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 concentration of 3.7%. Prepare this solution just before use. 
	Caution: Use gloves to handle the formaldehyde solution.</li>
	<li>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.</li>
	<li>To prepare BODIPY 10 mM stock solution, dissolve 10 mg of BODIPY 493/503 in 3.8 mL of dimethyl sulfoxide (DMSO). Keep 100 &#181;L aliquots in the dark at -70 &#176;C.</li>
	<li>To prepare BODIPY 5 &#181;M-quenching solution, add 1 &#181;L of 10 mM BODIPY 493/503 to 2 mL of 500 mM quenching solution. For the LFR assay, a final concentration of 5 &#181;M of BODIPY is required.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
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 &#176;C for up to one month if dehydration is avoided leaving a thin layer of water.
<ol>
	<li>Grow U. maydis cells under culture conditions relevant for the study. Start the culture with an initial OD600 of 0.05 (1.15 &#215; 106 cells/ mL). Incubate the cells at 28 &#176;C, 180 rpm for 24 h. An OD600 of 1 corresponds to 2.3 &#215; 107 cells/ mL.</li>
	<li>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.</li>
	<li>Measure the optical density at 600 nm of each aliquot.</li>
	<li>Centrifuge the cell suspension at 14,000 x g for 1 min at 4 &#176;C using a tabletop centrifuge. Discard the supernatant.</li>
	<li>Suspend the pellet of the cells in 1 mL of PBS-formaldehyde 3.7% buffer. Incubate the cells for 15 min at room temperature.</li>
	<li>After the incubation, centrifuge the cell suspension at 14,000 x g for 1 min at 4 &#176;C. Discard the supernatant.</li>
	<li>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 &#176;C. Discard the supernatant after each washing step.</li>
	<li>Adjust the cell pellet to 5 OD600 (1.15 x 108 cell/ mL) with distilled water (Equation 1). 
	<img alt="Equation 1" src="/files/ftp_upload/57279/57279eq1.jpg" />&#160;&#160;&#160;&#160;Equation 1.</li>
	<li>Keep the sample at 4 &#176;C until its use.</li>
</ol>
3. Liquid Fluorescence Recovery Assay (LFR)
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>Add 200 &#181;L of the BODIPY 5 &#181;M- quenched solution to each well of a 96 well black-clear bottom plate.</li>
	<li>Place the plate inside the spectrophotometer chamber and incubate 5 min at 30 &#176;C. From this point, protect the plate from light as much as possible.</li>
	<li>Click the START button and read the fluorescence (excitation 485/emission 510) and the OD600 corresponding to the blanks.</li>
	<li>Add 5 &#181;L 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.</li>
	<li>Repeat three successive additions of 5 &#181;L of cell suspension. Make sure that the cells do not precipitate. (Figure 1).</li>
</ol>
4. Calculations of The LD Index
<ol>
	<li>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.</li>
	<li>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: 
	<img alt="Equation 2" src="/files/ftp_upload/57279/57279eq2.jpg" />&#160;&#160;&#160;&#160;Equation 2 
	Where y= fluorescence or optical density, x = volume of sample (0, 5, 10, 15, 20 &#181;L), m = slope of the fluorescence or optical density line, and b = y-intercept.</li>
	<li>Divide the slope of the fluorescence line by the slope of the optical density line to get the LD index, equation 3. 
	<img alt="Equation 3" src="/files/ftp_upload/57279/57279eq3.jpg" />&#160;&#160;&#160;&#160;Equation 3 
	The LD index corresponds to the quotient of fluorescence and optical density slopes.</li>
</ol>
5. Data Quality Analysis
<ol>
	<li>Calculate the correlation coefficient of each straight line: click on the FUNCTION WIZARD (fx) and choose CORREL. If r &#60;0.9, discard the data and repeat the readings. If r &#8805; 0.9, readings are reliable.</li>
</ol>

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The authors have nothing to disclosure

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</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, &amp;ge; 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&amp;eacute;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...

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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 ...

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Research

JoVE Journal

Biochemistry

7.6K Views.  Instituto Politécnico Nacional. Hier beschrijven we een protocol om te verkrijgen van de lipide druppel index (LD index) om te bestuderen van de dynamiek van triacylglycerolen in cellen gekweekt in high-throughput experimenten. De LD index bepaling is een gemakkelijke en betrouwbare methode die gebruikmaakt van BODIPY 493/503. Deze test hoeft niet dispendious lipide extractie of microscopie analyse.

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

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

Chemistry

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

Immunology and Infection

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Biology

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

Bioengineering

Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are used as models in lipid metabolic research or for industrial lipid production. Lipid droplets are highly dynamic storage bodies and their cellular content represents a convenient readout of the lipid metabolic state. Fluorescence microscopy is a method of choice for quantitative analysis of cellular lipid droplets, as it relies on widely available equipment and allows analysis of individual lipid droplets. Furthermore, microscopic image analysis can be automated, greatly increasing overall analysis throughput. Here, we describe an experimental and analytical workflow for automated detection and quantitative description of individual lipid droplets in three different model yeast species: the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus, and the budding yeast Saccharomyces cerevisiae. Lipid droplets are visualized with BODIPY 493/503, and cell-impermeable fluorescent dextran is added to the culture media to help identify cell boundaries. Cells are subjected to 3D epifluorescence microscopy in green and blue channels and the resulting z-stack images are processed automatically by a MATLAB pipeline. The procedure outputs rich quantitative data on cellular lipid droplet content and individual lipid droplet characteristics in a tabular format suitable for downstream analyses in major spreadsheet or statistical packages. We provide example analyses of lipid droplet content under various conditions that affect cellular lipid metabolism.

Here, we present a MATLAB implementation of automated detection and quantitative description of lipid droplets in fluorescence microscopy images of fission and budding yeast cells.

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

Quantifying Retinal Pigment Epithelial Phagocytosis and Lipofuscin Accumulation

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.

Author Spotlight: Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures  

This protocol describes a lipofuscin accumulation model in highly differentiated and polarized human retinal pigment epithelial (RPE) cultures and an improved outer segment (OS) phagocytosis assay to detect the total OS consumption/degradation capacity of the RPE. These methods overcome the limitations of previous lipofuscin models and classical pulse-chase outer segment phagocytosis assays.

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging ...

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

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