as matrix, intermembrane space, and two membranes, as well as focal structures, such as nucleoids, RNA granules, and mitoribosomes. Comprehensive studies of the spatial distribution of proteins and RNAs inside the mitochondria are necessary to understand organellar gene expression processes and macromolecule targeting pathways. Here we give examples of distribution analysis of mitochondrial proteins and transcripts by conventional microscopy and the super-resolution technique 3D STORM. We provide detailed protocols and discuss limitations of immunolabeling of mitochondrial proteins and newly synthesized mitochondrial RNAs by bromouridine incorporation and single-molecule RNA FISH in hepatocarcinoma cells.


Assuntos
Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Microscopia Confocal/métodos , Proteínas Mitocondriais/metabolismo , Bromouracila/análogos & derivados , Bromouracila/química , Células Hep G2 , Humanos , Processamento de Imagem Assistida por Computador/métodos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , RNA Mitocondrial/química , Imagem Individual de Molécula/métodos , Uridina/análogos & derivados , Uridina/química
4.
Methods Mol Biol ; 2277: 187-201, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080153

RESUMO

Mitochondria, similar to living cells and organelles, have a negative membrane potential, which ranges between (-108) and (150) mV as compared to (-70) and (-90) mV of the plasma membrane. Therefore, permeable lipophilic cations tend to accumulate in the mitochondria. Those cations which exhibit fluorescence activity after accumulation into energized systems are widely used to decipher changes in membrane potential by imaging techniques. Here we describe the use of two different dyes for labeling mitochondrial membrane potential (Δψm) in live cells. One is the lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide (JC-1), which alters reversibly its color from green (J-monomer, at its low concentration in the cytosol) to red (J-aggregates, at its high concentration in active mitochondria) with increasing mitochondrial membrane potential (Δψm). The other is MitoTracker® Orange, a mitochondrion-selective probe which passively diffuses across the plasma membrane and accumulates in active mitochondria depending on their Δψm. We show that in addition to changes in Δψm, these specific dyes can be used to follow alterations in mitochondrial distribution and mitochondrial network connectivity. We suggest that JC-1 is a preferable probe to compare between different cell types and cell state, as a red to green ratio of fluorescence intensities is used for analysis. This ratio depends only on the mitochondrial membrane potential and not on other cellular and/or mitochondrial-dependent or independent factors that may alter, for example, due to treatment or disease state. However, in cells labeled either with green or red fluorescence protein, JC-1 cannot be used. Therefore, other dyes are preferable. We demonstrate various applications of JC-1 and MitoTracker Orange staining to study mitochondrial abnormalities in different cell types derived from schizophrenia patients and healthy subjects.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Esquizofrenia/metabolismo , Benzimidazóis/química , Carbocianinas/química , Técnicas de Cultura de Células , Fibroblastos/metabolismo , Fibroblastos/patologia , Corantes Fluorescentes/química , Humanos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Mitocôndrias/patologia , Estudo de Prova de Conceito , Esquizofrenia/patologia , Xantenos/química
5.
Methods Mol Biol ; 2274: 385-389, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050487

RESUMO

Confocal microscopy is a simple, super-resolution technique, which does not produce a marked increase in resolution compared to other advanced techniques, such as super-resolution nanoscopy. Here, we present a simple protocol to acquire "slightly, but easily resolved" images by pinhole closure (<1 airy unit) in a conventional confocal scanning microscope equipped with an avalanche photodiode, a detector with high sensitivity. We use murine neuroblastoma Neuro2a cells to demonstrate the image resolution obtained via this protocol without the use of any special software to enhance image quality.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/instrumentação , Microscopia de Fluorescência/instrumentação , Imagem Molecular/métodos , Neuroblastoma/patologia , Software , Animais , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Células Tumorais Cultivadas
6.
Curr Opin Ophthalmol ; 32(4): 369-378, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33989235

RESUMO

PURPOSE OF REVIEW: This review will discuss the utility of high-resolution anterior segment optical coherence tomography (HR-OCT), in-vivo confocal microscopy (IVCM) and ultrasound biomicroscopy (UBM) in characterizing and diagnosing various ocular surface tumors, namely ocular surface squamous neoplasia (OSSN), conjunctival lymphoma and conjunctival melanoma. The strengths and limitations of each imaging modality will be discussed along with the characteristics findings of each lesion on each imaging platform. RECENT FINDINGS: HR-OCT can consistently be utilized in the clinic setting to distinguish between epithelial ocular surface tumors such as OSSN as compared with subepithelial tumors such as conjunctival lymphoma and conjunctival melanoma given their distinctive findings. IVCM can be used as an adjunct to HR-OCT to obtain cellular and surface characteristics, whereas UBM can be used to assess tumor depth and thickness for larger and highly pigmented lesions as well as to detect intraocular invasion. SUMMARY: HR-OCT, IVCM and UBM are all helpful imaging modalities to diagnose and characterize various ocular surface tumors and can serve as valuable adjuncts to monitor treatment response and assess for recurrence ocular surface tumors.


Assuntos
Túnica Conjuntiva/patologia , Neoplasias da Túnica Conjuntiva/diagnóstico , Diagnóstico por Imagem , Humanos , Microscopia Acústica/métodos , Microscopia Confocal/métodos , Tomografia de Coerência Óptica/métodos
7.
Nat Commun ; 12(1): 3148, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035309

RESUMO

Structured Illumination Microscopy enables live imaging with sub-diffraction resolution. Unfortunately, optical aberrations can lead to loss of resolution and artifacts in Structured Illumination Microscopy rendering the technique unusable in samples thicker than a single cell. Here we report on the combination of Adaptive Optics and Structured Illumination Microscopy enabling imaging with 150 nm lateral and 570 nm axial resolution at a depth of 80 µm through Caenorhabditis elegans. We demonstrate that Adaptive Optics improves the three-dimensional resolution, especially along the axial direction, and reduces artifacts, successfully realizing 3D-Structured Illumination Microscopy in a variety of biological samples.


Assuntos
Imageamento Tridimensional/métodos , Microscopia Intravital/métodos , Iluminação/instrumentação , Animais , Artefatos , Ascomicetos , Caenorhabditis elegans , Linhagem Celular , Imageamento Tridimensional/instrumentação , Microscopia Intravital/instrumentação , Camundongos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Oryza/microbiologia , Reprodutibilidade dos Testes
8.
Nat Commun ; 12(1): 2977, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016996

RESUMO

When exploring new environments animals form spatial memories that are updated with experience and retrieved upon re-exposure to the same environment. The hippocampus is thought to support these memory processes, but how this is achieved by different subnetworks such as CA1 and CA3 remains unclear. To understand how hippocampal spatial representations emerge and evolve during familiarization, we performed 2-photon calcium imaging in mice running in new virtual environments and compared the trial-to-trial dynamics of place cells in CA1 and CA3 over days. We find that place fields in CA1 emerge rapidly but tend to shift backwards from trial-to-trial and remap upon re-exposure to the environment a day later. In contrast, place fields in CA3 emerge gradually but show more stable trial-to-trial and day-to-day dynamics. These results reflect different roles in CA1 and CA3 in spatial memory processing during familiarization to new environments and constrain the potential mechanisms that support them.


Assuntos
Região CA1 Hipocampal/fisiologia , Região CA3 Hipocampal/fisiologia , Células de Lugar/fisiologia , Percepção Espacial/fisiologia , Memória Espacial/fisiologia , Animais , Técnicas de Observação do Comportamento , Comportamento Animal/fisiologia , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/diagnóstico por imagem , Região CA3 Hipocampal/citologia , Região CA3 Hipocampal/diagnóstico por imagem , Craniotomia , Microscopia Intravital/instrumentação , Microscopia Intravital/métodos , Masculino , Camundongos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Modelos Animais , Imagem Óptica/instrumentação , Imagem Óptica/métodos
9.
J Vis Exp ; (170)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33938876

RESUMO

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Pâncreas/metabolismo , Animais , Camundongos , Pâncreas/citologia
10.
Nat Commun ; 12(1): 2650, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976192

RESUMO

Live cell imaging using fluorescent DNA markers are an indispensable molecular tool in various biological and biomedical fields. It is a challenge to develop DNA probes that avoid UV light photo-excitation, have high specificity for DNA, are cell-permeable and are compatible with cutting-edge imaging techniques such as super-resolution microscopy. Herein, we present N-aryl pyrido cyanine (N-aryl-PC) derivatives as a class of long absorption DNA markers with absorption in the wide range of visible light. The high DNA specificity and membrane permeability allow the staining of both organelle DNA as well as nuclear DNA, in various cell types, including plant tissues, without the need for washing post-staining. N-aryl-PC dyes are also highly compatible with a separation of photon by lifetime tuning method in stimulated emission depletion microscopy (SPLIT-STED) for super-resolution imaging as well as two-photon microscopy for deep tissue imaging, making it a powerful tool in the life sciences.


Assuntos
Núcleo Celular/química , DNA/química , Corantes Fluorescentes/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Organelas/química , Animais , Arabidopsis/citologia , Benzimidazóis/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , DNA/genética , DNA/metabolismo , Fluorescência , Células HeLa , Humanos , Camundongos , Microscopia Confocal/métodos , Estrutura Molecular , Células NIH 3T3 , Organelas/metabolismo , Coloração e Rotulagem/métodos , Imagem com Lapso de Tempo/métodos
11.
J Vis Exp ; (170)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33970138

RESUMO

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.


Assuntos
Dendritos/fisiologia , Microscopia Confocal/métodos , Tecido Nervoso/fisiologia , Neurônios/fisiologia , Animais , Camundongos
12.
Nat Commun ; 12(1): 2861, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001891

RESUMO

Hair cells detect sound, head position or water movements when their mechanosensory hair bundle is deflected. Each hair bundle has an asymmetric architecture that restricts stimulus detection to a single axis. Coordinated hair cell orientations within sensory epithelia further tune stimulus detection at the organ level. Here, we identify GPR156, an orphan GPCR of unknown function, as a critical regulator of hair cell orientation. We demonstrate that the transcription factor EMX2 polarizes GPR156 distribution, enabling it to signal through Gαi and trigger a 180° reversal in hair cell orientation. GPR156-Gαi mediated reversal is essential to establish hair cells with mirror-image orientations in mouse otolith organs in the vestibular system and in zebrafish lateral line. Remarkably, GPR156-Gαi also instructs hair cell reversal in the auditory epithelium, despite a lack of mirror-image organization. Overall, our work demonstrates that conserved GPR156-Gαi signaling is integral to the framework that builds directional responses into mechanosensory epithelia.


Assuntos
Epitélio/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas de Homeodomínio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição/metabolismo , Animais , Polaridade Celular/genética , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Células Ciliadas Auditivas/citologia , Proteínas de Homeodomínio/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal/métodos , Receptores Acoplados a Proteínas G/genética , Fatores de Transcrição/genética , Peixe-Zebra/metabolismo
13.
Int J Mol Sci ; 22(7)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807275

RESUMO

High mobility group box 1 (HMGB1) has been demonstrated to promote the migration and invasion of non-small cell lung cancer (NSCLC). However, the mechanism of action of HMGB1 in regulating tumor mobility remains unclear. Therefore, we aimed to investigate whether HMGB1 affects mitochondria distribution and regulates dynamin-related protein 1 (DRP1)-mediated lamellipodia/filopodia formation to promote NSCLC migration. The regulation of mitochondrial membrane tension, dynamics, polarization, fission process, and cytoskeletal rearrangements in lung cancer cells by HMGB1 was analyzed using confocal microscopy. The HMGB1-mediated regulation of DRP1 phosphorylation and colocalization was determined using immunostaining and co-immunoprecipitation assays. The tumorigenic potential of HMGB1 was assessed in vivo and further confirmed using NSCLC patient samples. Our results showed that HMGB1 increased the polarity and mobility of cells (mainly by regulating the cytoskeletal system actin and microtubule dynamics and distribution), promoted the formation of lamellipodia/filopodia, and enhanced the expression and phosphorylation of DRP1 in both the nucleus and cytoplasm. In addition, HMGB1 and DRP1 expressions were positively correlated and exhibited poor prognosis and survival in patients with lung cancer. Collectively, HMGB1 plays a key role in the formation of lamellipodia and filopodia by regulating cytoskeleton dynamics and DRP1 expression to promote lung cancer migration.


Assuntos
Dinaminas/metabolismo , Proteína HMGB1/metabolismo , Neoplasias Pulmonares/metabolismo , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Dinaminas/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Proteínas HMGB/metabolismo , Proteína HMGB1/fisiologia , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos SCID , Microscopia Confocal/métodos , Mitocôndrias/genética , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Fosforilação , Pseudópodes/metabolismo
14.
Methods Mol Biol ; 2267: 1-6, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33786781

RESUMO

Many proteins involved in the DNA damage pathway shuttle between the cytoplasm and nucleus, and their localizations are important for functions. In that regard, immunofluorescence microscopy has been widely used to delineate the temporal and spatial regulation of proteins. Here, we describe an unconventional method for studying the cellular localization of CHK1, a cell cycle checkpoint kinase that undergoes shuttling from the cytoplasm to the nucleus in response to genotoxic stress. In this study, we included an acid extraction step to better reveal the nuclear localization of CHK1.


Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Imunofluorescência/métodos , Células HCT116 , Humanos , Microscopia Confocal/métodos , Transporte Proteico
15.
J Vis Exp ; (169)2021 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-33749674

RESUMO

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 µm (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.


Assuntos
Retina/metabolismo , Coloração e Rotulagem/métodos , Células Amácrinas/fisiologia , Animais , Dendritos/fisiologia , Neurônios Dopaminérgicos/fisiologia , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia Confocal/métodos , Proteínas do Tecido Nervoso/metabolismo , Receptores de AMPA/metabolismo , Refratometria
16.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672992

RESUMO

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.


Assuntos
Cromossomos de Plantas/genética , Hordeum/genética , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Cromossomos de Plantas/química , Cromossomos de Plantas/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Corantes Fluorescentes/química , Hordeum/citologia , Indóis/química , Metáfase/genética , Reprodutibilidade dos Testes
17.
Methods Mol Biol ; 2265: 47-63, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704704

RESUMO

In order to protrude within a dense tissue, tumor cells have to develop the ability to digest the extracellular matrix (ECM). Melanoma cells, similarly to other types of tumor cells, form invadopodia, membranous invaginations rich in filamentous actin and several other proteins including matrix metalloproteinases (MMPs). MMPs degrade ECM structural proteins such as collagens, fibronectin, or laminin. Here we describe an assay that allows the detection of gelatinase activity exhibited by tumor cells under 2D conditions and methods to present obtained data in both a quantitative and a qualitative manner.


Assuntos
Matriz Extracelular/enzimologia , Gelatina/metabolismo , Melanoma/enzimologia , Microscopia Confocal/métodos , Actinas/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fluorescência , Gelatinases/metabolismo , Humanos , Metaloproteinases da Matriz/metabolismo , Melanoma/patologia , Imagem Óptica , Podossomos/enzimologia , Podossomos/metabolismo , Podossomos/patologia
18.
Nat Commun ; 12(1): 1901, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33772008

RESUMO

The trans-Golgi network (TGN) has been known as a key platform to sort and transport proteins to their final destinations in post-Golgi membrane trafficking. However, how the TGN sorts proteins with different destinies still remains elusive. Here, we examined 3D localization and 4D dynamics of TGN-localized proteins of Arabidopsis thaliana that are involved in either secretory or vacuolar trafficking from the TGN, by a multicolor high-speed and high-resolution spinning-disk confocal microscopy approach that we developed. We demonstrate that TGN-localized proteins exhibit spatially and temporally distinct distribution. VAMP721 (R-SNARE), AP (adaptor protein complex)-1, and clathrin which are involved in secretory trafficking compose an exclusive subregion, whereas VAMP727 (R-SNARE) and AP-4 involved in vacuolar trafficking compose another subregion on the same TGN. Based on these findings, we propose that the single TGN has at least two subregions, or "zones", responsible for distinct cargo sorting: the secretory-trafficking zone and the vacuolar-trafficking zone.


Assuntos
Arabidopsis/metabolismo , Microscopia Confocal/métodos , Vacúolos/metabolismo , Rede trans-Golgi/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clatrina/genética , Clatrina/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Eletrônica de Transmissão , Plantas Geneticamente Modificadas , Transporte Proteico , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Vacúolos/ultraestrutura , Rede trans-Golgi/ultraestrutura
19.
Nat Commun ; 12(1): 1913, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33772014

RESUMO

Diffusion is a major molecular transport mechanism in biological systems. Quantifying direction-dependent (i.e., anisotropic) diffusion is vitally important to depicting how the three-dimensional (3D) tissue structure and composition affect the biochemical environment, and thus define tissue functions. However, a tool for noninvasively measuring the 3D anisotropic extracellular diffusion of biorelevant molecules is not yet available. Here, we present light-sheet imaging-based Fourier transform fluorescence recovery after photobleaching (LiFT-FRAP), which noninvasively determines 3D diffusion tensors of various biomolecules with diffusivities up to 51 µm2 s-1, reaching the physiological diffusivity range in most biological systems. Using cornea as an example, LiFT-FRAP reveals fundamental limitations of current invasive two-dimensional diffusion measurements, which have drawn controversial conclusions on extracellular diffusion in healthy and clinically treated tissues. Moreover, LiFT-FRAP demonstrates that tissue structural or compositional changes caused by diseases or scaffold fabrication yield direction-dependent diffusion changes. These results demonstrate LiFT-FRAP as a powerful platform technology for studying disease mechanisms, advancing clinical outcomes, and improving tissue engineering.


Assuntos
Córnea/metabolismo , Espaço Extracelular/metabolismo , Recuperação de Fluorescência Após Fotodegradação/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Tendões/metabolismo , Animais , Anisotropia , Colágeno/química , Colágeno/metabolismo , Difusão , Análise de Fourier , Microscopia Confocal/métodos , Microscopia Eletrônica de Varredura/métodos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Suínos , Engenharia Tecidual/métodos , Tecidos Suporte/química
20.
PLoS One ; 16(2): e0244034, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33591984

RESUMO

Confocal microscopes can reject out-of-focus and scattered light; however, widefield microscopes are far more common in biological laboratories due to their accessibility and lower cost. We report confocal imaging capacity on a widefield microscope by adding a spatial light modulator (SLM) and utilizing custom illumination and acquisition methods. We discuss our illumination strategy and compare several procedures for postprocessing the acquired image data. We assessed the performance of this system for rejecting out-of-focus light by comparing images taken at 1.4 NA using our widefield microscope, our SLM-enhanced setup, and a commercial confocal microscope. The optical sectioning capability, assessed on thin fluorescent film, was 0.85 ± 0.04 µm for our SLM-enhanced setup and 0.68 ± 0.04 µm for a confocal microscope, while a widefield microscope exhibited no sectioning capability. We demonstrate our setup by imaging the same set of neurons in C. elegans on widefield, SLM, and confocal microscopes. SLM enhancement greatly reduces background from the cell body, allowing visualization of dim fibers nearby. Our SLM-enhanced setup identified 96% of the dim neuronal fibers seen in confocal images while a widefield microscope only identified 50% of the same fibers. Our microscope add-on represents a very simple (2-component) and inexpensive (<$600) approach to enable widefield microscopes to optically section thick samples.


Assuntos
Microscopia Confocal/métodos , Neurônios/metabolismo , Animais , Caenorhabditis elegans , Processamento de Imagem Assistida por Computador
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