Name: detection of phospholipase c activity in the brain homogenate from the honeybee
Uploaded: 2018-09-14T13:30:00.000+00:00
Duration: 8 min 30 s
Description: Watch this Scientific Journal Video about Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee at JoVE.com

Figure 4B - 4D was modified from Suenami et al.24 with the permission of Biology Open. The authors are grateful to the publisher for the permission. This work was supported by the Human Frontier Science Program (RGY0077/2016) to Shota Suenami and Ryo Miyazaki.

1. Capture of Foraging Honeybees
<ol>
	<li>Purchase honeybee colonies from a local distributor.</li>
	<li>Using an insect net, catch forager bees which return to the hive with pollen bags on their hind legs. Transfer the bees to a standard 50-mL plastic conical tube and cap the tube (Figure 1). Put the tube on ice to anesthetize the bees. 
	NOTE: Wear the designated jackets for beekeeping to avoid bee stings. Nursing bees can also be collected depending on the experiment. To catch the nurse bees, observe the bee&#39;s behavior on the wax comb and catch nurse bees, which poke their heads into brood cells to feed the larvae, by the wing or thorax using tweezers. Then, place the bees into a 50-mL plastic tube, cap the tube, and put it on ice as mentioned in step 1.2.</li>
	<li>Capture 12 foragers randomly. 
	NOTE: This protocol uses two bees per lot, resulting in six lots. Multiple bees can be caught in one tube, and the number of the bees in a single tube is not important, as bees in each lot are combined later (see step 3.1.1). Keep the tube cool in the summer, as the high temperature in the tube injures the bees.</li>
</ol>
2. Dissection of the Honeybee Brain
<ol>
	<li>In the laboratory, place the 50-mL tube on ice for at least 30 min6 to anesthetize the bees.</li>
	<li>To prepare the dissection stage, fold a piece of dental wax in half (the resulting size is about 3.5 x 7.5 cm) and push it into a plastic dish (60 x 15 mm). 
	NOTE: The folded wax firmly holds the insect pins.</li>
	<li>Under a binocular microscope, separate the bee&#39;s head from its body with tweezers and fix it on the dental wax by inserting insect pins at the base of each antenna to hold the anterior of the head in an upward position (Figure 2A). 
	NOTE: Wash the tweezers before use, using ethanol or sterilized water.</li>
	<li>Pour enough saline solution (0.13 mol/L NaCl, 5 mmol/L KCl, and 1 mmol/L CaCl2-2H2O) on the head to cover it. Pierce the head again with the pins if the head floats in the solution. Use an ice-cold saline solution, if necessary. 
	NOTE: However, this protocol works without the cold saline solution.</li>
	<li>Remove the antennae using tweezers (Figure 2B).</li>
	<li>Make a horizontal cut near the bases of the antennae, and longitudinally at the border of the compound eyes, using a scalpel. Then, make a horizontal cut at the top of the head. Remove the cuticle to open a window over the brain (Figure 2C). 
	NOTE: If necessary, wash the scalpel with ethanol or sterilized water before use.</li>
	<li>Remove the retinae from the ocelli and the tracheae on the anterior surface of the brain, using tweezers (Figure 2D).</li>
	<li>Expand the cut at the border of the compound eyes dorsally and ventrally, using the scalpel (Figure 2E). Next, remove the connective tissue between the cuticle of the compound eyes and retinae by inserting the tweezers directly under the eye cuticle (Figure 2F).</li>
	<li>Discard the cuticle of the compound eyes. Pick the dorsal tips of the retinae of the compound eyes with tweezers and move the tweezers in the ventral direction to carefully peel off the retinae (Figure 2G).</li>
	<li>Carefully remove the remaining tracheae on the anterior surface of the brain, using tweezers (Figure 2H).</li>
	<li>Cut the connective tissue between the brain and around the tissues, using tweezers, and dissect the brain from the head capsule (Figure 2H).</li>
	<li>Peel off the tracheae on the posterior surface of the brain, using the tweezers (Figure 2I).</li>
	<li>Using a scalpel, cut the connection between the MBs and other brain regions next to the vertical lobes, which are a part of the MBs (Figure 2J).</li>
	<li>Place the dissected MBs and remaining brain tissues into 1.5-mL tubes and rapidly freeze them with dry ice or liquid nitrogen (Figure 2K). Store the frozen tissues at -80 &#176;C until use. 
	NOTE: Handle liquid nitrogen with the designated gloves and ventilation to avoid cold burn and suffocation. Dry ice should also be handled carefully with gloves. The protocol can be paused here. The dissection and storage of the brain tissues should be performed one bee at a time to avoid protein degradation.</li>
</ol>
3. Preparation of Brain Homogenates
<ol>
	<li>Add 10 &#181;L of homogenization buffer comprising of 50 mmol/L HEPES-KOH (pH 7.2), 70 mmol/L KCl, and 1.0 mmol/L CaCl2 to the frozen brain tissue and homogenize the tissue in the 1.5-mL tube by manually applying pressure with a plastic pestle. Stroke the tissue at least 200x. 
	&#8203;NOTE: Perform the manipulations described in section 3 on ice. Use a pestle fitting right in the tube to sufficiently smash the tissues, which easily float in the homogenization buffer.
	<ol>
		<li>Transfer the homogenized tissue solution to the next tissue in the lot and homogenize the tissue in the same way. 
		NOTE: The honeybees in each lot are combined at this step. This protocol uses six lots.</li>
	</ol>
	</li>
	<li>Add 30 &#181;L of the homogenization buffer.</li>
	<li>Centrifuge the homogenized tissue sample for 10 min at approximately 900 x g27 and 4 &#176;C.</li>
	<li>Transfer the supernatant to a new 1.5-mL tube and centrifuge it again for 20 min at approximately 9,500 x g27 and 4 &#176;C.</li>
	<li>Place the supernatant in a new 1.5-mL tube. Store the homogenate at -20 &#176;C until use. 
	&#8203;NOTE: The protocol can be paused here.</li>
	<li>Determine the protein concentration using the bicinchoninic acid (BCA) assay.
	<ol>
		<li>Dilute 2.5 &#181;L of the homogenate with 17.5 &#181;L of sterilized water (thus diluting the homogenate eightfold) and use all the diluted homogenate. 
		NOTE: Sampling homogenate using a micropipette must be performed carefully because of its high viscosity.</li>
		<li>Perform the BCA assay using 0, 0.1, 0.2, 0.4, 1.0, and 2.0 mg/mL of bovine serum albumin (BSA) in 20 &#181;L as standards. 
		NOTE: Change the scale of the BCA assay as necessary, based on the volume of the homogenate and standard samples. The protocol can be paused here.</li>
	</ol>
	</li>
</ol>
4. PLC Reaction in Brain Homogenates
<ol>
	<li>Prepare the stock solution of the fluorogenic substrate WH-1526 dissolved in sterilized water at 50 &#181;mol/L and store it at -80 &#176;C. 
	NOTE: Cover the solutions containing the substrate with foil to prevent fading.</li>
	<li>Prepare the reaction premixture in the required volume to achieve the following composition in 10 &#181;L of reaction mixture: 50 mmol/L HEPES-KOH (pH 7.2), 70 mmol/L KCl, 1.0 mmol/L CaCl2, 2.0 mmol/L dithiothreitol, 50 mg/L BSA, and 12.5 &#181;mol/L WH-15.</li>
	<li>Dilute the homogenate with homogenization buffer, if required.</li>
	<li>Mix the reaction premixture and homogenate containing 1.3 &#181;g of proteins in a polymerase chain reaction (PCR) tube to achieve a final volume of 10 &#181;L of reaction mixture.
	<ol>
		<li>Prepare two types of control mixtures for the statistical analysis: put the homogenate but not WH-15 into&#160;mixture (control mixture 1), and vice versa (control mixture 2). 
		NOTE: Prepare the reaction mixtures and control mixtures on ice to prevent protein degradation and reaction of PLC. Control mixtures 1 and 2 are used to detect fluorescence from the homogenate and free substrate, respectively.</li>
	</ol>
	</li>
	<li>Flush the tube and incubate it in a thermal cycler for 30 min at 25 &#176;C.</li>
	<li>Stop the reaction by adding 2 &#181;L of 25 mmol/L ethylene glycol bis (&#946;-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid. 
	NOTE: Appropriate protein amounts and reaction times vary among experiments. Thus, to optimize the reaction condition, it might be necessary to repeat the procedures described in steps 4.1 - 5.3 in the preliminary experiments by changing the protein amount and incubation time until the fluorescence increases linearly. For reference, when&#160;the homogenate derived from the other brain regions is used, the reaction usually works linearly with 1.3 &#181;g or less of proteins within 30 min, but the linearity decreases either with at least 2.7 &#181;g of proteins or with an incubation time of 90 min or longer.</li>
</ol>
5. Detection of Fluorescence Resulting from PLC Activity
<ol>
	<li>Centrifuge the reaction mixture and control mixtures for 5 min at approximately 310 x g28 and transfer 10 &#181;L of the supernatant to different wells on a 384-well microplate applicable for fluorescence detection. 
	NOTE: To prevent fading and a contamination with dust, cover the plate with foil.</li>
	<li>Flush the microplate using a centrifuge for the microplate.</li>
	<li>Set the plate into a microplate reader and detect fluorescence with a technical triplicate. 
	NOTE: The excitation and emission wavelengths are 344 nm and 530 nm, respectively. If applicable (depending on the microplate reader), mix the sample mixtures for 5 s before each detection. The protocol can be stopped here.</li>
</ol>
6. Test of the Inhibitory Action of Pharmacologic Agents
<ol>
	<li>Prepare stock solutions of edelfosine and neomycin at the appropriate concentrations and store them until use: edelfosine at 5.0 mmol/L and -20 &#176;C; neomycin at 550 mmol/L and 4 &#176;C.</li>
	<li>When preparing the reaction premixture as described in step 4.2., add inhibitors to the premixture to achieve the appropriate final concentrations: edelfosine, 1.0 mmol/L; neomycin, 0.55 mmol/L.</li>
	<li>Add the homogenate to the reaction premixture and appropriate control mixtures as in step 4.4.
	<ol>
		<li>Prepare three types of control mixtures: put the homogenate and inhibitor, but not the substrate into control mixture 1; add the substrate and inhibitor but not the homogenate into control mixture 2; and put the inhibitor, but not the homogenate or substrate into control mixture 3.</li>
	</ol>
	</li>
	<li>Perform the PLC reaction and detection of fluorescence as described in steps 4.5 - 5.3.</li>
</ol>
7. Statistical Analysis
<ol>
	<li>For the detection of PLC activity using the mixtures described in section 4, calculate the fluorescence derived from the reaction between PLC and the substrate by subtracting the fluorescence emitted from the homogenate or free substrate as follows. 
	 
	Fl (PLC activity) = Fl (reaction mix) - {Fl (ctrl 1) + Fl (ctrl 2)} 
	&#8203; 
	NOTE: Fl (PLC activity), Fl (reaction mix), Fl (ctrl 1), and Fl (ctrl 2) denote bona fide fluorescence derived from PLC activity, fluorescence detected in the reaction mixture, and control mixtures 1 and 2, respectively.
	<ol>
		<li>After the measurement of fluorescence in the mixtures as described in steps 4.1 - 5.3, calculate Fl (PLC activity) according to the equation in step 7.1, and then the mean Fl (PLC activity) of the technical triplicate for each homogenate.</li>
		<li>Using the mean Fl (PLC activity) of the technical triplicate obtained in step 7.1.1, calculate again the mean Fl (PLC activity) of biological replicates of the MBs and other brain regions and compare the values between the brain tissues.</li>
	</ol>
	</li>
	<li>For the examination of PLC activity in the presence of inhibitors using the mixtures described in section 6, calculate the fluorescence derived from the reaction among the homogenate, substrate, and inhibitor as follows. 
	 
	Fl (PLC activity with inhibitor) = Fl (reaction mix) - {Fl (ctrl 1) + Fl (ctrl 2)} + Fl (ctrl 3) 
	 
	&#8203;NOTE: Here, Fl (PLC activity with inhibitor) is bona fide fluorescence resulting from PLC activity in the presence of the inhibitor. Fl (reaction mix), Fl (ctrl 1), Fl (ctrl 2), and Fl (ctrl 3) are fluorescence signals detected in the reaction mixture, control mixture 1, control mixture 2, and control mixture 3, respectively.
	<ol>
		<li>After the measurement of fluorescence in the mixtures as described in steps 6.1 - 6.4, calculate Fl (PLC activity with inhibitor) according to the equation in step 7.2. Then, obtain the mean Fl (PLC activity with inhibitor) of the technical triplicate for each homogenate.</li>
		<li>Using the mean value calculated in step 7.2.1, calculate the mean Fl (PLC activity with inhibitor) of biological replicates derived from each brain tissue. Compare the Fl (PLC activity with inhibitor) and the Fl (PLC activity) obtained in step 7.1.2 in each brain tissue.</li>
	</ol>
	</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+expression+of+phospholipase+C+encoded+by+the+norpA+gene+of+Drosophila+melanogaster.">Tissue-specific expression of phospholipase C encoded by the norpA gene of Drosophila melanogaster.</a> The Journal of Biological Chemistry. 268 (21), 15994-16001 (1993).</li><li>Huang, W., Hicks, S. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLC&#946;3+mediates+cortactin+interaction+with+WAVE2+in+MCP1-induced+actin+polymerization+and+cell+migration.">PLC&#946;3 mediates cortactin interaction with WAVE2 in MCP1-induced actin polymerization and cell migration.</a> Molecular Biology of the Cell. 26 (25), 4589-4606 (2015).</li><li>Fiala, A., M&#252;ller, U., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+downregulation+of+protein+kinase+A+during+olfactory+learning+using+antisense+technique+impairs+long-term+memory+formation+in+the+honeybee,+Apis+mellifera.">Reversible downregulation of protein kinase A during olfactory learning using antisense technique impairs long-term memory formation in the honeybee, Apis mellifera.</a> The Journal of Neuroscience. 19 (22), 10125-10134 (1999).</li><li>Thamm, M., Scheiner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKG+in+honey+bees:+spatial+expression,+Amfor+gene+expression,+sucrose+responsiveness,+and+division+of+labor.">PKG in honey bees: spatial expression, Amfor gene expression, sucrose responsiveness, and division of labor.</a> The Journal of Comparative Neurology. 522 (8), 1786-1799 (2014).</li><li>Balfanz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+transmembrane+adenylyl+cyclases+from+the+honeybee+brain.">Functional characterization of transmembrane adenylyl cyclases from the honeybee brain.</a> Insect Biochemistry and Molecular Biology. 42 (6), 435-445 (2012).</li><li>Lopez, I., Mak, E. C., Ding, J., Hamm, H. E., Lomasney, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+bifunctional+phospholipase+C+that+is+regulated+by+G&#945;12+and+stimulates+the+Ras/mitogen-activated+protein+kinase+pathway.">A novel bifunctional phospholipase C that is regulated by G&#945;12 and stimulates the Ras/mitogen-activated protein kinase pathway.</a> The Journal of Biological Chemistry. 276 (4), 2758-2765 (2001).</li><li>Huang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+membrane-associated,+fluorogenic+reporter+for+mammalian+phospholipase+C+isozymes.">A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes.</a> The Journal of Biological Chemistry. 293 (5), 1728-1735 (2018).</li></ol>

The authors have nothing to disclose.

The biochemical examination of protein activity is profoundly important for understanding molecular signaling in the brain, because the activity of an enzyme is affected by various molecules, such as substrates and inhibitors, and can, thus, change along with animal behavior (e.g., learning and memory)5. In honeybee studies, enzymes such as cyclic AMP-dependent protein kinase A, cyclic GMP-dependent protein kinase, PKC, phosphorylated CaMKII, and adenylate cyclase are reported to be differentially expressed in various brain regions based on immunohistochemistry5,10,29,30,31. Differences in enzymatic activity among brain regions, however, are only partially reported6. Here, we described a detailed protocol for detecting PLC activity and assessing the inhibitory effects of pharmacologic agents in the MBs and other brain regions.
There are some possible factors interfering with fluorescence detection. First, it is important to protect the protein from degradation when performing biochemical assays. In the protocol described here, a quick dissection and freezing of the brain are required. It is recommended that tissue samples are frozen and stored soon after each cycle of dissection. A minimization of the thaw/freeze cycle of the homogenate is also crucial to prevent the deterioration of the protein.
In addition to protein degradation, background fluorescence and absorbance in the reaction mixture may affect the results in this assay system, because PLC activity is detected by a fluorescence signal. For example, neomycin dissolved in water has a yellow color that affects fluorescence detection. Although we used 0.55 mmol/L neomycin, a 1000-fold dilution of the stock solution, a further optimization of the concentration might be required. Moreover, contamination by other tissues may also influence the result. The retina, which detects visual stimuli and contains pigments, comprises one tissue type that potentially interferes with the assay.
With the present protocol, we detected higher PLC activity in other brain regions than in the MBs24. This was inconsistent with the result of quantitative reverse-transcription PCR analysis, which revealed that the MBs express higher levels of PLC genes than other brain tissues24. This contradiction might be due to WH-15, which is a free-floating substrate26, and the fact that we did not distinguish the membrane and cytosolic fractions of the brain homogenates. Considering that PLC&#946; and PLC&#949; are membrane-associated enzymes32 and can interact with the endogenous PIP2 substrate, the amount of the membrane fraction in the homogenate likely affects the reaction between PLC and WH-15. Another possible explanation is that the PLC concentration might be higher in the other brain regions than in the MBs due to differences in protein production and/or degradation rates. Hence, bona fide PLC activity in the brain must be clarified further by additional experiments, such as by using a recently reported new substrate incorporated into the membrane33, analyzing the activity of membrane-associated and cytosolic PLCs separately, or quantifying the content of PLCs or PIP2 in each brain tissue.
Taking the above points into account, the PLC assay system described here can be expanded to measure PLC activity in different tissues not assessed here, such as the digestive tract, muscle, and reproductive organ. It is also possible to compare PLC activity between the brains of forager and nurse bees to evaluate the involvement of PLC activity in different social roles.
Overall, the assay system presented here is a feasible option for detecting PLC activity in tissue homogenates, because it can be performed with standard laboratory equipment, while a traditional approach using radiolabeled PIP2 requires specialized facilities, training, and equipment for radioisotopes. Taking advantage of the system with further modifications will deepen our understanding of the molecular mechanisms underlying the honeybee&#39;s complex behavior.

The European honeybee (Apis mellifera L.) is a eusocial insect, and female bees show caste-dependent reproduction and&#160;age-dependent division of labor. For example, in the sterile caste of bees referred to as &#39;workers&#39;, younger individuals feed the broods while older ones&#160;forage nectar and pollen outside the hive1. Learning and memory ability is critically important in the life of the honeybee, because foragers must repeatedly go back and forth between food sources and their nest and then communicate the locations of good food sources to their nestmates through dance communication1. Previous studies demonstrated that the MB, a higher brain center in insects, is involved in the learning and memory ability of the honeybee2,3,4. Differentially expressed genes and proteins have been identified in various brain regions of the honeybee5,6,7,8,9,10,11, suggesting that they are related to the unique functions of each brain region. Although the pharmacologic inhibition or activation of a protein of interest is a well-used approach to reveal the function of the protein in honeybee behavior12,13,14, it is unknown whether all drugs have functional effects in different regions of the honeybee brain. The validation of the functions of such drugs will strengthen conclusions in studies of behavioral pharmacology.
Here, we focus on PLC, one of the enzymes implicated in mouse cognition15,16,17,18. PLC triggers calcium signaling by degrading phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)19,20,21. IP3 opens IP3 receptors on the endoplasmic reticulum (ER), leading to the release of calcium ions from the ER. The released calcium activates both calcium/calmodulin-dependent protein kinase II (CaMKII) with calmodulin and protein kinase C (PKC) in the presence of DAG. Both protein kinases are involved in learning and memory22,23, consistent with the involvement of PLC in this process. PLCs are categorized into subtypes, including PLC&#946;, PLC&#947;, and PLC&#949;, based on their structures20. Each PLC subtype is activated in a different context20, and genes encoding those subtypes are differentially expressed in different tissues. We previously demonstrated that honeybee MBs express genes encoding PLC&#946; and PLC&#949; subtypes at higher levels than the remaining brain regions24, and that two pan-PLC inhibitors (edelfosine and neomycin sulfate [neomycin]) decrease PLC activity in different brain regions and, indeed, affect the learning and memory ability of the honeybee24.
Traditionally, the enzymatic activity of PLC has been measured using radiolabeled PIP225, which requires appropriate training, equipment, and facilities. Recently, a synthetic fluorogenic substrate of PLC has been established26, making it easy to assess PLC activity in the standard laboratory. Here, we present a detailed protocol to detect PLC activity in different brain regions of the honeybee using the fluorogenic substrate and to subsequently test the inhibitory effects of edelfosine and neomycin on PLC in these tissues. Because the protocol requires only basic manipulations, it may be applicable to studies of PLC activity in other tissues or brain areas in bees allocated to different social tasks.

<table><tbody><tr><td>Pierce BCA Protein Assay Kit</td><td>ThermoFisher Scientific</td><td>23227</td><td>The reagent kit for measurement of protein concentration</td></tr><tr><td>Pierce Bovine Serum Albumin Standard Ampules 2mg/mL</td><td>ThermoFisher Scientific</td><td>23209</td><td>The standard samples used in BCA assay</td></tr><tr><td>Paraffin wax</td><td>GC</td><td>13B1X00155000141</td><td>Dental wax used as dissection stage</td></tr><tr><td>Insect pin</td><td>Shiga</td><td>No. 0</td><td>Stainless, solid head</td></tr><tr><td>PLCglow</td><td>KXT Bio</td><td>KCH-0001</td><td>A fluorogenic substrate of PLC</td></tr><tr><td>384-well microplate</td><td>Corning</td><td>4511</td><td>Low-volume, round-bottom plate in black color</td></tr><tr><td>Gemini EM microplate reader</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Edelfosine</td><td>Santa Cruz Biotechnology</td><td>sc-201021</td><td>pan-PLC inhibitor</td></tr><tr><td>Neomycin sulfate</td><td>Santa Cruz Biotechnology</td><td>sc-3573</td><td>pan-PLC inhibitor</td></tr></tbody></table>

detection of phospholipase c activity in the brain homogenate from the honeybee

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.
We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

Protein Concentrations in Brain Homogenates: 
We prepared homogenates using forager bees. The calculated protein concentrations in the original homogenates are shown in Figure 3. The approximate protein concentrations in the original homogenate were as follows: 1.5 mg/mL in the MBs and 2.3 mg/mL in other brain regions. We used two bees per lot and six lots were analyzed.
Detection of PLC Activity in the Brain Homogenates: 
In the pilot experiment, we detected higher fluorescence in other brain regions than in the MBs. Therefore, we repeated the preliminary experiments using the homogenate of the other brain regions and determined the reaction time (i.e., 30 min) and protein amount (i.e., 1.3 &#181;g). The result of the reaction under these conditions is shown in Figure 4A. The reaction mixtures containing both tissue homogenate and the fluorogenic substrate exhibited &#62; 4.2-fold higher fluorescence than the control mixtures containing either the tissue homogenate or the fluorogenic substrate, suggesting that PLC activity was detected in the reaction mixtures. Relative fluorescence was approximately 3.4-fold higher in other brain regions than in the MBs (Figure 4B).
Analysis of the Inhibitory Effects of Pharmacologic Agents on PLC Activity: 
To examine the effects of the pan-PLC inhibitors edelfosine and neomycin on PLC activity, we performed the reaction in the presence of 1.0 mmol/L edelfosine or 0.55 mmol/L neomycin, using the same homogenate as analyzed above. In the presence of edelfosine, the fluorescence level decreased to approximately 6.0% and 5.4% in the MBs and other brain regions, respectively, compared to controls without edelfosine (Figure 4C). Neomycin treatment reduced the fluorescence level to 44% and 20% that of untreated controls in the MBs and other brain regions, respectively (Figure 4D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58173/58173fig1.jpg" /> 
Figure 1: Capturing a forager honeybee. A forager returning to her hive was captured in an insect net, and she was confined into a 50-mL plastic conical tube. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58173/58173fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58173/58173fig2.jpg" /> 
Figure 2: Schematic representation of the dissection procedure. (A) Axes of the honeybee brain mentioned in the protocol are shown. The bee from the head to the anterior thorax is viewed from the lateral side. (B - K) Photos and illustrations of the dissection procedures are shown. See the main text for details. Only the head of the honeybee is presented. An illustration of the tracheae is omitted. MBs = mushroom bodies. The scale bars correspond to 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58173/58173fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58173/58173fig3.jpg" /> 
Figure 3: Protein concentrations in brain tissue homogenates. Protein concentrations in the original homogenates were measured by BCA assay and calculated. The mean concentrations with standard deviations are shown. Two forager bees were used for each lot, and six lots were analyzed. MBs = mushroom bodies. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58173/58173fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58173/58173fig4.jpg" /> 
Figure 4: Fluorescence detected in reactions. (A) This panel shows fluorescence in each tissue with or without the fluorogenic substrate. The reaction was performed for 30 min using 1.3 &#181;g of protein. Fluorescence was measured by the microplate reader. The values of the sample wells were corrected by empty wells and shown in arbitrary units (AUs). (B) This panel shows a comparison of fluorescence between brain tissues. The data in panel A were corrected for control mixtures by subtraction and normalized by the calculated fluorescence in the MBs. * P &#60; 0.005, Mann-Whitney&#39;s U test. Panels C and D show relative fluorescence in the presence of (C) 1.0 mmol/L edelfosine and (D) 0.55 mmol/L neomycin. The same homogenates used in panels A and B were analyzed. The fluorescence values were normalized by the results of the control experiment without drug treatment for each tissue. The data of the control reactions in panel C are the same as in panel B. In panel D, all homogenates were analyzed again in a different experiment. The mean fluorescence values with standard deviations are shown. Two bees were used for each lot, and six lots were analyzed. * P &#60; 0.05, Wilcoxon signed-ranks test. No Hg = control mixture not containing homogenate; MBs = mushroom bodies. Panels B - D were modified from Suenami et al.24 with the publisher&#39;s permission. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58173/58173fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee at JoVE.com

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

detection-phospholipase-c-activity-brain-homogenate-from

Nanyang Technological University

Research

JoVE Journal

Biochemistry

7.7K Views.  National Institute of Advanced Industrial Science and Technology. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van neurowetenschappen, zoals of en hoe PLC-activiteiten in verschillende hersengebieden betrokken zijn bij de hersenfunctie van de honingbij. Het belangrijkste voordeel van deze techniek is dat onderzoekers gemakkelijk een experiment met de standaard laboratoriumfaciliteiten kunnen voorvormen aangezien dit protocol slechts basismanipulaties vereist. Om te beginnen plaatst u een 50 ml buis met honingbijen 30 min...

Video: Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. Alternatively, other (phospho)lipases may be physically associated with the producing organism causing a turnover of intrinsic lipids and frequently giving rise to a remodeling of the cellular membranes. Although potential (phospho)lipases can be predicted with a number of algorithms when the gene/protein sequence is available, experimental proof of the enzyme activities, substrate specificities, and potential physiological functions has frequently not been obtained. This manuscript describes the optimization of assay conditions for prospective (phospho)lipases with unknown substrate specificities and how to employ these optimized conditions in the search for the natural substrate of a respective (phospho)lipase. Using artificial chromogenic substrates, such as p-nitrophenyl derivatives, may help to detect a minor enzymatic activity for a predicted (phospho)lipase under standard conditions. Having encountered such a minor enzymatic activity, the distinct parameters of an enzyme assay can be varied in order to obtain a more efficient hydrolysis of the artificial substrate. After having determined the conditions under which an enzyme works well, a variety of potential natural substrates should be assayed for their degradation, a process that can be followed employing distinct chromatographic methods. The definition of substrate specificities for new enzymes, often provides hypotheses for a potential physiological role of these enzymes, which then can be tested experimentally. Following these guidelines, we were able to identify a phospholipase C (SMc00171) that degrades phosphatidylcholine to phosphocholine and diacylglycerol, in a crucial step for the remodeling of membranes in the bacterium Sinorhizobium meliloti upon phosphorus-limiting conditions of growth. For two predicted patatin-like phospholipases (SMc00930 and SMc01003) of the same organism, we could redefine their substrate specificities and clarify that SMc01003 is a diacylglycerol lipase.

Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Neuroscience

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...

Anti-RDL and Anti-mGlutR1 Receptors Antibody Testing in Honeybee Brain Sections using CRISPR-Cas9

Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing technique widely used in studies of gene function. We use this method in this study to check for the specificity of antibodies developed against the insect GABAA receptor subunit Resistance to Dieldrin (RDL) and a metabotropic glutamate receptor mGlutR1 (mGluRA). The antibodies were generated in rabbits against the conjugated peptides specific to fruit flies (Drosophila melanogaster) as well to honeybees (Apis mellifera). We used these antibodies in honeybee brain sections to study the distribution of the receptors in honeybee brains. The antibodies were affinity purified against the peptide and tested with immunoblotting and the classical method of preadsorption with peptide conjugates to show that the antibodies are specific to the corresponding peptide conjugates against which they were raised. Here we developed the CRISPR-Cas9 technique to test for the reduction of protein targets in the brain 48 h after CRISPR-Cas9 injection with guide RNAs designed for the corresponding receptor. The CRISPR-Cas9 method can also be used in behavioral analyses in the adult bees when one or multiple genes need to be modified.

Presented here is a protocol to use the CRISPR-Cas9 system for reducing the production of a protein in the adult honeybee brain to test antibody specificity.

Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing technique widely used in studies of ...

Histology Basics and Cell Death Detection in Honeybee Tissue

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather changes, various pesticides, pathogens, and malnutrition, mainly entering through the mouth and primarily affecting the digestive tracts of adult bees. To understand and prevent the effects of such external and internal stressors on honeybees, one useful research method is the immunohistochemical method. A basic protocol is described to prepare the midgut (ventriculus) and hypopharyngeal glands (HPGs) of adult bees for histological analysis. A detailed methodology is described to assess the level of cell damage and distinguish necrosis from programmed cell death (apoptosis) as a natural process of tissue regeneration. The results of adult honeybee treatment with oxalic acid and pesticides (insecticide and acaricide) and the determination of cell death in the ventriculus and HPGs are presented. The pros and cons of the methodology are also discussed.

Immunohistochemical methods are useful in honeybee research to detect and assess the level of apoptosis and necrosis in the midgut and hypopharyngeal glands of adult bees.

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather ...

Biology

Phospholipase Activity Assay: A Gel Diffusion Assay to Determine Phospholipase Activity in Crude Venom Extract from Anthopleura dowii

Source: Ram&#237;rez-Carreto, S., et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/63630">Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays.</a> J. Vis. Exp. (2022)
In this video, we demonstrate an assay to establish the presence of phospholipase in the crude venom extract from Anthopleura dowii&#8212;a marine sea anemone. The assay utilizes an agarose gel diffusion technique, in which the enzyme phospholipase diffuses and breaks down phospholipids incorporated in the solid media in a radial manner.

Source: Ram&#237;rez-Carreto, S., et al., Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward ...

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

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