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....

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-sol....

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