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Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry
In This Article
Summary
The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.
Abstract
The activation of phospholipase Cβ (PLCβ) is an essential step during sensory transduction in Drosophila photoreceptors. PLCβ activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLCβ also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.
Introduction
Phototransduction in Drosophila is mediated by a G-protein-coupled PLCβ cascade leading to the activation of the light-activated channels TRP and TRPL1. PLCβ hydrolyzes the membrane-bound phospholipid, phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] and generates diacylglycerol (DAG), and inositol 1,4,5 trisphosphate (IP3). DAG is then phosphorylated by DAG-kinase to generate phosphatidic acid (PA). Subsequently, through a series of reactions that involve the generation of lipid intermediates, PI(4,5)P2 is regenerated2. Several components of this PI(4,5)P2 ....
Protocol
1. Rearing flies and preparation of chemicals
- Rear flies (Drosophila melanogaster) on standard fly food in an incubator with 50% relative humidity at 25 °C without internal illumination. Prepare fly food by adding 80 g/L of corn flour, 20 g/L of D-glucose, 40 g/L of sucrose, 8 g/L of agar, 15 g/L of yeast extract, 4 mL of propionic acid, 0.7 g/L of TEGO (methyl para hydroxybenzoate), and 0.6 mL of orthophosphoric acid as described in34 and are also availa.......
Representative Results
Determination of linearity of measurement in MS. Linearity is the MS method's ability to provide results which are directly proportional to the concentration of the lipid analyte. Linearity depends on (a) ionization efficiency of the lipid analyte and (b) ionization behavior of lipid analyte at different concentrations depends on the used ion source. In electrospray ionization (ESI) that is used in this study, linearity holds at lower concentrations depending on (a) ion transport from ESI source to t.......
Discussion
A number of lines of evidence converge on multiple roles of signaling lipids in regulating the organization and function of Drosophila photoreceptors. In addition to the well-studied role of lipids in regulating phototransduction3, signaling lipids have also been implicated in protein trafficking and sub-cellular organization23,30,39,40,
Acknowledgements
The work described in this manuscript was supported by Department of Atomic Energy, Government of India (Project Identification No. RTI 4006), the Department of Biotechnology, Government of India (BT/PR4833/MED/30/744/2012) and an India Alliance Senior Fellowship (IA/S/14/2/501540) to PR. We thank the NCBS Mass Spectrometry Facility, especially Dr. Dhananjay Shinde and members of the PR lab for their contributions to developing these methods.
....Materials
| Name | Company | Catalog Number | Comments |
| 0.1 N methanolic HCL | For total lipid isolation | ||
| 0.88% KCl | Sigma Aldrich | P9541 | For total lipid isolation |
| 1.5 ml / 2ml LoBind Eppendorf tubes | Eppendorf, | 022431081/022431102 | For total lipid isolation |
| 2.3.18 16:0/18:1 Diether PE | Avanti polar lipids | 999974 | Lipid Internal Standard |
| 37% pure HCl | Sigma Aldrich | 320331 | For total lipid isolation |
| 96-well plate | Total Organic Phosphate assay | ||
| Acetone | Fisher Scientific | 32005 | For dissections |
| Ammonium molybdate | Total Organic Phosphate assay | ||
| Ascorbic Acid | Total Organic Phosphate assay | ||
| Bath sonicator | |||
| BEH300 C18 column [1.0 mm x 100mm x 1.7 mm] | Waters India Pvt. Ltd. | 186002352 | LC |
| Blade holder | Fine Scientific Tools | 10052-11 | For dissections |
| BOD incubator | Total Organic Phosphate assay | ||
| Breakable blades | Fine Scientific tools | 10050-00 | For dissections |
| Butter paper | GE healthcare | 10347671 | For dissections |
| C4, 300 A0, [1.7 μm x1 mm x 100 mm] column | Waters India Pvt. Ltd. | 186004623 | LC |
| Chromatography amber color glass vials with inserts | Merck | 27083-U | |
| d18:1/17:0) | Avanti polar lipids | 860517 | Lipid Internal Standard |
| d5-Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2]-16:0/16:0 | Avanti polar lipids | 850172 | Lipid Internal Standard |
| Dissecting microscopes | Olympus | SZ51 | For dissections |
| Dry heat bath. | |||
| Eluent A | Hexane:Isopropyl alcohol:100 mM aqueous ammonium acetate (68:30:2) , for LC | ||
| Eluent B | Hexane:Isopropyl alcohol:100 mM aqueous ammonium acetate (70:20:10), for LC | ||
| Filter paper | Indica-HM2 | 74039 | For dissections |
| Flasks | Borosil | For dissections | |
| Flies | NA | NA | Raghu Padinjat lab |
| Fly food | NA | NA | NCBS lab kitchen, composition: corn flour 80 g/L, D-glucose 20 g/L, sucrose 40 g/L, agar 8 g/L, yeast extract 15 g/L, propionic acid 4 mL, TEGO (methyl para hydroxybenzoate) 0.7 g/L, orthophosphoric acid 0.6 mL) |
| Forceps | Fine Scientific Tools | 11254-20 | For dissections |
| Fume hood | |||
| Funnel | Borosil | For dissections | |
| Glacial acetic acid | Fisher Scientific | A35-500 | For derivatization |
| Glass bottles: transparent and amber color | For total lipid isolation | ||
| High-temperature-resistant phosphate-free glass tubes. | Total Organic Phosphate assay | ||
| Homogenization tubes with zirconium oxide beads | For total lipid isolation | ||
| Homogenizer instrument | Precellys | ||
| Humidified CO2 connected to fly pads | For fly pushing | ||
| Illumination controlled incubators | Panasonic Sanyo | MIR-553 | For fly rearing |
| Initial organic mixture | methanol:chloroform (2:1), For total lipid isolation | ||
| LC-MS grade Chloroform | Sigma Aldrich | 650498 | For total lipid isolation |
| LC-MS grade Methanol | Sigma Aldrich | 34860 | For total lipid isolation |
| LC-MS grade water | Sigma Aldrich | 34877 | For total lipid isolation |
| Light meter | HTC instruments | LX-103 | |
| Low retention tips | Eppendorf | 0030072006/72014/72022/72030 | For total lipid isolation |
| LTQ Orbitrap XL instrument | Thermo Fisher Scientific, Bremen, Germany | ||
| Lysophosphatidic acid (LPA)- 13:0 | Avanti polar lipids | LM-1700 | Lipid Internal Standard |
| Lysophosphatidic acid (LPA)- 17:1 | Avanti polar lipids | LM 1701 | Lipid Internal Standard |
| Lysophosphatidylcholine (LPC) -13:0 | Avanti polar lipids | LM-1600 | Lipid Internal Standard |
| Lysophosphatidylcholine (LPC) -17:1 | Avanti polar lipids | 855677 | Lipid Internal Standard |
| Lysophosphatidylcholine (LPC)- 19:0 | Avanti polar lipids | 855776 | Lipid Internal Standard |
| Perchloric acid. | Total Organic Phosphate assay | ||
| Phosphate standard potassium dihydrogen phosphate | Total Organic Phosphate assay | ||
| Phosphate-buffered saline (PBS) | NA | NA | Composition: 137mMNaCl, 2.7mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4 |
| Phosphatidic acid (PA)- 12:0/13:0 | Avanti polar lipids | , LM-1400 | Â Lipid Internal Standard |
| Phosphatidic acid (PA)- 17:0/14:1 | Avanti polar lipids | LM-1404 | Lipid Internal Standard |
| Phosphatidic acid (PA)-(17:0/17:0) | Avanti polar lipids | 830856 | Lipid Internal Standard |
| Phosphatidic acid (PA)-16:0-D31/18:1 | Avanti polar lipids | 860453 | Lipid Internal Standard |
| Phosphatidylcholine (PC) -12:0/13:0 | Avanti polar lipids | LM-1000 | Lipid Internal Standard |
| Phosphatidylcholine (PC)- 17:0/14:1 | Avanti polar lipids | LM-1004 | Lipid Internal Standard |
| Phosphatidylethanolamine (PE) - 17:0/14:1 | Avanti polar lipids | LM-110 | Lipid Internal Standard |
| Phosphatidylinositol (PI) - 17:0/14:1 | Avanti polar lipids | LM-1504 | Lipid Internal Standard |
| Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2)]-17:0/20:4 | Avanti polar lipids | LM-1904 | Lipid Internal Standard |
| Phosphatidylinositol 4-phosphate (PI4P) - 17:0/20:4 | Avanti polar lipids | LM-1901 | Lipid Internal Standard |
| Robotic nanoflow ion source | TriVersa NanoMate (Advion BioSciences, Ithaca, NY, USA) | ||
| Rotospin instrument | Tarsons | 3090X | |
| Silicone pads | For dissections | ||
| solvent A | 0.1% formic acid in water, for LC | ||
| solvent B | 0.1% formic acid in acetonitrile, for LC | ||
| Table-top centrifuge | |||
| Thermo-mixer | |||
| TMS-diazomethane | Acros | AC385330050 | For derivatization |
| Triple quadrupole mass spectrometer | AB Sciex | QTRAP 6500 | |
| UPLC system | Waters Acquity | ||
| Vacuum centrifugal concentrator | Scanvac , Labogene | ||
| Vortex machine |
References
- Hardie, R. C. C., Raghu, P. Visual transduction in Drosophila. Nature. 413 (6852), 186-193 (2001).
- Raghu, P., Yadav, S., Mallampati, N. B. N. Lipid signaling in Drosophila photoreceptors. Biochimica et Biophysica Acta. 1821 (8)....
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