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1.
Nat Commun ; 12(1): 2005, 2021 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-33790271

RESUMEN

Förster resonant energy transfer (FRET) is a powerful mechanism to probe associations in situ. Simultaneously performing more than one FRET measurement can be challenging due to the spectral bandwidth required for the donor and acceptor fluorophores. We present an approach to distinguish overlapping FRET pairs based on the photochromism of the donor fluorophores, even if the involved fluorophores display essentially identical absorption and emission spectra. We develop the theory underlying this method and validate our approach using numerical simulations. To apply our system, we develop rsAKARev, a photochromic biosensor for cAMP-dependent protein kinase (PKA), and combine it with the spectrally-identical biosensor EKARev, a reporter for extracellular signal-regulated kinase (ERK) activity, to deliver simultaneous readout of both activities in the same cell. We further perform multiplexed PKA, ERK, and calcium measurements by including a third, spectrally-shifted biosensor. Our work demonstrates that exploiting donor photochromism in FRET can be a powerful approach to simultaneously read out multiple associations within living cells.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Proteínas Luminiscentes/química , Algoritmos , Animales , Técnicas Biosensibles/métodos , Células COS , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente/métodos , Imagen de Lapso de Tiempo/métodos
2.
Nat Commun ; 12(1): 1990, 2021 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-33790272

RESUMEN

A crucial phase in the infection process, which remains poorly understood, is the localization of suitable host cells by bacteria. It is often assumed that chemotaxis plays a key role during this phase. Here, we report a quantitative study on how Salmonella Typhimurium search for T84 human colonic epithelial cells. Combining time-lapse microscopy and mathematical modeling, we show that bacteria can be described as chiral active particles with strong active speed fluctuations, which are of biological, as opposed to thermal, origin. We observe that there exists a giant range of inter-individual variability of the bacterial exploring capacity. Furthermore, we find Salmonella Typhimurium does not exhibit biased motion towards the cells and show that the search time statistics is consistent with a random search strategy. Our results indicate that in vitro localization of host cells, and also cell infection, are random processes, not involving chemotaxis, that strongly depend on bacterial motility parameters.


Asunto(s)
Algoritmos , Adhesión Bacteriana/fisiología , Células Epiteliales/metabolismo , Salmonella typhimurium/metabolismo , Línea Celular Tumoral , Quimiotaxis/fisiología , Células Epiteliales/microbiología , Interacciones Huésped-Patógeno , Humanos , Locomoción/fisiología , Microscopía/métodos , Movimiento (Física) , Salmonella typhimurium/fisiología , Imagen de Lapso de Tiempo/métodos
3.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-33799598

RESUMEN

We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC50) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC50 value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC50 values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement.


Asunto(s)
Aflatoxina B1/toxicidad , Efecto Fundador , Mediciones Luminiscentes/métodos , Primaquina/toxicidad , Imagen de Lapso de Tiempo/métodos , Xenobióticos/toxicidad , Animales , Línea Celular Tumoral , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Luciferasas/genética , Luciferasas/metabolismo , Luminiscencia , Ratones
4.
Science ; 372(6538)2021 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-33833095

RESUMEN

During multicellular development, spatial position and lineage history play powerful roles in controlling cell fate decisions. Using a serine integrase-based recording system, we engineered cells to record lineage information in a format that can be read out in situ. The system, termed integrase-editable memory by engineered mutagenesis with optical in situ readout (intMEMOIR), allowed in situ reconstruction of lineage relationships in cultured mouse cells and flies. intMEMOIR uses an array of independent three-state genetic memory elements that can recombine stochastically and irreversibly, allowing up to 59,049 distinct digital states. It reconstructed lineage trees in stem cells and enabled simultaneous analysis of single-cell clonal history, spatial position, and gene expression in Drosophila brain sections. These results establish a foundation for microscopy-readable lineage recording and analysis in diverse systems.


Asunto(s)
Linaje de la Célula , Expresión Génica , Células Madre Embrionarias de Ratones/citología , Neuronas/citología , Análisis de la Célula Individual , Animales , Encéfalo/citología , Línea Celular , Células Clonales/citología , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Perfilación de la Expresión Génica , Respuesta al Choque Térmico , Hibridación Fluorescente in Situ , Integrasas/metabolismo , Ratones , Mutagénesis , Análisis Espacial , Imagen de Lapso de Tiempo , Transcripción Genética
5.
J Vis Exp ; (168)2021 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-33645588

RESUMEN

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an "eat-me" signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.


Asunto(s)
Bioensayo/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Macrófagos/metabolismo , Neuroblastoma/patología , Fagocitosis , Animales , Muerte Celular , Línea Celular Tumoral , Análisis de Datos , Colorantes Fluorescentes/química , Células Madre Embrionarias Humanas/citología , Humanos , Concentración de Iones de Hidrógeno , Células Madre Pluripotentes Inducidas/citología , Control de Calidad , Reproducibilidad de los Resultados , Imagen de Lapso de Tiempo
6.
Nat Commun ; 12(1): 1413, 2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33658493

RESUMEN

pH-sensitive fluorescent proteins (FPs) are highly advantageous for the non-invasive monitoring of exocytosis events. Superecliptic pHluorin (SEP), a green pH-sensitive FP, has been widely used for imaging single-vesicle exocytosis. However, the docking step cannot be visualized using this FP, since the fluorescence signal inside vesicles is too low to be observed during docking process. Among the available red pH-sensitive FPs, none is comparable to SEP for practical applications due to unoptimized pH-sensitivity and fluorescence brightness or severe photochromic behavior. In this study, we engineer a bright and photostable red pH-sensitive FP, named pHmScarlet, which compared to other red FPs has higher pH sensitivity and enables the simultaneous detection of vesicle docking and fusion. pHmScarlet can also be combined with SEP for dual-color imaging of two individual secretory events. Furthermore, although the emission wavelength of pHmScarlet is red-shifted compared to that of SEP, its spatial resolution is high enough to show the ring structure of vesicle fusion pores using Hessian structured illumination microscopy (Hessian-SIM).


Asunto(s)
Exocitosis/fisiología , Proteínas Luminiscentes/metabolismo , Animales , Línea Celular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Humanos , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/genética , Mutación , Neuronas/citología , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vesículas Sinápticas/fisiología , Imagen de Lapso de Tiempo , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismo
7.
Nat Commun ; 12(1): 1411, 2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33658500

RESUMEN

Genetically programmed circuits allowing bifunctional dynamic regulation of enzyme expression have far-reaching significances for various bio-manufactural purposes. However, building a bio-switch with a post log-phase response and reversibility during scale-up bioprocesses is still a challenge in metabolic engineering due to the lack of robustness. Here, we report a robust thermosensitive bio-switch that enables stringent bidirectional control of gene expression over time and levels in living cells. Based on the bio-switch, we obtain tree ring-like colonies with spatially distributed patterns and transformer cells shifting among spherical-, rod- and fiber-shapes of the engineered Escherichia coli. Moreover, fed-batch fermentations of recombinant E. coli are conducted to obtain ordered assembly of tailor-made biopolymers polyhydroxyalkanoates including diblock- and random-copolymer, composed of 3-hydroxybutyrate and 4-hydroxybutyrate with controllable monomer molar fraction. This study demonstrates the possibility of well-organized, chemosynthesis-like block polymerization on a molecular scale by reprogrammed microbes, exemplifying the versatility of thermo-response control for various practical uses.


Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Ingeniería Metabólica/métodos , Polihidroxialcanoatos/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Fermentación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hidroxibutiratos/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microorganismos Modificados Genéticamente , Poliésteres/metabolismo , Temperatura , Imagen de Lapso de Tiempo
8.
J Vis Exp ; (168)2021 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-33645552

RESUMEN

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.


Asunto(s)
Movimiento Celular , Separación Celular/métodos , Embrión de Mamíferos/citología , Mesodermo/citología , Paladar (Hueso)/citología , Imagen de Lapso de Tiempo , Animales , Rastreo Celular , Células Cultivadas , Criopreservación , Modelos Animales de Enfermedad , Disección , Femenino , Ratones , Cicatrización de Heridas
9.
J Vis Exp ; (169)2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33749676

RESUMEN

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins - α-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) - and one M-line protein - Myomesin-2 (Myom2) - were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.


Asunto(s)
Colorantes Fluorescentes/metabolismo , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Sarcómeros/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Dependovirus/metabolismo , Cuerpos Embrioides/citología , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Miocitos Cardíacos/citología , Coloración y Etiquetado , Imagen de Lapso de Tiempo
10.
Nat Commun ; 12(1): 1807, 2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33753743

RESUMEN

Mitochondria-lysosome contacts are recently identified sites for mediating crosstalk between both organelles, but their role in normal and diseased human neurons remains unknown. In this study, we demonstrate that mitochondria-lysosome contacts can dynamically form in the soma, axons, and dendrites of human neurons, allowing for their bidirectional crosstalk. Parkinson's disease patient derived neurons harboring mutant GBA1 exhibited prolonged mitochondria-lysosome contacts due to defective modulation of the untethering protein TBC1D15, which mediates Rab7 GTP hydrolysis for contact untethering. This dysregulation was due to decreased GBA1 (ß-glucocerebrosidase (GCase)) lysosomal enzyme activity in patient derived neurons, and could be rescued by increasing enzyme activity with a GCase modulator. These defects resulted in disrupted mitochondrial distribution and function, and could be further rescued by TBC1D15 in Parkinson's patient derived GBA1-linked neurons. Together, our work demonstrates a potential role of mitochondria-lysosome contacts as an upstream regulator of mitochondrial function and dynamics in midbrain dopaminergic neurons in GBA1-linked Parkinson's disease.


Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Glucosilceramidasa/genética , Lisosomas/genética , Mitocondrias/genética , Mutación , Enfermedad de Parkinson/genética , Células Cultivadas , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/ultraestructura , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Glucosilceramidasa/metabolismo , Humanos , Hidrólisis , Lisosomas/metabolismo , Lisosomas/ultraestructura , Microscopía Confocal , Microscopía Electrónica de Transmisión , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Enfermedad de Parkinson/metabolismo , Imagen de Lapso de Tiempo/métodos , Proteínas de Unión al GTP rab/metabolismo
11.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-33672911

RESUMEN

The Gram-negative bacterium Flavobacterium johnsoniae employs gliding motility to move rapidly over solid surfaces. Gliding involves the movement of the adhesin SprB along the cell surface. F. johnsoniae spreads on nutrient-poor 1% agar-PY2, forming a thin film-like colony. We used electron microscopy and time-lapse fluorescence microscopy to investigate the structure of colonies formed by wild-type (WT) F. johnsoniae and by the sprB mutant (ΔsprB). In both cases, the bacteria were buried in the extracellular polymeric matrix (EPM) covering the top of the colony. In the spreading WT colonies, the EPM included a thick fiber framework and vesicles, revealing the formation of a biofilm, which is probably required for the spreading movement. Specific paths that were followed by bacterial clusters were observed at the leading edge of colonies, and abundant vesicle secretion and subsequent matrix formation were suggested. EPM-free channels were formed in upward biofilm protrusions, probably for cell migration. In the nonspreading ΔsprB colonies, cells were tightly packed in layers and the intercellular space was occupied by less matrix, indicating immature biofilm. This result suggests that SprB is not necessary for biofilm formation. We conclude that F. johnsoniae cells use gliding motility to spread and maturate biofilms.


Asunto(s)
Adhesinas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Flavobacterium/fisiología , Locomoción/fisiología , Proteínas Bacterianas/genética , Flavobacterium/genética , Flavobacterium/ultraestructura , Locomoción/genética , Microscopía Electrónica de Transmisión/métodos , Microscopía Fluorescente/métodos , Mutación , Imagen de Lapso de Tiempo/métodos
12.
Nat Commun ; 12(1): 1762, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741910

RESUMEN

Time-resolved studies of biomacromolecular crystals have been limited to systems involving only minute conformational changes within the same lattice. Ligand-induced changes greater than several angstroms, however, are likely to result in solid-solid phase transitions, which require a detailed understanding of the mechanistic interplay between conformational and lattice transitions. Here we report the synchronous behavior of the adenine riboswitch aptamer RNA in crystal during ligand-triggered isothermal phase transitions. Direct visualization using polarized video microscopy and atomic force microscopy shows that the RNA molecules undergo cooperative rearrangements that maintain lattice order, whose cell parameters change distinctly as a function of time. The bulk lattice order throughout the transition is further supported by time-resolved diffraction data from crystals using an X-ray free electron laser. The synchronous molecular rearrangements in crystal provide the physical basis for studying large conformational changes using time-resolved crystallography and micro/nanocrystals.


Asunto(s)
Conformación de Ácido Nucleico , Transición de Fase , ARN/química , Riboswitch , Adenina/química , Aptámeros de Nucleótidos/química , Cristalografía por Rayos X , Microscopía de Fuerza Atómica/métodos , Microscopía de Polarización/métodos , Modelos Moleculares , Imagen de Lapso de Tiempo/métodos
13.
Methods Mol Biol ; 2218: 137-155, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33606229

RESUMEN

Oocyte production is crucial for sexual reproduction. Recent findings in zebrafish and other established model organisms emphasize that the early steps of oogenesis involve the coordination of simultaneous and tightly sequential processes across cellular compartments and between sister cells. To fully understand the mechanistic framework of these coordinated processes, cellular and morphological analysis in high temporal resolution is required. Here, we provide a protocol for four-dimensional live time-lapse analysis of cultured juvenile zebrafish ovaries. We describe how multiple-stage oocytes can be simultaneously analyzed in single ovaries, and several ovaries can be processed in single experiments. In addition, we detail adequate conditions for quantitative image acquisition. Finally, we demonstrate that using this protocol, we successfully capture rapid meiotic chromosomal movements in early prophase for the first time in zebrafish oocytes, in four dimensions and in vivo. Our protocol expands the use of the zebrafish as a model system to understand germ cell and ovarian development in postembryonic stages.


Asunto(s)
Cromosomas/fisiología , Meiosis/fisiología , Oogénesis/fisiología , Ovario/fisiología , Imagen de Lapso de Tiempo/métodos , Pez Cebra/fisiología , Animales , Femenino , Oocitos , Diferenciación Sexual/fisiología
14.
J Vis Exp ; (167)2021 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-33522506

RESUMEN

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to. Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres. This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.


Asunto(s)
Drosophila melanogaster/citología , Imagenología Tridimensional , Microscopía Fluorescente/métodos , Animales , Técnicas de Cultivo de Célula , Supervivencia Celular , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Microtúbulos/metabolismo , Células Madre/citología , Fracciones Subcelulares/metabolismo , Testículo/citología , Imagen de Lapso de Tiempo , Tubulina (Proteína)/metabolismo
15.
J Vis Exp ; (167)2021 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-33522507

RESUMEN

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.


Asunto(s)
Vasos Sanguíneos/crecimiento & desarrollo , Neovascularización Fisiológica , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Actinas/metabolismo , Biomarcadores/metabolismo , Movimiento Celular , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/efectos de los fármacos , Fibronectinas/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Impresión Tridimensional , Imagen de Lapso de Tiempo
16.
Anticancer Res ; 41(1): 137-149, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33419807

RESUMEN

BACKGROUND/AIM: Conventional viability tests, help to screen the cellular effects of candidate molecules, but the endpoint of these measurements lacks sufficient information regarding the molecular aspects. A non-invasive, easy-to-setup live-cell microscopic method served to in-depth analysis of mechanisms of potential anticancer drugs. MATERIALS AND METHODS: The proposed method combining the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test with time-lapse scanning microscopy (TLS), provided additional data related to the cell-cycle and the dynamic properties of cell morphology. Apoptotic and necrotic events became detectable with these methods. RESULTS: Quantification of the results was assisted by image analysis of the acquired image sequences. After demonstrating the potential of the TLS method, a series of experiments compared the in vitro effect of a known and a newly synthesized nucleoside analogue. CONCLUSION: The proposed approach provided a more in-depth insight into the cellular processes that can be affected by known chemotherapeutic agents including nucleoside analogues rather than applying repeated individual treatments.


Asunto(s)
Antineoplásicos/farmacología , Nucleósidos/farmacología , Sales de Tetrazolio , Tiazoles , Imagen de Lapso de Tiempo , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Microscopía , Nucleósidos/análogos & derivados , Imagen de Lapso de Tiempo/métodos
17.
Nat Commun ; 12(1): 669, 2021 01 28.
Artículo en Inglés | MEDLINE | ID: mdl-33510146

RESUMEN

Plants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. Here, we establish long-term live-cell imaging of single Arabidopsis cells undergoing proto-xylem trans-differentiation, resulting in spiral wall patterns, to understand microtubule re-organization. We find that the re-organization requires local microtubule de-stabilization in band-interspersing gaps. Using microtubule simulations, we recapitulate the process in silico and predict that spatio-temporal control of microtubule nucleation is critical for pattern formation, which we confirm in vivo. By combining simulations and live-cell imaging we further explain how the xylem wall-deficient and microtubule-severing KATANIN contributes to microtubule and wall patterning. Hence, by combining quantitative microscopy and modelling we devise a framework to understand how microtubule re-organization supports wall patterning.


Asunto(s)
Arabidopsis/metabolismo , Pared Celular/metabolismo , Microtúbulos/metabolismo , Xilema/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Hipocótilo/citología , Hipocótilo/genética , Hipocótilo/metabolismo , Microscopía Fluorescente/métodos , Plantas Modificadas Genéticamente , Análisis de la Célula Individual/métodos , Imagen de Lapso de Tiempo/métodos , Xilema/citología , Xilema/genética
18.
Methods Mol Biol ; 2130: 207-219, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33284447

RESUMEN

Live imaging of the molecular clockwork within the circadian pacemaker neurons offers the unique possibility to study complex interactions between the molecular clock and neuronal communication within individual neurons and throughout the entire circadian circuitry. Here we describe how to establish brain explants and dissociated neuron culture from Drosophila larvae, guidelines for time-lapse fluorescence microscopy, and the method of image analysis. This approach enables the long-term monitoring of fluorescence signals of circadian reporters at single-cell resolution and can be also applicable to analyze real-time expression of other fluorescent probes in Drosophila neurons.


Asunto(s)
Ritmo Circadiano , Neuronas/citología , Imagen de Lapso de Tiempo/métodos , Animales , Células Cultivadas , Drosophila melanogaster , Microscopía Fluorescente/métodos , Neuronas/fisiología , Cultivo Primario de Células/métodos
19.
Methods Mol Biol ; 2196: 235-244, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32889726

RESUMEN

Live-cell imaging is widely used by researchers to study cellular dynamics and obtain a deep understanding of cell biological processes. Keeping cells in the proper growing environment and immobilizing the cells are essential for the imaging of live yeast cells. Here we describe a protocol for monitoring cytoophidia in Saccharomyces cerevisiae and Schizosaccharomyces pombe using inverted confocal fluorescence microscopy. This protocol includes yeast culture, sample preparation, fluorescence imaging, and data analysis.


Asunto(s)
Microscopía Confocal , Imagen de Lapso de Tiempo , Levaduras/fisiología , Citoplasma , Análisis de Datos , Procesamiento de Imagen Asistido por Computador , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/fisiología , Levaduras/citología
20.
Methods Mol Biol ; 2179: 183-197, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32939722

RESUMEN

The neural tube in amniotic embryos forms as a result of two consecutive events along the anteroposterior axis, referred to as primary and secondary neurulation (PN and SN). While PN involves the invagination of a sheet of epithelial cells, SN shapes the caudal neural tube through the mesenchymal-to-epithelial transition (MET) of neuromesodermal progenitors, followed by cavitation of the medullary cord. The technical difficulties in studying SN mainly involve the challenge of labeling and manipulating SN cells in vivo. Here we describe a new method to follow MET during SN in the chick embryo, combining early in ovo chick electroporation with in vivo time-lapse imaging. This procedure allows the cells undergoing SN to be manipulated in order to investigate the MET process, permitting their cell dynamics to be followed in vivo.


Asunto(s)
Electroporación/métodos , Células Epiteliales/citología , Mesodermo/citología , Neurulación , Imagen de Lapso de Tiempo/métodos , Animales , Embrión de Pollo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Epiteliales/metabolismo , Mesodermo/embriología , Mesodermo/metabolismo
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