<ol><li> Wählen Sie eine saubere Präparation Gericht, das nicht an Fixiermittel ausgesetzt wurde. </li><li> Dissect ein wandernder 3 rd instar Drosophila Larve in Schneiders Drosophila Medium mit Ca 2 + und L-Glutamin, (nicht schneiden alle Nerven oder Schäden Muskelfasern Nr. 7, 6, 13 oder 12). </li><li> Wählen Sie ein Glas füllen-Pipette mit einer 12 Mikrometer Spitze (Innendurchmesser). </li><li> Mit einer Spritze und Schlauch (Unterdruck an die Pipette gelten) sicherzustellen, dass die Pipettenspitze nicht behindert wird. </li><li> Wählen Sie eine feine Kunststoff-Füllung-Filament, das untergliedert werden können die Länge der Glaspipette eingebracht. </li><li> Draw ~ 1 cm von 5 mM Dextran-konjugierten Ca 2 +-Indikator in der Kunststoff-Filament. </li><li> Schneiden Sie alle Segment Nerven. </li><li> Unterstützung der Pipette auf eine Rampe, damit die Pipettenspitze auf die ventrale Mittellinie des seziert Larve Ansatz. </li><li> Zeichnen Sie das abgeschnittene Ende eines Nerven zu segmentieren No.4, ohne Quetschung der Nerven, in das Ende der Pipette (mit einer kleinen Menge von Schneider Medium). </li><li> Entfernen Sie die Schläuche und die Kunststoff-Faden in die Pipette bis zum Ende des Fadens wird innerhalb von 50 Mikron das abgeschnittene Ende des Nervs (berühren Sie nicht die Nerven). </li><li> Eject ausreichende Ca 2 +-Indikator auf die Nervenenden, um die Lautstärke des Schneiders Medium um etwa 33% (Endkonzentration sollte &lt;2mm) zu erhöhen. Wichtig - Dies muss innerhalb von 5 Minuten des Schneidens den Nerv abgeschlossen sein. </li><li> Ort der Vorbereitung im Dunkeln bei Raumtemperatur während die Nerven Lasten. </li><li> Nach 40 Minuten entfernen Sie die Ca 2 +-Indikator mit dem Filament. </li><li> Verlassen Sie die Pipette in Ort und füllen Sie es vollständig mit frischen Schneider-Medium, da dies zur stimulierende Impulse, um den Nerv zu beantragen. </li><li> Lassen Sie die Ca 2 +-Indikator in den Nerv für mindestens 60 Minuten ausgleichen, aber nicht mehr als 4 Stunden vor Beginn der Ca 2 +-Imaging. </li><li> Spülen Sie die Zubereitung mit frischen Schneider Medium alle 30 Minuten, während es ausgleichenden ist. </li><li> 20 Minuten vor dem Imaging ersetzen Schneider-Medium mit Hämolymphe-Like No.6 Lösung (HL6; Macleod et al 2002;. 2003). </li><li> L-Glutaminsäure oder Glutamat kann zu HL6 Lösung bei 7mm hinzugefügt werden, um postsynaptische Glutamatrezeptoren desensibilisieren, um Nerven-evozierten Muskelkontraktion verhindern (Macleod et al 2004;. Reiff et al 2002;. 2005). </li></ol>

<ol>
	<li>Macleod, G. T., Hegstrom-Wojtowicz, M., Charlton, M. P., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+calcium+signals+in+Drosophila+motor+neuron+terminals.">Fast calcium signals in Drosophila motor neuron terminals.</a> J. Neurophysiol. 88, 2659-2663 (2002).</li><li>Macleod, G. T., Suster, M. L., Charlton, M. P., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+neuron+activity+in+the+Drosophila+larval+CNS+detected+with+calcium+indicators.">Single neuron activity in the Drosophila larval CNS detected with calcium indicators.</a> J. Neurosci. Methods. 127, 167-178 (2003).</li><li>Macleod, G. T., Marin, L., Charlton, M. P., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicles:+test+for+a+role+in+presynaptic+calcium+regulation.">Synaptic vesicles: test for a role in presynaptic calcium regulation.</a> J. Neurosci. 24, 2496-2505 (2004).</li><li>Reiff, D. F., Thiel, P. R., Schuster, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+active+zone+density+during+long-term+strengthening+of+Drosophila+neuromuscular+junctions.">Differential regulation of active zone density during long-term strengthening of Drosophila neuromuscular junctions.</a> J. Neurosci. 22, 9399-9409 (2002).</li><li>Reiff, D. F., Ihring, A., Guerrero, G., Isacoff, E. Y., Joesch, M., Nakai, J., Borst, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+performance+of+genetically+encoded+indicators+of+neural+activity+in+flies.">In vivo performance of genetically encoded indicators of neural activity in flies.</a> J. Neurosci. 25, 4766-4778 (2005).</li></ol>

The authors have nothing to disclose.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Schneider&#x2019;s Insect Medium</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0146</td>
<td>must contain L-glutamine and calcium</td>
</tr>
<tr>
<td>L-glutamic acid monosodium salt hydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G1626</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
					</div>

loading drosophila nerve terminals with calcium indicators

Calcium spielt viele Rollen im Nervensystem aber keiner eindrucksvoller als als Auslöser für die Freisetzung von Neurotransmittern, und niemand tiefer als wie der Bote essentiell für die synaptische Plastizität, dass Lernen und Gedächtnis unterstützt. Zur weiteren Aufklärung der molekularen Grundlagen der Ca 2 +-abhängige synaptische Mechanismen, ist ein Modell-System benötigt, das sowohl genetisch formbar und physiologisch zugänglich. Drosophila melanogaster bietet ein solches Modell. In diesem System sind genetisch kodierte fluoreszierende Indikatoren zur Verfügung Ca 2 + Änderungen in Nervenendigungen zu erkennen. Allerdings haben diese Indikatoren Empfindlichkeit gegenüber Ca 2 + beschränkt und zeigen oft ein nicht-lineares Ansprechverhalten. Synthetische fluoreszierenden Indikatoren sind besser für die Messung der schnellen Ca 2 + ändert sich mit Nerven-Aktivität assoziiert geeignet. Hier zeigen wir eine Technik zum Be-Dextran-konjugierten synthetischen Ca 2 +-Indikatoren in den Live-Nervenendigungen in Drosophila-Larven. Besonderes Augenmerk wird auf jene Aspekte des Protokolls am wichtigsten für die Technik für den Erfolg, wie, wie elektrostatische Entladungen entlang der isolierten Nerven zu vermeiden, die Erhaltung der Gesundheit der Zubereitung bei längeren Ladezeiten gelegt, und die Gewährleistung Axon Überleben durch Ca 2 + zur Förderung der Abdichtung von abgetrennten Axonendigungen. Geringe Affinität Dextran-konjugierten Ca 2 +-Indikatoren, wie Fluo-4 und rhod, zur Verfügung, die ein hohes Signal-Rausch-Verhältnis zu zeigen, während minimal stören präsynaptischen Ca 2 + Dynamik. Dextran-Konjugation hilft zu verhindern, Ca 2 +-Indikatoren werden in Organellen wie Mitochondrien sequestriert. Die Belastung Technik kann auch für Larven, Embryonen und Erwachsenen angewendet werden.

Watch this Scientific Journal Video about Loading Drosophila Nerve Terminals with Calcium Indicators at JoVE.com

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium ist ein allgegenwärtiges Botenstoff im Nervensystem, wichtig für die Auslösung die Freisetzung von Neurotransmittern und Veränderungen der synaptischen Stärke. Hier zeigen wir eine Technik zum Be-Ca 2 +-Indikatoren in Drosophila Nervenendigungen. Wir zeigen auch die Herstellung der erforderlichen Geräte und betonen Punkte von entscheidender Bedeutung für die Technik für den Erfolg.

Loading Drosophila Nerve Terminals mit Calcium-Indikatoren

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

loading-drosophila-nerve-terminals-with-calcium-indicators

Research

JoVE Journal

Biology

10.0K Aufrufe.  University of Texas Health Science Center at San Antonio (UTHSCSA).  Calcium ist ein allgegenwärtiges Botenstoff im Nervensystem, wichtig für die Auslösung die Freisetzung von Neurotransmittern und Veränderungen der synaptischen Stärke. Hier zeigen wir eine Technik zum Be-Ca 2 +-Indikatoren in Drosophila Nervenendigungen. Wir zeigen auch die Herstellung der erforderlichen Geräte und betonen Punkte von entscheidender Bedeutung für die Technik für den Erfolg. 

Serie: Loading Drosophila Nerve Terminals with Calcium Indicators

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

Ex vivo mechanische Belastung der Sehnen

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

Forschung

Biologie

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Visualisierung der Live- Drosophila Glial-neuromuskulären Synapse mit Fluoreszenzfarbstoffen

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Montage-, Lade-und Alignment einer analytischen Ultrazentrifuge Messkammerdirektanschluss

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Hui Zhang and Niko G. Gubernator contributed equally to this work.

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

Freisetzung von Dopamin an den einzelnen präsynaptischen visualisiert FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.
These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.
Building on the classic inverted motility assay 2, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.
The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4, quantum dots 5 or a wide variety of antigens via biotinylated antibodies 4-6.

Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described.

Frachtladesysteme auf Kinesin Powered Molekulare Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated Calcium (CRAC) channels. CRAC channels play important role in regulation of T cell proliferation and gene expression. Abnormal CRAC channel function in T cells has been linked to severe combined immunodeficiency and autoimmune diseases 1, 2 . Studying CRAC channel function in human T cells may uncover new molecular mechanisms regulating normal immune responses and unravel the causes of related human diseases. Electrophysiological recordings of membrane currents provide the most accurate assessment of functional channel properties and their regulation. Electrophysiological assessment of CRAC channel currents in Jurkat T cells, a human leukemia T cell line, was first performed more than 20 years ago 3, however, CRAC current measurements in normal human T cells remains a challenging task. The difficulties in recording CRAC channel currents in normal T cells are compounded by the fact that blood-derived T lymphocytes are much smaller in size than Jurkat T cells and, therefore, the endogenous whole-cell CRAC currents are very low in amplitude. Here, we give a step-by-step procedure that we routinely use to record the Ca2+ or Na+ currents via CRAC channels in resting human T cells isolated from the peripheral blood of healthy volunteers. The method described here was adopted from the procedures used for recording the CRAC currents in Jurkat T cells and activated human T cells 4-8.

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived human T lymphocytes.

Whole-Cell Aufnahme von Calcium Release-Activated Calcium (CRAC) Ströme in menschlichen T-Lymphozyten

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Mess-Fast-Flux Calcium in Kardiomyozyten

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Direct Imaging von Calcium ER mit Targeted-Esterase Induzierte Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

Monitoring ER / SR Calcium Release mit dem gezielten Ca<sup&gt; 2+</sup&gt; Sensor Catcher<sup&gt; +</sup

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...

Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca2+) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca2+ is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M1) and screening for the phenotype of interest was carried out in the segregating (M2) population. Using a 96-well high-throughput Ca2+ measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca2+ reporter background and identify novel Ca2+ regulated targets.

A forward genetic screen based on Ca2+ elevation as a read-out leads to identification of genetic components involved in calcium dependent signaling pathways in plants.

Forward Genetic Screen Mit transgenem CalciumReporter Aequorin, um neuartige Ziele bei der Calciumsignalisierung zu identifizieren

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis ...