Quantitation of Cellular Components in Cryptococcus neoformans for System Biology Analysis

Abstract

Methods and procedures in molecular biology used to study fungal pathogenesis have significantly improved during the last decade. In this chapter, we provide step-by-step procedures for performing genetics and biochemical studies in the human pathogenic fungal microorganism Cryptococcus neoformans (Cn). These methods are employed for studying the pathobiology of Cn and for experimental validation of theoretical models of fungal pathogenicity.

1. Introduction

Cryptococcus neoformans (Cn) is the causative agent of cryptococcosis, a fungal disease acquired by inhalation of infectious particles from the environment. Cryptococcosis is a relatively frequent disease in immunocompromised subjects and in certain regions of the world such as sub-Saharan Africa in which the estimated number of deaths associated with cryptococcal disease, at half a million per year, is comparable with the number attributed to tuberculosis (1, 2). In the USA, the prevalence of cryptococcosis in HIV positive patients is 5–10%, which is approximately the same as that for meningococcal meningitis (3). Emerging groups at risk include patients suffering from chronic lymphatic leukemia, Hodgkin’s disease, chronic myelogenous leukemia, and multiple myeloma (4). The median overall survival of patients with lymphoproliferative disorders affected by cryptococcosis is 2 months, which is significantly shorter than the 9-month median survival of an AIDS patient with cryptococcosis (5). Cryptococcosis is also associated with organ transplantation (6, 7), and was documented in 2.8% of organ transplant recipients with an overall death rate of 42% (8). Some cases of cryptococcosis occur in patients with apparently normal immune function (9–12).

One area of investigation that has significantly improved in the last 2 decades is the molecular biology of this microorganism. The development of molecular epidemiology and phylogeny and molecular technology for clinical diagnosis have significantly helped the clinicians to better manage this life-threatening disease. However, it was the advent of genetics and biochemistry of this microorganism that allowed basic and clinical investigators to address mechanistic questions and study the pathophysiology of cryptococcosis. This was (and still is) an essential step to define fungal features and characteristics necessary for the organism to cause disease (13). These fungal factors can then be exploited for the understanding of fungal pathogenicity and fungal interaction with the host cells and, ultimately, and for the development of new therapeutic strategies. With the rise of its importance as a human pathogen, there has been a concurrent rise in the ability to molecularly study its physiopathology.

In Chapter 9, we provide a mathematical model of the regulation of melanin production by the sphingolipid pathway. In particular, we show that a specific enzyme of the sphingolipid pathway, inositol phosphoryl ceramide synthase 1 (Ipc1), regulates melanin formation in Cn through the production of diacylglycerol (DAG) and the consequent activation of protein kinase C 1 (Pkc1). Thus, the downregulation or/and deletion of IPC1 or/and PKC1 genes by homologous recombination should produce mutant strains that make less or no melanin. We would expect IPC and DAG lipid measurements to be decreased in the mutant in which Ipc1 is downregulated. Also in this mutant, Pkc1 enzymatic activity should be decreased. This experimental approach is necessary to validate the changes in the network behavior simulated by the mathematical model. Therefore, the deletion of the gene of interest by homologous recombination and confirmation by Southern or/and Northern blot of the isolated genomic DNA or total RNA, respectively, and the analysis of protein and lipid levels regulated by those genes are useful methods to refine and validate the mathematical model.

In this chapter, we provide basic molecular methods for performing genetics and biochemistry studies in Cn. These methods can be employed to validate hypotheses and theoretical models of Cn pathogenicity or simply to study the pathobiology of this important human pathogen.

2. Materials

2.1. DNA Isolation from Cryptococcus neoformans

• 1Yeast Peptone Dextrose (YPD) agar plates and YPD broth (see Note 1).
• 2Sterile PBS 1×.
• 31 M Tris–HCl pH 7.5.
• 40.5 M, EDTA pH 8.0.
• 55 M NaCl.
• 6100% Triton X-100.
• 720% SDS.
• 8TENTS: 10 mM Tris–HCl, pH 7.5, 1 mM EDTA, pH 8.0, 100 mM NaCl, 2% Triton X-100, 1% SDS.
• 9Acid washed 0.425–600 µm glass beads (SIGMA).
• 10Phenol:chloroform:isoamyl alchohol =25:24:1 (SIGMA).
• 113 M Sodium acetate (NaOAC).
• 12TE buffer, pH 8.0, sterile.

2.2. Biolistic Delivery in Cryptococcus neoformans

• 1YPD agar + 1 M Sorbitol plates.
• 2YPD agar + Nourseothricin/Hygromycin (100 µg/ml) plates.
• 30.6 µm Gold beads (BIORAD).
• 4MacroCarriers (BIORAD).
• 5Rupture Disks, 1,350 psi, (BIORAD).
• 6Stopping Screens (BIORAD).
• 72.5 M CaCl₂ sterile.
• 81.0 M spermidine (filter sterilize) (SIGMA), can be stored at −20°C.
• 9100% Ethanol.
• 10Isopropanol.
• 11This instruction assumes the use of PDS-1000/He Biolistic Particle Delivery System from BIORAD.

2.3. Southern Hybridization of DNA Extracted from Cryptococcus neoformans

• 1Denaturing solution: 1.5 M NaCl, 0.5 M NaOH.
• 2Neutralizing Solution: 1 M Tris–HCl pH 8.0, 1.5 M NaCl.
• 3Nytran SPC (0.45 µm Nylon Transfer Membrane) (Whatman).
• 4Whatman 3MM Blotting Paper.
• 5Paper towels, preferably single fold.
• 620 × SSC: 175.3 g of NaCl, 88.2 g of sodium citrate in 800 ml of double distilled water. Adjust pH with NaOH pellets and adjust the total volume to 1 L. Autoclave. Can be stored at room temperature.
• 720 × SSPE : 175.3 g of NaCl, 27.6 g of NaH₂PO₄·H₂O, 7.4 g EDTA in 800 ml of double distilled water. Adjust the pH with NaOH pellets to 7.4. Final volume made up to 1 L. Autoclave. Can be stored at room temperature.
• 820% SDS.
• 9Nonfat dry milk.
• 10Prehybridizing solution: 10 ml 20× SSPE, 10 ml 20% SDS, 2 ml 10% nonfat dry milk in a total volume of 40 ml. Can be stored at 4°C with 0.02% sodium azide for 2–3 days.
• 11Random Primers DNA labeling system kit (Invitrogen).
• 12³²P dCTP (Perkin Elmer).
• 13Microspin G-25 column (Amersham Biosciences).
• 14Sterile TE buffer, pH 8.0.

2.4. Highly Pure Total RNA Isolation from Cryptococcus neoformans (e.g., for Microarray Studies)

• 1YPD agar plate and YPD broth.
• 2Phosphate Buffered Saline (PBS) 1× sterile.
• 3Tri Reagent (Molecular Research Centre).
• 4BAN as a phase separation reagent, molecular biology grade (Molecular Research Centre).
• 5RNeasy Mini Kit and RNeasy MinElute Cleanup Kit (Qiagen).
• 6RNase Zap for removing RNase contamination from external surface (Ambion).
• 7RNase/DNase free plastic wares.

2.5. Protein Extraction from Cryptococcus neoformans

• 1YPD media and agar plates (made from YPD 50 g/L and agar 20 g/L).
• 2Buffer for Cn cell lysis: 1 ml 1 M Tris–HCl pH 8, 9 ml H₂O, 1.5 ml glycerol (13% v/v), 10 µl CLAP: chymostatin, leupeptin, antipain, and pepstatin A (each at 10 mg/ml in DMSO and stored at −20°C), and 20 µl 100 mM solution phenylmethylsulfonyl fluoride (PMSF) in isopropanol.
• 3Glass beads, acid washed, 425 µm (30–40 US sieve) (Sigma).

2.6. Lipid Extraction

• 1Mandala lipid extraction buffer: 150 ml ethanol, 150 ml distilled water, 50 ml diethyl ether, 10 ml pyridine, and 180 µl 14.2 N ammonium hydroxide.
• 2Use glass tubes for all extraction steps (VWR) fit best in the ThermoSavant SPD2010 SpeedVac system we use).
• 3Waters Sep-Pak Classic Silica cartridges (WAT 051900, 690 mg) for analytical scale or WAT036930 200 cc, 5 g cartridges for semipreparative scale lipid isolation and purification.
• 410″ × 10″ glass tank for thin layer chromatography (TLC).
• 53MM Whatman chromatography paper (Fisher).
• 6TLC chromatography plates (Fisher M5628-5 or 05-713-329 depending on analytical or semipreparative purposes).
• 7Soy glucosylceramide standard (Avanti Polar Lipids) made up to 3.5 mM (2.5 µg/µl) in chloroform/methanol (2:1).
• 8Prepare 70% H₂SO₄ by adding 14 ml H₂SO₄ slowly to 6 ml water on ice, with mixing. Add 40 mg resorcinol to 20 ml 70% H₂SO₄. Stir well at room temperature with a magnetic stirrer bar. Pour solution into a glass TLC sprayer.

2.7. In Vitro Enzyme Activity Assay

• 1NBD-C6-ceramide (Avanti Polar Lipids).
• 2Lysis buffer: 25 mM Tris–HCl pH 7.4, 5 mM EDTA, 1 mM PMSF, and CLAP: chymostatin, leupeptin, antipain, and pepstatin A (each at 10 mg/ml in DMSO and stored at −20°C).
• 3Silica gel 60 TLC plates (EM Sciences, Fisher).

2.8. Mass Spectrometry of Lipids

• 1Commercially available synthetic lipid standards.

3. Methods

3.1. DNA Isolation (14, 15)

• 1Inoculate a 10–15 ml YPD broth with a single colony from a fresh YPD agar plate and grow them for 20–24 h at 30°C with constant shaking. Pellet cells from this culture at 1,200 × g 4°C for 10 min.
• 2Wash with sterile PBS 1× twice and resuspend in 1 ml of sterile double distilled water and transfer to a 2-ml screw cap tube (see Note 2).
• 3Pellet cells in a Microcentrifuge for 30 s at 1,200 × g at room temperature.
• 4Pour off the water; add 0.5 ml of TENTS and vortex at 7–8 speed for three times, 45 s each. This step assumes the use of Vortex Genie 2 from Scientific Industries.
• 5Add two cups (1 cup =400–500 µl) of acid washed 0.45 µm glass beads (see Note 3) and 0.5 ml phenol–chloroform–Isoamyl alcohol (see Note 4).
• 6This step assumes the uses of Bead Beater 16 from Scientific Industries. Tubes were vortexed/homogenized in a Bead Beater three times, 45 s each, with a gap of 45 s on ice, in between each cycle (see Note 5).
• 7After homogenizing (or lysing) of the cells, centrifuge the cells for 10 min at 8,000 × g at room temperature to separate the cell debris and unbroken cells.
• 8Remove the upper aqueous phase which now contains the DNA, to a fresh 1.5-ml Eppendorf and add 1 ml of ethanol 100% and keep at −20°C overnight (see Note 6).
• 9Centrifuge the tube at 8,000 × g for 30 min at 4°C. Remove the supernatant, dissolve the pellet in 200 µl of TE containing RNase A at a concentration of 100 µg/ml and then incubate at 37°C for 20 min.
• 10After incubation, add equal volume of phenol–chloroform–isoamyl alcohol and mix gently by inverting 4–6 times. Centrifuge at 8,000 × g, 10 min, 4°C. Remove the aqueous phase and repeat the step with the aqueous phase.
• 11Add 20 µl of 3M NaOAC and 400 µl of ethanol (100%) to the final aqueous phase and incubate at −20°C for 30–60 min for complete precipitation.
• 12After precipitation, centrifuge the tube at 8,000 × g at 4°C for 5 min.
• 13Wash the DNA pellet twice with 200 µl of ice-cold 70% ethanol and air dry the pellet (see Note 7).
• 14Dissolve the pellet in 30–50 µl of sterile TE gently and store at −20°C.

3.2. Biolistic Delivery in Cryptococcus neoformans (14, 16)

• 1Spin down 19–20 h grown culture (15 ml) of the recipient strain and throw off 12 ml of the supernatant (see Note 8).
• 2Plate 200–250 µl of this cell suspension on prewarmed YPD agar + 1 M sorbitol plates and let them dry for 4–5 h at 30°C (see Note 9). This should include a “non-shot” control plate.
• 3During this time of incubation, prepare the shot. For preparing a stock of Gold Beads – 60 mg/ml, 30 mg was weighed out and dissolved in 100% ethanol, vortexed vigorously for 3 min, incubated at room temperature for 15 min and spun for 1 min. Discard the supernatant and suspend the gold beads in 1 ml of sterile water. Incubate or allow the particles to settle down, pellet and discard the supernatant. Add 500 µl of 50% Glycerol to make a final concentration of 60 mg/ml. This stock can be stored in 4°C.
• 4
  Each Shot should be prepared as follows in the same sequence:

  • 10 µl of 60 mg/ml of gold beads
  • 1 µl of ≥1 µg/µl of DNA
  • 10 µl of 2.5 M CaCl₂
  • 2 µl of 1.0 M Spermidine
  • Vortex the mix for 3–5 min and let it settle for 5 min at room temperature. Spin for 20 s and take off the supernatant. Wash the Bead-DNA mix once with 500 µl of 100% ethanol by vortexing and spin down the Bead-DNA. Throw off the supernatant. Finally, resuspend the Bead-DNA in 25 µl of 100% ethanol (see Note 10).
• 5The Macrocarriers should be prepared inside a Laminar Hood to prevent contamination. Dip the macrocarriers (one for each shot) in 100% ethanol. Blot off the excess liquid on a sterile wiper and keep in a sterile Petri dish until completely dry.
• 6Vortex the Bead-DNA well so that the beads are uniformly coated with the DNA (see Note 11). Spread 10 µl of this mix, first onto the center of the macrocarrier then working outward, within 5mmto the edge in a slow circular motion. Let it dry. If there is any extra Bead-DNA, it can be added to each macrocarrier in the same fashion (see Note 12).
• 7The machine (PDS-1000/He Biolistic Particle Delivery System) should be sterilized with 70% ethanol and dried before shooting. The chamber should be kept closed as much as possible. Open the Helium tank pressure valve and set the pressure regulator at 1,800–2,100 psi.
• 8Soak the rupture disk, 1,350 psi in isopropanol, place in the retaining cap and screw the unit onto the gas acceleration tube of the machine with the retaining cap torque wrench (see Note 13).
• 9Unscrew the macrocarrier cover lid and place a stopping screen on the stopping screen support. Place the macrocarrier on top (Bead-DNA side up) of the macrocarrier holder, invert and place on the fixed nest. The dried microcarriers should face toward the stopping screen. Screw the macrocarrier cover lid to the assembly until tightened and place this in the top slot inside the chamber.
• 10Place the target shelf on the second to bottom shelf (see Note 14). Place the YPD agar + 1Msorbitol Petri dishes with cells, on this shelf without the lid on.
• 11Close the chamber and set the vacuum switch at “VAC” position till the desired vacuum of 28.5–29″ is reached. Hold the vacuum chamber at this level of vacuum by quickly pressing the switch to “HOLD” position and press the “FIRE” switch to bombard the sample into the plate until the rupture disk pops. Vent the chamber and immediately cover the Petri dish with lid and remove it from the chamber.
• 12Repeat the shooting until all the macrocarriers coated with Bead-DNA were utilized. All the parts should be cleaned and surface sterilized with 70% ethanol between two different DNA samples.
• 13Incubate the “shot” along with a “non-shot control” plates for 2 h at 30°C (see Note 15).
• 14Label Falcon 2054 tubes, one for each of the shot and non-shot plates.
• 15Aliquot 1 ml of prewarmed YPD broth onto each plate. Rub the liquid broth across the whole surface of the plate with sterile hockey stick and scrape off the cells. Tilt the plate and pipette the liquid into the labeled Falcon tubes.
• 16Plate 200–250 µl of the scraped liquid and spread uniformly onto prewarmed YPD Nourseothricin/Hygromycin plates. Incubate the plates at 30°C for several days.

3.3. Southern Hybridization (17)

• 1After taking picture of the Gel (see Note 16), denature in the Denaturing Solution (use fresh) for 1 h at room temperature with constant shaking.
• 2Neutralize the gel in Neutralizing Solution for 1–2 h.
• 3Wash the gel with double distilled water.
• 4Wet the membrane and the 3MM Whatman paper in 2× SSC until complete wet and assemble the transfer. Transfer overnight at room temperature or at least for 16–18 h.
• 5Before removing the gel, mark with pencil the wells on the membrane (see Note 17). Keep the membrane on a filter paper presoaked with 6× SSC at room temperature and semidry. Auto cross-link for 1 min at 1,200 (µJ × 100; this instruction assumes the use of UV Stratalinker 1800 from Stratagene). The membrane, if not set for hybridization can be stored at 4°C for 2–3 days in a sealed bag.
• 6Prehybridize the membrane in prehybridizing solution for 1–2 h at 65°C.
• 7Labeling of probes: Spun down the contents of the Random Primer Labeling kit for 30 s in microcentrifuge after thawing. Boil 9 µl of DNA (for probe) for 5 min and cool it down on ice for 1 min. Add to the DNA 1 µl each of dATP, dGTP, dTTP, 2 µl of Random Primers, 5 µl of ³²P dCTP, and lastly 1 µl of Klenow. Incubate the mix for 30 min at 37°C. After incubation, add 2 µl of Stop buffer and 20 µl of TE. Snap the tip of a microspin G 25 column and put in a 1.5-ml Eppendorf and spin for 30 s in a centrifuge inside the Laminar Hood and then run the probe through the column (see Note 18). Boil the probe for 5 min and cool it on ice for 1 min. Add 1 ml of 5× SSPE with a syringe into the probe and transfer it to the hybridizing chamber carefully. Hybridize overnight and wash sequentially with 50 ml of 0.1% SDS in 2× SSC for 20–30 min at 65°C, 50 ml of 0.5% SDS in 0.1× SSC thrice, each for 20–30 min at 65°C.
• 8Dry the membrane over a filter paper and saran wrap and tape it on a cassette. Inside the darkroom put the film on top of the membrane and expose the film at −80°C overnight or at the least 4–5 h before developing.

3.4. Isolation of Total RNA (18)

• 1Harvest cells (20–24 h) grown in the required media by pelleting down at 1,200 × g at 4°C for 10 min (see Note 19).
• 2Wash the pelleted cells with sterile PBS twice and spin down at 1,200 × g, 4°C for 5 min. Drain out the PBS on a sterile wipe and flash freeze in a dry ice – ethanol bath and set for lyophilization (see Note 20).
• 3Aliquot ~100 µl (about 50–75 mg) of lyophilized cells in a 2-ml screw cap tube and grind or smash the cells to powder form with the help of the spatula used to scoop out the lyophilized cells (see Note 21). Add 1–1.25 ml of Tri reagent. Cap the tubes properly and homogenize in Bead Beater 16 with pulses as follows 45 s thrice, 30 s once with a gap of 45 s between each cycle on ice.
• 4Incubate the tubes for 10 min at room temperature. Centrifuge for 10 min at 4°C at 8,000 × g to pellet the cell debris and unbroken cells.
• 5Transfer the supernatant to a fresh tube and add 50–60 µl of BAN (50 µl of BAN/ml of Tri reagent added) and shake vigorously for 20–30 s. Incubate for 5 min at room temperature and centrifuge at 8,000 × g at 4°C for 10 min.
• 6Transfer the aqueous phase to a new tube and add equal volume of 70% Ethanol and mix gently and properly (see Note 22).
• 7Load the aqueous phase (700 µl at a time) onto an RNeasy isolation column and spin for 30 s in a centrifuge at 8,000 × g at room temperature. If the volume exceeds 700 µl, the same column can be reloaded until the whole aqueous phase had passed through it.
• 8Discard the flow-through and wash the column with RW1 buffer provided with the kit. Discard the flow-through and wash with 500 µl of RPE twice, spin for 30 s at 8,000 × g and discard the flow-through. Transfer the RNA isolation column to a new 2-ml collection tube and spin for 2 min at 8,000 × g at room temperature.
• 9Elute the RNA in 50 µl of RNase free water in a fresh 1.5-ml Eppendorf. Re-elute the residual RNA in another aliquot of 50 µl of RNase free water in the same tube.
• 10Concentrate the RNA with the column from RNeasy MinElute Cleanup kit following instructions of the manufacturer. Elute in a final volume of 20 µl of DNase–RNase free water.

3.5. Protein Extraction from Cn

• 1These instructions assume the use of a Bead Beater 8.
• 2Streak out Cn strains of interest (e.g., wt or mutant) onto YPD agar plate and incubate at 30°C for 48 h.
• 3Pick a single colony into a 50-ml Corning Centrifuge tube containing 10 ml YPD media and allow to grow at 30°C with shaking for 24 h.
• 4Centrifuge the culture 10 min at 1,200 × g at ambient temperature (20°C), wash once with doubly distilled water and then resuspend into 7 ml doubly distilled water.
• 5Aliquot 1 ml each into 1.5-ml conical tubes with screw caps and centrifuge at 3,500 × g for 10 min at 25°C.
• 6Meanwhile prepare the lysis buffer.
• 7Following centrifugation, discard the supernatant and resuspend each pellet into 200 µl lysis buffer.
• 8Add one “cupful” glass beads (see Note 3), then vortex and place on ice.
• 9Place each tube into the beadbeater in a 4°C coldroom and beadbeat for 40 s, followed by 1 min on ice. Repeat this four times (see Note 23).
• 10Centrifuge each tube at 3,500 × g for 12 min at 4°C.
• 11Collect the supernatant and carry out Bio-Rad protein assay to determine the amount of protein.

3.6. Lipid Extraction

• 1Under sterile conditions, fill 50-ml tube with 9 ml yeast-peptone (YP) and 1 ml 20% glucose. Add a single colony of the strain of interest (in this case Cn Gcs1^REC) and incubate 48 h at 30°C, 250 rpm.
• 2Centrifuge at 1,200 × g for 10 min at 4°C. Wash pellet twice with water then resuspend in 9 ml sterile water.
• 3Count the cells after appropriate serial dilution and aliquot 5 × 10⁸ cells per tube. Centrifuge 10 min 1,200 × g at 4°C. Suction out water carefully (see Note 24).

3.6.1. Mandala Extraction (for Extraction of Inositol-Containing Phospholipids and Phosphatidylcholine) (see Note 25)

• 4Add 1.5 ml Mandala extraction buffer (19) to each tube. Vortex and sonicate 20 s each.
• 5Incubate at 60°C in a water bath for 15 min, vortex and sonicate for 20 s each then reincubate at 60°C for 15 min.
• 6Sonicate 20 s then centrifuge 10 min at 1,200 × g at 4°C. Using a glass Pasteur pipette, combine supernatant from two tubes together into a clean tube.
• 7Evaporate the solvent in the Speedvac (see Note 25).

3.6.2. Bligh and Dyer Lipid Extraction (for Determination of Neutral Lipids)

• 8Following evaporation, add 2 ml methanol and vortex. Sonicate if necessary.
• 9Add 1 ml chloroform and vortex. Ensure there is one phase, even if turbid (see Note 26).
• 10Incubate the samples at 37°C for 1 h. During this period, vortex each sample twice for 30 s.
• 11Centrifuge at 1,200 × g for 5 min at room temperature, then transfer the lower phase to a clean tube with a glass Pasteur pipette. Add 1 ml Chloroform and 1 ml water and vortex twice for 30 s each. Recentrifuge samples at 1,200 × g for 5 min at room temperature.
• 12Once again, using a glass Pasteur pipette transfer lower phase to a clean tube. Up to three tubes can be combined into one to lessen the amount of tubes being handled.
• 13Evaporate the solvent in the Speedvac (see Note 25).

3.6.3. Additional Purification Steps (e.g., Isolation of Glucosylceramide Using a Silica Column)

3.6.3.1. Silica Column Purification 1

• 14Resuspend the lipids in 1 ml chloroform/acetic acid (99:1).
• 15Wash the SepPak cartridges with 15 ml chloroform (see Note 27). Apply sample (in 1 ml) and rinse with 1.5 ml chloroform/acetic acid (99:1). Collect flow-through after 0.5 ml has been allowed to collect into waste.
• 16Add 15 ml chloroform/acetic acid (99:1) and collect 5 ml per tube.
• 17Add 15 ml acetone and collect 5 ml per tube. Evaporate acetone from these tubes in the SpeedVac then resuspend in minimum amount acetone to combine into one tube. Reevaporate (see Note 28).

3.6.4. Base Hydrolysis

• 18Add 0.5 ml chloroform, followed by 0.5 ml 0.6 M KOH in methanol to each sample. Vortex well and leave at room temperature for 1 h.
• 19Add 0.325 ml 1 M HCl followed by 0.125 ml distilled water. Vortex well then centrifuge at 1,200 × g for 10 min at room temperature. Transfer lower organic phase to a clean tube.
• 20Evaporate solvent in the SpeedVac. You should have a small dark brown pellet at this stage (see Note 25).

3.6.5. Silica Column Purification 2

• 21Resuspend the pellet in 1 ml chloroform/acetic acid (99:1). Repeat steps 16 and 17.
• 22Change eluting solvent to chloroform/methanol (95:5); add 10 ml and collect in two tubes.
• 23Change eluting solvent to chloroform/methanol (90:10); add 15 ml and collect into 3 tubes. These are the tubes that will contain glucosylceramide, the lipid of interest for this example. Evaporate solvent using a SpeedVac. Do not combine the tubes (see Note 29).
• 24Wash the column with 15 ml methanol and collect in case needed.

3.6.6. Thin Layer chromatography

• 25Prepare a 10′ × 10′ glass TLC tank by adding chloroform/methanol/water (97.5:37.5:6) to a clean, dry tank lined with white chromatography paper. Apply a thin layer of vacuum grease around the top lip of the tank to ensure a good seal (see Note 30). Leave until paper is well saturated, usually at least 5 h to overnight.
• 26Spot the soy standard onto a TLC plate 1.5 cm from the bottom using a 10 µl pipette, using 1, 2, and 3 µl in three separate lanes (equivalent to 2.5, 5, and 7.5 µg, respectively).
• 27Resuspend the dried lipid from step 24 in 30 µl chloroform/methanol (2:1), and spot either 30 µl (analytical) or 5 µl (semipreparative scale) onto the TLC plate into a fourth lane. Allow solvent to evaporate in fume hood (~1–2 min) before placing the TLC plate in the tank.
• 28Make sure the TLC tank is tightly closed. Allow the solvent front to migrate up to 1 cm from the top of the plate, before removing the plate from the tank.
• 29Dry the TLC plate in the hood at room temperature prior to placing it in another tank containing only iodine crystals to allow visualization of the lipids. Alternatively, the plate can be sprayed with resorcinol in 70% H₂SO₄, and then placed in an oven for 10 min to allow a dark purple color to develop wherever sugar moieties are located on the lipids.

3.7. In Vitro Enzyme Activity Assay

• 1This protocol describes the in vitro activity assay of Ipc1 (20) but could be adapted for assaying any enzyme from Cn. Ipc1 activity is measured by using the fluorescent ceramide analog NBD-C6-ceramide as substrate and monitoring the formation of NBD-C6-IPC, as described by Fischl et al. (21) with some modifications.
• 2Grow wt and mutant Cn strains in YPD media in a shaker incubator for 24 h at 30°C. Harvest the cells by centrifugation and wash with sterile distilled water (see Note 24).
• 3Resuspend the pellets in lysis buffer, add acid-washed glass beads for a volume equal to ¾ of the cell suspension and homogenize three times for 45 s, followed by 1 min on ice each time, using the Bead Beater 8.
• 4Centrifuge at 2,500 × g for 10 min at 4°C, then transfer the supernatant (~100 µl) to a sterile 1.5-ml microcentrifuge tube for protein quantification.
• 5Following protein determination, incubate 100 µg protein from the cell lysates for 30 min at 30°C in 50 mM bis-Tris–HCl buffer (pH 6.5) containing 1 mM phosphatidyl inositol, 5 mM Triton X-100, 1 mM MnCl₂, 5 mM MgCl₂, and 20 µM NBD-C6-ceramide in a final reaction volume of 100 µl.
• 6Terminate the reaction by addition of 0.5 ml 0.1 N HCl in methanol.
• 7Add 1 ml chloroform and 1.5 ml 1 M MgCl₂, mix well and centrifuge at 1,000 × g for 10 min to separate the phases.
• 8Analyze the chloroform-soluble product, NBD-IPC, by TLC on silica gel 60 plates (EM Science) as described above using chloroform/methanol/water (65:25:4).
• 9Identify and quantify NBD-IPC by direct fluorescence using a Molecular Dynamics 840 Storm unit.

3.8. Mass Spectrometry of Lipids (22, 23)

• 1This protocol describes MS and MS/MS of Cn glucosylcersamide but is applicable to any Cn lipid molecule.
• 2Following Bligh and Dyer extraction described above under lipid extraction, MS and MS/MS scans of glucosylceramide were carried out on a Thermo Finnigan TSQ7000 triple quadrupole mass spectrometer equipped with electrospray ionization as described in ref. 6.
• 3A 31 min method was used with A; water/0.2% formic acid/2 mM ammonium formate and B: methanol/0.2% formic acid/1 mM ammonium formate, on a 150 × 3 mm Spectra 3 µm C8SR column (Peeke Scientific) using gradient elution and addition of internal standards.
• 4Include multiple reaction monitoring (MRM) for the characteristic production m/z 276.2.
• 5Quantify Cn glucosylceramide using soy glycosylceramide (Avanti Polar lipids) for standard curve generation.
• 6Normalize mass spectral data to inorganic phosphate determination.