Name: purification of endogenous drosophila transient receptor potential channels
Uploaded: 2021-12-28T14:00:00.000+00:00
Duration: 8 min 39 s
Description: Watch this Scientific Journal Video about Purification of Endogenous Drosophila Transient Receptor Potential Channels at JoVE.com

This work was supported by the National Natural Science Foundation of China (No. 31870746), Shenzhen Basic Research Grants (JCYJ20200109140414636), and Natural Science Foundation of Guangdong Province, China (No. 2021A1515010796) to W. L. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

1. Purification of GST-tagged TRP and His-tagged NORPA fragments
<ol>
	<li>Purify GST-tagged TRP 1261-1275 fragment
	<ol>
		<li>Transform the pGEX 4T-1 TRP 1261-1275 plasmid10 into Escherichia coli (E. coli) BL21 (DE3) cells using the CaCl2 heat-shock transformation method21. Inoculate a single colony in 10 mL of Luria Bertani (LB) medium and grow overnight at 37 &#176;C. Then, amplify the 10 mL of seeding culture in 1 L of LB medium at 37 &#176;C.</li>
		<li>After the optical density (OD600) of the cells reaches 0.5, cool down the cells to 16 &#176;C and add 0.1 mM isopropyl &#946;-D-1-thiogalactopyranoside (IPTG; final concentration) to induce the overexpression of the target protein and incubate at 16 &#176;C for 18 h.</li>
		<li>After overexpression, pellet 1 L of cultured cells by centrifugation at 3,993&#160;&#215; g&#160;for 20 min and resuspend in 40 mL of phosphate-buffered saline (PBS) buffer.</li>
		<li>Load the resuspended cells in a high-pressure homogenizer pre-cooled at 4 &#176;C. Slowly increase the homogenizer pressure to 800 bar. Open the inlet tap and let the resuspended cells circularly pass through a valve with very narrow slits. 
		NOTE: The cells are homogenized by the high shearing forces caused by a large pressure drop and cavitation.</li>
		<li>Load 5 mL of glutathione beads to a gravity flow column and wash the beads with 50 mL of PBS buffer for a total of three times.</li>
		<li>Centrifuge the cell lysate from the high-pressure homogenizer at 48,384 x g. Add the supernatant of the centrifuged cell lysate (40 mL) to the equilibrated glutathione beads in the gravity flow column and incubate for 30 min at 4 &#176;C. Resuspend the glutathione beads every 10 min.</li>
		<li>After 30 min of incubation, open the column outlet tap to separate the beads and flow-through fraction. Discard the flow-through fraction. Rinse the remaining glutathione beads twice with 50 mL of PBS buffer.</li>
		<li>Add 15 mL of elution buffer to the glutathione beads and incubate for 30 min. Resuspend the beads every 10 min.</li>
		<li>After 30 min of incubation, elute the GST-tagged TRP 1261-1275 fragment in a 50 mL conical tube and load in a size-exclusion column (preparation grade), which is equilibrated using 50 mM Tris (pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT) buffer.</li>
		<li>Keep the elution flow rate of the size-exclusion column to 3 mL/min. Collect the eluted proteins at the rate of 5 mL/tube.</li>
		<li>Identify the peak of the target protein in the size-exclusion column by analyzing the UV absorption signals at 280 nm and verify by SDS-PAGE gel analysis (electrophoresis parameters: 150 V for the stacking gel; 200 V for the resolving gel). Stain the gel with Coomassie blue R250.</li>
		<li>Concentrate the purified GST-tagged TRP 1261-1275 fragment from the size-exclusion column to 1 mL using a 15 mL ultrafiltration spin column centrifuged at 3,000 x g at 4 &#176;C in a desk-top refrigerated centrifuge.</li>
		<li>Determine the concentration of concentrated protein using the Beer-Lambert Law. Measure the UV absorption of GST-tagged TRP 1261-1275 fragment at 280 nm using a spectrophotometer.</li>
		<li>Obtain the extinction coefficient at 280 nm by importing the protein sequences into the Protparam program (https://web.expasy.org/protparam/). Typically, 1 L culture of GST-tagged TRP 1261-1275 yields 1 mL of 600 &#956;M of protein (6 x 10-7 mol). See Table 1 for materials needed.</li>
	</ol>
	</li>
	<li>Purification of His-tagged NORPA 863-1095 fragment
	<ol>
		<li>Transform the pETM.3C NORPA 863-1095 plasmid20 into E. coli BL21 (DE3) cells using the CaCl2 heat-shock transformation method21. Inoculate a single colony in 10 mL of LB medium and grow overnight at 37 &#176;C. Then, amplify the 10 mL seeding culture in 1 L of LB medium at 37 &#176;C.</li>
		<li>After the OD600 of the cells reaches 0.5, cool down the cells to 16 &#176;C and add 0.1 mM IPTG (final concentration) to induce the overexpression of target protein and incubate at 16 &#176;C for 18 h.</li>
		<li>After overexpression, pellet 1 L of cultured cells by centrifugation at 3,993&#160;x g&#160;for 20 min and resuspend in 40 mL of binding buffer. Next, lyse the resuspended cells in a high-pressure homogenizer at 4 &#176;C as described in step 1.1.4.</li>
		<li>Load 5 mL of Ni-beads into a gravity flow column and wash three times with 50 mL of binding buffer.</li>
		<li>Centrifuge the cell lysate from the high-pressure homogenizer at 48,384 x g. Add the supernatant of the centrifuged cell lysate to the equilibrated Ni-beads in the gravity flow column and incubate for 30 min at 4 &#176;C. Resuspend the Ni-beads every 10 min.</li>
		<li>After 30 min of incubation, open the column outlet tap to separate the beads and flow-through fraction. Discard the flow-through fraction and wash the remaining Ni-beads twice with 50 mL of wash buffer.</li>
		<li>Add 15 mL of elution buffer to the Ni-beads and incubate for 30 min. Resuspend the Ni-beads every 10 min.</li>
		<li>After 30 min of incubation, collect the eluted His-tagged NORPA 863-1095 fragment in a 50 mL conical tube and load into a size-exclusion column (preparation grade), which is equilibrated using 50 mM Tris (pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT).</li>
		<li>Keep the elution flow rate of the size-exclusion column to 3 mL/min. Collect the eluted protein at the rate of 5 mL/tube.</li>
		<li>Identify the peak of the target protein in the size-exclusion column by analyzing the UV absorption signals at 280 nm and verify by SDS-PAGE gel analysis (electrophoresis parameters: 150 V for the stacking gel; 200 V for the resolving gel). Stain the gel using Coomassie blue R250.</li>
		<li>Concentrate the purified His-tagged NORPA 863-1095 fragment from the size-exclusion column to 1 mL using a 15 mL ultrafiltration spin column centrifuged at 3,000 x g at 4 &#176;C in a desk-top refrigerated centrifuge.</li>
		<li>Determine the concentration of concentrated protein using the Beer-Lambert Law. Measure the UV absorption of His-tagged NORPA 863-1095 fragment at 280 nm using a spectrophotometer.</li>
		<li>Obtain the extinction coefficient at 280 nm by importing the protein sequences into the Protparam program (https://web.expasy.org/protparam/). Typically, 1 L culture of His-tagged NORPA 863-1095 fragment yields 1 mL of 600 &#956;M of protein (6 x 10-7 mol). See Table 2 for materials needed.</li>
	</ol>
	</li>
</ol>
2. Preparation of Drosophila heads
<ol>
	<li>Collect adult flies in 50 mL conical centrifugation tubes using the CO2 anaesthetization method22,23; immediately freeze in liquid nitrogen for 10 min and store in a -80&#176;C freezer.</li>
	<li>After collecting a sufficient number of flies, vigorously shake the frozen 50 mL conical tubes by hand to separate the flies&#39; legs, heads, wings, and bodies. Transfer the mixture to three sequentially stacked pre-cooled stainless-steel sieves (20/30/40 mesh size, respectively) and shake the sieves.</li>
	<li>Next, since the heads cannot pass through the 40-mesh sieve, use a brush to sweep the fly heads off the 40-mesh sieve, transfer them into 50 mL conical tubes, and store them at &#8722;80 &#176;C.</li>
	<li>Continuously collect the flies and their heads and store them in a -80 &#176;C freezer until they reach the required amount needed for experimentation (0.5 g). Typically, to collect 0.5 g of heads, 35 mL of flies in a 50 mL conical tube are needed. See Table 3 for materials needed.</li>
</ol>
3. Drosophila TRP channel purification
<ol>
	<li>Weigh a total of 0.5 g of heads and completely homogenize in liquid nitrogen using a pre-cooled mortar-pestle. Dissolve the homogenized heads in 10x v/w lysis buffer (5 mL), incubate in a shaker at 4 &#176;C for 20 min, and then centrifuge at 20,817 x g for 20 min at 4 &#176;C.</li>
	<li>Collect the spin-down supernatant (&#34;20817 g S&#34;, Figure 4) and further centrifuge it at 100,000 x g for 60 min at 4 &#176;C. Use the spin-down supernatant (&#34;100,000 g S&#34;, Figure 4) for the following pull-down assay.</li>
	<li>Add 1 mL of Ni-beads into the gravity flow column and wash the beads with 10 mL of double-distilled H2O (ddH2O) at 4 &#176;C for a total of three times. Equilibrate the beads with 10 column volumes of lysis buffer three times at 4 &#176;C.</li>
	<li>Add 500 &#181;L of 600 &#181;M purified His-tagged NORPA 863-1095 protein (3 x 10-7 mol) into the Ni-column and incubate for 30 min at 4 &#176;C. Resuspend the beads every 10 min.</li>
	<li>Open the column outlet tap to separate the beads and flow-through fraction. Take the flow-through fraction for SDS-PAGE analysis (NORPA F, Figure 4). In this section, the bait proteins are immobilized on the Ni-beads.</li>
	<li>Wash the Ni-beads with 10 column volumes of lysis buffer (10 mL) at 4 &#176;C and keep the washing fraction for SDS-PAGE analysis (Wash 1, Figure 4A). Repeat the above steps and keep the sample for SDS-PAGE analysis (Wash 2, Figure 4A). In this section, the excessive bait proteins on the Ni-beads are removed.</li>
	<li>Add the supernatant of Drosophila head homogenate after 100,000 x g centrifugation into the Ni-column at 4 &#176;C, where the His-tagged NORPA 863-1095 fragment has been immobilized.</li>
	<li>Incubate the supernatant with the Ni-beads at 4 &#176;C for 30 min. Resuspend the beads every 10 min. Then, open the column outlet tap to separate the beads and flow-through fraction.</li>
	<li>Collect the supernatant for SDS-PAGE analysis (Dro head lysis F, Figure 4A). In this section, the INAD protein complexes (INAD/TRP/ePKC) in the head homogenates are captured by the immobilized NORPA 863-1095 fragments on the Ni-beads.</li>
	<li>Wash the Ni-beads with 10 column volumes of lysis buffer (10 mL) at 4 &#176;C and keep the supernatant from gravity precipitation for SDS-PAGE analysis (Wash 3, Figure 4A). Repeat the above steps and collect the supernatant for SDS-PAGE analysis (Wash 4, Figure 4A). In this section, the unbound proteins on the Ni-beads are removed.</li>
	<li>Add 500 &#181;L of 600 &#181;M of GST-tagged TRP 1261-1275 protein (3 x 10-7 mol) into the Ni-beads and incubate for 20 min at 4 &#176;C. Resuspend the beads every 10 min.</li>
	<li>Collect the eluted fraction from the gravity column (TRP E1, Figure 4B), which contains the endogenous Drosophila TRP channel. Repeat the above stepsand collect the elution fraction (TRP E2, Figure 4B). In this step, by using the GST-tagged TRP 1261-1275 fragments as the competitor, the TRP channels are eluted from the captured INAD protein complexes (INAD/TRP/ePKC) on the Ni-beads.</li>
	<li>Wash the Ni-beads with 10 column volumes of binding buffer (10 mL; Table 1) at 4 &#176;C and collect the washing fraction for SDS-PAGE analysis (Wash 5, Figure 4B).</li>
	<li>Add 500 &#181;L of elution buffer (Table 1) into the Ni-beads and incubate for 20 min at 4 &#176;C. Collect the elution fraction from the gravity flow column (NORPA E1, Figure 4B). Repeat the above steps, and collect the elution fraction (NORPA E2, Figure 4B).</li>
	<li>Using the elution buffer, elute the His-tagged NORPA 863-1095 fragment accompanied with the INAD/ePKC protein complexes. Next, resuspend the Ni-beads in 500 &#181;L of binding buffer.</li>
	<li>Take the resuspended Ni-beads to run the SDS-PAGE (stained by Coomassie blue R250) to analyze the efficiency of the elution and evaluate whether the elute buffer works (beads, Figure 4B). See Table 4 for materials needed.</li>
</ol>
4. Size-exclusion column purification of Drosophila TRP channel
<ol>
	<li>Install a size-exclusion column (analytical grade) on the protein purification system. Equilibrate the column with the column buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM DTT, 0.75 mM DDM), which is filtered by a 0.45 &#181;m filter.</li>
	<li>Concentrate the TRP E1 and E2 fraction from step 3.14 using a 4 mL ultrafiltration spin column, centrifuged at 3,000 x g at 4 &#176;C in a refrigerated centrifuge.</li>
	<li>Rinse the sample loop with the column buffer and load the sample into the sample loop. Inject the sample into the size-exclusion column and elute the proteins with a proper flow rate (0.5 mL/min).</li>
	<li>Identify the peak of the target protein by absorption at 280 nm and run an SDS-PAGE gel to detect the purified endogenous Drosophila TRP channel (Figure 5). See Table 5 for materials needed.</li>
</ol>

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The authors have nothing to disclose.

INAD, which contains five PDZ domains, is the core organizer of Drosophila phototransduction machinery. Previous studies showed that INAD PDZ3 binds to the TRP channel C-terminal tail with exquisite specificity (KD = 0.3 &#181;M)18. INAD PDZ45 tandem interacts with NORPA 863-1095 fragment with an extremely high binding affinity (KD = 30 nM). These findings provide a solid biochemical basis to design the affinity purification plus competition strategy, which enables t...

Phototransduction is a process where absorbed photons are converted into electrical codes of neurons. It exclusively relays opsins and the following G protein-coupled signaling cascade in both vertebrates and invertebrates. In Drosophila, by using its five PDZ domains, scaffold protein inactivation-no-after-potential D (INAD) organizes a supramolecular signaling complex, which consists of a transient receptor potential (TRP) channel, phospholipase C&#946;/No receptor potential A (PLC&#946;/NORPA), and eye-specific protein kinase C (ePKC)1. The formation of this supramolecular signaling complex guarantees the correct subcellular localization, high efficiency, and specificity of Drosophila phototransduction machinery. In this complex, light-sensitive TRP channels act as downstream effectors of NORPA and mediate calcium influx and the depolarization of photoreceptors. Previous studies showed that the opening of the Drosophila TRP channel is mediated by protons, disruption of the local lipid environment, or mechanical force2,3,4. The Drosophila TRP channel also interacts with calmodulin5 and is modulated by calcium by both positive and negative feedback6,7,8.
So far, electrophysiology studies on the gating mechanism of Drosophila TRP and TRP-like (TRPL) channels were based on excised membrane patches, whole-cell recordings from dissociated wild-type Drosophila photoreceptors, and hetero-expressed channels in S2, SF9, or HEK cells2,9,10,11,12,13, but not on purified channels. The structural information of the full-length Drosophila TRP channel also remains unclear. In order to study the electrophysiological properties of purified protein in a reconstituted membrane environment and to gain structural information of the full-length Drosophila TRP channel, obtaining purified full-length TRP channels is the necessary first step, similar to the methodologies used in mammalian TRP channel studies14,15,16,17.
Recently, based on the assembling mechanism of INAD protein complex18,19,20, an affinity purification plus competition strategy was first developed to purify the TRP channel from Drosophila head homogenates by streptavidin beads5. Considering the low capacity and expensive cost of streptavidin beads, an improved purification protocol is introduced here that uses His-tagged bait protein and corresponding low-cost Ni-beads with much higher capacity. The proposed method will help to study the gating mechanism of the TRP channel from structural angles and to measure the electrophysiological properties of the TRP channel with purified proteins.

<table><tbody><tr><td>&lt;strong&gt;Bacterial strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;BL21(DE3) Competent Cells&lt;/em&gt;</td><td>Novagen</td><td>69450</td><td>Protein overexpression</td></tr><tr><td>&lt;strong&gt;Experiment models&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>D.melanogaster: W&lt;sup&gt;1118&lt;/sup&gt; strain</td><td>Bloomington Drosophila Stock Center</td><td>BDSC:3605</td><td>&lt;em&gt;Drosophila &lt;/em&gt;head preparation</td></tr><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>20/30/40 mesh stainless steel sieves</td><td>Jiufeng metal mesh company</td><td>GB/T6003.1</td><td>&lt;em&gt;Drosophila &lt;/em&gt;head preparation</td></tr><tr><td>30% Acrylamide-N,N&amp;prime;-Methylenebisacrylamide(29:1)</td><td>Lablead</td><td>A3291</td><td>SDS-PAGE gel preparation</td></tr><tr><td>Ammonium Persulfate</td><td>Invitrogen</td><td>HC2005</td><td>SDS-PAGE gel preparation</td></tr><tr><td>Cocktail protease inhibitor</td><td>Roche</td><td>05892953001</td><td>Protease inhibitor</td></tr><tr><td>Coomassie brilliant blue R-250</td><td>Sangon Biotech</td><td>A100472-0025</td><td>SDS-PAGE gel staining</td></tr><tr><td>DL-Dithiothreitol (DTT)</td><td>Sangon Biotech</td><td>A620058-0100</td><td>Size-exclusion column buffer preparation</td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt (EDTA)</td><td>Sangon Biotech</td><td>A500838-0500</td><td>Size-exclusion column buffer preparation</td></tr><tr><td>Glycine</td><td>Sangon Biotech</td><td>A610235-0005</td><td>SDS-PAGE buffer preparation</td></tr><tr><td>Glutathione Sepharose 4 Fast Flow beads</td><td>Cytiva</td><td>17513202</td><td>Affinity chromatography</td></tr><tr><td>Imidazole</td><td>Sangon Biotech</td><td>A500529-0001</td><td>Elution buffer preparation for Ni-column</td></tr><tr><td>Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td><td>Sangon Biotech</td><td>A600168-0025</td><td>Induction of protein overexpression</td></tr><tr><td>LB Broth Powder</td><td>Sangon Biotech</td><td>A507002-0250</td><td>&lt;em&gt;E.coli.&lt;/em&gt; cell culture</td></tr><tr><td>L-Glutathione reduced (GSH)</td><td>Sigma-aldrich</td><td>G4251-100G</td><td>Elution buffer preparation for Glutathione beads</td></tr><tr><td>Ni-Sepharose excel beads</td><td>Cytiva</td><td>17371202</td><td>Affinity chromatography</td></tr><tr><td>N-Dodecyl beta-D-maltoside (DDM)</td><td>Sangon Biotech</td><td>A610424-001</td><td>Detergent for protein purification</td></tr><tr><td>N,N,N&#039;,N&#039;-Tetramethylethylenediamine (TEMED)</td><td>Sigma-aldrich</td><td>T9281-100ML</td><td>SDS-PAGE gel preparation</td></tr><tr><td>PBS</td><td>Sangon Biotech</td><td>E607008-0500</td><td>Homogenization buffer for &lt;em&gt;E.coli.&lt;/em&gt; cell</td></tr><tr><td>PMSF</td><td>Lablead</td><td>P0754-25G</td><td>Protease inhibitor</td></tr><tr><td>Prestained protein marker</td><td>Thermo Scientific</td><td>26619/26616</td><td>Prestained protein ladder</td></tr><tr><td>Size exclusion column (preparation grade)</td><td>Cytiva</td><td>28989336</td><td>HiLoad 26/60 Superdex 200 PG column</td></tr><tr><td>Size exclusion column (analytical grade)</td><td>Cytiva</td><td>29091596</td><td>Superose 6 Increase 10/300 GL column</td></tr><tr><td>Sodium chloride</td><td>Sangon Biotech</td><td>A501218-0001</td><td>Protein purification buffer preparation</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Sangon Biotech</td><td>A500228-0001</td><td>SDS-PAGE gel/buffer preparation</td></tr><tr><td>Tris base</td><td>Sigma-aldrich</td><td>T1503-10KG</td><td>Protein purification buffer preparation</td></tr><tr><td>Ultrafiltration spin column</td><td>Millipore</td><td>UFC901096/801096</td><td>Protein concentration</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Analytical Balance</td><td>DENVER</td><td>APX-60</td><td>Metage of &lt;em&gt;Drosophila &lt;/em&gt;head</td></tr><tr><td>Desk-top high-speed refrigerated centrifuge for 15mL and 50mL conical centrifugation tubes</td><td>Eppendorf</td><td>5810R</td><td>Protein concentration</td></tr><tr><td>Desk-top high-speed refrigerated centrifuge 1.5mL centrifugation tubes</td><td>Eppendorf</td><td>5417R</td><td>Centrifugation of &lt;em&gt;Drosophila&lt;/em&gt; head lysate after homogenization</td></tr><tr><td>Empty gravity flow column (Inner Diameter=1.0cm)</td><td>Bio-Rad</td><td>738-0015</td><td>TRP protein purification</td></tr><tr><td>Empty gravity flow column (Inner Diameter=2.5cm)</td><td>Bio-Rad</td><td>738-0017</td><td>Bait and competitor protein purification from &lt;em&gt;E.coli.&lt;/em&gt;</td></tr><tr><td>Gel Documentation System</td><td>Bio-Rad</td><td>Universal Hood II Gel Doc XR System</td><td>SDS-PAGE imaging</td></tr><tr><td>High-speed refrigerated centrifuge</td><td>Beckman coulter</td><td>Avanti J-26 XP</td><td>Centrifugation of &lt;em&gt;E.coli.&lt;/em&gt; cells/cell lysate</td></tr><tr><td>High pressure homogenizer</td><td>UNION-BIOTECH</td><td>UH-05</td><td>Homogenization of &lt;em&gt;E.coli.&lt;/em&gt; cells</td></tr><tr><td>Liquid nitrogen tank</td><td>Taylor-Wharton</td><td>CX-100</td><td>&lt;em&gt;Drosophila &lt;/em&gt;head preparation</td></tr><tr><td>Protein purification system</td><td>Cytiva</td><td>AKTA purifier</td><td>Protein purification</td></tr><tr><td>Refrigerator (-80&amp;deg;C)</td><td>Thermo</td><td>900GP</td><td>&lt;em&gt;Drosophila &lt;/em&gt;head preparation</td></tr><tr><td>Spectrophotometer</td><td>MAPADA</td><td>UV-1200</td><td>OD600 measurement of &lt;em&gt;E.coli.&lt;/em&gt; cells</td></tr><tr><td>Spectrophotometer</td><td>Thermo Scientific</td><td>NanoDrop 2000c</td><td>Determination of protein concentration</td></tr><tr><td>Ultracentrifuge</td><td>Beckman coulter</td><td>Optima XPN-100 Ultracentrifuge</td><td>Ultracentrifugation</td></tr></tbody></table>

purification of endogenous drosophila transient receptor potential channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase C&#946;/No receptor potential A (PLC&#946;/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

In this article, a protein purification method is demonstrated to purify endogenous Drosophila TRP channel (Figure 1).
First, recombinant protein expression and purification are applied to obtain the bait and competitor proteins. Then, a GST-tagged TRP 1261-1275 fragment is expressed in E. coli BL21 (DE3) cells in LB medium and purified using glutathione beads and a size-exclusion column (Figure 2). The samples were ...

Watch this Scientific Journal Video about Purification of Endogenous Drosophila Transient Receptor Potential Channels at JoVE.com

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...

purification-endogenous-drosophila-transient-receptor-potential

Research

JoVE Journal

Biochemistry

1.6K Views.  Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center. In dit protocol demonstreren we het zuiveringsproces om gezuiverde TRP-kanalen over de volledige lengte te verkrijgen, wat de noodzakelijke eerste stap is om elektrofysiologische eigenschappen te bestuderen en structurele informatie te verkrijgen over het Drosophila TRP-kanaal over de volledige lengte. Op basis van het bemonsteringsmechanisme van het INAD-eiwitcomplex en met behulp van goedkope nikkelparels met een veel hogere capaciteit, kan voldoende hoeveelheid TRP-kanalen uit vliegenkoppe...

Video: Purification of Endogenous Drosophila Transient Receptor Potential Channels

Purification of Low-abundant Cells in the Drosophila Visual System

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

Genetics

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

Neuroscience

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Biology

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between target neurons. The method relies on co-expression of a transgenic activator with the tetrodotoxin-resistant sodium channel, NaChBac, in a specific presynaptic neuron. Connections with putative post-synaptic partners are determined by whole-cell recordings in the presence of TTX, which blocks electrical activity in neurons that do not express NaChBac. This approach can be modified to work with any activator or calcium imaging as a reporter of connections. TERPS adds to the growing set of tools available for determining connectivity within networks. However, TERPS is unique in that it also reliably reports bulk or volume transmission and spillover transmission.

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

This article features a method to test the monosynaptic connections between neurons by employing tetrodotoxin and the tetrodotoxin-resistant sodium channel, NaChBac.

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study introduces easy-to-implement setups for applying optogenetic methods in Drosophila melanogaster. Two optogenetic tools, CsChrimson, a red-light-activated cation channel, and GtACR2, a blue-light-activated anion channel, were employed in four experimental approaches. Three of these approaches involve single-fly experiments: (1) a blue-light optogenetic thermotactic positional preference assay targeting temperature-sensitive heating cells, (2) a red-light optogenetic positional preference assay activating bitter sensing neurons, and (3) a proboscis extension response assay activating the sweet-sensing neurons. The fourth approach (4) is a fly maze setup to assess avoidance behaviors using multiple flies. The ability to manipulate neural activity temporally and spatially offers powerful insights into sensory processing and decision-making, underscoring the potential of optogenetics to advance our knowledge of neural function. These methods provide an accessible and robust framework for future research in neuroscience to enhance the understanding of specific neural pathways and their behavioral outcomes.

This paper presents methods for optogenetic manipulation in Drosophila melanogaster, utilizing CsChrimson and GtACR2 to activate and silence specific neurons. Four experiments are described to utilize optogenetics to explore thermotactic and gustatory behaviors, providing insights into the underlying neural mechanisms governing these processes.

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study ...

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally important for cell function and cell integrity of neurons. Drosophila photoreceptor cells have become a model for studying membrane protein trafficking. Besides rhodopsin, which upon illumination becomes internalized from the photoreceptor membrane and is degraded, the transient receptor potential-like (TRPL) ion channel in Drosophila exhibits a light-dependent translocation between the rhabdomeral photoreceptor membrane (where it is located in the dark) and the photoreceptor cell body (to which it is transported upon illumination). This intracellular transport of TRPL can be studied in a simple and non-invasive way by expressing eGFP-tagged TRPL in photoreceptor cells. The eGFP fluorescence can then be observed either in the deep pseudopupil or by water immersion microscopy. These methods allow detection of fluorescence in the intact eye and are therefore useful for high-throughput assays and genetic screens for Drosophila mutants defective in TRPL translocation. Here, the preparation of flies, the microscopic techniques, as well as quantification methods used to study this light-triggered translocation of TRPL are explained in detail. These methods can be applied also for trafficking studies on other Drosophila photoreceptor proteins, for example, rhodopsin. In addition, by using eGFP-tagged rhabdomeral proteins, these methods can be used to assess the degeneration of photoreceptor cells.

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Here, non-invasive methods are described for localization of photoreceptor membrane proteins and assessment of retinal degeneration in the Drosophila compound eye using eGFP fluorescence.

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally ...

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells

<a href='https://app.jove.com/v/63375/studying-membrane-protein-trafficking-drosophila-photoreceptor-cells'>Source: Wagner, K. et al., Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins.&#160;J. Vis. Exp. (2022).</a>This video demonstrates the visualization of membrane protein trafficking in Drosophila photoreceptor cells using eGFP-tagged TRPL proteins. It details the preparation of transgenic flies for imaging, the capture of fluorescence images of photoreceptor cells, and the quantification of TRPL trafficking by measuring relative eGFP fluorescence in the rhabdomeres.

Purification of Endogenous Drosophila Transient Receptor Potential Channels

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