Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis

Summary

Here, we describe a protocol to obtain the lipid droplet index (LD index) to study the dynamics of triacylglycerols in cells cultured in high-throughput experiments. The LD index assay is an easy and reliable method that uses BODIPY 493/503. This assay does not need dispendious lipid extraction or microscopy analysis.

Abstract

The article shows how to implement the LD index assay, which is a sensitive microplate assay to determine the accumulation of triacylglycerols (TAGs) in lipid droplets (LDs). LD index is obtained without lipid extraction. It allows measuring the LDs content in high-throughput experiments under different conditions such as growth in rich or nitrogen depleted media. Albeit the method was described for the first time to study the lipid droplet metabolism in Saccharomyces cerevisiae, it was successfully applied to the basidiomycete Ustilago maydis. Interestingly, and because LDs are organelles phylogenetically conserved in eukaryotic cells, the method can be applied to a large variety of cells, from yeast to mammalian cells. The LD index is based on the liquid fluorescence recovery assay (LFR) of the BODIPY 493/503 under quenching conditions, by the addition of cells fixed with formaldehyde_. Potassium iodine is used as a fluorescence quencher. The ratio between the fluorescence and the optical density slopes is named LD index. Slopes are calculated from the straight lines obtained when BODIPY fluorescence and optical density at 600 nm (OD₆₀₀) are plotted against sample addition. Optimal data quality is reflected by correlation coefficients equal or above 0.9 (r ≥ 0.9). Multiple samples can be read simultaneously as it can be implemented in a microplate. Since BODIPY 493/503 is a lipophilic fluorescent dye that partitions into the lipid droplets, it can be used in many types of cells that accumulate LDs.

Introduction

Lipid droplets (LDs) are ubiquitous intracellular fat bodies composed of a core of neutral lipids, mainly TAGs and sterol esters (SE). The core is surrounded by a monolayer of phospholipids that interacts with proteins like perilipin and enzymes involved in the synthesis of neutral lipids, such as the diacylglycerol acyl-transferase, acetyl-CoA carboxylase and acyl-CoA synthase¹. Due to its dynamic behavior, the phospholipid monolayer also contains triacylglycerol lipases that hydrolyze TAGs and SE². Depending on the cell type and organism, stored neutral lipids can be used to generate energy, or synthesize phospholipids and signaling molecules. In yeast and other fungi, the content of LDs changes in response to variations in the nitrogen/carbon ratio in the culture media, indicating that decreased nitrogen availability might be the key to increase neutral lipid production^3,4,5. The production of high amounts of TAGs in yeast has a potential use in biotechnology as source of biofuels and in the food industry. Some stored lipids contain high proportions of polyunsaturated fatty acids, like omega 3 and omega 6, with nutritional and dietary importance ^{6,7,8,9,10,11}. LDs of mammalian cells, including human cells, also contain TAGs and cholesterol esters. The phospholipid monolayer interacts with the mammalian homologous of the proteins described in the yeast LDs, and with an additional protein, perilipin, which is absent in S. cerevisiae¹². One proposed role for the phospholipid monolayer and its associated proteins is to stabilize the LDs structure and to allow the interaction of LDs with organelles as mitochondria, endoplasmic reticulum, peroxisomes, and vacuoles, mainly for lipid exchange ^13,14. Interestingly, in humans, LDs appear to be involved in pathologies like type 2 diabetes, atherosclerosis, steatohepatitis, and coronary heart disease, in which there is an increase in their number ^15,16,17. Some types of viruses use LDs as platforms to assemble the virions ^2,18,19.

Due to the implications of the LDs in human pathologies and their potential biotechnological use, the exact experimental determination of LDs formation is an important task. This article describes a reliable assay based on the recovery of the fluorescence (LFR) of BODIPY 493/503 (4, 4-difluoro-1, 3, 5, 7, 8-pentamethyl-4-bora-3a, 4a-diaza-s-indacene), to get a relative value of the content of neutral lipids in cells. With this assay, it is possible to follow the dynamics of the accumulation of neutral lipids in fungi like U. maydis and S. cerevisiae, and also in mammalian cells, without the need of lipid extraction ²⁰. The method was applied for the first time in S. cerevisiae to identify the protein phosphatases and kinases involved in the regulation of lipid metabolism. This was possible without lipid extraction or protein purification^21,22. LFR also has been used to establish the dynamics of LDs formation in peritoneal macrophages²³. The use of BODIPY 493/503 has some advantages over other neutral lipid dyes as Nile Red. BODIPY 493/503 is highly specific for neutral lipids and has a narrow emission spectrum, facilitating the simultaneous detection of signals from dyes like red fluorescent protein or Mito-tracker, when the samples are analyzed by confocal microscopy. Unfortunately, BODIPY 493/503 is sensitive to photobleaching, but this process can be avoided by using an antiquenching reagent during exposure to light²⁴.

To carry out the LFR assay in yeast cells, they are cultured under the desired nutritional conditions, and aliquots are withdrawn at different times. Next, cells are fixed with formaldehyde, which preserves the integrity of LDs for months when cells are stored at 4 °C. Other fixation techniques should be avoided, especially those using methanol or cold acetone, since they lead to the degradation of LDs within the cells²⁴. To measure the LD index, formaldehyde-fixed cells are suspended in water to obtain a defined concentration. Then, they are added to a solution containing the fluorophore BODIPY 493/503 quenched by KI, and when the fluorophore enters the cell and associates with the LDs, there is a recovery of its fluorescence. Concomitant to the fluorescence measurement (485 nm/510 nm), the concentration of cells is quantified by measuring the optical density at 600 nm. Each sample is read four times by adding subsequent 5 µL aliquots of a formaldehyde-fixed cell suspension to the same well. Blanks of fluorescence and absorbance are acquired before the addition of cells. The quality of the fluorescence and the absorbance data are evaluated by determining the linearity of the measurements: if r <0.9, data are discarded. The r value is important because basically the intensity of fluorescence should produce a linear response to increasing cell concentrations. If the cell concentration is too high, the linearity is lost. The LFR assay provides a fast, simple and economic method to select the desired LD phenotype in high-throughput experiments. After selecting the desired conditions, the LD content of individual cells can be studied by confocal microscopy, using the same formaldehyde-fixed cells stained with BODIPY, providing an image of the LDs in the cell. Their TAGs and SE content can now be further analyzed by thin layer chromatography.

Protocol

1. Preparation of Buffers and Solutions

1. To prepare 1 L of phosphate buffered saline (PBS, pH 7), dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na₂HPO₄, 0.24 g of KH₂PO₄ in 800 mL of distillated water. Adjust the pH to 7.0 with HCl, and then add water for a final volume of 1 L. PBS can be made as a 10x stock solution and stored at room temperature.
2. To prepare 10 mL of fixing solution, add 1 mL of 37% formaldehyde to 9 mL of PBS to reach a final concentration of 3.7%. Prepare this solution just before use.
   Caution: Use gloves to handle the formaldehyde solution.
3. To prepare the quenching-solution (500 mM), dissolve 8.3 g of KI in 100 mL of distilled water. Prepare this solution before use and store it at room temperature.
4. To prepare BODIPY 10 mM stock solution, dissolve 10 mg of BODIPY 493/503 in 3.8 mL of dimethyl sulfoxide (DMSO). Keep 100 µL aliquots in the dark at -70 °C.
5. To prepare BODIPY 5 µM-quenching solution, add 1 µL of 10 mM BODIPY 493/503 to 2 mL of 500 mM quenching solution. For the LFR assay, a final concentration of 5 µM of BODIPY is required.
6. To prepare the mineral solution, add 0.06 g of H₃BO₃, 0.14 g of MnCl₂-4H₂O, 0.4 g of ZnCl₂, 0.04 g of Na₂MoO₄-2H₂O, 0.1 g of FeCl₃-6H₂O and 0.4 g of CuSO₄-5H₂O to 1 L of distilled water.
7. To prepare the salt solution, add 16 g of KH₂PO₄, 4 g of Na₂SO₄, 8 g of KCl, 2 g of MgSO₄, 1 g of CaCl₂, and 8 mL of mineral solution to 1 L of distilled water.
8. To prepare the minimal medium without a nitrogen source for U. maydis FB2 (a2b2), add 10 g of glucose and 62.5 mL of salt solution to 1 L of distilled water, according to Holliday, et al.³¹. Adjust the pH to 7.0 and then dispense 100 mL of the media in 250 mL flasks and sterilize them. Autoclave for 20 min at 15 psi (1.05 kg/cm²) on liquid cycle or filter sterilize.
9. To prepare the yeast peptone dextrose medium (YPD), add 5 g of yeast extract, 2.5 g of peptone, and 5 g of glucose to 1 L of distilled water. Dispense 100 mL of the solution in 250 mL flasks and sterilize them. Autoclave for 20 min at 15 psi (1.05 kg/cm²) on liquid cycle or filter sterilize.

2. Culture Condition and Cell Fixation

NOTE: Preserve the structure of the LDs by fixing the cells as soon as possible. The fixation can be done in microcentrifuge tubes. Fixed cells can be kept at 4 °C for up to one month if dehydration is avoided leaving a thin layer of water.

1. Grow U. maydis cells under culture conditions relevant for the study. Start the culture with an initial OD₆₀₀ of 0.05 (1.15 × 10⁶ cells/ mL). Incubate the cells at 28 °C, 180 rpm for 24 h. An OD₆₀₀ of 1 corresponds to 2.3 × 10⁷ cells/ mL.
2. At the selected times, withdraw an aliquot of the cells. For U. maydis, withdraw 5 mL every 2 h during the first 8 h. After 8 h of growth, aliquots of 2 mL are enough.
3. Measure the optical density at 600 nm of each aliquot.
4. Centrifuge the cell suspension at 14,000 x g for 1 min at 4 °C using a tabletop centrifuge. Discard the supernatant.
5. Suspend the pellet of the cells in 1 mL of PBS-formaldehyde 3.7% buffer. Incubate the cells for 15 min at room temperature.
6. After the incubation, centrifuge the cell suspension at 14,000 x g for 1 min at 4 °C. Discard the supernatant.
7. Wash the cells pellet twice with a similar volume of distilled water (1 mL). Centrifuge the cell suspension at 14,000 x g for 1 min at 4 °C. Discard the supernatant after each washing step.
8. Adjust the cell pellet to 5 OD₆₀₀ (1.15 x 10⁸ cell/ mL) with distilled water (Equation 1).
       Equation 1.
9. Keep the sample at 4 °C until its use.

3. Liquid Fluorescence Recovery Assay (LFR)

1. Turn on the spectrophotometer and open the Skanlt software. Click on NEW SESSION, and choose START and then Varioskan Lux. Select PROTOCOL from the session tree, enter the settings for the fluorescence wavelengths (excitation 485 nm/emission 510 nm; optical density 600 nm) and choose automatic photomultiplier gain. For excitation bandwidth, select 12 nm. In the optics, select top (excitation of the sample is from the top of the well).
2. Enter a gentle and continuous agitation (300 rpm). Select RUN PLATE OUT, and PAUSE UNTIL THE USER ACTION (time used for the addition of the sample to the wells). Repeat the protocol five times. Click SAVE and write a name for the session on the SESSION NAME FIELD. The protocol is now ready for the samples analyses.
3. Add 200 µL of the BODIPY 5 µM- quenched solution to each well of a 96 well black-clear bottom plate.
4. Place the plate inside the spectrophotometer chamber and incubate 5 min at 30 °C. From this point, protect the plate from light as much as possible.
5. Click the START button and read the fluorescence (excitation 485/emission 510) and the OD₆₀₀ corresponding to the blanks.
6. Add 5 µL of the formaldehyde-fixed cell suspension to the wells during the pause time. Mix the samples carefully with the pipette. Put the plate inside the spectrophotometer and click CONTINUE. Then, the samples will be processed according to the protocol designed above.
7. Repeat three successive additions of 5 µL of cell suspension. Make sure that the cells do not precipitate. (Figure 1).

4. Calculations of The LD Index

1. For each sample in the microplate, plot a graph of fluorescence and absorbance against the volume of each successive addition (5 points including the blank). In this study, use Microsoft Excel software for the calculations. To plot the data, enter the volume, fluorescence, and absorbance data in the first, second and third column of the datasheet, respectively. Then, select the whole data with the cursor, click INSERT and select (XY) SCATTER.
2. Select the points for each line in the graph and insert the respective TREND LINE with the SHOW EQUATION box selected. Write down the values of the slopes of the two straight lines, according to equation 2:
       Equation 2
   Where y= fluorescence or optical density, x = volume of sample (0, 5, 10, 15, 20 µL), m = slope of the fluorescence or optical density line, and b = y-intercept.
3. Divide the slope of the fluorescence line by the slope of the optical density line to get the LD index, equation 3.
       Equation 3
   The LD index corresponds to the quotient of fluorescence and optical density slopes.

5. Data Quality Analysis

1. Calculate the correlation coefficient of each straight line: click on the FUNCTION WIZARD (fx) and choose CORREL. If r <0.9, discard the data and repeat the readings. If r ≥ 0.9, readings are reliable.

Results

LFR, with BODIPY 493/503 as the fluorescent probe is a reliable and easy method to study the dynamic accumulation of LDs in U. maydis, regardless the growth conditions. When cells were cultured in YPD-medium, there was an increase in the LD index during the exponential phase, followed by a decline in the stationary phase (Figure 2A). In contrast, in cells grown under nitrogen starvation, there was a steady increase in LD index in both the exponential...

Discussion

LFR is a novel method that was applied for the first time to study the lipid metabolism in S. cerevisiae and later it was successfully implemented in U. maydis^20,21. Although the LD index does not give an absolute value of the TAGs accumulated in cells, it provides a fast idea of the lipid content of cells, and the interpretation of the data is straightforward when LD index from two or more experimental conditions are compared. BODIPY 493/503 is...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Spectrophotometer	Varioskan Lux multimode microplate reade	D5879	Filter 493/503 or monochromator detector
Plate costar	Corning Inc.	3615	96 well black-wall/clear bottom
Plate costar	Corning Inc	3598	96 well cell culture plate costar
BODIPY 493/503	Invitrogen/ Thermofisher	D3922	4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene
Formaldehyde solution	Sigma Aldrich	252549	ACS reagent, 37 wt % in H2O, contains 10-15% methanol to prevent polymerization
Potassium iodide	Sigma Aldrich	60399	BioUltra, ≥ 99.5% (AT)
FB2 Ustilago maydis	ATCC	201384	Basidiomycete-Yeast
Sodium chloride	Sigma Aldrich	746398	ACS reagent, inorganic salt
Potassium phosphate monobasic	Sigma Aldrich	P0662	ACS reagent
Select Yeast Extract	Sigma Aldrich	Y100	Mixture of amini acids, peptides, water- soluble, vitamins and carbohydrates for culture media
N-Z case plus	Sigma aldrich	N4642	Casein enzymatic hydrolyzate from bovine milk
Glucose	Sigma Aldrich	G-8270	D (+) glucose
Skaker flask	Pirex	CLS4450250-6EA	Borosiicate glass
Shaker	SEV México	INO650V-7	Orbital shaker
Centrifuge table	Eppendorf	5415C	Centrifuge
Microtubes	Sigma Aldrich	Z606340	Eppendorf
Pipet tips	Axygen scientific	T-200-Y	Universal Pipet Tips with Bevelled End, 200 microliter, non sterile
mLine pipette	Biohit	725130	8 channels, volume range 5-100 uL