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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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 diffe...

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 border="0" cellpadding="0" cellspacing="0" style="width:479px;" width="478">
	<tbody>
		<tr height="80">
			<td height="80" style="height:80px;width:113px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:101px;">ThermoFisher Scientific</td>
			<td style="width:139px;">23227</td>
			<td style="width:125px;">The reagent kit for measurement of protein concentration</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:113px;">Pierce Bovine Serum Albumin Standard Ampules 2mg/mL</td>
			<td style="width:101px;">ThermoFisher Scientific</td>
			<td>23209</td>
			<td style="width:125px;">The standard samples used in BCA assay</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:113px;">Paraffin wax</td>
			<td style="width:101px;">GC</td>
			<td>13B1X00155000141</td>
			<td style="width:125px;">Dental wax used as dissection stage</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:113px;">Insect pin</td>
			<td style="width:101px;">Shiga</td>
			<td>No. 0</td>
			<td style="width:125px;">Stainless, solid head</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:113px;">PLCglow</td>
			<td style="width:101px;">KXT Bio</td>
			<td>KCH-0001</td>
			<td style="width:125px;">A fluorogenic substrate of PLC</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:113px;">384-well microplate</td>
			<td style="width:101px;">Corning</td>
			<td>4511</td>
			<td style="width:125px;">Low-volume, round-bottom plate in black color</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:113px;">Gemini EM microplate reader</td>
			<td style="width:101px;">Molecular Devices</td>
			<td></td>
			<td style="width:125px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:113px;">Edelfosine</td>
			<td style="width:101px;">Santa Cruz Biotechnology</td>
			<td>sc-201021</td>
			<td style="width:125px;">pan-PLC inhibitor</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:113px;">Neomycin sulfate</td>
			<td style="width:101px;">Santa Cruz Biotechnology</td>
			<td>sc-3573</td>
			<td style="width:125px;">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.
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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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

7.7K Views.  National Institute of Advanced Industrial Science and Technology. 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.

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

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

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SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer