Name: lipid index determination by liquid fluorescence recovery in the fungal pathogen ustilago maydis
Uploaded: 2018-04-03T15:00:00
Description: Watch this Scientific Journal Video about Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis at JoVE.com

This work was supported by grants Instituto Polit&#233;cnico Nacional-Secretaria de Investigaci&#243;n y Posgrado (IPN-SIP-20170864), Programa de Apoyo a Proyectos de Investigaci&#243;n e Innovaci&#243;n Tecnol&#243;gica (PAPIIT IN222117)- Universidad Nacional Aut&#243;noma de M&#233;xico (UNAM), and Consejo Nacional de Ciencia y Tecnolog&#237;a (CONACyT 254904-JPP and 256520-GGS). Funda&#231;&#227;o de Amparo a Pesquisa do Rio de Janeiro (FAPERJ-Cientistas do Nosso Estado: E 26/103.353/2011). We thanks to QFB. Oscar Iv&#225;n Luque&#241;o Bocardo for the schematic overview. We thank Dr. Miguel Tapia Rodr&#237;guez for the valuable help in the confocal microscopy of LDs.&#160;We wish to thank Dr. Bruno Bozaquel Morais for his pioneering work in developing the technique in S. cerevisiae that we adapted for U. maydis.&#160;

1. Preparation of Buffers and Solutions
<ol>
	<li>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.</li>
	<li>To prepare 10 mL of fixing solution, add 1 mL of 37% formaldehyde to 9 mL of PBS to reach a final concentratio...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+lipid+droplets+in+metabolic+disease+in+rodents+and+humans.">The role of lipid droplets in metabolic disease in rodents and humans.</a> Journal of Clinical Investigation. 121 (6), 2102-2110 (2011).</li><li>Aguilar, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+accumulation+and+other+biochemical+changes+induced+in+the+fungal+pathogen+Ustilago+maydis+under+nitrogen-starvation.">Lipid droplets accumulation and other biochemical changes induced in the fungal pathogen Ustilago maydis under nitrogen-starvation.</a> Archives of Microbiology. , (2017).</li><li>Bozaquel-Morais, B. L., Madeira, J. B., Maya-Monteiro, C. M., Masuda, C. A., Montero-Lomeli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+fluorescence-based+method+identifies+protein+phosphatases+regulating+lipid+droplet+metabolism.">A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.</a> PLoS One. 5 (10), e13692 (2010).</li><li>Madeira, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TORC1+Inhibition+Induces+Lipid+Droplet+Replenishment+in+Yeast.">TORC1 Inhibition Induces Lipid Droplet Replenishment in Yeast.</a> Molecular and Cellular Biology. 35 (4), 737-746 (2015).</li><li>Maya-Monteiro, C. 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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. maydis20,21. 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...

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 production3,4,5. 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 purification21,22. 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 &#176;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 &#181;L 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 &#60;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.

<table>
	<tbody>
		<tr>
			<td>Spectrophotometer</td>
			<td>Varioskan Lux multimode microplate reade</td>
			<td>D5879</td>
			<td>Filter 493/503 or monochromator detector</td>
		</tr>
		<tr>
			<td>Plate costar</td>
			<td>Corning Inc.</td>
			<td>3615</td>
			<td>96 well black-wall/clear bottom</td>
		</tr>
		<tr>
			<td>Plate costar&#160;</td>
			<td>Corning Inc</td>
			<td>3598</td>
			<td>96 well cell culture plate costar</td>
		</tr>
		<tr>
			<td>BODIPY 493/503</td>
			<td>Invitrogen/ Thermofisher</td>
			<td>D3922</td>
			<td>4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene</td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma Aldrich</td>
			<td>252549</td>
			<td>ACS reagent, 37 wt % in H2O, contains 10-15% methanol to prevent polymerization</td>
		</tr>
		<tr>
			<td>Potassium iodide</td>
			<td>Sigma Aldrich</td>
			<td>60399</td>
			<td>BioUltra, &#8805; 99.5% (AT)</td>
		</tr>
		<tr>
			<td>FB2 Ustilago maydis</td>
			<td>ATCC</td>
			<td>201384</td>
			<td>Basidiomycete-Yeast</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>746398</td>
			<td>ACS reagent, inorganic salt</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>P0662</td>
			<td>ACS reagent</td>
		</tr>
		<tr>
			<td>Select Yeast Extract</td>
			<td>Sigma Aldrich</td>
			<td>Y100</td>
			<td>Mixture of amini acids, peptides, water- soluble, vitamins and carbohydrates for culture media</td>
		</tr>
		<tr>
			<td>N-Z case plus</td>
			<td>Sigma aldrich</td>
			<td>N4642</td>
			<td>Casein enzymatic hydrolyzate from bovine milk</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G-8270</td>
			<td>D (+) glucose</td>
		</tr>
		<tr>
			<td>Skaker flask</td>
			<td>Pirex</td>
			<td>CLS4450250-6EA</td>
			<td>Borosiicate glass</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>SEV M&#233;xico</td>
			<td>INO650V-7</td>
			<td>Orbital shaker</td>
		</tr>
		<tr>
			<td>Centrifuge table</td>
			<td>Eppendorf</td>
			<td>5415C</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>Microtubes</td>
			<td>Sigma Aldrich</td>
			<td>Z606340</td>
			<td>Eppendorf</td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td>Axygen scientific</td>
			<td>T-200-Y</td>
			<td>Universal Pipet Tips with Bevelled End, 200 microliter, non sterile</td>
		</tr>
		<tr>
			<td>mLine pipette</td>
			<td>Biohit</td>
			<td>725130</td>
			<td>8 channels, volume range 5-100 uL</td>
		</tr>
	</tbody>
</table>

lipid index determination by liquid fluorescence recovery in the fungal pathogen ustilago maydis

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 &#8805; 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.

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...

Watch this Scientific Journal Video about Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis at JoVE.com

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

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.

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

The article shows how to implement the LD index assay, which is a sensitive microplate assay to determine the accumulation of triacylglycerols (TAGs) in ...

The overall goal of this procedure is to determine the Lipid Droplet Index of cells cultured under different nutritional conditions. This method can help answer key questions in the Lipid Droplet and Lipid Field, such as the dynamic of accumulation and the different nutritional conditions. The main advantage of this technique is that multiple samples can be read simultaneously, because it can be implemented in the microplate.With the start of this method when we observe refractance of Ustilago maydis cells cultured under nitrogen starvation and then realize that this might mean that these instructors might be lipid droplets. So we contacted Monica Montero from Universidade Federale do Rio de Janeiro in Brazil who implemented the Liquid Florescence Recovery Assay of lipid droplets in Saccharomyces cerevisiae to start a collaboration to adapt the procedure for Ustilago maydis. The first step in this protocol is the preparation of the required buffers and solutions.To prepare a 10 millimolar BO-DP stock solution, dissolve 10 milligrams of BO-DP 493-503 and 3.8 milliliters of dimethyl sulphoxide. Divide into 100 microliter aliquots. Then keep in the dark a minus 70 degrees Celsius.Prepare a five micromolar BO-DP quenching solution for the liquid florescence recover, or LFR-Assay by adding one microliter of 10 millimolar BO-DP 493-503 to two milliliters of 500 millimolar quenching solution. Grow the umeda cells for this study by starting a culture with an initial optical density of 600 nanometers of 05. Incubate the cells at 28 degrees Celsius and 180 RPM for 24 hours.At selected times, withdraw an aliquot of the cells. During the first eight hours, withdraw five milliliters every two hours. After eight hours of growth aliquots of two milliliters are enough.Measure the optical density at 600 nanometers of each aliquot. Centrifuge the cell suspension at 14, 000 times G for one minute at four degrees Celsius. And, discard the supernatant.Suspend the cell pellet in one milliliter of fixing solution containing 3.7%formaldehyde in PBS. Incubate the cells at room temperature for 15 minutes. After 15 minutes, centrifuge the cell suspension at 14, 000 times G and four degrees Celsius for one minute.Discard the supernatant. Wash the cell pellet with one milliliter of distilled water. Centrifuge the cell suspension for one minute.Discard the supernatant and add one milliliter of distilled water for a second wash. Centrifuge again. Remove the supernatant from the second wash.Using distilled water, adjust the optical density of the cell pellet at 600 nanometers to five, which is equivalent to 1.5 times 10 to the eighth cells per milliliter. Keep the samples at four degrees Celsius until use. Begin this procedure by turning on the spectrophotometer.Open the SkanIt software. Click on new session. From the session tree select protocol enter the settings for the florescence wavelength and choose automatic photo multiplayer gain.For the excitation bandwidth select 12 nanometers. In the optics, select top for excitation of the sample from the top of the well. Enter 300 RPM and low for a gentle and continuous agitation.Select run plate out, pause, and until the user action, repeat the protocol five times. Click save and in the session name field, write a name for the session. The protocol is now ready for sample analysis.Add 200 microliters of the 5 micromolar BO-DP quenching solution to each well of a black 96 well clear-bottomed plate. Place the plate inside the spectrophotometer chamber and incubate at 30 degrees Celsius for five minutes. From here on, protect the plate from light as much as possible.Click the start button and read the florescence and the optical density at 600 nanometers, corresponding to the blanks. During the pause time, add five microliters of the formaldehyde fixed cell suspension to the wells and mix the samples carefully with a pipette. Put the plate back inside the spectrophotometer, and click continue.Repeat three successive additions of five microliters of cell suspension. Mix with a pipette to make sure the cells do not precipitate before continuing with the read. In this demonstration, Excel software is used to calculate and plot a graph of florescence and absorbance against the volume of each successive addition for each sample in the microplate.Enter the volume, florescence and absorbance data in the data sheet. Then select the whole data with the cursor, click insert and X Y scatter. 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 the equation. To get the lipid droplet, or LD index, divide the slope of the fluorescence line by the slope of the optical density line. To analyze the quality of the data, calculate the correlation coefficient of each straight line.Click on the function wizard and choose coral. The readings are reliable if R is equal to or greater than 0.9. It is important to corroborate the sensitivity of this method by confocal microscope.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. In contrast cells grown under nitrogen starvation showed a steady increase in LD index in both the exponential and stationary phases. Under nitrogen starvation, the increase in the LD index was inhibited by Soraphen A, a specific inhibitor of the Acetyl-CoA carboxylase enzyme, essential for the synthesis of fatty acids, contained in the triacylglycerols stored in lipid droplets.Since both cultures were started with the same optical density, the results show the capacity of the assay to detect small changes in the lipid content. The sensitivity of this method was corroborated by confocal microscopy. YPD cells contain many small LDs throughout the whole cell, while nitrogen starvation produced large LDs, typically four to five per cell.To further investigate the mobilization of LDs in U.Maydis, cells grown under nitrogen starvation for 72 hours were transferred to YPD medium. As shown, the LD index decreased following an exponential function. Since the cells were able to mobilize the LDs accumulated during nitrogen starvation, the result suggests a reduction in the triacylglycerol content.Once mastered, this technique can be done in one hour if it is performed properly. When attempting this procedure, it's important to remember to protect the cells from light and to mix between additions. Following this procedure, other methods for example triacylglycerol or total lipid content determination can be performed to answer additional questions, like the exact lipid content in the cells.After its development, this technique paved the way for researchers in the field of lipid biotechnology to explore new or alleged organisms in order to increase the production of biofuels or lipids in pharmaceutical applications. Even malignant cancer cells can be studied with this technique to explore the participation of lipid metabolism in disease development. After watching this video, you too have a good understanding of how to determine the lipid droplet index of these cells.Don't forget that working with formaldehyde can be extremely hazardous and precautions such as wearing gloves should always be taken while performing this procedure.

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