Nous remercions Emily Jackson, Joshua Zeidman, et Nicholas Wren pour les commentaires sur le logiciel. Ce travail a été financé par GM95733 (au Mexique), BBBE 103316 et fonds de démarrage du MIT (à NM).

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L., Volfson, D., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+dynamics+of+single-cell+gene+expression+over+multiple+cell+cycles.">Monitoring dynamics of single-cell gene expression over multiple cell cycles.</a> Molecular Systems Biology. 1 (1), 2005.0024 (2005).</li><li>Paliwal, S., Iglesias, P. A., Campbell, K., Hilioti, Z., Groisman, A., Levchenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAPK-mediated+bimodal+gene+expression+and+adaptive+gradient+sensing+in+yeast.">MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast.</a> Nature. 446 (7131), 46-51 (2007).</li><li>Charvin, G., Cross, F. R., Siggia, E. 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Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Le protocole ci-dessus décrit une méthode simple pour obtenir et analyser des données de séries chronologiques de fluorescence avec une expérience limitée en microfluidique ou dans le développement de logiciels. Il permet d&#39;obtenir time-lapse films de fluorescence des cellules de levure simples; extraire la taille des cellules pertinentes et les mesures d&#39;expression; suivi curé et missions de la lignée, et analyser le comportement des cellules entières dans le temps en utilisant un dispositif de cultur...

Analyse unicellulaire de l&#39;expression génique a permis de mieux comprendre de nombreux aspects de la régulation des gènes. Instantanés statiques d&#39;expression rapporteur fluorescent par cytométrie en flux ou microscopie fournissent des informations utiles sur la distribution de l&#39;expression seule cellule, mais n&#39;ont pas l&#39;histoire et l&#39;évolution des données de séries chronologiques nécessaires pour informer directement la dynamique de l&#39;expression des gènes. Microscopie time-lapse fluorescence présente un moyen d&#39;obtenir deux mesures unicellulaires et leur histoire. Diverses techniques expérimentales et analytiques ont été développées afin d&#39;obtenir et de quantifier les films d&#39;expression rapporteur fluorescent, conférant ainsi un aperçu des caractéristiques de régulation des gènes (cf. 1 pour une revue) tels que la variation de cellule de la cellule 2,3, la formation de persister bactérienne 4, transcription initiation et d&#39;élongation 5, transcription éclatement 6,7, la dépendance du cycle cellulaire 8,9 et héritabilité 10. Cependant, obtaining série temporelle de fluorescence unicellulaire qualité implique des difficultés techniques importantes dans la culture d&#39;une monocouche de cellules dans un environnement contrôlable dans la quantification et à haut débit des films de fluorescence acquises. Ici, nous décrivons une procédure pour obtenir et analyser des films de fluorescence de S. cerevisiae avec aucune expérience requise dans la fabrication du dispositif de culture cellulaire ou dans le développement de logiciels (Figure 1). Tout d&#39;abord, nous détaillons un protocole d&#39;exemple pour produire des films de séries chronologiques de fluorescence pour levure bourgeonnante exprimant un ou plusieurs rapporteurs fluorescents. Bien que des chambres de culture microfluidiques personnalisés ont été construits et utilisés avec succès précédemment 11-13, nous utilisons un dispositif microfluidique disponible dans le commerce à partir de CellAsic (Hayward, CA). Le système confine à la croissance des cellules de la monocouche et permet un contrôle continu du milieu de perfusion. Le protocole de microscopie que nous présentons est un moyen simple d&#39;obtenir Déessfilms rence de levure bourgeonnante, mais tout protocole modifié expérimental (un dispositif de culture personnalisée, les conditions de médias alternatifs, etc.) fournissant des données de films similaires fluorescence de cellules de levure simples peuvent être substitués. Ensuite, nous présentons l&#39;analyse des films en utilisant une interface utilisateur graphique (GUI) basée sur logiciel dans MATLAB (The MathWorks, Natick, MA), surnommé le GUI pour l&#39;analyse rapide des séries chronologiques Fluorescence (greffes), pour extraire des données de séries chronologiques pour des cellules individuelles. GREFFES a des caractéristiques similaires à la polyvalente, logiciel open-source paquet Cell-ID 14 dans la segmentation et le suivi des cellules et dans l&#39;extraction de l&#39;intensité de fluorescence et de l&#39;information géométrique. Toutefois, les greffes fournit d&#39;importantes fonctionnalités supplémentaires. Tout d&#39;abord, il offre une édition interactive facile de segmentation et le suivi des résultats afin de vérifier l&#39;exactitude des données, plutôt que de simplement gating statistique des traces de la région aberrantes après analyse. En outre, elle étend l&#39;analyse aux automatically désigné lignée et les points du cycle cellulaire d&#39;intérêt de la levure bourgeonnante. Déterminer le moment où mère et fille se divisent pour former deux régions de cellules indépendantes est cruciale pour déterminer les mesures à travers le cycle cellulaire 8 cellules entières (y compris toute mère bourgeon connecté). La suite se compose de trois modules pour accomplir ces tâches. Les premières régions cellulaires segments basé sur le contraste entre les images de champ lumineux ciblés et non ciblés, et permet à l&#39;utilisateur de définir et de tester visuellement les paramètres de segmentation. La seconde pistes (. Utilisant Blair et la mise en œuvre de la MATLAB Crocker et al IDL routine de Dufresne, disponible sur: <a href="http://physics.georgetown.edu/matlab/" target="_blank">http://physics.georgetown.edu/MATLAB/</a> ) et les mesures régions cellulaires à travers le temps; attribue automatiquement lignées et permet une inspection visuelle et la correction d&#39;erreur. Une interface graphique tracé simple est inclus aux propriétés unicellulaires requête. Le troisième module attribue bourgeon émergence et la division times, et délivre des données de séries chronologiques de cellules entières ainsi que leurs dérivées premières et secondes de temps (tel que discuté dans 9). Le module d&#39;analyse fournit en sortie les données dans un fichier de texte délimité par des espaces pour l&#39;étude ultérieure dans le logiciel de statistique de choix. Ainsi, le forfait permet à l&#39;utilisateur d&#39;extraire des données de séries chronologiques de haute qualité par le biais d&#39;une interface graphique. Nous avons utilisé cette méthode pour estimer les taux de transcription en temps réel dans des cellules de levure bourgeonnante simples en fonction du cycle cellulaire 9. Alors que les modules ont été optimisés pour levure bourgeonnante, les paramètres ou, le cas échéant, le code librement disponible peut être adapté à d&#39;autres organismes et les types d&#39;images. Segmentation, le suivi, et la lignée des algorithmes d&#39;affectation peuvent être spécifiques à types d&#39;imagerie affecté et l&#39;organisme en question. Les algorithmes existants pourraient être remplacés, mais conservent l&#39;interface graphique qui permet une inspection visuelle conviviale et la correction des erreurs de segmentation et de suivi qui invariablement occupantsr avec n&#39;importe quel algorithme.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Y04C Yeast Perfusion Plate</td>
			<td>CellAsic</td>
			<td>Y04C-02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ONIX Microfluidic Perfusion Platform</td>
			<td>CellAsic</td>
			<td>EV-262</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Axio Observer.Z1 Microscope</td>
			<td>Zeiss</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plan-Apochromat 63X/1.40 Oil DIC objective</td>
			<td>Zeiss</td>
			<td>440762-9904-000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cascade II EMCCD camera</td>
			<td>Photometrics</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lumen 200 metal-halide arc lamp</td>
			<td>PRIOR Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triple-bandpass dichroic filter cube and excitation and emission filter set</td>
			<td>Chroma Technology Corp</td>
			<td>set #89006</td>
			<td>Used for YFP (Venus/Citrine), CFP (Cerulean), RFP (mCherry/tdTomato)</td>
		</tr>
		<tr>
			<td>MAC 5000 controller and filter wheels</td>
			<td>Ludl Electronic Products</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MATLAB R2011a</td>
			<td>Mathworks</td>
			<td>&nbsp;</td>
			<td>64-bit version handles large data files better than 32-bit</td>
		</tr>
	</tbody>
</table>

acquiring fluorescence time-lapse movies of budding yeast and analyzing single-cell dynamics using grafts

Microscopie time-lapse fluorescence est devenu un outil puissant dans l&#39;étude de nombreux processus biologiques au niveau d&#39;une seule cellule. En particulier, les films dépeignant la dépendance temporelle de l&#39;expression des gènes permettent de mieux comprendre la dynamique de sa réglementation, mais il ya beaucoup de défis techniques à obtenir et à analyser les films de fluorescence des cellules individuelles. Nous décrivons ici un protocole simple en utilisant un dispositif de culture microfluidique disponible dans le commerce pour produire ces données, et une interface graphique basée sur MATLAB utilisateur (GUI) basée sur logiciel pour quantifier les images de fluorescence. Les segments des logiciels et des cellules de pistes, permet à l&#39;utilisateur de vicaire visuellement erreurs dans les données, et attribue automatiquement lignage et des temps de division. L&#39;interface graphique analyse en outre la série temporelle afin de produire des traces de cellules entières, ainsi que leurs dérivées première et seconde de temps. Bien que le logiciel a été conçu pour S. cerevisiae, sa modularité et sa polyvalence devrait lui permettre de to servir de plate-forme pour l&#39;étude d&#39;autres types cellulaires avec quelques modifications.

Une expérience réalisée et analysée avec succès donnera série surtout à temps continu pour les cellules entières simples avec réalisme attribué apparition des bourgeons et des temps de division. À titre d&#39;exemple, nous avons réalisé le protocole ci-dessus avec une souche de levure haploïde exprimant une copie intégrée de la protéine fluorescente Cerulean (CFP) entraînée par le promoteur constitutif ADH1 d&#39;observer comment la croissance et l&#39;expression globale peuvent vari...

Watch this Scientific Journal Video about Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS at JoVE.com

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Nous présentons un protocole simple d&#39;obtenir des films de microscopie par fluorescence de cellules de levure en croissance, et un logiciel GUI basée à extraire des données de séries chronologiques unicellulaires. L&#39;analyse comprend lignée automatisé et l&#39;affectation du temps de division intégrée à l&#39;inspection visuelle et la conservation manuelle de données à chenilles.

L&#39;acquisition de fluorescence Time-lapse films de levure bourgeonnante et analyse de la dynamique de cellules uniques en utilisant des greffes

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

acquiring-fluorescence-time-lapse-movies-budding-yeast-analyzing

Research

JoVE Journal

Biology

12.7K vues.  Massachusetts Institute of Technology. Nous présentons un protocole simple d'obtenir des films de microscopie par fluorescence de cellules de levure en croissance, et un logiciel GUI basée à extraire des données de séries chronologiques unicellulaires. L'analyse comprend lignée automatisé et l'affectation du temps de division intégrée à l'inspection visuelle et la conservation manuelle de données à chenilles.

Vidéo: Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Mesure Life Span réplicative dans la levure bourgeonnante

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Recherche

Biologie

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Imagerie time-lapse de la mitose après transfection de siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Analyse quantitative des migrations au hasard des cellules en utilisant la microscopie vidéo time-lapse

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Bourgeonnement de levure extraction et purification des protéines pour l&#39;étude de la fonction, Interactions, et des modifications post-traductionnelles

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Protein Purification Technique qui permet la détection de sumoylation et Ubiquitination de levure bourgeonnante kinétochoriens protéines Ndc10 et Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Haute résolution Time-lapse d&#39;imagerie et d&#39;analyse automatisée des microtubules Dynamics Living ombilicale humaine Cellules endothéliales de veine

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

Applications de pHluorin pour Quantitative, Kinetic et à haut débit Analyse des endocytose dans la levure bourgeonnante

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

L&#39;utilisation des dispositifs microfluidiques pour mesurer et cellulaire Lifespan dans Phenotypes simples cellules de levure de bourgeonnement

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

L&#39;imagerie à haute résolution et d&#39;analyse de l&#39;individu Astral microtubules Dynamique dans la levure bourgeonnante

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Examen de la dynamique nucléaire de levure de fission mitotique et méiotique par fluorescence Microscopie à cellules vivantes

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...