Name: Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons (Video) | JoVE Journal
Uploaded: 2011-04-06T11:00:00.000+00:00
Duration: 9 min 54 s
Description: Learn live-imaging of PKC translocation in Sf9 cells and Aplysia sensory neurons using fluorescently tagged proteins and laser scanning microscopy. This method reveals differential isoform regulation by second messengers in real time.

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This work was supported by Canadian Institutes of Health Research (CIHR) Grant to Wayne S. Sossin. Carole A. Farah is the recipient of a postdoctoral fellowship from the Fonds de la Recherche en Sant&eacute; du Qu&eacute;bec (FRSQ) and a Conrad Harrington fellowship. The authors would like to thank Joanna Bougie, Margaret Hastings and Margaret Labban for help with the video shoot and Madeline Richmond Pool for help with the graphic overview.

1. Preparation and Maintenance of Sf9 Cell Monolayer Cultures 
<ol>
 <li>Sf9 cell culture and maintenance is performed in a tissue culture hood.</li>
 <li>Sf9 cells are derived from Spodoptera frugiperda ovarian cells (Sf21 cells) and can be purchased as frozen cells in Grace's media from Invitrogen.</li>
 <li>Place 8 mL of Grace's insect medium, supplemented (1X), to which 30% fetal bovine serum (FBS) has been added in a 75 cm2 cell culture flask with a canted neck.</li>
 <li>Thaw the frozen tube of Sf9 cells rapidly in a 37&deg;C water bath by moving the tube back and forth (about 1-2 minutes).</li>
 <li>Transfer Sf9 cells to the 75 cm2 cell culture flask and rock plate gently by hand to distribute the cells evenly.</li>
 <li>Incubate 1-2 hours at 27&deg;C until cells have attached.</li>
 <li>Remove old medium and replace with 10 mL of freshGrace's insect medium with 30% FBS.</li>
 <li>Incubate at 27&deg;C until the cells are confluent.</li>
 <li>To maintain monolayer cultures, remove old medium from the confluent flask of Sf9 cells and add 5 mL of Grace's insect medium with 10% FBS.</li>
 <li>Lightly tap on the side of the flask several times to detach Sf9 cells.</li>
 <li>Using a 10 mL pipette and a pipettor, pipette the media up and down once gently, spraying flask wall while pipetting down. Transfer the cells to a sterile 15 mL Polysterene tube.</li>
 <li>Add 2 mL of Sf9 cell suspension to 8 mL of Grace's insect medium with 10% FBS (1:5 dilution to maintain log phase growth) in new 75 cm2 cell culture flask and rock plate gently by hand.</li>
 <li>Incubate at 27&deg;C until the cells are confluent.</li>
</ol>
2. Expression of Fluorescently Tagged PKCs in Sf9 Cells
<ol>
 <li>For live-imaging experiment, culture Sf9 cells on 35 mm MatTek glass bottom culture disheswith a glass surface of 14 mm and a coverslip thickness of 0.16 to 0.19 mm.</li>
 <li>PKCs tagged with fluorescent proteins are expressed in Sf9 cells using Cellfectin II transfection reagent following recommendation of the manufacturer with the modifications detailed below.</li>
 <li>Day 1: Prior to plating the cells, treat each MatTek dish with Grace's insect medium with 10% FBS for at least 30 minutes.</li>
 <li>Count Sf9 cells from step 11 (above) using a hemacytometer.</li>
 <li>Plate 0.05 x 106 cells on each MatTek glass coverslip in a total volume of 150 &mu;L. Allow the cells to attach for 30 minutes at room temperature.</li>
 <li>Add 2 mL of Grace's supplemented with 10% FBS to each dish 1 mL at a time slowly to avoid detaching the cells.</li>
 <li>Incubate at 27&deg;C overnight.</li>
 <li>From this point on, use manufacturer's recommendation for transfection of Sf9 cells with plasmid DNA.</li>
 <li>Day 2: Transfect Sf9 cells with fluorescently tagged PKCs using Cellfectin II transfection reagent. We have often co-expressed in this system PKCs tagged with enhanced Green Fluorescent Protein (eGFP) and monomeric Red Fluorescent Protein (mRFP) (details for plasmid construction have been previously described 2,5). Expression of one fluorescent protein at a time yields about 70-80% expressing cells 72 hours post-transfection. Co-expression of two fluorescent proteins decreases transfection efficiency to about 30% co-expressing cells. When co-expressing eGFP-tagged PKC and mRFP-tagged PKC, use 2x more plasmid DNA for the mRFP-tagged protein for optimal protein expression.</li>
 <li>Perform live-imaging 48-72 hours post-transfection (for optimal protein expression, wait 72 hours).</li>
</ol>
3. Expression of Fluorescently Tagged PKCs in Aplysia Sensory Neurons
<ol>
 <li>Preparation of Aplysia neuronal cell cultures has been described in a video article by Zhao and colleagues 4.</li>
 <li>PKCs tagged with fluorescent proteins are expressed in Aplysia sensory neurons using microinjection procedure detailed below.</li>
 <li>Prepare a stock solution of 1% Fast Green (FCF) in distilled water, filter using a syringe and a 28 mm syringe filter (0.20 &mu;m). Aliquot and store at -20&deg;C.&nbsp;</li>
 <li>Microinjection of the sensory neurons can be performed on day 1 or day 2 after isolating the neurons but we find that waiting 2 days allows for better cell adherence to the coverslip.</li>
 <li>On day 2 after isolation of the sensory neurons, thaw an aliquot of Fast Green and filter it again using a syringe and a 28 mm syringe filter (0.20 &mu;m).</li>
 <li>Prepare a solution of plasmid DNA in distilled water containing 0.5% Fast Green. Concentrations of plasmid DNA as high as 0.4 &mu;g/&mu;L can be used but it is not recommended to go above these values.&nbsp;</li>
 <li>Filter the plasmid DNA / Fast Green solution using a 1 mL syringe and a 4 mm syringe filter (0.20 &mu;m).</li>
 <li>Centrifuge the plasmid DNA / Fast Green solution at 16110 x g for 15 minutes using a microcentrifuge.</li>
 <li>Prepare sharp electrodes for microinjection of neurons using a microelectrode puller (Sutter Flaming/Brown Micropipette Puller, Model P-97). Using a box filament, pull glass microelectrodes with 1 mm outer diameter, 0.75 mm inside diameter and 100 mm length using thin wall glass capillaries with a filament inside (World Precision Instruments TW100F-4). For more information about pulling microinjection electrodes please refer to Sutter electrode cookbook.</li>
 <li>Fill each electrode with 2 &mu;L of plasmid DNA / Fast Green solution using microloader pipette tip (eppendorf CA32950-050).</li>
 <li>Transfer the MatTek glass bottom culture dish containing Aplysia neuronal cultures to the microinjection station.</li>
 <li>The microinjection station consists of the following: a home-made large glass stage to hold the culture dishes on a vibration isolation table (Kinetic Systems vibration isolation workstation), a stereo-scope with external halogen illumination, a micromanipulator (Sutter Huxley Wall Type Micromanipulator which allows both coarse and ultra-fine positioning in the three-axis), a microelectrode holder connected to a pneumatic pump (World Precision Instruments PV820 Pneumatic Pico-Pump). The pressure Input of the pneumatic pump is connected to a compressed Nitrogen tank.&nbsp;&nbsp;&nbsp;</li>
 <li>Insert a microelectrode into the microelectrode holder.</li>
 <li>Viewing under a stereo-microscope, insert the tip of the micropipette into the cell nucleus.</li>
 <li>Deliver short pressure pulses of Nitrogen (10 to 100 ms in duration; 20 lb/in2) until the nucleus becomes uniformely green.</li>
 <li>Incubate the cells for 4 hours at room temperature and keep at 4&deg;C until use. For optimal PKC translocation, perform live-imaging experiment 20 - 24 hours post-injection.</li>
</ol>
4. Visualization of PKC Translocation in Living Sf9 Cells and in Aplysia Sensory Neurons
<ol>
 <li>For imaging, we use a Zeiss LSM510 inverted confocal microscope with an Axiovert 200.</li>
 <li>Imaging protocol is the same for Sf9 cells and for Aplysia sensory neurons except for the following: Sf9 cells are imaged with a x63 oil immersion objective whereas Aplysia sensory neurons are imaged with a x40 oil immersion objective. Sf9 cells are imaged in a temperature-controlled chamber maintained at 26-27oC whereas Aplysia sensory neurons are imaged at room temperature maintained around 18-20oC.</li>
 <li>We use a 30 mW Argon laser (excitation at 488nm) with 50% laser output to image PKC tagged with eGFP protein and a HeNe laser (excitation at 543nm) to image PKC tagged with mRFP protein. The Argon and HeNe laser lines are attenuated to 4% and 50% transmission output respectively prior to imaging and the pinhole is adjusted to one.</li>
 <li>It is important to have the culture dish stabilized on the imaging stage and to avoid movement or vibration.</li>
 <li>Remove 1 mL of culture media from the dish gently using a 10 mL pipette leaving 1 mL total volume in the dish.</li>
 <li>Take pictures in the middle section of the cells where the nucleus can be seen.</li>
 <li>Set up a time series of 10-20 confocal images taking a picture every 30 seconds.</li>
 <li>Start the time series.</li>
 <li>After 2 pictures are taken (after the 30 seconds time point), add 1 mL of the drug (2X concentration) gently drop-by-drop on top of the cells using a P1000 pipette. The drug is added continuously for about 30 seconds.</li>
 <li>Some drugs induce a fast translocation which can be visible immediately following drug treatment (at the 60 seconds time point) while others induce a slow translocation which is only optimal 5-10 minutes post-treatment (Movie 1 and Movie 2).</li>
 <li>If the drug needs to be washed away, use 2 x 10 mL pipets to do the wash, pipetting the wash buffer down gently with one pipette on one side of the dish and pipeting up the media on the other side.</li>
 <li>If the wash has been effective and if the effect of the drug is reversible, PKC reverts back to the cytosol within 30-60 seconds.</li>
 <li>Save the movie as an LSM file.</li>
 <li>Use NIH Image J software to open the LSM files and quantify the images Pre and Post drug treatment. Quantification has been described elsewhere 1.</li>
</ol>

<ol><li>Farah, C. A., Nagakura, I., Weatherill, D., Fan, X. &amp, Sossin, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiological+role+for+phosphatidic+acid+in+the+translocation+of+the+novel+protein+kinase+C+Apl+II+in+Aplysia+neurons%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Physiological role for phosphatidic acid in the translocation of the novel protein kinase C Apl II in Aplysia neurons.</a> Mol Cell Biol. 28, 4719-4733 (2008).</li>
<li>Zhao, Y., Leal, K., Abi-Farah, C., Martin, K. C., Sossin, W. S. &amp, Klein, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((M.Isoform+specificity+of+PKC+translocation+in+living+Aplysia+sensory+neurons+and+a+role+for+Ca2%2B-dependent+PKC+APL+I+in+the+induction+of+intermediate-term+facilitation%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">M.Isoform specificity of PKC translocation in living Aplysia sensory neurons and a role for Ca2+-dependent PKC APL I in the induction of intermediate-term facilitation.</a> J Neurosci. 26, 8847-8856 (2006).</li>
<li>Farah, C. A., Weatherill, D., Dunn, T. W. &amp, Sossin, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PKC+differentially+translocates+during+spaced+and+massed+training+in+Aplysia%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">PKC differentially translocates during spaced and massed training in Aplysia.</a> J Neurosci. 29, 10281-10286 (2009).</li>
<li>Zhao, Y., Wang, D. O., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+of+Aplysia+sensory-motor+neuronal+cell+cultures%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Preparation of Aplysia sensory-motor neuronal cell cultures.</a> J Vis Exp. , (2009).</li>
<li>Manseau, F., Fan, X., Hueftlein, T., Sossin, W. S., Castellucci, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ca2%2B-independent+PKC+Apl+II+mediates+the+serotonin+induced+facilitation+at+depressed+synapses+in+Aplysia%5BTitle%5D)+AND+%22J.+Neuroscience%22%5BJournal%5D)">Ca2+-independent PKC Apl II mediates the serotonin induced facilitation at depressed synapses in Aplysia.</a> J. Neuroscience. 21, 1247-1256 (2001).</li>
</ol>

No conflicts of interest declared.

We have described a technique to image translocation of fluorescently tagged PKCs in real time in Sf9 cells and in Aplysia sensory neurons. Sf9 cells provide a simple system to image PKC translocation since culturing and transfecting them is pretty straight-forward. In contrast, microinjection of Aplysia sensory neurons takes some time to master; up to a few months. Difficulties related to this technique include electrode clogging. Filtering the injection solution and centrifuging it usually helps but s...

<table><tbody><tr><td>Sf9 frozen cells</td><td>&lt;em&gt;Spodoptera frugiperda&lt;/em&gt; ovarian cells</td><td>Invitrogen</td><td>B825-01</td><td></td></tr><tr><td>Grace&amp;rsquo;s Insect Medium, Supplemented (1X), liquid</td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>11605-094</td><td>Use to culture Sf9 cells</td></tr><tr><td>Corning 75cm&lt;sup&gt;2&lt;/sup&gt; canted neck cell culture flask</td><td>Tool</td><td>Corning</td><td>430720</td><td>Use to culture Sf9 cells</td></tr><tr><td>Fetal Bovine Serum</td><td>Reagent</td><td>CanSera</td><td>CS-C08-500</td><td>Supplement Grace&amp;rsquo;s media with FBS prior to use</td></tr><tr><td>Syringe</td><td>Tool</td><td>BD Biosciences</td><td>309604</td><td>Use to filter Fast Green solution</td></tr><tr><td>Syringe</td><td>Tool</td><td>BD Biosciences</td><td>309602</td><td>Use to filter plasmid DNA / Fast Green solution</td></tr><tr><td>Filter</td><td>Tool</td><td>Corning</td><td>431229</td><td>Use to filter Fast Green solution</td></tr><tr><td>Filter</td><td>Tool</td><td>Corning</td><td>431212</td><td>Use to filter plasmid DNA / Fast Green solution</td></tr><tr><td>Glass Bottom Dish</td><td>Tool</td><td>MatTek Corp.</td><td>P35G-1.5-14-C</td><td>Use to culture Sf9 cells prior to transfection and imaging</td></tr><tr><td>Cellfectin II Reagent</td><td>Reagent</td><td>Invitrogen</td><td>10362-100</td><td>Used to express fluorescently tagged PKCs in Sf9 cells</td></tr><tr><td>Fast Green FCF</td><td>Reagent</td><td>Sigma-Aldrich</td><td>F-7252</td><td>Use to visualize microinjection of plasmid DNA solution into &lt;em&gt;Aplysia&lt;/em&gt; sensory neurons</td></tr><tr><td>Thin wall glass capillaries</td><td>Tool</td><td>World Precision Instruments, Inc.</td><td>TW100F-4</td><td>Use to make micr&amp;#8211;lectrodes for microinjection</td></tr><tr><td>Microloader pipette tip</td><td>Tool</td><td>Eppendorf</td><td>CA32950-050</td><td>Autoclave before using to fill up the micr&amp;#8211;lectrodes for microinjection</td></tr><tr><td>PV820 Pneumatic PicoPump </td><td>Tool</td><td>World Precision Instruments, Inc.</td><td>SYS-PV820</td><td>Part of microinjection station</td></tr><tr><td>Micr&amp;#8211;lectrode holder</td><td>Tool</td><td>World Precision Instruments, Inc.</td><td>MPH6S</td><td>Use to hold micr&amp;#8211;lectrode </td></tr><tr><td>Micromanipulator</td><td>Tool</td><td>Sutter Instrument Co.</td><td>MP-85</td><td>Use to position micr&amp;#8211;lectrode in the three-axis</td></tr></tbody></table>

live-imaging of pkc translocation in sf9 cells and in aplysia sensory neurons

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

The aim of this procedure is to visualize fluorescently tagged PKC translocation in living cells. This is accomplished by first preparing SF nine cell cultures or cultures of sensory neurons from a Plegia California. The second step is to express fluorescently tagged pkcs in the cells.The final step is to image PKC translocation in response to different activators using laser scanning microscopy. Ultimately, results can be obtained that show differential regulation of the different PKC isoforms by second messengers such as diol glycerol through imaging of PKC translocation in real time using live cells. Hi, my name is Carol Ferra.I'm a research associate in Dr.Wayne Sawn lab at the Montreal Neurological Institute. Today I will be demonstrating live imaging of PKC translocation in Aplysia sensory neurons. This method can help answer key questions in the synaptic plasticity field, such as how are the different PKC isoforms activated during memory formation.To prepare the SF nine cell monolayer cultures in a tissue culture hood at eight milliliters of grace's insect medium with 30%fetal bovine serum to a 75 centimeter squared cell culture flask then thaw the frozen tube of SF nine cells rapidly in a 37 degree Celsius water bath by moving the tube back and forth for about one to two minutes. Next, transfer the SF nine cells to the cell culture flask and rock the flask gently by hand to distribute the cells evenly. Incubate the cells for one to two hours at 27 degrees Celsius until they have attached.Then remove the old medium from the flask and replace it with another 10 milliliters of fresh graces insect medium with 30%FBS incubate the cells at 27 degrees Celsius until they are cofluent. To maintain monolayer cultures, remove the old medium from the confluent flask of the SF nine cells and add five milliliters of grace's insect medium with 10%FBS lightly tap on the side of the flask several times to detach the SF nine cells. Then using a 10 milliliter pipette gently pipette the media up and down.Once spritz in the flask wall while pipetting down, transfer the cells to a sterile 15 milliliter polystyrene tube. Add two milliliters of SF nine cell suspension to eight milliliters of grace's insect medium with 10%FBS in the new 75 centimeter squared cell culture flask with the dilution ratio of one to five. To maintain log phase growth, rock the flask gently by hand.Next, incubate the cells again at 27 degrees Celsius until they're co fluent. Following incubation, the SF nine cells are ready to use for the expression of fluorescently tagged pkcs as described in the written procedure prior to the expression of fluorescently tagged pkcs Plegia neuronal cell cultures are prepared in matech glass bottom culture dishes as previously described. Two days after isolation of the sensory neurons filter a previously prepared quat of fast green using a syringe and a 20 millimeter syringe filter with a 0.2 micron pore size.Prepare a solution of plasmid DNA encoding for E-G-F-P-P KC API two in distilled water containing 0.5%fast green at a concentration no higher than 0.4 micrograms per microliter. Filter the plasmid DNA fast green solution using a one milliliter syringe and a four millimeter syringe filter with a 0.2 micron pore size. Then centrifuge the plasmid DNA fast green solution and 16, 110 Gs for 15 minutes using a micro centrifuge.Next, prepare sharp electrodes for micro injection of neurons using a micro electrode puller. Pull glass micro electrodes using thin wall glass capillaries with filaments inside. Fill each electrode with two microliters of plasmid DNA fast green solution.Using a micro load pipette tip, microinjection is performed at the microinjection station. The microinjection station consists of a homemade large glass stage to hold the culture dishes on a vibration isolation table. A stereoscope with external halogen illumination, a micro manipulator and a micro electrode holder connected to a pneumatic pump.The pressure input of the pneumatic pump is connected to a compressed nitrogen tank. Transfer the matech glass bottom culture dish containing the plegia neuron cultures to the micro injection station and insert a micro electrode into the micro electrode holder. View the neurons under a stereo microscope and insert the tip of the micro pipette into the cell nucleus.Deliver short pressure pulses of nitrogen of 10 to 100 milliseconds in duration and 20 pounds per square inch until the nucleus becomes uniformly green. Incubate the cells for four hours at room temperature and keep at four degrees Celsius until use a Zeiss LSM five 10 inverted confocal microscope with an axia avert. 200 is used for imaging of both SF nine cells and plegia sensory neurons.Imaging of plegia sensory neurons is demonstrated in this video. Details for imaging of SF nine cells can be found in the written protocol. Use a 30 milliwatts argonne laser with 50%laser output to image PKC tagged with EGFP protein.Next, attenuate the Argonne laser line to 4%transmission output. Then adjust the pinhole to one, stabilize the culture dish on the imaging stage and avoid movement or vibration. Gently remove one milliliter of culture media from the dish using a 10 milliliter pipette and leave one milliliter total volume.In the dish. Locate the middle section of the cells where the nucleus can be seen. Set up a time series of 10 to 20 confocal images at time intervals of 30 seconds.Start the movie and after the second picture is taken, induce translocation by gently adding one milliliter of drug at two times concentration. Add the drug drop by drop on top of the cells continuously for about 30 seconds. Here serotonin is used to induce a fast translocation, which can be visible immediately following drug treatment.Some drugs induce a slow translocation, which is only optimal five to 10 minutes post-treatment, wash the drug away with two 10 milliliter pipettes, pipetting the wash buffer down gently with one pipette on one side of the dish and pipetting the media up on the other side. If the wash is effective and the effect of the drug is reversible, PKC will revert back to the cytosol within 30 to 60 seconds. Save the movie as an LSM file shown.Here are the representative results of live imaging of PKC translocation in plegia sensory neurons. E-G-F-P-P KC APL two was expressed in a sensory neuron and a series of 10 confocal images were recorded at time intervals of 30 seconds. In this example, serotonin was added to the dish after 30 seconds of recording.In this example, live imaging of PKC translocation is shown in SF nine cells. E-G-F-P-P KC APL one and M-R-F-P-P KC APL two were co-expressed In SF nine cells and a series of 10 confocal images were recorded at time intervals of 30 seconds. One two DIOCTYL SN glycerol.A cell permeable analog of DYL glycerol was added to the dish after 30 seconds of recording. After watching this video, you should have a good understanding of how to express fluorescently tagged pkcs in aplasia sensory neurons, and how to image PKC translocation in live cells.

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Learn live-imaging of PKC translocation in Sf9 cells and Aplysia sensory neurons using fluorescently tagged proteins and laser scanning microscopy. This method reveals differential isoform regulation by second messengers in real time.

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

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