Wir bedanken uns bei Dr. R. Howard Berg, Leiter der integrierten Mikroskopie-Einrichtung bei Donald Danforth Plant Science Center, für seine Beratung und Hilfe bei der konfokalen Mikroskopie. Die Autoren haben keinen Interessenkonflikt zu erklären.

1. Kennzeichnung der Defensine mit Fluorophore
<ol>
	<li>Wählen Sie eine Fluorophore, die minimale Auswirkungen auf die antimikrobiellen Eigenschaften sowie die Aufnahme und Lokalisierung von defensin innerhalb der lebenden Zelle hat. Hinweis: Optimale Fluorophor Auswahl richtet sich nach experimentellen Einzelziele. Spektrale und chemischen Eigenschaften, Photostabilität, Größe und Ladung der Fluorophor sollten berücksichtigt werden.</li>
	<li>Label defensin mit den ausgewählten Fluorophor mit einer entsprechenden Peptid labeling Kit von kommerziellen Anbietern erhältlich. Alternativ synthetisieren Sie chemisch Defensine Fluorophore beschriftet. In dieser Studie beschriften MtDef5 defensin mit DyLight550 mit einer kommerziellen Kennzeichnung gemäß des Herstellers Protokoll und Tetramethyl Rhodamin (TAMRA) kit-beschrifteten MtDef4 defensin kommerziell chemisch synthetisiert wurde. Hinweis: Es wird empfohlen, dass chemisch synthetisierten Fluorophor getaggt Peptide (vor allem für kurze Peptide von bis zu 50 Aminosäuren) verwendet werden, da die N - oder C-terminalen Rückstände des Peptids zu gewährleisten minimale Auswirkungen auf das Fluorophor befestigt werden kann seine antimikrobielle Aktivität. Synthese von langen Peptiden mit mehr als drei Disulfid-Bindungen ist eine Herausforderung. In diesem Fall empfiehlt es sich das Peptid mit einer optimalen Fluorophor mit ein geeigneten handelsüblichen Peptid labeling Kit gekennzeichnet werden.</li>
	<li>In-vitro- antimikrobielle Aktivität des beschrifteten defensin testen und bestimmen der minimalen inhibitorischen Konzentration (MIC) gegen die Pilze untersucht werden. Hinweis: In dieser Studie wurde die MIC der DyLight550-MtDef5 und TMR-MtDef4 gegen Neurospora Crassa und Fusarium Graminearumermittelt.</li>
</ol>
(2) Pilzkulturen und Wachstumsmedium
<ol>
	<li>Übertragen Sie Konidien aus einer N. Crassa Lager Kultur schräg Röhrchen mit Vogels Agar mittlere21 und 5 Tage bei Raumtemperatur unter Licht inkubieren.</li>
	<li>Kultur F. Graminearum PH-1-Stamm auf Tellern mit komplette mittlere (CM)22 auf 28 ° c für 5 Tage. Impfen Sie für die Produktion der Konidien 4 (10 mm Durchmesser) Stecker 5 Tage alten Kultur der F. Graminearum PH-1 in 50 mL Carboxymethylcellulose Zellulose (CMC) Medium (15 g Carboxymethylcellulose Zellulose, 1 g Hefeextrakt, 0,5 g MgSO4.7 H2O, 1 g NH 4 KEINE3und 1 g KH2PO4) und Kultur für 4-7 Tage bei 28 ° c in einem Dreh Shaker bei 180 Umdrehungen pro Minute.</li>
</ol>
3. Vorbereitung der Conidial Suspension
<ol>
	<li>F. Graminearum conidial Aufhängung
	<ol>
		<li>Wirbel der Flüssigkultur F. Graminearum und sammle Konidien durch das Filtern von 1,5 mL pilzartige Kultur durch zwei Schichten von Filtermaterial (z. B. Miracloth) zu einem 2 mL Microcentrifuge Schlauch. Zentrifugieren Sie die conidial Aufhängung an 13.226 x g Geschwindigkeit in einem Microcentrifuge für 2 min.</li>
		<li>Verwerfen Sie den Überstand, und fügen Sie 1 mL sterilem Wasser um die Pellets zu waschen.</li>
		<li>Zentrifugieren Sie conidial Aufhängung an 13.226 x g Geschwindigkeit in einem Microcentrifuge für 2 min. verwerfen überstand und Aufschwemmen Sie das Pellet in 1 mL 2 X SFM (synthetische Pilz Medien)23.</li>
		<li>Zählen Sie die Konidien, die mit einem Hemocytometer unter einem Lichtmikroskop.</li>
		<li>Passen Sie die conidial Aufhängung 105 Konidien/mL mit 2 X SFM.</li>
	</ol>
	</li>
	<li>N. Crassa conidial Aufhängung
	<ol>
		<li>Eine kleine Menge (5 Tage alt) wachsende Kultur von N. Crassa mit einer Impfung-Schleife zu einem Microcentrifuge Schlauch mit 2 mL des flüssigen Mediums Vogels zu übertragen.</li>
		<li>Wirbel conidial Federung und Filter durch Filtermaterial zu einem neuen Microcentrifuge Schlauch.</li>
		<li>Zentrifugieren der conidial Suspension mit maximaler Geschwindigkeit in einem Microcentrifuge für 2 min. verwerfen des Überstands und conidial Pellet in 1 mL des flüssigen Mediums Vogels aufzuwirbeln.</li>
		<li>Zählen Sie die Konidien, die mit einem Hemocytometer unter einem Lichtmikroskop.</li>
		<li>Passen Sie die conidial Aufhängung 105 Konidien/mL mit Vogels flüssigen Mediums.</li>
	</ol>
	</li>
</ol>
4. Probieren Sie Vorbereitung und konfokale Mikroskopie
<ol>
	<li>Subzelluläre Lokalisation der MtDef4 defensin in Pilzzellen
	<ol>
		<li>Pipette 50 µL der einzelnen conidial Suspension (105 Konidien/mL) in 10 mm Microwell von 35 mm unten Microwell Glasschalen. Für die Beobachtung der Konidien direkt fahren Sie fort mit dem nächsten Schritt fort, oder für Germlings Vorbereitung, Pipette 50 µL N. Crassa und F. Graminearum Konidien in die 35 mm-Kultur-Gerichte und für 3-6 h bei Zimmertemperatur Keimen zu lassen.</li>
		<li>Fügen Sie 50 µL Eindringmittel beschrifteten defensin am MIC zu jedem Microwell-Gericht und 2,5 h inkubieren. Das Mikrofon des Fluorophore beschriftet Defensine hier verwendet (Schritt 1.3) ist 3 µM für N. Crassa und F. Graminearum und das Mikrofon des TMR-MtDef4 für N. Crassa und F. Graminearum betrug 1 µM und 12 µM bzw..</li>
		<li>Fügen Sie 2 µL der Membran-selektiven färben FM4-64 (Endkonzentration: 5 µM) in Kultur Gericht und 30 min inkubieren und sofort auf die confocal Mikroskop für die Bildgebung zu montieren.</li>
		<li>Wählen Sie den weiße Licht-Laser (WLL). Verwenden Sie die zwei Laser Quellen 488 nm und 550 nm, TMR-MtDef4 und FM4-64 Farbstoff bzw. begeistern. Fluoreszenz des TMR-MtDef4 bei 580-700 nm zu erkennen und FM4-64 Farbstoff bei 690-800 nm zu erkennen. Hinweis: Führen Sie konfokalen Mikroskopie in den dunklen Raum.</li>
	</ol>
	</li>
	<li>Time Lapse Bildgebung der Internalisierung von MtDef5 defensin
	<ol>
		<li>Pipette 50 µL N. Crassa und F. Graminearum conidial Suspension (105 Konidien/mL) in 10 mm Microwell von 35 mm unten Microwell Glasschalen. Die 10 mm Microwell ist unbeschichtet und hat eine Nr. 1.5 Deckglas an der Unterseite. Fortfahren Sie für die Beobachtung der Konidien direkt mit dem nächsten Schritt. Inkubieren Sie für die Beobachtung von Germlings Konidien für 3-6 h bei Raumtemperatur bevor Sie mit der Mikroskopie.</li>
		<li>Schalten Sie WLL. Wählen Sie die Laserlinie bei 550 nm begeistern die Fluorophore beschriftet Defensine hier verwendet (Schritt 1.3) und FM4-64 mit 1,00 % Intensität und aktivieren Sie die entsprechende Detektoren. Fluorophore beschriftet Defensine hier verwendet (Schritt 1.3) wurde gefunden bei 560-600 nm und die FM4-64dye bei 690-800 nm erkannt wurde. Hinweis: Verwenden Sie niedrige Laserintensität für live Cell imaging, da hohe Laserintensität live Pilzzellen beschädigen kann.</li>
		<li>Montieren Sie die Microwell-Schüssel am Mikroskop. Die Zellen mit 10 X Ziele zunächst zu finden, wechseln Sie dann zu 100 X / 1,44 Öl Ziel für höhere Vergrößerung. Hinweis: Standardmäßig den Scan-Modus mit Xyzt (Kombination von Z-Stapel und Zeitraffer-Modus), Z-Position, Zoom, Häufigkeit der Bilderfassung, etc..</li></ol></li></ol>bevor Sie mit dem nächsten Schritt fortfahren.
		<li>Jedes Gericht Microwell 50 µL der Fluorophore beschriftet Defensine hier verwendet (Schritt 1.3) der an das Mikrofon von 3 µM und 2 µL der Membran-selektive Farbstoff FM4-64 (Endkonzentration 5 µM) hinzu, und starten Sie Zeitraffer Imaging. Legen Sie kleine Stücke von nassen Filterpapiere in der Microwell Gerichte um Verdunstung zu verhindern. Die Häufigkeit der Bilderfassung war 3 min 30 s und die Gesamtdauer 2 h 30 min. Hinweis: Hinzufügen von defensin und Fluorophor nach Montage Microwell Gericht im Mikroskop des Interessenbereichs defocus kann. Daher muss nach der Microwell defensin und Fluorophor hinzufügen, Interessenbereich neu ausgerichtet werden die Verzögerung bei der Bildaufnahme hervorrufen können.</li>
		<li>Verwenden Sie ein confocal Mikroskop für alle konfokale Bildgebung und der Mikroskopie bei Raumtemperatur in einem dunklen Raum durchführen.</li>

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Die Autoren haben nichts preisgeben.

In dieser Studie wurde eine zuverlässige live-Cell Image Methodik mit dem Einsatz von Eindringmittel beschrifteten antimykotische Defensine beschrieben, die Kinetik der Internalisierung dieser Peptide in Pilzzellen zu studieren und ihre subzelluläre Ziele zu bestimmen. Diese Methode ist ein mächtiges Werkzeug, um die Dynamik der Interaktion zwischen Defensine und Pilzzellen zeitlich und räumlich zu visualisieren.
Verschiedene Methoden wurden zur Untersuchung der Verinnerlichung und intraze...

Pflanzen haben eine anspruchsvolle angeborene Immunsystem zur Abwehr der mikrobiellen Werk Krankheitserreger1entwickelt. Sie äußern zahlreiche Gen kodiert Host Verteidigung Peptide mit vermeintlichen antimikrobielle Aktivität2. In der Tat, viele dieser Peptide antimikrobielle Aktivität in Vitro3anzeigen Defensine bilden eine der größten Gruppen von Host Verteidigung Peptiden in der Pflanze Königreich4. Diese reich an Cystein, kationischen Peptide zeigen starkes Wachstum hemmende Aktivität gegen Pilz- und Eipilz Krankheitserreger in mikromolaren Konzentrationen und repräsentieren eine der ersten Linien der Verteidigung gegen diese Erreger5,6. Wegen ihrer starken antimykotische Aktivität kann Defensine ausgenutzt werden, in Agribiotechnological Anwendungen um Krankheiten resistente Pflanzen zu erzeugen. Konstitutive Überexpression von mehreren pflanzlichen Defensine hat sich gezeigt, Resistenz gegen Krankheiten in Gewächshaus und Freiland-Tests von transgenen Pflanzen6zu verbessern. Es ist wichtig, die Mechanismen der Wirkung (MOA) dieser antimykotische Peptide zu entwirren, um ihr Potenzial voll und ganz als wirksame Instrumente für den Pflanzenschutz zu erschließen. Die MOA diese Pflanze Defensine sind jedoch kaum erforscht. Aktuelle Hinweise darauf, dass sie verschiedene MOA5,6,7,8aufweisen. Einige Defensine wirken extrazellulär auf Pilze und gezielt bestimmte Zellwand/Plasma Membran resident Sphingolipide, Membran Integrität stören und aktivieren zelluläre Toxizität Wege9,10,11. Jedoch vor kurzem, wurden antimykotische Defensine, die in Pilzzellen translozieren entdeckten12,13,14. Einige von diesen Defensine binden an Membran-Resident bioaktive Phospholipide, bilden Oligomere komplexe und Plasmamembranen15,16,17permeabilize. So haben einige Aspekte des MOA von pflanzlichen Defensine aufgeklärt. Allerdings beinhalten die MOA von pflanzlichen Defensine wahrscheinlich eine komplexe Reihe von Ereignissen, die noch nicht identifiziert und in ein umfassendes Modell integriert wurden. Insbesondere bleibt eine große Lücke in unserem Verständnis der zellulären Ziele dieser Peptide.
Mit jüngsten Fortschritte in der Mikroskopie-Technologien und die Entwicklung von neuen fluoreszierende Sonden sind Leben-Zelle bildgebende Verfahren jetzt häufig verwendet, um die MOA antimikrobielle Peptide (AMPs) zu studieren. Diese Techniken ergänzen verbreitete Methoden wie Immunolocalization, Elektronenmikroskopie, Rasterkraftmikroskopie oder Röntgen-Tomographie18, die angewandt wurden, vor allem auf die Auswirkungen von Antimykotika Peptiden auf die Morphologie zu analysieren und Wachstum von Pilzzellen einschließlich der Untersuchung der Zellwand Integrität, Veränderungen in der Zelle Wachstum/Verzweigung Muster sowie Permeabilisierung der Plasmamembran und töten. Allerdings wurden diese Studien beschränkt Imaging Zellen zu einem bestimmten Zeitpunkt nach der Behandlung mit der Peptide anstatt Zeitraffer-Aufnahme die gleichen Zellen, ihre dynamischen Veränderungen in Reaktion auf defensin Herausforderung zu überwachen. In den letzten Jahren hat die Verwendung von Eindringmittel beschriftete Peptide in Verbindung mit live Cell imaging mit der konfokalen Mikroskopie Echtzeit-Visualisierung der Dynamik des AMP-Mikroben-Interaktionen ermöglicht. Beide natürlich gereinigt und chemisch synthetisierten antimykotische Peptide können mit Fluoreszenzmarkierungen markiert werden (z.B., DyLight, Rhodamin, BODIPY oder Alexa Fluor basierend Farbstoffe) und direkt während ihrer Interaktion mit Zellen von Time-Lapse beobachtet Live Cell Imaging. Die Verwendung dieser Peptide beschriftet unser Verständnis für die verschiedenen Aspekte der ihre MOA einschließlich Modus Eintritt, subzelluläre Lokalisation, intrazellulären Menschenhandel und Websites antimykotische Maßnahmen im lebenden Pilzzellen deutlich gestiegen 18.
Vor kurzem, haben mehrere Studien gezeigt, dass verschiedene Antimykotika Peptide einschließlich Anlage Defensine Leben Pilzzellen12,14,19,20internalisiert werden. Die MOA diese Defensine wahrscheinlich beinhalten Interaktion mit intrazellulären Ziele. Wir haben vor kurzem die antimykotische Wirkung von einer Pflanze defensin MtDef4 in zwei Schlauchpilz Pilzen, Neurospora Crassa und Fusarium Graminearumberichtet. MtDef4 wurde gezeigt, um verschiedene Wege für die Pilzzelle ein- und subzelluläre Lokalisation in diesen Pilzen14zu verwenden. Diese Studie verwendet chemisch synthetisiert Tetramethyl Rhodamin (TAMRA)-mit der Bezeichnung MtDef4 in Kombination mit lebenswichtigen Fluoreszenzfarbstoffen (Membran-permeationsfähigen Farbstoff, SYTOX Green, der Membran selektiv Farbstoff, FM4-64; der Zelle Tod Reporter Farbstoff, Propidium Jodid) und metabolische Inhibitoren. Diese Analysen zeigten die Kinetik der Internalisierung von MtDef4, die Mechanismen der intrazellulären Transport und seine subzelluläre Ziele14.
Hier ist ein Protokoll für live Cell Imaging mit der konfokalen Mikroskopie vorgestellt. Das Protokoll nutzt Eindringmittel beschriftete Peptide in Kombination mit lebenswichtigen Fluoreszenzfarbstoffen Pflanze defensin-Pilz-Interaktionen, insbesondere zu studieren, die Pfade der Translokation und die intrazellulären Ziele der Defensine in Pilzzellen.

<table>
	<tbody>
		<tr>
			<td>FM4-64 Dye</td>
			<td>Life Technologies</td>
			<td>T13320</td>
			<td></td>
		</tr>
		<tr>
			<td>DyLight 550 Antibody Labeling Kit</td>
			<td>Thermo Scientific</td>
			<td>84530</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>Mat TeK</td>
			<td>P35G-1.5-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Mira cloth</td>
			<td>EMD Millipore Corp</td>
			<td>475855-1R</td>
			<td></td>
		</tr>
		<tr>
			<td>SP8-X confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>FiJi</td>
			<td></td>
			<td>For Image analysis</td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td></td>
			<td>For Image analysis</td>
		</tr>
	</tbody>
</table>

live-cell imaging of fungal cells to investigate modes of entry and subcellular localization of antifungal plant defensins

Kleinen Cystein-reiche Defensine sind eine der größten Gruppen von Host Verteidigung Peptiden in allen Pflanzen vorhanden. Viele pflanzlichen Defensine potente in Vitro antimykotische Aktivität gegen ein Breitspektrum Pilzerreger auszustellen und haben daher das Potenzial, als Antimykotika in transgenen Pflanzen verwendet werden. Um das volle Potenzial der Pflanze Defensine für Krankheiten Kontrolle zu erschließen, ist es wichtig, ihre Wirkmechanismen (MOA) aufzuklären. Mit dem Aufkommen der modernen Mikroskopiertechniken ist live Cell Imaging ein leistungsfähiges Werkzeug für das Verständnis der Dynamik von Antimykotika MOA von pflanzlichen Defensine geworden. Hier wird ein konfokalen Mikroskopie basierend live-Cell imaging Verfahren beschrieben mit zwei Eindringmittel bezeichnete Pflanze Defensine (MtDef4 und MtDef5) in Kombination mit lebenswichtigen Fluoreszenzfarbstoffen. Diese Technik ermöglicht Echtzeit-Visualisierung und Analyse der dynamischen Ereignisse der MtDef4 und MtDef5 Internalisierung in Pilzzellen. Wichtig ist, erzeugt dieser Assay eine Fülle von Informationen, einschließlich der Internalisierung Kinetik, Modus für die Einreise und subzelluläre Lokalisation dieser Peptide. Zusammen mit anderen biologischen Zelle-Tools haben diese Methoden kritische Einblicke in die Dynamik und Komplexität der MOA dieser Peptide zur Verfügung gestellt. Diese Tools können auch verwendet werden, die MOA dieser Peptide gegen verschiedene Pilze zu vergleichen.

Live Cell Imaging wurde durchgeführt, um zu verfolgen und vergleichen Sie die Internalisierung und subzelluläre Lokalisation der zwei Defensine, MtDef4 und MtDef5 von Medicago Truncatula; in Pilzzellen. TMR-MtDef4 wurde chemisch synthetisiert, während MtDef5 mit Dylight550 beschriftet war (Dylight550-MtDef5). Konidien inkubiert mit entweder defensin in Kombination mit der Membran selektiv Farbstoff FM4-64. Abbildung 1 zeigt, dass TMR-MtDef4 versch...

Watch this Scientific Journal Video about Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins at JoVE.com

Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

Pflanzlichen Defensine spielen eine wichtige Rolle in der Verteidigung der Pflanze gegen Krankheitserreger. Für effektive Nutzung dieser antimykotische Peptide als Antimykotika, verstehen ihre Wirkungsweisen (MOA) entscheidend ist. Hier ist eine live-Cell-imaging-Methode beschrieben, um kritische Aspekte der MOA dieser Peptide zu studieren.

Live Cell Imaging von Pilzzellen Modi des Eintrags und subzelluläre Lokalisation der antimykotische Pflanze Defensine zu untersuchen

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro ...

live-cell-imaging-fungal-cells-to-investigate-modes-entry-subcellular

Research

JoVE Journal

Biology

6.6K Aufrufe.  Donald Danforth Plant Science Center. Pflanzlichen Defensine spielen eine wichtige Rolle in der Verteidigung der Pflanze gegen Krankheitserreger. Für effektive Nutzung dieser antimykotische Peptide als Antimykotika, verstehen ihre Wirkungsweisen (MOA) entscheidend ist. Hier ist eine live-Cell-imaging-Methode beschrieben, um kritische Aspekte der MOA dieser Peptide zu studieren.

Serie: Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Live-Cell-Imaging und Quantitative Analyse der embryonalen Epithelzellen in Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Forschung

Biologie

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Fluoreszenz-basierte Messung von Calcium-Shop-Eintrag in lebenden Zellen: aus kultivierten Krebszelle zu Skelettmuskelfaser

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Zwei-und dreidimensionale Live Cell Imaging der DNA-Reparatur-Proteine

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induktion und Analyse von epithelialen zu mesenchymalen Transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

Rasterelektronenmikroskopie (SEM) Protokolle für Problematische Pflanze, Oomycete und Pilzproben

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

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 ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

Live-Zelle Bildgebung von Platelet Degranulation und Sekretion unter Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

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 ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

Live-Cell Imaging of Single-Cell Arrays (LISCA) - eine vielseitige Technik zur Quantifizierung der zellulären Kinetik

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...