Wir sind dankbar, dass Dr. Kozo Tanaka der Tohoku University, Japan für den Austausch von HeLa-Zellen stabil mit dem Ausdruck mCherry-Histon H2B und GFP-α-Tubulin. Diese Arbeit wurde durch ein NCI Zuschuss an DV (R00CA178188) und Starthilfe von der Northwestern University unterstützt.

1. doppelter Thymidin sperren und freigeben: Reagenz Vorbereitungen
<ol><li>Machen 500 mL Dulbecco geändert Eagle Medium (DMEM) Medium mit 10 % FBS, Penicillin und Streptomycin ergänzt.</li>
	<li>Machen Sie 100 mM Lager von Thymidin in sterilem Wasser oder Store in Aliquote bei-80 ° C.</li>
</ol>2. Protokoll für feste Cell Imaging der mitotischen Progression (Abbildung 1A)
<ol><li>Am Tag 1, Samen ~ 2 x 105 HeLa-Zellen in die Vertiefungen einer 6-Well-Platte mit einem Deckglas (mit 70 % Ethanol und UV-Bestrahlung sterilisiert) und 2 mL DMEM Medium. Wachsen Sie die Zellen in einem befeuchteten Inkubator für 24 h bei 37 ° C und 5 % CO2.</li>
	<li>1St Thymidin Block: am 2. Tag, mischen Sie die erforderliche Menge Thymidin in frischen DMEM Medien (100 mM Lager, 2 mM Endkonzentration).</li>
	<li>Fügen Sie 2 mL Thymidin-haltigen Medien auf die Zellen in jede Vertiefung des 6-Well Platte und 18 h inkubieren.</li>
	<li>Am 3. Tag aspirieren Sie das Medium und waschen Sie die Zellen zweimal mit 2 mL 1 X PBS und einmal mit frischen vorgewärmte DMEM; wachsen Sie Zellen in frisch vorgewärmte DMEM Medium für 9 h Zellen aus Block freizugeben.</li>
	<li>2Nd -Thymidin-Block: erneut hinzufügen 2 mL Thymidin-haltigen Medien (2 mM Endkonzentration) auf die Zellen in jede Vertiefung des 6-Well-Platte.</li>
	<li>Ein weiterer 18 h inkubieren.</li>
	<li>Am Tag 4 aspirieren Sie das Medium und waschen Sie die Zellen zweimal mit 2 mL 1 X PBS und einmal mit frischen vorgewärmte DMEM.</li>
	<li>Verdünnte SiRNAs (Kontrolle und Cdt1) und Transfection Reagens in Serum frei Wachstumsmedium für 10 min. separat mischen sie zusammen und 20 min bei RT inkubieren Die Endkonzentration von SiRNA verwendet in jeder Transfektion betrug 100 nM. Fügen Sie das Reaktionsgemisch auf die Zellen, die aus Thymidin gewaschen worden sind.</li>
	<li>Inkubieren Sie Zellen bei 37 ° C für 9-10 h von der Blockierung lösen und befestigen der Zellen auf der Abdeckung rutscht mit 4 % PFA für 20 min bei RT Achtung: PFA ist giftig, angemessenen Schutz zu tragen.</li>
	<li>
Immunostain Zellen, nach dem permeabilizing mit 0,5 % (V/V) Reinigungsmittel für 10 min bei RT und die Zellen zweimal mit 1 X PBS für 5 min waschen.
	<ol>
<li>Sperren von Zellen mit 1 % BSA (w/V) mit 1 X PBS-Puffer für 1 h bei RT gefolgt von Behandlung von Zellen mit 50 µL Primärantikörper (Maus Anti-α-Tubulin Antikörper verdünnt 1:1, 000, Kaninchen-Anti-Zwint1-Antikörper verdünnt 1: 400 und Maus, die Anti-Phospho-ɣ H2AX bei 1: 300 verdünnt) bei 1 % (w/V) BSA mit 1 X PBS-Puffer für 1 h bei 37 oC.</li>
		<li>Nach dem Waschen der Zellen mit 1 X PBS dreimal, Behandlung von Zellen mit 50 µL Sekundärantikörper für jedes Cover-Glas (Alexa 488 und Rhodamine Red bei 1: 250 Verdünnungen in 1 % (w/V) BSA mit 1 X PBS-Puffer) für 1 h bei RT</li>
		<li>Nach dem Waschen der Zellen mit 1 X PBS zweimal für jeweils 5 min., Behandlung von Zellen mit DAPI (0.1µg / mL in 1 X PBS) für 5 min bei RT</li>
		<li>Legen Sie nach dem Waschen der Zellen mit 1 X PBS zweimal für 5 min, das Deckglas mit Blick auf Zellen an die entsprechenden Montage-Medien auf einer klaren mikroskopische Folie.</li>
	</ol>
</li>
	<li>Bild der Zellen für die Immunostained Proteine mit 60 X oder 100 X 1,4 NA Plan-apochromatische DIC Öl eintauchen Ziel auf eine invertierte hochauflösende confocal Mikroskop, ausgestattet mit einer geeigneten Kamera als notwendig für die Qualität der Bilder erforderlich montiert.</li>
	<li>Abrufen von Bildern bei Raumtemperatur Z-Stapel von 0,2 µm Dicke mit Software für das Mikroskop nach Bedarf.</li>
</ol>3. Protokoll für Live Cell Imaging der mitotischen Progression (Abbildung 3A)
<ol><li>Am 1. Tag Saatgut ca. 0,5-1 x 105 HeLa-Zellen stabil mCherry-H2B und GFP-α-Tubulin (leicht variabel je nach Zelle Art und die Proteine, die sie zum Ausdruck bringen) in 35 mm unten Glasschalen mit 1,5 mL DMEM Medium ausdrücken und in eine befeuchtete wachsen Inkubator für 24 h bei 37 oC und 5 % CO2.</li>
	<li>1St Thymidin Block: am 2. Tag, fügen Sie 1,5 mL Thymidin-haltigen Medien, um die Zellen in der Schale (100 mM Thymidin, 2 mM Endkonzentration).</li>
	<li>18 h inkubieren.</li>
	<li>Am 3. Tag aspirieren Sie Thymidin-haltigem Medium und waschen Sie die Zellen dreimal, zweimal mit 2 mL 1 X PBS und einmal mit frischen vorgewärmte DMEM.</li>
	<li>Verdünnte SiRNAs (Kontroll- und Hec1) und Transfection Reagens mit Serum frei Wachstumsmedium für 10 min. separat mischen sie zusammen und 20 min bei RT inkubieren Die Endkonzentration von SiRNA verwendet in jeder Transfektion betrug 100 nM. Fügen Sie das Reaktionsgemisch auf die Zellen, die aus Thymidin gewaschen worden sind.</li>
	<li>Wachsen Sie Zellen in 1,5 mL frisch vorgewärmte DMEM Medium für 8 h, Zellen aus Block freizugeben.</li>
	<li>2Nd -Thymidin-Block: Fügen Sie 1,5 mL Thymidin-haltigen Medien (2 mM Endkonzentration) auf die Zellen in der Schale.</li>
	<li>Ein weiterer 18 h inkubieren.</li>
	<li>Am 4. Tag aspirieren Sie das Medium und waschen Sie die Zellen dreimal, zweimal mit 2 mL 1 X PBS und einmal mit frischen vorgewärmte DMEM zu. Dann wachsen Zellen in frisch vorgewärmte Leibovitz ist (L-15) Medium ergänzt mit 10 % FBS und 20 mM HEPES bei pH 7,0 für 8 h, Zellen aus dem Block freizugeben.</li>
	<li>Legen Sie die Schale in die Temperatur Kontrollkammer inmitten der hochauflösende confocal Mikroskop, die bereits auf mindestens 30 min vor Beginn der Bildgebung zur Stabilisierung der Temperaturen auf der Bühne und experimentelle gewechselt hat.</li>
	<li>Fokussieren im Hellfeld mit Objektiv 60 X bis Zellen sichtbar sind, und klicken Sie dann manuell in die Region der Wahl der auf der Bühne zu sichten.</li>
	<li>Licht und Kraft/Laserbelichtung, Erwerb Bildparameter und Dauer des Experiments mit der Mikroskop-Bild-Datenerfassungs-Software der Wahl einrichten. Verwenden Sie Filter für GFP (488 Erregung; 385 nm Emission) und mCherry (561 nm Anregung und 385 nm Emission) Bilder zu erwerben.</li>
	<li>Führen Sie die Zeitraffer-Experiment durch gepulste Durchlicht separat zu erwerben und Fluoreszenz Bilder alle 10 Minuten für einen Zeitraum bis zu 16 h.</li>
	<li>Abrufen von Bildern zwölf 1,0 µm getrennt Z-Ebenen um 9 Uhr nach der Entlassung aus der doppelten Thymidin-Block mit Software das Mikroskop nach Bedarf.</li>
	<li>Analysieren Sie die Verfolgung einzelner Zellen für mitotischen Progression Bilder zu und schließlich montieren Sie den entsprechenden Film mit den Fidschi-Inseln/ImageJ-Source-Software.</li>
</ol>

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Die Autoren erklären, dass sie keine konkurrierendes finanzielles Interesse haben.

Der wichtigsten Vorteil der doppelten Thymidin-Synchronisation ist, dass es eine höhere Stichprobengröße der mitotischen Zellen in einem kurzen Zeitfenster viele dieser Zellen in Mitose unisono bietet, wodurch auch Analysen von Chromosom Ausrichtung, bipolar Spindel-Bildung und Chromosom Segregation mit wesentlich höheren Wirkungsgrad.
Viele regulatorische Proteinkomplexe und Signalwege sind dafür normale Progression durch Mitose gewidmet und Deregulierung dieses Prozesses kann führte zu...

In den Zellzyklus durchlaufen Zellen eine Reihe von stark regulierten und zeitlich gesteuerte Events für die genaue Vervielfältigung ihrer Genom und Verbreitung. Bei Säugetieren bestehend aus den Zellzyklus Interphase und M-Phase. In Interphase, bestehend aus drei Stufen - G1, S und G2, die Zelle seines Genoms dupliziert und Wachstum, die notwendig ist für normale Zellzyklus Progression1,2erfährt. In der M-Phase, bestehend aus Mitose (Prophase, prometaphase, Metaphase, Anaphase und Telophase) und Cytokinese, produziert eine elterliche Zelle zwei genetisch identische Tochterzellen. In Mitose, Schwester Chromatiden des doppelten Genoms sind verdichtete (Prophase) und sind an ihren Kinetochore von Mikrotubuli der montierten mitotischen Spindel (prometaphase) erfasst, das treibt ihre Ausrichtung an der Metaphase-Platte (Metaphase) gefolgt von ihrer gleich Trennung, wenn Schwester Chromatiden sind aufgeteilt in Richtung und transportiert zum gegenüberliegenden Spindel (Anaphase) Polen. Die zwei Tochterzellen sind räumlich getrennt von der Aktivität eines Actin-basierte kontraktilen Rings (Telophase und Cytokinese). Das Kinetochor ist eine spezialisierte proteinhaltige Struktur, die in der Zentromer-Region der Schwesterchromatiden montiert und dienen als Befestigung Standorte für Spindel Mikrotubuli. Seine Hauptfunktion ist zu fahren Chromosom erfassen, Ausrichtung, und Hilfe bei der Korrektur unsachgemäße Spindel Mikrotubuli Anlage, bei der Vermittlung des Spindel Baugruppe Prüfpunkt um die Treue des Chromosom Abtrennung3,4zu erhalten.
Die Technik der Zelle Synchronisation dient als ideales Werkzeug für die molekulare und strukturelle Ereignisse im Zellzyklus Fortschreiten zu verstehen. Dieser Ansatz wurde zur Zell-Populationen in bestimmten Phasen für verschiedene Arten von Analysen, einschließlich der gen-Expression profiling Analysen der zelluläre biochemische Prozesse und Erkennung der subzellulären Lokalisierung von Proteinen zu bereichern. Synchronisierte Säugerzellen können nicht nur für das Studium der einzelnen Gen-Produkte, sondern auch für Ansätze mit Analyse der ganzer Genome Microarray Analyse des Gens Ausdruck5, MiRNA Ausdruck Muster6einschließlich verwendet werden, Translational Regelung7und Proteomic Analyse von Protein-Modifikationen-8. Synchronisation kann auch verwendet werden, Studie zu den Auswirkungen der gen-Expression oder Protein Knock-down oder Knock-out- oder Chemikalien auf Zellzyklus Fortschreiten.
Zellen können in den verschiedenen Phasen des Zellzyklus synchronisiert werden. Physikalische und chemische Methoden sind für die Zelle Synchronisation verbreitet. Die wichtigsten Kriterien für die Synchronisierung der Zelle sind, dass Synchronisation noncytotoxic und reversibel sein sollte. Wegen der potenziellen nachteiligen zellulären folgen Zellen durch pharmakologische Wirkstoffe zu synchronisieren können chemisch-abhängige Methoden für die Untersuchung wichtigen Zellzyklus Veranstaltungen vorteilhaft sein. Z. B. Hydroxyurea, Amphidicolin, Mimosine und Lovastatin, einsetzbar für Zelle Synchronisation am G1/S-Phase aber, wegen ihrer Wirkung auf die biochemischen Bahnen, die sie hemmen sie Zellzyklus Checkpoint Mechanismen zu aktivieren und töten einen wichtigen Bruchteil der Zellen9,10. Auf der anderen Seite kann Feedback-Hemmung der DNA-Replikation durch Zugabe von Thymidin, Wachstumsmedien, bekannt als "Thymidin-Block" Zellzyklus bei bestimmten Punkten11,12,13verhaften. Zellen können auch durch die Behandlung mit Nocodazole und RO-33069,14in G2/M-Phase synchronisiert werden. Nocodazole, die Montage von Mikrotubuli verhindert, hat eine relativ hohe Zytotoxizität. Darüber hinaus können Nocodazole verhaftet Zellen zurück altklug durch mitotische Schlupf interphase. Doppelte Thymidin Block Verhaftung-Zellen am G1/S-Phase und nach der Entlassung aus dem Block, sind Zellen gefunden, synchron durch G2 und in Mitose vorzugehen. Die normale Progression des Zellzyklus für Zellen befreit Thymidin-Block kann unter hochauflösenden konfokalen Mikroskopie durch Zelle Fixierung oder live Imaging beobachtet werden. Die Auswirkungen der Störung der mitotischen Proteine kann studiert werden, besonders dann, wenn Zellen eingeben und fahren Sie durch Mitose nach Entlassung aus dem doppelten Thymidin-Block. Cdt1, ein multifunktionales Protein ist beteiligt an der DNA-Replikation Herkunft Lizenzierung in der G1-Phase und ist auch während der Mitose15für Kinetochor-Mikrotubuli-Anlagen erforderlich. Um die Funktion des Cdt1 während der Mitose zu studieren, braucht man eine Methode zu verabschieden, die die Wirkung von seiner Erschöpfung zur Replikation Lizenzierung während der G1-Phase, während gleichzeitig bewirken seine Erschöpfung speziell in der G2/M-Phase nur vermeidet. Hier präsentieren wir Ihnen ausführliche Protokolle basierend auf der doppelten Thymidin-Block zur Untersuchung der mitotischen Rolle von Proteinen, die mehrere Funktionen in verschiedenen Phasen des Zellzyklus von festen und live-Cell Imaging.

<table><tbody><tr><td>DMEM (1x)</td><td>Life Technologies</td><td>11965-092</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>DPBS (1x)</td><td>Life Technologies</td><td>14190-144</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Leibovitz&amp;rsquo;s (1x) L-15 medium</td><td>Life Technologies</td><td>21083-027</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Serum reduced medium (Opti-MEM)</td><td>Life Technologies</td><td>319-85-070</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Penicillin and streptomycin</td><td>Life Technologies</td><td>15070-063 (Pen Strep)</td><td>1:1,000 dilution</td></tr><tr><td>Dharmafect2</td><td>GE Dharmacon</td><td>T-2002-02</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Thymidine</td><td>MP Biomedicals LLC</td><td>103056</td><td>Dissolved in sterile distiled water</td></tr><tr><td>Cdt1 siRNA</td><td>Life Technologies</td><td></td><td>Ref 10</td></tr><tr><td>Hec1 siRNA</td><td>Life Technologies</td><td></td><td>Ref 13</td></tr><tr><td>HeLa cells expressing GFP-H2B</td><td></td><td></td><td></td></tr><tr><td>HeLa cells expressing GFP-&amp;alpha;-tubulin and mCherry H2B</td><td>Generous gift from Dr. Kozo Tanaka of Tohoku university, Japan</td><td></td><td></td></tr><tr><td>Formaldehyde solution</td><td>Sigma-Aldrich Corporation</td><td>F8775</td><td>Toxic, needs caution</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich Corporation</td><td>D9542</td><td>Toxic, needs caution</td></tr><tr><td>Mouse anti-&amp;alpha;-tubulin</td><td>Santa Cruz Biotechnology</td><td>Sc32293</td><td>1:1,000 dilution</td></tr><tr><td>Rabbit ant-Zwint1</td><td>Bethyl</td><td>A300-781A</td><td>1:400 dilution</td></tr><tr><td>Mouse anti-phospho-&amp;gamma;H2AX (Ser139)</td><td>Upstate Biotechnology</td><td>05-626, clone JBW301</td><td>1:300 dilution</td></tr><tr><td>Alexa 488</td><td>Jackson ImmunoResearch</td><td></td><td>1:250 dilution</td></tr><tr><td>Rodamine Red-X</td><td>Jackson ImmunoResearch</td><td></td><td>1:250 dilution</td></tr><tr><td>BioLite 6 well multidish</td><td>Thermo Fisher Scientific</td><td>130184</td><td></td></tr><tr><td>35 mm Glass bottom dish</td><td>MatTek Corporation</td><td>P35GCOL-1.5-14-C</td><td></td></tr><tr><td>Nikon Eclipse TiE inverted microscope</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Spinning disc for confocal</td><td>Yokagawa</td><td>CSU-X1</td><td></td></tr><tr><td>Ultra 888 EM-CCD Camera</td><td>Andor</td><td>iXon Ultra EMCCD</td><td></td></tr><tr><td>4 wave length laser</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>Incubation System for Microscopes</td><td>Tokai Hit</td><td>TIZB</td><td></td></tr><tr><td>NIS-elements software</td><td>Nikon Instruments</td><td></td><td></td></tr></tbody></table>

combining mitotic cell synchronization and high resolution confocal microscopy to study the role of multifunctional cell cycle proteins during mitosis

Studie der verschiedenen Ereignisse, Regulierung des Zellzyklus in einer Phase-abhängigen Weise liefert ein klares Verständnis über Zelle Wachstum und Teilung. Die Synchronisation von Zell-Populationen in bestimmten Phasen des Zellzyklus wurde festgestellt, als sehr nützlich in diesem experimentellen bemühen. Synchronisation von Zellen durch Behandlung mit Chemikalien, die relativ weniger toxisch sind kann vorteilhaft sein, über die Verwendung von pharmakologischen hemmenden Medikamenten für die Studie der konsequente Zellzyklus Ereignisse und spezifische Anreicherung von ausgewählten mitotischen Phasen zu erhalten. Hier beschreiben wir das Protokoll für die Synchronisierung menschliche Zellen in den verschiedenen Phasen der Zelle Zyklus, einschließlich der beiden in S und M-Phase mit einem doppelten Thymidin-Block und Verfahren zur Untersuchung der Funktionalität der mitotischen Proteine im Chromosom Ausrichtung lassen und Segregation. Dieses Protokoll wurde äußerst nützlich für das Studium der mitotischen Rollen multifunktionale Proteine, die etablierten Interphase Funktionen besitzen. In unserem Fall kann die mitotische Rolle der Cdt1, entscheidend für die Replikation Herkunft Lizenzierung in G1-Phase, ein Protein effektiv studiert werden, nur wenn G2/M-spezifischen Cdt1 aufgebraucht werden können. Wir beschreiben das ausführliche Protokoll für Abbau der G2/M-spezifischen Cdt1 mit doppelten Thymidin-Synchronisation. Wir erklären das Protokoll der Zelle Fixierung auch live Cell Imaging mit hochauflösenden konfokalen Mikroskopie nach Thymidin-Release. Die Methode ist auch nützlich für die Analyse der Funktion der mitotischen Proteine unter physiologischen und gestörten Bedingungen wie z. B. für Hec1, ein Bestandteil der Ndc80 Komplex, da es eine zu große Stichproben der mitotischen Zellen für feste und live Zelle ermöglicht Analyse wie wir hier zeigen.

Studie der mitotischen Progression und Mikrotubuli Stabilität in den Zellen fest nach Entlassung aus dem Thymidin-block Cdt1 engagiert sich bei der Lizenzierung der DNA-Replikation Ursprünge in der G1-Phase. Es wird abgebaut, während der S-Phase aber wieder sammelt sich im G2/M-Phase. Um ihre Rolle bei der Mitose zu untersuchen, muss der endogenen Cdt1 speziell auf die mit der am besten geeigneten Zelle Synchronisation Technik, die doppelte Thymidin Block<sup class=...

Watch this Scientific Journal Video about Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis at JoVE

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Wir präsentieren Ihnen ein Protokoll für doppelte Thymidin-Synchronisation von HeLa-Zellen, gefolgt von Analyse mit hochauflösenden konfokalen Mikroskopie. Diese Methode ist Schlüssel zu große Anzahl von Zellen, die synchron von S-Phase, Mitose gehen, Studien auf mitotischen Rollen multifunktionale Proteine, die auch Interphase Funktionen besitzen.

Kombination von mitotische Zelle Synchronisation und hochauflösenden konfokalen Mikroskopie zur Untersuchung der Rolle von multifunktionalen Zelle Zyklus Proteine während der Mitose

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

combining-mitotic-cell-synchronization-high-resolution-confocal

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.2K Aufrufe.  Northwestern University. Wir präsentieren Ihnen ein Protokoll für doppelte Thymidin-Synchronisation von HeLa-Zellen, gefolgt von Analyse mit hochauflösenden konfokalen Mikroskopie. Diese Methode ist Schlüssel zu große Anzahl von Zellen, die synchron von S-Phase, Mitose gehen, Studien auf mitotischen Rollen multifunktionale Proteine, die auch Interphase Funktionen besitzen.

Serie: Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

Das Studium der Zellzyklus-regulierten Genexpression durch zwei komplementäre Zellsynchronisationsprotokolle

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Forschung

Genetik

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Live Cell Imaging der Segregation der Chromosomen während der Mitose

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Entwicklungsbiologie

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Live-Imaging der Mitose in der Entwicklung von embryonalen Maus-Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Neurowissenschaften

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.

Zeitraffer-Imaging der Mitose Nach siRNA-Transfektion

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

Biologie

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

Säugetier-Zellteilung in 3D Matrizen über Quantitative konfokale Reflexion Mikroskopie

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Biotechnik

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

Studieren mitotischen Checkpoint durch Veranschaulichung Dynamische Kinetochor Protein Verhalten und Chromosome Motion in Lebens-<em&gt; Drosophila</em&gt; Syncytial Embryonen

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Mitochondrial fusion was measured by tracking the equilibration of photoconverted matrix-targeted GFP across the mitochondrial network over time. Thus far, only one cell could be subjected to an hour long kinetic analysis at a time. We present a method that simultaneously measures multiple cells, thereby speeding up the data collection process.

Der schnellere, Hohe Auflösung, MTPA-GFP-basierte Mitochondriale Fusion Assay Acquiring Kinetische Daten mehrerer Zellen parallel mit Hilfe der konfokalen Mikroskopie

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Studieren Proteolyse von Cyclin B an der Single Cell Level in Whole Cell Populationen

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

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 ...

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification&#160;and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

Live Cell Imaging zur Bewertung der Dynamik von Metaphasen-Timing und Zellschicksal nach mitotischen Spindelstörungen

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...