Wir danken Kerry Bloom (University of North Carolina), Kyung Lee (NCI), Steven Markus (Colorado State University), und Elmar Schiebel (Universität Heidelberg) für verschiedene FP-TUB1 Plasmide zu teilen. Wir sind dankbar, dass Melissa Gardner (University of Minnesota) für die Ausbildung uns in der Schieberkammer Herstellungsverfahren. Diese Arbeit wurde von den National Institutes of Health (NIH) gewähren R01GM112893-01A1 (bis JKM) und T32GM008730 (CE) unterstützt.

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinating+mitosis+with+cell+polarity:+Molecular+motors+at+the+cell+cortex.">Coordinating mitosis with cell polarity: Molecular motors at the cell cortex.</a> Semin Cell Dev Biol. 21 (3), 283-289 (2010).</li><li>Gartz Hanson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+alpha-tubulin+mutation+disrupts+neural+development+and+tubulin+proteostasis.">Novel alpha-tubulin mutation disrupts neural development and tubulin proteostasis.</a> Dev Biol. 409 (2), 406-419 (2016).</li><li>Fees, C. P., Aiken, J., O'Toole, E. T., Giddings, T. H. J., Moore, J. 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Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Die Bäckerhefe Modell bietet große Vorteile für die in einem in - vivo - Einstellung hochauflösende Messungen der Dynamik der Mikrotubuli sammeln, einschließlich der Fähigkeit, einzelne Bild Mikrotubuli über die Zeit und die Fähigkeit , Tubulinen und Mikrotubuli Regulatoren mit den Werkzeugen der Hefegenetik zu manipulieren. Die Concanavalin A-beschichtete Kammern bieten eine Reihe von Vorteilen gegenüber den zuvor beschriebenen Vorrichtungen, einschließlich der g...

Mikrotubuli sind Polymere aus Zytoskelett-αβ-Tubulin-Protein-Untereinheiten und werden in einer großen Vielzahl von zellulären Zusammenhängen, einschließlich intrazellulären Transport, die Zellteilung, die Morphogenese und Motilität verwendet. Um Mikrotubuli Netzwerke für diese unterschiedlichen Rollen zu erstellen, müssen Zellen sorgfältig regeln, wann und wo Mikrotubuli Form. Diese Regelung wird durch die Steuerung der Reaktionen erreicht, daß entweder αβ-Tubulin-Untereinheiten in Mikrotubuli-Polymere oder disassemble Mikrotubuli in freie Untereinheiten zusammenzubauen; dies wird als die Dynamik der Mikrotubuli bekannt. Ein Hauptziel des Mikrotubuli-Feld ist, die molekularen Mechanismen aufzuklären, die Dynamik der Mikrotubuli, einschließlich Studien der αβ-Tubulin-Untereinheiten und extrinsische Regulatoren regulieren, die an Tubulin und / oder an Mikrotubuli binden. Ein etablierter experimenteller Ansatz ist dieses System in vitro unter Verwendung von gereinigtem αβ-Tubulin - Protein rekonstituieren, die oft in combination mit gereinigtem extrinsischen Regulatoren. Obwohl dies ein sinnvoller Ansatz ist, ist es klar, dass in rekonstituierten Systemen die Dynamik der Mikrotubuli stark von der in lebenden Zellen beobachtet unterscheidet. Zum Beispiel wächst Mikrotubuli schneller und schrumpft langsamer in vivo als in vitro. Diese Unterschiede können auf die Verfügbarkeit von bekannten extrinsischen Regler 1 sowie auf noch-nicht definierte Faktoren in Zellen zurückgeführt werden. Daher ist es wichtig, die Aktivitäten von Mikrotubulus-Regulierungsbehörde und Mutanten zu bestimmen, die Dynamik in einem nativen zellulären Kontext zu stören. Obwohl metazoan Modelle die vorherrschenden Systeme werden für die Untersuchung von Mikrotubuli-Funktion und höherer Ordnung Organisation, mehrere praktische Bedenken schränken den Nutzen dieser Modelle für die präzise Messung der Dynamik der Mikrotubuli bewährt haben. Erstens ist die hohe Anzahl von Mikrotubuli, die von Dutzenden bis Tausenden pro Zelle, macht es schwierig, sicherverfolgt einzelne Mikrotubuli im Laufe der Zeit. Viele Studien befassen sich diese Herausforderung durch Abbilden Proteine, die an Mikrotubuli-Enden, wie beispielsweise Proteine ​​in der End-Bindung (EB) Familie selektiv lokalisieren. Jedoch werden diese Proteine bekannt, nur an den Enden des Wachsens Mikrotubuli in Metazoen 2 zu lokalisieren. Daher wird die Nützlichkeit dieser Proteine ​​beschränkt auf Wachstumsraten direkt zu messen, während nur indirekt andere Aspekte der Dynamik zu messen, wie Frequenz der Katastrophe. Zweitens, trotz der Fortschritte in der Genombearbeitungstechnologie, Zellen schaffen, die fluoreszierend markierte Tubulin oder Einführung von Mutationen stabil exprimieren, selektiv Tubulin oder Mikrotubulus-Regula zu manipulieren bleibt eine große Herausforderung. Außerdem confounds die Anwesenheit von vielen Tubulin-Isotypen in metazoans die Studie, wie Mutationen Gene einzelne Tubulin auswirken. Die Bäckerhefe System bietet mehrere wichtige Vorteile für die in - vivo - Messung microtubule Dynamik. Hefe hat ein vereinfachtes Mikrotubuli-Netzwerk, das die Visualisierung der einzelnen Mikrotubuli erlaubt. In Hefe stammen Mikrotubuli von Zentren bekannt als Spindel Polkörper (SPBs) der Organisation, die 3 in der Kernhülle eingebettet ist. Die SPBs dienen als Gerüste für γ-Tubulin kleine Komplexe , die Mikrotubuli 4, 5 nukleieren. SPBs nucleate zwei Klassen von Mikrotubuli, Spindel Mikrotubuli und astralen Mikrotubuli. Spindel Mikrotubuli Projekt in Nukleoplasma und sind wichtig für die Befestigung an Chromosomen über kinetochore Mikrotubuli und zur Stabilisierung der Spindel über überlappende inter Mikrotubuli 6. Im Gegensatz dazu, nach außen vorstehen Astral Mikrotubuli in das Cytosol und relativ selten ist mit dem dichten Netz von Mikrotubuli Spindel verglichen. Während der Mitose haben pre-Anaphase Zellen nur 1-2 Astral Mikrotubuli entweder von SPB ausgeht; diese existieren, wie ichNDIVIDUELLE Mikrotubuli anstatt als Bündel 7. Die Rolle des astralen Mikrotubuli während der Mitose ist den Kern und Spindel in die Verbindung zwischen der Mutter und Knospe Abteilen, bekannt als Keim Hals zu bewegen. Diese Bewegung beinhaltet gut definierte Wege , die Kraft , die auf astralen Mikrotubuli erzeugen, ziehen den Kern und Spindel in Richtung und schließlich in die Knospe Hals 8. Ein weiterer Vorteil des Hefe-Systems ist das Nutzen ihrer Genetik, die verwendet werden kann Mikrotubuli Regler und Tubulin-Untereinheiten mit bisher unerreichter Genauigkeit zu untersuchen. Hefe besitzt auch ein vereinfachtes Repertoire von Tubulin - Isotypen: eine einzelne β-Tubulin - Gen (TUB2) und zwei α-Tubulin - Gene (TUB1 und TUB3). Mutationen können leicht in diese Gene eingeführt werden und damit in einer homogenen Population Tubulin 9 untersucht, 10. Es gibt eine Reihe von weitverfügbar Konstrukte zur Markierung von Mikrotubuli, und diese können für die Integration in die chromosomale Loci für eine stabile Expression ausgerichtet werden (siehe Diskussion). Das Gesamtziel dieses Verfahrens ist, die das Bild einzelnes Mikrotubuli in lebenden Hefezellen in vier Dimensionen (X, Y, Z und T) für hochauflösende Messungen der Dynamik des Mikrotubuli. Verfahren zum Konstrukten für die konstitutive, Low-Level-Expression von fluoreszenzmarkierten Tubulin in Hefezellen Integration beschrieben. Vor der Bebilderung werden lebenden Zellen in Gleitberührung Kammern montiert mit dem Lectin Concanavalin A beschichtet ist, die Zellen für die Langzeitbilderzeugung zu stabilisieren. Die optimalen Parameter für die Bildaufnahme, sowie ein Workflow für die Datenanalyse, werden ebenfalls beschrieben.

<table><tbody><tr><td>DOB (dropout bases)</td><td>Sunrise science&amp;nbsp;</td><td>1650</td><td></td></tr><tr><td>CSM-Leu</td><td>Sunrise science&amp;nbsp;</td><td>1005</td><td></td></tr><tr><td>Agar</td><td>Ameresco</td><td>N833</td><td></td></tr><tr><td>100 mm polystyrene plates</td><td>Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>ssDNA (Samon Sperm)&amp;nbsp; in sterile DiH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich&amp;nbsp;</td><td>&amp;nbsp;D7656</td><td>resuspend at 10 mg/mL in DiH2O. Store aliquots at -20 &amp;ordm;C</td></tr><tr><td>Synthetic Complete Media&amp;nbsp;</td><td>Sunrise science&amp;nbsp;</td><td>1459-100</td><td></td></tr><tr><td>Concanavalin A</td><td>Sigma-Aldrich&amp;nbsp;</td><td>L7647</td><td>resuspend at 2 mg/mL in DiH2O. Store aliquots at -20 &amp;ordm;C</td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>12-544-1</td><td></td></tr><tr><td>Microscope Coverslips</td><td>Fisher Scientific</td><td>12-541-B</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-12</td><td>paraffin film</td></tr><tr><td>VALAP (Equal parts of Vaseline, lanolin and paraffin)</td><td></td><td></td><td>melt at 75 &amp;ordm;C before use</td></tr><tr><td>forceps&amp;nbsp;</td><td>Fisher Scientific</td><td>16-100-106</td><td></td></tr><tr><td>Poyethylene glycol (PEG) 3350</td><td>Sigma-Aldrich&amp;nbsp;</td><td>202444</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscope&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ti E inverted Perfect Focus microscope</td><td>Nikon Instruments</td><td>MEA53100</td><td></td></tr><tr><td>1.45 NA 100X CFI Plan Apo objective</td><td>Nikon Instruments</td><td>MRD01905</td><td></td></tr><tr><td>Piezo electric stage&amp;nbsp;</td><td>Physik Instrumente</td><td>P-736</td><td></td></tr><tr><td>Spinning disk scanner</td><td>Yokogawa</td><td>CSU10</td><td></td></tr><tr><td>Laser combiner module</td><td>Agilent Technologies</td><td>MCL400B</td><td></td></tr><tr><td>EMCCD camera</td><td>Andor Technology</td><td>iXon Ultra 897&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NIS Elements software</td><td>Nikon Instruments</td><td>MQS31100</td><td></td></tr><tr><td>Microsoft Excel software</td><td>Microsoft</td><td></td><td></td></tr><tr><td>MATLAB software</td><td>MathWorks, Inc</td><td></td><td></td></tr><tr><td>ImageJ64</td><td>NIH</td><td></td><td>Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016.</td></tr><tr><td>Bio-Formats Importer plug-in</td><td>Open Microscopy Environment</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pUC19-LEU2::GFP-TUB1</td><td></td><td>pSK1050</td><td>reference: Song, S. and Lee, K. S. A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis. J Cell Biol. 152 (3), 451-469 (2001)</td></tr></tbody></table>

high-resolution imaging and analysis of individual astral microtubule dynamics in budding yeast

Dynamische Mikrotubuli sind von grundlegender Bedeutung für viele zelluläre Prozesse, und genaue Messungen der Dynamik der Mikrotubuli können Einblick, wie Zellen regulieren diese Prozesse und wie genetische Mutationen Auswirkungen Regulierung. Die Quantifizierung der Dynamik der Mikrotubuli in metazoan Modelle hat eine Reihe von Herausforderungen im Zusammenhang, einschließlich einer hohen Dichte von Mikrotubuli und Einschränkungen auf genetische Manipulationen. Im Gegensatz dazu bietet das Bäckerhefe Modell Vorteile, die diese Herausforderungen zu meistern. Dieses Protokoll beschreibt ein Verfahren, die Dynamik des einzelnen Mikrotubuli zu messen in lebenden Hefezellen. Zellen fluoreszierend markierte Tubulin exprimieren, werden zu montierten Schieber Kammer eingehalten, so dass für eine stabile Zeitraffer-Bildaufnahme. Eine ausführliche Anleitung für die Hochgeschwindigkeits, vierdimensionale Bildaufnahme wird auch als auch als ein Protokoll vorgesehen ist, um die Eigenschaften des dynamischen Mikrotubuli in konfokaler Bildstapeln zu quantifizieren. Dieses Verfahren, mit herkömmlicher Hefegenetik kombiniert, proveinen Ansatz ides, die in einzigartiger Weise geeignet ist für die quantitativ die Effekte von Mikrotubulus-Regulator oder Mutationen der Beurteilung, die die Aktivität von Tubulin-Untereinheiten zu verändern.

Messung der Dynamik der Mikrotubuli in lebenden Hefezellen liefert ein überzeugendes Werkzeug zu beurteilen, wie Mutationen in Genen, die für Mikrotubulus-Regulatoren oder Tubulin-Untereinheiten Auswirkungen Polymerisation und Depolymerisation Raten sowie die Häufigkeit des Übergangs zwischen diesen Zuständen. Abbildung 1 zeigt eine Zeitreihe von Astral Dynamik der Mikrotubuli in einer Wildtyp - Zelle und eine mutante Zelle mit einer Mutation in β-Tubulin (tub2...

Watch this Scientific Journal Video about High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast at JoVE.com

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

Hefe Angehende ist ein vorteilhaftes Modell die Dynamik der Mikrotubuli in vivo aufgrund seiner leistungsstarken Genetik und die Einfachheit der Mikrotubuli - Zytoskelett für das Studium. Das folgende Protokoll beschreibt, wie zu transformieren und Kultur Hefezellen, konfokale Mikroskopie Bilder erfassen und quantitativ die Dynamik der Mikrotubuli in lebenden Hefezellen analysieren.

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 ...

high-resolution-imaging-analysis-individual-astral-microtubule

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.2K Aufrufe.  University of Colorado School of Medicine.  Hefe Angehende ist ein vorteilhaftes Modell die Dynamik der Mikrotubuli in vivo aufgrund seiner leistungsstarken Genetik und die Einfachheit der Mikrotubuli - Zytoskelett für das Studium. Das folgende Protokoll beschreibt, wie zu transformieren und Kultur Hefezellen, konfokale Mikroskopie Bilder erfassen und quantitativ die Dynamik der Mikrotubuli in lebenden Hefezellen analysieren. 

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

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 ...

Forschung

Biologie

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Simultane Visualisierung der Dynamik von vernetzten und einzelnen Mikrotubuli in vitro mittels TIRF-Mikroskopie

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

Biochemie

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

Mikroskopie von Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

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.

Hochauflösende Zeitraffer-Imaging und automatische Analyse von Dynamik der Mikrotubuli in lebenden menschlichen Endothelzellen aus der Nabelvene

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

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

Rekonstitution von Grund Mitosespindeln in Sphärische Emulsionströpfchen

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

Räumlich-zeitliche Analyse von zytokinetischen Events in Spalthefe

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

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 ...

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.

Untersuchung der Mitotic and Meiotic Fission Yeast Nuclear Dynamics durch Fluoreszenz-Livezellmikroskopie

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 ...

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

Messung der Mikrotubuli-Dynamik durch Spinnen der Disk-Mikroskopie in Monopolar-Mitotischen Spindeln

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

Super-Resolution Live Cell Imaging of Subcellular Structures

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.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

Hochauflösende Live-Cell-Bildgebung subzellulärer Strukturen

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...