We are grateful to B. Stevenson for technical assistance and Dr Marc R. Knight for providing Aequorin transgenic line. This work was funded by the National Institutes of Health Grant R01 GM059138.

1. Aequorin Based Ca2 +-Imaging-System Mit der FAS <ol><li> Bereiten Pflanzgut für Lumineszenz-Bildgebung. Sterilisieren Samen von Arabidopsis-Pflanzen, Aequorin mit 10% Bleichlösung, die 0,01% Triton-100. Säen die Samen steril auf einer quadratischen Platte (10 x 10 cm im Quadrat Petrischale mit Gitter,) mit voller Kraft MS (Murashige und Skoog Basalsalzmischung), 1% Saccharose und 1,2% Agar. Ort Platten vertikal in einer Wachstumskammer nach einer Schichtung bei 4 ° C für 2 Tage (1A). </li><li> Übertragen Sie die Sämlinge auf einen Film. Ein Klebefilm (1B) auf der Oberseite der 7-10 Tage alten Sämlinge wachsen auf der Platte. Schieben Sie die Folie mit der Hand, um sicherzustellen, dass Keimlinge auf den Film (1C) einzuhalten. Schälen die Folie vorsichtig, so dass die Sämlinge bleiben an der Folie haften (1D und 1E) </li><li> Bebrüten die Sämlinge mit Cofaktor. Legen Sie die einedhered Sämlinge auf den Platz Platte (10 x 10 cm) mit 15 ml 2 pg / ml h-CTZ (Coelenterazins) in Wasser. Die Sämlinge bei Raumtemperatur inkubiert für 4 h bis über Nacht (1F). </li><li> Bereiten Sie sich für Lumineszenz-Bildgebung. Nehmen Sie den Film aus der h-CTZ Lösung und schneiden Sie es in der Mitte und bilden zwei Stücke. Legen Sie jedes Stück Film mit Jungpflanzen stehen in zwei verschiedenen Platten. Lassen Sie die Platten im Dunkeln für 5 min. </li><li> Erwerben Lumineszenz Bilder. Im Dunkeln, legen die beiden Platten nebeneinander auf der Bühne der Lumineszenz-Abbildungssystem (Figur 1G). Erwerben Bilder sofort nach Zugabe von 20 ml Lösung von Stimuli an die Platten gleichzeitig. </li><li> Analysieren Lumineszenz Bilder. Wählen Sie die gleichen Anzeigebereich für alle Lumineszenz Bilder. Beschneiden die Region of Interest (ROI) und erzeugen die Bilder als JPEG-Dateien (Abbildung 2a). Alternativ exportieren Sie die Bilder als SPE-Format-Dateien und importieren Sie sie in dieImageJ Bildanalyse-Software. Set-Messungen für die Berechnung der mittleren Grauwert der ROI. Wählen Sie die gleiche Größe der ROI-Bereich und messen die mittlere Grau und Darstellung von Daten als Balkendiagramme (Abbildung 2B). </li></ol> 2. Live Cell Imaging konfokale Ca 2 + <ol><li> Bereiten Setzlinge für die konfokale Bildgebung. Sterilisieren Samen von Arabidopsis-Pflanzen case12 ausdrücken mit 10% Bleichlösung, die 0,01% Triton. Aussaat der Samen auf einem sterilen Platte mit Vollkraft MS-Salze, 1% Saccharose und 1,2% Agar. Ort Platten vertikal in einer Wachstumskammer nach einer Schichtung bei 4 ° C für 2 Tage. </li><li> Setup Imaging Chambers <ol><li> Montieren Sie eine Imaging-Kammer mit einer Rutsche und Deckglas (Raum A). Legen Sie ein Stück Watte Wasser eingeweicht, auf der Mitte der Folie. Dann Transfer ein oder zwei 5 Tage alten Keimlingen von der Platte auf die Oberseite des Wasser eingeweicht Watte. Halten Sie kleine Stücke von Ton zu jeder Ecke eines Deckglases, eind Platz Deckglas auf der Oberseite der Setzlinge, um eine Lücke (Kammer) zwischen dem Deckglas und Objektträger (3A, links) zu erstellen. Das eine Ende des Polyethylenschlauch (0,58 mm Durchmesser) auf eine 1 ml-Spritze und das andere Ende des Rohrs unmittelbar angrenzend an die Kammer. Halten Sie den Schlauch mit Klebeband (3A, rechts). </li><li> Montieren Sie eine Imaging-Kammer mit einer Plexiglaskammer (Chamber B). <ol><li> Verteilen Sie eine dünne Schicht Silikonfett rund zwei Polyethylen-Rohre (0,58 mm Durchmesser), und drücken Sie die beschichteten Rohre in die Kanäle auf jeder Seite der Plexiglaskammer (3B links). Verteilen Sie eine dünne Schicht Silikonfett auf die Oberfläche der Kammer, dass das Rohr Nuten enthält und drücken Sie eine Deckglas in das Fett auf der einen Seite der Kammeröffnung (ausgeschnitten) zu versiegeln. </li><li> Übertragen einer 5 Tage alten Keimling von der Platte in die Kammer und setzen Sie ein Stück Watte mit Wasser getränkt auf der Oberseite of des Keimlings. Verbreiten Silikonfett auf die andere Oberfläche der Kammer, und drücken Sie eine andere Deckglas zu versiegeln. Verbinden des Endes eines der Rohre an eine 1 ml Spritze. Lassen Sie das Ende der anderen Röhre offen (3B rechts). </li></ol></li><li> Bereiten Sie eine Imaging-Kammer mit einem Kammerdeckglas (Kammer C). Sterilisieren Kammerdeckglas mit 70% Ethanol und lassen Sie es auf der Motorhaube bis trocken. 0,6 ml oder 0,2 ml Vollkraft MS-Medium mit 1% Saccharose und 0,5% Phytagel in jede Vertiefung einer 2-Kammer oder auch ein 8-Well-Kammer (3C rechts) auf. Sterilisieren Samen und säen die Samen direkt in einem Kammerdeckglas, das eine dünne Schicht klares Gel und lassen Sie sie vertikal für 5 Tage (Abbildung 3C) wachsen. </li></ol></li><li> Erwerben Sie Bilder mit einem konfokalen Mikroskop. Um Stress Reize, wie Salz, Kälte oder Peroxid gelten, langsam injizieren etwa 200 ul von 150 mM NaCl, Eis-cold Wasser oder 1 mM H 2 O 2-Lösung in die Kammer (Kammer A oder B) kurz vor der Aufnahme von Bildern oder sanft hinzufügen 500 ul oder 100 ul der Reiz Lösung für die Vertiefung einer 2-oder 8-Well-Kammer-bzw. . Erfassen von Bildern unmittelbar nach der Anwendung des Stimulus-Lösung unter Verwendung eines invertierten Nikon A1R konfokalen Laser-Scanning-Mikroskop mit einem 20x Wasserimmersionsobjektiv (numerische Apertur 0.75). Sammeln einer Zeitserie von Bildern bei 4 sec Intervallen mit Anregungs-und Emissionswellenlängen von 488 nm und 500 bis 550 nm und bei einer Pixelauflösung von 512 x 512. </li><li> Bildanalyse Mit Nikon-Elemente wurden ROIs um jede Zelle (oder Fläche) von Interesse gezogen. Gesamtintensität innerhalb jedes ROI wurde im Laufe der Zeit über das Dialogfeld Zeitmessung (ImageJ stattdessen verwendet werden könnten) gemessen. Gesamtintensität Messungen wurden exportiert und in Datagraph verarbeitet. Ca 2 + Spike Amplitude wurde als Spitzenintensität minus Ruhe Intensität, Dauer, wie th definierte Zeit zwischen Initiierung und Abschluss einer Spitze, und Zeit als das Zeitintervall zwischen benachbarten Dorn Spitzen von zwei Spikes. t-test wurde verwendet, um die Mittel zu vergleichen. </li></ol>

<ol>
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The authors have nothing to disclose.

Wir haben ein FAS-System zur Aufzeichnung der räumlichen-zeitlichen Ca2 + Antwort von Arabidopsis Keimlingen nachgewiesen. Diese FAS Ca 2 +-Aufzeichnungssystem ist eine einfache, empfindliche und stabile Ansatz zur Messung Ca2 + Dynamik durch verschiedene Stimuli neben den abiotischen Stressreize, die hier vorgestellt werden ausgelöst, angepasst werden kann. Mit diesem System können wir einfach vergleichen gewebespezifische Reize oder räumlich-zeitliche Dynamik Ca 2 + in d...

Die Pflanzenzelle reagiert auf die Umgebung über die Signalisierungszelle Aktionen koordiniert. Ein frühes Zellsignalisierungsereignis als Reaktion auf Umweltreize ist eine vorübergehende Ca 2 + zu erhöhen. Das Muster oder der Signatur einer transienten Zunahme der freien Ca 2 +-Konzentration wird durch seine Amplitude, Frequenz und Dauer ist. Unterschiedliche Raum-Zeit-Ca 2 +-Signaturen regulieren verschiedene Zellaktivitäten 1. Spezifische Reize, wie Hitze, Kälte, Salz, Trockenheit, Licht oder Pflanzenhormone, kann eine Feinabstimmung des räumlichen und zeitlichen Aktivität des Membran-lokalisierten Ca 2 +-Kanäle und / oder Transporteure, was in bestimmten Ca2 +-Signaturen. Obwohl Ca 2 +-Transporter sind gut gekennzeichnet, ist nur wenig über die molekularen Identitäten und Funktionen der Ca2 +-Kanäle in Pflanzen 1 bekannt. Genetische-Mutanten mit veränderten Ca 2 + Reaktion auf Reize betonen, kann eine wirksame ca. seinoach für die Identifizierung der Komponenten, die Ca 2 +-Signaturen zu komponieren. Kürzlich mehrere Aequorin basierend Ca 2 + Detektionssysteme entwickelt, die genetischen Screens erleichtern für Ca2 + Signalkomponenten in Reaktion auf Krankheitserreger angreifen und abiotischen Stress 2-4. Äquorin wurde zuerst verwendet, um in den frühen 1990er Jahren 5 Ca 2 +-Signale zu erkennen in Pflanzen. Seitdem hat Aequorin zu verschiedenen Zellkompartimenten wie dem Zytoplasma 5, Kern 6, 7, Chloroplasten, tonoplast 8, 9 Mitochondrien und Stroma 10 sowie an verschiedenen Zelltypen in der Stammzell spezifische Ca 2 überwachen gezielt worden + Signale 11. Äquorin basierend Ca 2 +-Messungen zeigen, die räumliche und zeitliche Ca2 + Antwort einer Population von Zellen auf Reize zu betonen. In den meisten Fällen sind jedoch die Ca 2 +-Antworten einzelner Zellen unsynchroniim antwort Gewebe 4 zed. Daher Aequorin Ca2 + Aufnahme nicht unbedingt die Ca 2 +-Signal in einzelnen Zellen zu melden. In den letzten Jahren genetisch kodierte Fluoreszenzprotein (FP) basierenden Ca 2 +-Indikatoren, wie gelb Cameleon (YCS) 12 und 13 CASEs12 verwendet worden, um Ca 2 + studieren Signalisierung mit hoher Auflösung subzellulärer. Ycs sind Fluoreszenz-Resonanz-Energie-Transfer (FRET) basierenden Ca 2 +-Indikatoren, CFP und YFP-Varianten enthalten, durch die Ca 2 + gebunden bindende Protein Calmodulin und Calmodulin-Bindungspeptid M13. Calmodulin eine Konformationsänderung wie sie Ca 2 + bindet, wodurch bewirkt CFP und YFP näher zusammen, was zu einem erhöhten Energie-Transfer (FRET verstärkt). Die FRET-Level über die Zeit, in etwa dem Verhältnis der an GFP YFP Signalintensitäten berechnet, spiegelt die intrazelluläre Ca 2 + Dynamik. YC mehrere Versionen in Pflanzen verwendet worden. YC3.6 war tin das Cytosol 14,15, 16 Kern, Mitochondrien 17 und 18 Plasmamembran und YC4.6 und D4ER argeted wurden in die Notaufnahme 15,19 gezielte und D3cpv wurde den Peroxisomen 20 ausgerichtet. Transgene Pflanzen, die YCS ermöglichen die Live-Cell-Imaging von Ca2 + Dynamik in verschiedenen zellulären Kompartimenten verschiedener Zelltypen. Fällen (vermutlich Ca lcium se nsor) sind Einzel zirkular permutierten fluoreszierende Proteine ​​(cpFPs) beherbergen eine Calmodulin und Calmodulin-Bindungspeptid M13. Nach der Bindung an Ca 2 +, Koffer unterziehen Konformationsänderungen, die zu einer Erhöhung der Fluoreszenzintensität. Die Korrelation zwischen Fluoreszenzantwort der Fall und Ca 2 +-Konzentration ermöglicht die intrazelluläre Ca2 + Dynamik quantitativ gemessen werden. Die Case12 Variante hat 12-fach erhöht Fluoreszenz in den Ca 2 + -gesättigtem Formen. N. benthiminana Pflanzen vorübergehend ausdrEssing Case12 oder Arabidopsis-Pflanzen stabil exprimieren Fall 12 wurden verwendet, um Ca2 +-Signalisierung in der Verteidigung und abiotische Stress 4,21 studieren. Asynchrone räumliche und zeitliche Ca2 + Oszillationen in Zellen, die auf Pathogenbefall oder Austrocknung Stress haben mit Case12 basierend Ca 2 +-Bildgebung offenbart worden. Hier präsentieren wir detaillierte Anweisungen für Aequorin Lumineszenz-Bildgebung von Gewebe-und Reize spezifischen Ca2 + Dynamik in Arabidopsis-Keimlinge, und für die konfokale Bildgebung von zytosolischen und Kern Ca 2 + Dynamik in Arabidopsis Wurzelzellen, die Fall 12. Lumineszenz-Bildgebung von FAS äußern könnten, die den Stress-induzierten Ca2 + Dynamik in ganzen Pflanzen oder Gewebe hier nicht beschrieben zu analysieren oder mutagenisierten Arabidopsis Pflanzenpopulationen nach Mutanten mit veränderten Stress induziert Ca2 +-Signale zu screenen. Die Lebendzell Ca2 + Imaging-Setup könnte angepasst Ca analysieren2 + Dynamik innerhalb verschiedener subcelluar Kammern oder in verschiedenen Zelltypen mit anderen Ca 2 +-Indikatoren.

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			<td height="21" style="height:21px;">10X10 cm square petri dish&#160;</td>
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			<td height="21" style="height:21px;">1 mL syringe&#160;</td>
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			<td height="21" style="height:21px;">2-well chambered cover glass&#160;</td>
			<td>Nalge Nunc international</td>
			<td>155379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">8-well chambered cover glass</td>
			<td>Nalge Nunc international</td>
			<td>155409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">luminescence imaging system&#160;</td>
			<td>Princeton Instruments</td>
			<td style="width:175px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">inverted confocal laser-scanning microscope&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td style="width:175px;">N/A</td>
			<td style="width:184px;">Nikon A1R&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Imaging software</td>
			<td>Nikon Instruments Inc.</td>
			<td style="width:175px;">N/A</td>
			<td>Nikon Elements&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">ImageJ</td>
			<td style="width:172px;">NIH</td>
			<td style="width:175px;">N/A</td>
			<td>Public domain, Java-based image processing program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DataGraph</td>
			<td>Visual Data Tools Inc</td>
			<td style="width:175px;">N/A</td>
			<td>DataGraph 3.1.1 is the newest version</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">coelenterazine</td>
			<td>NanoLight Technolgies</td>
			<td style="width:175px;">#301B NF-BCTZ-FB</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:356px;">All purpose Bleach</td>
			<td style="width:172px;">Any local store</td>
			<td style="width:175px;">N/A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Triton X-100</td>
			<td style="width:172px;">Fisher</td>
			<td style="width:175px;">BP151500</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:356px;">MS salt</td>
			<td style="width:172px;">Phytotechnology Labs</td>
			<td style="width:175px;">M524-50L</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:356px;">sucrose</td>
			<td style="width:172px;">VWR</td>
			<td style="width:175px;">BDH8029-12KG</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Agar</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:175px;">A1296-5KG</td>
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			<td height="21" style="height:21px;width:356px;">Phytagel</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:175px;">P8169-1KG</td>
			<td></td>
		</tr>
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measuring spatial and temporal ca2+ signals in arabidopsis plants

Entwicklungs-und Umweltreize auslösen Ca 2 + Schwankungen in Pflanzenzellen. Reiz-spezifischen räumlichen-zeitlichen Ca 2 + Muster werden durch zelluläre Ca2 +-bindende Proteine, die Ca 2 +-Signalkaskaden initiieren abgetastet. Allerdings wissen wir noch wenig darüber, wie bestimmte Reiz Ca 2 +-Signale erzeugt. Die Spezifität eines Ca 2 +-Signal kann der ausgereiften Regulierung der Aktivitäten von Ca 2 +-Kanäle und / oder Transporteure als Antwort auf einen gegebenen Reiz zurückzuführen. Diese Zellkomponenten zu identifizieren und ihre Funktionen zu verstehen, ist es wichtig, Systeme, die eine empfindliche und stabile Aufnahme der Ca2 +-Signale sowohl auf dem Gewebe und zellulärer Ebene zu ermöglichen verwenden. Genetisch kodierte Ca 2 +-Indikatoren, die zu verschiedenen zellulären Kompartimenten richtet sind haben eine Plattform für Live Cell konfokale Bildgebung von zellulären Ca 2 +-Signale zur Verfügung gestellt. Hier beschreiben wir Unterrichts für die Verwendung von zwei Erfassungssystemen Ca 2 +: Aequorin basierend FAS (Klebefilm Sämlinge) Ca 2 +-Lumineszenz-Bildgebung und case12 basierend Lebendzell konfokalen Fluoreszenz Ca 2 +-Bildgebung. Lumineszenz-Bildgebung mit der FAS-System bietet eine einfache, robuste und sensitive Detektion der räumlichen und zeitlichen Ca 2 +-Signale in der Gewebeebene, während Live Cell Imaging mit konfokalen Case12 bietet simultane Detektion von zytosolischen und Kern Ca 2 +-Signale mit einer hohen Auflösung.

Mannit, NaCl und H 2 O 2 wurden als Stellvertreter für die Dehydratisierung, Salz und oxidativer Stress Stimuli verwendet wurden. Um zu überprüfen, ob die Schwermetallionen Cu 2 + Synergie mit jeder dieser drei Spannungsreize, verglichen wir die Ca2 + Antwort auf jeden Stimuli in Gegenwart oder Abwesenheit von Cu 2 +. Wie in 2 gezeigt, zeigte FAS Lumineszenzbildgebung dass Arabidoposis Sämlinge zu Dehydrierung, Salz und oxidativen Stress reagier...

Watch this Scientific Journal Video about Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants at JoVE.com

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2 + Signalweg reguliert verschiedene biologische Prozesse in Pflanzen. Hier präsentieren wir Ansätze zur Überwachung abiotischen Stress induziert räumliche und zeitliche Ca 2 +-Signale in Arabidopsis Zellen und Geweben unter Verwendung der genetisch codierten Ca2 +-Indikatoren Aequorin oder Case12.

Mess räumliche und zeitliche Ca<sup&gt; 2 +</sup&gt; Signale in Arabidopsis-Pflanzen

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

measuring-spatial-and-temporal-ca2-signals-in-arabidopsis-plants

Research

JoVE Journal

Biology

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

12.0K Aufrufe.  Purdue University.  Ca2 + Signalweg reguliert verschiedene biologische Prozesse in Pflanzen. Hier präsentieren wir Ansätze zur Überwachung abiotischen Stress induziert räumliche und zeitliche Ca 2 +-Signale in Arabidopsis Zellen und Geweben unter Verwendung der genetisch codierten Ca2 +-Indikatoren Aequorin oder Case12. 

Serie: Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

Modellierung biologischer Membranen mit Leiterplatten und Messung elektrischer Signale in Axone: Schülerlabor Übungen

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Forschung

Biologie

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

Ice-Cap: Ein Verfahren zur Züchtung von Arabidopsis Und Tomatenpflanzen in 96-well-Platten für High-Throughput-Genotypisierung

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Mess-intrazellulären Ca<sup&gt; 2 +</sup&gt; Änderungen in menschlichen Spermien mit vier Techniken: Konventionelle Fluorometrie, Stopped Flußfluorometrie, Durchflusszytometrie und Single Cell Imaging

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Automatisierte Analyse von dynamischen Ca<sup&gt; 2 +</sup&gt; Signale in Bildsequenzen

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imaging Lokale Ca<sup&gt; 2+</sup&gt; Signale in kultivierten Säugetierzellen

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

In Echtzeit In Vivo Aufnahme von Arabidopsis Kalziumsignale während der Fütterung mit einer fluoreszierenden Biosensor Insekt

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

Anwendungen der räumlich-zeitliche Zuordnung und Partikel-Analyse-Techniken zu quantifizieren, intrazellulären Ca2 + Signalisierung In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

Laser-Capture Microdissection RNA-Sequenzierung für räumliche und zeitliche gewebespezifische Genexpressionsanalyse in Pflanzen

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

Weitfeld-Echtzeit-Bildgebung lokaler und systemischer Wundsignale bei Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Eine einfache und flexible Impfmethode zur genauen Beurteilung von Phänotypen von Arabidopsis und anderen Pflanzen bei Mehltauinfektionen

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...