We would like to thank Gero Miesenb&#246;ck and Tim Ryan for sharing pHluorin cDNA used to generate reagents in this study, Nathan Wright, Joanna Poprawski and Lydia Nyasae for excellent technical assistance, members of the Wendland lab for helpful discussions, and Michael McCaffery and Erin Pryce at the Integrated Imaging Center (Johns Hopkins) for advice and assistance with microscopy and flow cytometry. This work was supported by grants from the National Institutes of Health (to B.W., GM60979) and the National Science Foundation (to B.W., MCB 1024818). K.W. was supported in part by a training grant from the National Institutes of Health (T32-GM007231). A.F.O. was supported by developmental funds from the Department of Biological Sciences at Duquesne University and National Science Foundation CAREER grant 553143.

The authors declare no competing interests.

Anwendungen von pHluorin zur Quantifizierung von endocytischen Ereignisse in Hefezellen macht von Transmembran- cargos in der die fluoreszierende Markierung an den zytoplasmatischen Schwanz des Proteins (1A) fusioniert ist. Für die Tests hier beschriebenen Frachten sind hell fluoreszierende, wenn die pHluorin Tag neutralen Umgebungen ausgesetzt ist, aber abgeschreckt werden, wenn die pHluorin Tag sauren Bedingungen trifft. So a pHluorin-markierte endocytic Ladung leicht an der zytoplasmatischen Gesicht...

Vesikeltransport spielt eine wichtige Rolle Organell Identität und Funktion in eukaryontischen Zellen beibehalten wird, und ist ein Schlüsselmechanismus Protein und Membranzusammensetzung der einzelnen zellulären Kompartimenten zur Regulierung. An der Plasmamembran, Fusion von Vesikeln Exozytose liefert neue Proteine ​​und Membranen auf der Oberfläche der Zelle, während die erzeugten Bläschen durch Endozytose Membran und Proteine ​​von der Oberfläche zum Lysosom zur anschließenden Wiederverwertung oder Targeting entfernen. Somit ist Endozytose wichtig für die Nährstoffaufnahme und für Reaktionen auf die extrazelluläre Umgebung. Exozytose und Endozytose werden ausgewogen Plasmamembranoberfläche zu regulieren, und der Umsatz von beschädigten Proteinen zu ermöglichen. Studien in Hefe und Säugerzellen haben eine große Anzahl von Proteinen in der Endozytose, sowie mehrere endocytic Wegen beteiligt identifiziert, die zu einer Vielzahl von en Internalisierung spezifischer Frachten oder diese Handlung als Reaktion fördernbungsbedingungen. Die am besten untersuchten Weg ist Clathrin-vermittelte Endozytose (CME), in der Clathrin und zytosolischen akzessorischen Proteine ​​in einer Schichtstruktur zusammenstellen, die im Entstehen begriffenen endocytic Vesikel zu stabilisieren. Experimente in der Bäckerhefe Saccharomyces cerevisiae haben wichtige Einblicke in die Dynamik und die Reihenfolge der Rekrutierung ergab für viele endocytic Proteine 1-3. Bemerkenswerterweise wird die CME Maschinen stark durch die Evolution konserviert, so dass die Mehrheit der CME-verwandten Proteinen in Hefe menschliche Orthologe haben; Somit Hefe Knospung hat ein wichtiges Werkzeug für das Verständnis der Mechanismen der Endozytose gegeben, die in höheren Eukaryonten konserviert sind. Zum Beispiel Studien Bäckerhefe mit bestimmt, dass die Bildung und Reifung von CME-Strukturen (als kortikalen Aktin-Patches in Hefe bezeichnet) umfasst die schrittweise Einstellung vieler Proteine, beginnend mit Clathrin und Fracht-bindenden Adapterproteine, gefolgt von Einstellung zusätzlicher endocytic Zubehör Proteine,Aktivierung von Arp2 / 3-vermittelte Aktin - Polymerisation und die Rekrutierung von Proteinen in Vesikel scission 1,2 beteiligt. Die Rekrutierung von CME Maschinen Proteine ​​Aktin-Patches zu kortikalen ist eine hoch geordnete und stereotypisch Prozess, und die genaue Reihenfolge der Rekrutierung für viele Proteine ​​in Bezug auf andere Komponenten der CME Maschinen etabliert. Wichtig ist , dass die jüngsten Studien haben eine ähnliche Reihenfolge der Rekrutierung für CME Proteine bei Clathrin beschichteten Gruben in Säugetierzellen 4 bestätigt. Neben CME besitzen viele Zelltypen eine oder mehrere Clathrin-unabhängige endocytic (CIE) Wege , die auf alternative Mechanismen beruhen Vesikelbildung und Internalisierung von der Plasmamembran 5-7 zu fördern. In Hefe identifizierten wir vor kurzem eine CIE - Weg, der die kleine GTPase Rho1 und seine Aktivierung Guaninnukleotidaustauschfaktor (GEF), Rom1 8,9 verwendet. Rho1 aktiviert die formin Bni1 Actinpolymerisation 1 zu fördern0,11, die für diese Form der Clathrin-unabhängige Endozytose in Hefe 12 erforderlich ist. Außerdem rekrutieren die α-Arrestin Familie von Proteinen , die Ubiquitin - Ligase Rsp5 zur Ladung Ubiquitinierung und anschließende Internalisierung durch CME 13-17, und auch fördern Ladung Internalisierung über den CIE - Weg, möglicherweise durch direkte Interaktion mit Proteinen beteiligt in CIE 18 fördern. Eine Vielzahl von CIE Wege existieren auch in Säugerzellen, einschließlich Clathrin-unabhängige und phagozytischen Bahnen , die auf der Rho1 Ortholog verlassen, RhoA 9,19. Die Rolle von RhoA in Säuger CIE ist wenig bekannt; also Studien in Bäckerhefe kann zusätzliche mechanistische Erkenntnisse liefern, die auf Säugetier CIE anwendbar sind. Eine genaue Quantifizierung von endocytic Ereignisse können, Endozytose bei der Regulierung der wichtige Informationen über die Rollen von spezifischen Proteinen liefern und die Auswirkungen der Mutationen auf die Ladung Verinnerlichung oder progressio offenbarenn durch den endocytischen. Zu diesem Zweck können biochemische Verfahren verwendet werden, Liganden-Aufnahme zu überwachen oder Abbauraten von endocytischen Frachtproteinen zu messen. In lebenden Zellen Fusion des grün fluoreszierenden Proteins (GFP) und dessen Varianten Frachten von Interesse ermöglicht die direkte Visualisierung von Frachtverkehr. Allerdings GFP-markierten Frachten sind von begrenztem Nutzen für die Quantifizierung der Endozytose, weil GFP zu einem Abbau in den Lysosomen (oder Vakuole in Hefe) resistent ist. Außerdem ist GFP - Fluoreszenz nur teilweise empfindlich gegenüber Änderungen in pH und bleibt innerhalb der Vakuole Lumen 20,21 nachweisbar. Folglich Fluoreszenz des GFP-Tag besteht in der Vakuole, lange nachdem der Rest der Ladung abgebaut worden ist, und ganze zellbasierten Quantifizierung von Ladungsintensität kann durch langfristige GFP-Fluoreszenz in die Vakuole ungenau sein. Um die Nachteile von vakuolären GFP-Fluoreszenz zu überwinden, wir zuvor Verwendung superecliptic pH gemachtluorin, eine pH-sensitive Variante von GFP , die hell bei neutralem pH fluoresziert, verliert aber Fluoreszenz in sauren Umgebungen , wie beispielsweise das Lumen der Vakuole / Lysosomen 20,22,23. Eine pHluorin Tag auf der zytoplasmatischen Schwanz von endocytischen cargos Platzierung erlaubt die Visualisierung der Frachten an der Plasmamembran und am frühen Endosomen, in denen die pHluorin Tag in das Zytoplasma (1A) ausgesetzt bleibt. Bereits Endosomen reifen, die Endosomen Sortierkomplexe , die für Transport (ESCRT) Maschinen - Pakete oberflächen lokalisierte Frachten in Vesikeln, die in das Lumen des Endosomen Knospe, Erzeugung multivesicular Körper (MVBs) 24. Für Frachten, die in interne MVB Vesikel aufgenommen wurden, steht die pHluorin Tag in Richtung der Vesikel Lumen. Interne MVB Vesikel werden angesäuert; so verlieren pHluorin-markierte Frachten Fluoreszenz auf frühe Endosomen , wie sie reifen in MVBs 20. Die anschließende Verschmelzung der MVB mit der Vakuole liefert die MVB luminalen Inhaltefür den Abbau und pHluorin Tags bleiben in der sauren Umgebung der Vakuole Lumen gequencht. Dieses Dokument enthält detaillierte Beschreibungen der quantitativen endocytic Assays Cargos mit zytoplasmatischen pHluorin Tags in Hefe verwendet. Die Stämme pHluorin-markierte Frachten exprimieren, können in kinetische und / oder Endpunkt-Assays verwendet werden, abhängig von den Eigenschaften und den Handel Verhalten der spezifischen Ladung. Darüber hinaus sind einige pHluorin-tagged cargos zugänglich Hochdurchsatz-Analyseverfahren, einschließlich Durchflusszytometrie. Wichtig ist, dass die pHluorin Tag ein vielseitiges Werkzeug für die in lebenden Zellen endocytic Ereignisse zu studieren, so dass eine Quantifizierung der Endozytose und den Vergleich von endocytic Funktion in Wildtyp und Mutantenstämmen.

<table><tbody><tr><td>Adenine</td><td>Sigma</td><td>A8626-25G</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>Bacto-agar</td><td>Fisher</td><td>BP1423-2</td><td>Use for preparation of plate media</td></tr><tr><td>BD Difco Yeast Nitrogen Base (without amino acids)</td><td>BD</td><td>291920</td><td>Use for preparation of liquid and plate media</td></tr><tr><td>Concanavalin A</td><td>Sigma</td><td>C5275-5MG</td><td>Use for coating of chamber slides</td></tr><tr><td>Dextrose</td><td>Fisher</td><td>BP350-1</td><td>Use for preparation of liquid and plate media</td></tr><tr><td>L-Histidine</td><td>Fisher</td><td>BP382-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Leucine</td><td>Acros</td><td>125121000</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Lysine</td><td>Fisher</td><td>BP386-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Methionine</td><td>Fisher</td><td>BP388-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Tryptophan</td><td>Fisher</td><td>BP395-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Tyrosine</td><td>Acros</td><td>140641000</td><td>Use for preparation of amino acid mixture</td></tr><tr><td>Nunc Lab-Tek Chambered Coverglass (8-well)</td><td>Thermo Scientific</td><td>155411</td><td>Use for kinetic and endpoint assays of Mup1-pHluorin internalization</td></tr><tr><td>Uracil</td><td>Sigma</td><td>U0750-100G</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>Axiovert 200 inverted microscope</td><td>Carl Zeiss</td><td>Custom Build</td><td></td></tr><tr><td>100X/1.4 Plan-Apochromat Oil Immersion Objective Lens</td><td>Carl Zeiss</td><td></td><td>Objective should be 100X, 1.4NA or higher</td></tr><tr><td>Sensicam</td><td>Cooke Corporation</td><td></td><td>Camera should have 12-bit or higher dynamic range</td></tr><tr><td>X-Cite 120PC Q Illumination Source</td><td>Excelitas Technologies</td><td></td><td></td></tr><tr><td>Slidebook 5 software</td><td>Intelligent Imaging Innovations</td><td></td><td></td></tr><tr><td>ImageJ software</td><td>National Institutes of Health</td><td></td><td>http://imagej.nih.gov/ij/</td></tr></tbody></table>

applications of phluorin for quantitative, kinetic and high-throughput analysis of endocytosis in budding yeast

Das grün fluoreszierende Protein (GFP) und seine Varianten sind Werkzeuge zur Untersuchung von Proteinlokalisierung und Dynamik von Ereignissen wie Zytoskelett-Umbau und Vesikeltransport in lebenden Zellen weit verbreitet. Quantitative Methoden unter Verwendung von chimären GFP-Fusionen wurden für viele Anwendungen entwickelt; jedoch GFP ist etwas resistent gegen Proteolyse, wodurch seine Fluoreszenz bleibt im Lysosom / Vakuole, die Quantifizierung von Frachthandels in der endocytischen behindern können. Ein alternatives Verfahren zur Quantifizierung Endozytose und post-endocytic Handel Ereignisse nutzt superecliptic pHluorin, eine pH-sensitive Variante von GFP, die in sauren Umgebungen abgeschreckt wird. Chimäre Fusion von pHluorin zum Zytoplasmaschwanz von Transfracht-Proteine ​​führt zu einer Dämpfung der Fluoreszenz bei der Gründung der Ladung in multivesicular Körper (MVBs) und Auslieferung an die Lysosomen / Vakuole Lumen. Somit Löschung der Vakuole Fluoreszenz erleichtert quantifiKation der Endozytose und der frühen Ereignisse im endocytischen. Dieses Papier beschreibt Methoden pHluorin-markierte Frachten für die Quantifizierung der Endozytose über Fluoreszenzmikroskopie sowie populationsbasierten Assays Durchflusszytometrie verwendet wird.

Steady-State - Lokalisierung und Quantifizierung des konstitutiv internalisiert endocytic Fracht Protein STE3 Um zu demonstrieren, dass pHluorin-tagged cargos kann zur Quantifizierung von Endozytose in lebenden Zellen, Lokalisierung von chimären GFP und pHluorin Fusionen mit der zytoplasmatischen C-terminalen Schwanz von STE3 verwendet werden, die a-Faktor-Pheromon-Rezeptor in Hefe, wurden ve...

Watch this Scientific Journal Video about Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast at JoVE.com

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

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.

Anwendungen von pHluorin für Quantitative, Kinetic und Hochdurchsatz-Analyse von Endozytose in Bäckerhefe

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

applications-phluorin-for-quantitative-kinetic-high-throughput

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.8K Aufrufe.  The Johns Hopkins University. 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.

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

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

Verwendung von pHluorin zur Bewertung der Dynamik von Axon-Leitrezeptoren in der Zellkultur und im Küken-Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Forschung

Neurowissenschaften

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Die Analyse der Entwicklung eines morphologischen Phänotyp als Funktion der Protein-Konzentration in Hefezellen

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Biologie

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM und pH-sensitive GFP-Sonden an Neurotransmitter-Vesikel-Dynamics Werten in SH-SY5Y Neuroblastomzellen: Cell Imaging und Datenanalyse

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Messung der synaptischen Vesikel Endozytose in kultivierten Hippocampal Neuronen

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Quantitative Live Cell Fluoreszenz-Mikroskopie Analyse von Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

Messung von pH Vacuolar und Cytosolic<em&gt; In Vivo</em&gt; In Hefe Zellsuspensionen

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

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

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

Acquiring Fluoreszenz Zeitraffer-Filme von Bäckerhefe und Analysieren von Einzel-Zell-Dynamics mit Transplantaten

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

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

pHluorin-getaggt Receptors Verwendung zu überwachen subzelluläre Lokalisierung und Handel

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

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.

Hochauflösende Bildgebung und Analyse einzelner Astral Dynamik der Mikrotubuli in Bäckerhefe

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

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

4D-Mikroskopie der Hefe

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...