This work was supported by the National Key Research &#38; Development Program (2016YFD0200900) of China, the National Natural Scientific Foundation of China (No. 31772076 and No. 31270728), China Postdoctoral Science Foundation (2018M630700), and Open Fund of State Key Laboratory of Tea Plant Biology and Utilization (SKLTOF20180111).

1. Tea callus culture
NOTE: Sterile leaves were derived from in vitro-grown plantlet lines first developed in the research group17. All procedures up to section 5 were carried out in a sterile laminar flow hood, except for the culture time in an incubator.
<ol>
	<li>Adjust the pH of the two media (Murashige and Skoog [MS] basal medium and Gamborg&#39;s B5 liquid medium) to 5.8 prior to autoclaving (121 &#176;C, 20 min).</li>
	<li>Cut along the middle vein of a sterile leaf using scissors, and then subdivide each half into small pieces of about 0.3 cm x 0.3 cm in a petri dish.</li>
	<li>Place the sterile explants (the small leaf pieces) onto MS basal media containing the plant hormones 2,4-D (1.0 mg L-1) and KT (0.1 mg L-1). Six explants can be placed in a 300 mL flask containing 100 mL of MS basal media.</li>
	<li>&#160;Culture the above leaf explants at a constant temperature of 25 &#176;C in the dark. After 28 days, select the first generation of induced callus and transfer to fresh flask (a subculture). Acquire the loose and friable callus after 3&#8722;4 subcultures.</li>
</ol>
2. Tea cell suspension culture
<ol>
	<li>Cut the vigorous, friable and loose calluses from the solid medium into small pieces (range here 0.5&#8722;2 mm) using a sterile surgical blade under sterile conditions.</li>
	<li>Weigh about 3 g of the small pieces of callus. Place the callus into a 150 mL flask containing 20 mL of B5 liquid media supplemented with 2,4-D (1.0 mg L-1) and KT (0.1 mg L-1).</li>
	<li>Culture the liquid cell suspension at a constant temperature (25 &#177; 1 &#176;C) in a shaking incubator at 120 rpm in the dark.</li>
	<li>After 7 to 10 days of culturing, remove the culture flasks and let them stand for a few minutes.</li>
	<li>Take all the supernatant as seed material for subculture to fresh medium (subculture ratio of suspension mother liquid to fresh medium ranged between 1:1 and 1:2). Remove the precipitated, large calluses.</li>
	<li>Obtain the final well-grown cell suspension culture after 3&#8722;4 subculture cycles of 28 days each.</li>
</ol>
3. Triphenyl tetrazolium chloride assay of cell viability
<ol>
	<li>Kill a sample of living cells at 100 &#176;C for 10 min as a control cell before viability staining.</li>
	<li>Centrifuge all cell suspension culture for 8 min at 6000 x g. Remove the supernatant before suspending the cells in 2.5 mL of phosphate-buffered saline (PBS) buffer (pH 7.3), and shake it for 1 min by hand.</li>
	<li>Add 2.5 mL of the 0.4%triphenyl tetrazolium chloride (TTC) solution and shake by hand again.</li>
	<li>Incubate the mixture for 1 h in a standing incubator (30 &#176;C).</li>
</ol>
4. Treatment and sampling of tea cell suspension cultures with insecticides
<ol>
	<li>Add an aliquot of 400 &#181;L of filter-sterilized stock solution (500 &#181;g mL-1) of four neonicotinoids (thiamethoxam, acetamiprid, imidacloprid, and imidaclothiz) or two organophosphates (dimethoate and omethoate) into the cell suspension cultures, respectively. 
	NOTE: If the aim is to compare xenobiotic behaviors, use the same mother batch of cell suspensions to test the different compounds.</li>
	<li>Culture the samples of cell suspensions with insecticides at constant temperature (25 &#177; 1 &#176;C) and shaking incubator speed (120 rpm). Take the samples (see step 4.3 or 4.4) on 0, 3, 5 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 and 75 days.</li>
	<li>To test a sample containing a neonicotinoid, remove a 1 mL aliquot of the homogeneous cell culture, place it into a 1.5 mL plastic centrifuge tube, and centrifuge at 4000 x g for 2 min.
	<ol>
		<li>Pass the supernatants through a 0.22-&#181;m pore-size filter membrane before analysis by high-performance liquid chromatography-ultraviolet (HPLC-UV) and ultra-high performance liquid chromatography-quadrupole time-of-flight (UPLC-QTOF) mass spectrometry (Table of Materials).</li>
	</ol>
	</li>
	<li>To test a sample containing an organophosphate, remove a 500 &#181;L aliquot of the cell culture and place into a 35 mL centrifuge tube or a 1.5 mL plastic centrifuge tube (prepare the latter sample like that of neonicotinoid).
	<ol>
		<li>Add 0.1 g of sodium chloride and 5 mL of acetone/ethyl acetate (3:7, v/v) into the 35 mL centrifuge tube of the 500 &#181;L samples.</li>
		<li>Vortex the mixtures for 1 min, and then allow them to rest for 10 min.</li>
		<li>Take 2.5 mL of the supernatant into a 10 mL glass tube and evaporate to near-dryness using a nitrogen evaporator at 40 &#176;C.</li>
		<li>Dissolve the residue with 1 mL acetone, vortex for 1 min, pass it through a 0.22-&#181;m filter membrane before analysis by gas chromatography-flame photometric detector (GC-FPD).</li>
	</ol>
	</li>
</ol>
5. Sample preparation of intact tea plant with insecticides
NOTE: The intact tea plant trial was conducted in a hydroponic system using tea seedlings grown in 50 mL of a nutrient solution (30 NH4+, 10 NO3-, 3.1 PO4-, 40 K+, 20 Ca2+, 25 Mg2+, 0.35 Fe2+, 0.1 B3+, 1.0 Mn2+, 0.1 Zn2+, 0.025 Cu2+, 0.05 Mo+, and 10 Al3+, in mg L-1)18. An experimental greenhouse was under a light-dark cycle (12 h of light and 12 h of darkness) at 20 &#176;C at Anhui Agricultural University.
<ol>
	<li>Put five plants in a 4 L plastic pot for 15 days.</li>
	<li>Add 0 ppm (control) or 100 ppm of thiamethoxam or dimethoate into plastic pots, respectively.</li>
	<li>Prepare the intact plant sample according to the previous method, except for presoaking19, and then analyze with mass spectrometry for an accurate mass spectrum.</li>
</ol>
6. Instrument analysis
<ol>
	<li>HPLC analysis of the metabolic behavior of neonicotinoids
	<ol>
		<li>Use an HPLC-UV (Table of Materials) to detect the content and metabolic products of thiamethoxam and acetamiprid at a wavelength of 254 nm, and of imidacloprid and imidaclothiz at 270 nm in samples from section&#160;4.3. 
		NOTE: The HPLC-UV condition was the same as the previous study19.</li>
	</ol>
	</li>
	<li>GC analysis of the metabolic behavior of organophosphates
	<ol>
		<li>Detect the content of dimethoate and omethoate in samples from section&#160;4.4 by a GC-FPD using a chiral column (Table of Materials).</li>
		<li>Use nitrogen as the carrier gas and set the flow rate at 1.0 mL min-1.</li>
		<li>Set the initial temperature to 120 &#176;C, and hold it for 5 min. Increase the temperature to 150 &#176;C at 30 &#176;C min-1 and hold for 3 min. Increase to 170 &#176;C at 10 &#176;C min-1 and hold for 7 min. Finally increase to 210 &#176;C at 30 &#176;C min-1 and then hold for 5 min.</li>
		<li>Set the injection temperature to 200 &#176;C in splitless mode; Set the detector temperature to 250 &#176;C.</li>
		<li>Set the injection volume to 1 &#181;L.</li>
	</ol>
	</li>
	<li>UPLC-QTOF analysis of the insecticide metabolites in cell culture
	<ol>
		<li>Detect the metabolites of the insecticides in cell culture (samples from section&#160;4.3) using UPLC-QTOF with a C18 column (Table of Materials).</li>
		<li>Set the flow rate to 0.2 mL min-1. Set the injection volume to 10 &#181;L.</li>
		<li>For the neonicotinoid-treated samples, set the initial mobile phase to 85% A (5 mM ammonium formate water) and 15% B (acetonitrile). Over 10 min, increase mobile phase B to 38% and return to 15% over 1 min, hold for 9 min.</li>
		<li>For the organophosphate-treated samples, set the initial mobile phase to 55% A (0.1% formic acid water) and 45% B (acetonitrile). Over 5 min, increase mobile phase B to 70%, then return to 45% of B over 0.5 min, hold for 2.5 min.</li>
		<li>Set the QTOF operation parameters as follows: gas temperature, 325 &#176;C; drying gas (nitrogen), 10 L min-1; sheath gas temperature, 350 &#176;C; sheath gas flow, 11 L min-1; capillary voltage, 4000 V; nozzle voltage, 1000 V; fragmentor voltage, 100 V for neonicotinoid insecticides or 110 V for organophosphorus insecticides; skimmer voltage, 65 V; operating in positive ion mode.</li>
		<li>Set the instrument to the full scan spectrum and target MS/MS mode.</li>
		<li>Process the data using accurate mass tools; Infer the metabolites with no standard products from the MS/MS annotation as well as the literature12,15,20,21,22.</li>
	</ol>
	</li>
	<li>UPLC-Orbitrap analysis of the insecticide metabolites in intact plant extract
	<ol>
		<li>Detect the metabolites of insecticides in intact plant extract using UPLC-Orbitrap mass spectrometry (Table of Materials).</li>
		<li>Set the mass spectrometry (Table of Materials) operation parameters as follows: sheath gas pressure, 35 arb; gas temperature, 300 &#176;C; nozzle voltage, 3.5 KV; capillary temperature, 350 &#176;C.</li>
		<li>Set the elution programs as the above (steps 6.3.3 and 6.3.4) for UPLC-QTOF analysis of cell culture.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This article presents the detailed process of establishing a model of pesticide metabolism in tea plant tissue, including the selection of explants, the determination of cell viability, and the establishment of a tea cell suspension culture with high metabolic activity. Any parts of a plant tissue could be used to initiate callus in a sterilized environment25. Tea leaves were chosen for callus initiation in this study, not only because leaves to tend to be less contaminated than the parts below gr...

Tea is one of the most widely consumed non-alcoholic beverages in the world1,2. Tea is produced from the leaves and buds of the woody perennial Camellia sinensis L. Tea plants are grown in vast plantations and are susceptible to numerous insect pests3,4. Organophosphorus and neonicotinoid insecticides are often used as systemic insecticides5 to protect tea plants from pests such as whiteflies, leaf hoppers, and some lepidopteran species6,7. After application, these insecticides are absorbed or translocated into the plant. Within the plant, these systemic insecticides may be transformed through hydrolysis, oxidation or reduction reactions by plant enzymes. These transformation products can be more polar and less toxic than the parent compounds. However, for some organophosphates, the bioactivities of some products are higher. For example, acephate is metabolized into the more toxic methamidophos8,9, and dimethoate into omethoate10,11. Plant metabolic studies are thus important for determining the fate of a pesticide within a plant12.
Plant tissue cultures have been proven to be a useful platform for investigating the pesticide metabolism, with the identified metabolites similar to those found in intact plants13,14,15. The use of tissue cultures, particularly cell suspension cultures, has several advantages. Firstly, experiments can be carried out free of microorganisms, thus avoiding the interference of pesticide transformation or degradation by microbes. Secondly, tissue culture provides consistent materials for use at any time. Thirdly, the metabolites are easier to extract from tissue cultures than from intact plants, and tissue cultures often have fewer interring compounds and lower complexity of compounds. Finally, tissue cultures can more easily be used to compare a series of pesticides metabolism in a single experiment16.
In this study, a cell suspension derived from the leaves of sterile-grown tea plantlet was successfully established. The tea cell suspension culture was then used to compare the dissipation behaviors of six systemic insecticides.
This detailed protocol is intended to provide some guidance so that researchers can establish a plant tissue culture platform useful for studying the metabolic fate of xenobiotics in tea.

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			<td height="38" style="height:38px;width:188px;">Acetamiprid (99.8%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">46717</td>
			<td style="width:123px;">CAS No: 135410-20-7</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:188px;">Acetonitrile (CAN, 99.9%)</td>
			<td style="width:188px;">Tedia</td>
			<td style="width:175px;">AS1122-801</td>
			<td style="width:123px;">CAS No: 75-05-8</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Agar</td>
			<td style="width:188px;">Solarbio Science &#38; Technology</td>
			<td style="width:175px;">A8190</td>
			<td style="width:123px;">CAS No: 9002-18-0</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Clothianidin (99.8%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">525</td>
			<td style="width:123px;">CAS No: 210880-92-5</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:188px;">Dimethoate (98.5%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">109217</td>
			<td style="width:123px;">CAS No: 60-51-5</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Imidacloprid (99.8%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">91029</td>
			<td style="width:123px;">CAS No: 138261-41-3</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Imidaclothiz (99.5%)</td>
			<td style="width:188px;">Toronto Research Chemical</td>
			<td style="width:175px;">I275000</td>
			<td style="width:123px;">CAS No: 105843-36-5</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Kinetin (KT, &#62;98.0%)</td>
			<td style="width:188px;">Solarbio Science &#38; Technology</td>
			<td style="width:175px;">K8010</td>
			<td style="width:123px;">CAS No: 525-79-1</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:188px;">Omethoate (98.5%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">105491</td>
			<td style="width:123px;">CAS No: 1113-02-6</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Polyvinylpolypyrrolidone (PVPP)</td>
			<td style="width:188px;">Solarbio Science &#38; Technology</td>
			<td style="width:175px;">P8070</td>
			<td style="width:123px;">CAS No: 25249-54-1</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:188px;">Sucrose</td>
			<td style="width:188px;">Tocris Bioscience</td>
			<td style="width:175px;">5511</td>
			<td style="width:123px;">CAS No: 57-50-1</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Thiamethoxam (99.8%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">20625</td>
			<td style="width:123px;">CAS No: 153719-23-4</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Triphenyltetrazolium Chloride (TTC, 98.0%)</td>
			<td style="width:188px;">Solarbio Science &#38; Technology</td>
			<td style="width:175px;">T8170</td>
			<td style="width:123px;">CAS No: 298-96-4</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:188px;">2,4-Dichlorophenoxyacetic Acid (2,4-D, &#62;98.0%)</td>
			<td style="width:188px;">Guangzhou Saiguo Biotech</td>
			<td style="width:175px;">D8100</td>
			<td style="width:123px;">CAS No: 94-75-7</td>
		</tr>
		<tr height="77">
			<td height="77" style="height:77px;width:188px;">chiral column</td>
			<td style="width:188px;">Agilent CYCLOSIL-B</td>
			<td>112-6632</td>
			<td style="width:123px;">Chromatography column (30 m &#215; 0.25 mm &#215; 0.25 &#956;m)</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:188px;">Gas chromatography (GC)</td>
			<td style="width:188px;">Shimadu</td>
			<td>2010-Plus</td>
			<td style="width:123px;">Paired with Flame Photometric Detector (FPD)&#160;&#160;</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:188px;">High-performance liquid chromatography (HPLC)</td>
			<td>Agilent</td>
			<td>1260</td>
			<td style="width:123px;">Paired with Ultraviolet detector (UV)</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:188px;">HSS T3 C18 column</td>
			<td style="width:188px;">Waters</td>
			<td>186003539</td>
			<td style="width:123px;">Chromatography column (100 mm &#215; 2.1 mm &#215; 1.8 &#956;m)</td>
		</tr>
		<tr height="96">
			<td height="96" style="height:96px;width:188px;">Ultra-high-performance liquid chromatography (UPLC)</td>
			<td style="width:188px;">Agilent</td>
			<td>1290-6545</td>
			<td style="width:123px;">Tandem quadrupole time-of-flight mass spectrometer (QTOF)</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:188px;">Ultra-high-performance liquid chromatography (UPLC)</td>
			<td style="width:188px;">Thermo Scientific</td>
			<td>Ultimate 3000-Q Exactive Focus</td>
			<td style="width:123px;">Connected to a Orbitrap mass spectrometer</td>
		</tr>
	</tbody>
</table>

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.

The induction of callus from leaves harvested from field-grown tea trees and from leaves excised from tea plantlets grown in vitro in a sterile environment was compared by measuring contamination, browning, and induction after 28 days of cultivation on MS media (Figure 1A). Callus growth was recorded at 20, 37, 62 and 90 days of culture (Figure 1B). The callus derived from the in vitro-grown leaves showed more vigorous growth tha...

Watch this Scientific Journal Video about Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves at JoVE.com

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.

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 ...

study-on-metabolism-six-systemic-insecticides-newly-established-cell

Research

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Biochemistry

7.6K Views.  Anhui Province Key Lab of Analysis and Detection for Food Safety. 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.

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

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

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. ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.