Generation of Drosophila Heparan Sulfate Mutant Cell Lines from Existing Fly Strains

Abstract

Genetic studies using a model organism, Drosophila melanogaster, have been contributing to elucidating the in vivo functions of heparan sulfate proteoglycans (HSPGs). On the other hand, biochemical analysis of Drosophila glycosaminoglycans (GAGs) has been limited, mainly due to the insufficient amount of the material obtained from the animal. Recently, a novel in vitro system has been developed by establishing mutant cell lines for heparan sulfate (HS)-modifying enzyme genes. Metabolic radiolabeling of GAGs allows us to assess uncharacterized features of Drosophila GAGs and the effects of the mutations on HS structures and function. The novel in vitro system will provide us with a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic data obtained over the last two decades using this animal model.

1. Introduction

In vivo studies using a model organism, Drosophila have helped define functions of heparan sulfate proteoglycans (HSPGs) during development [1]. Drosophila has the complete set of heparan sulfate (HS) biosynthetic enzymes found in mammalian species and produces complex HS structures that are equivalent to mammalian HS. Drosophila has only one gene for each of these enzymes. This absence of genetic redundancy is advantageous to interpret phenotypic data. Furthermore, a number of genetic tools (mutations, RNAi transgenic animals, and overexpression constructs) for a complete set of genes of the HS biosynthetic machinery have been generated. These tools in combination with sophisticated molecular genetic techniques in this model enable us to manipulate HSPGs in vivo in a temporally and spatially controlled manner [2, 3].

Despite the many strengths of the Drosophila model for in vivo studies, information on Drosophila glycosaminoglycan (GAG) structure has been limited. Namely, it has been a challenge to determine several HS structural features, such as molecular size, net charge, domain organization, N-sulfation pattern, and the amount/distribution of iduronic acid residues. This is mainly due to the difficulty of metabolic radiolabeling of HS in vivo using Drosophila animals. One approach to overcome this issue is to establish an in vitro system using Drosophila cell lines. Recently, an efficient genetic method for generating continuous cell lines from already existing Drosophila strains has been developed [4-6]. The method uses expression of Ras^V12, a constitutively activated form of the oncogene Ras, to induce embryonic primary culture cells to progress to permanent cell lines. Using this technique, we are now able to establish cell lines that are mutant for any HSPG or HS biosynthetic genes [7].

The current method uses actin-Gal4, a ubiquitous Gal4 driver, which induces Ras^V12 expression in most cells of the embryo [5]. Therefore, this method should be able to create cell lines of various cell types. However, morphological and immunological analyses of the established lines have shown that many of them were derived from mesodermal origin [5, 7]. This probably reflects the proliferative advantage of this cell type under the condition of the current protocol. Development of modified protocols (e.g., the use of cell type-specific Gal4 drivers) should be able to facilitate establishing cell lines of desired cell types [4]. Control mesodermal cell lines (Ras-RMCE cells), which bear actin-Gal4/UAS-Ras^V12 but no other mutation, can be obtained from The Drosophila Genomics Resource Center. In this chapter, these cells are simply referred to as “wild-type.”

The established mutant cell lines are useful for metabolic radiolabeling of GAG for structural analysis [7]. GAG chains are often labeled with ³⁵S (Na₂³⁵SO₄). However, tissue culture media for insect cell lines, including Schneider’s Insect Medium and Shields and Sang M3 insect medium, contain very high levels of sulfate (e.g., 15.0 mM MgSO₄ in M3 medium), which makes it difficult to metabolically label the cells with ³⁵S-sulfate. To overcome this, one can use a custom-ordered, M3 insect medium without MgSO₄ (Life Technologies), to which a minimal amount of MgSO₄ (final concentration 0.15 mM) is supplemented [7]. The growth and health of Drosophila cells in this medium are indistinguishable from those using intact M3 medium.

These cell lines are also useful for cell signaling assays [7]. Bone morphogenetic proteins (BMPs) are one class of well-established HS-dependent growth factors. For example, Decapentaplegic (Dpp), one Drosophila ortholog of BMPs, requires an HSPG co-receptor, Dally, for its signal transduction in vitro and in vivo [8-13]. A library of HS mutant cell lines will allow us to study the structural features of GAGs required for specific signaling events, such as the pathways mediated by BMPs, FGFs, Wnt/wingless, and hedgehog.

In summary, these cells will offer an excellent system to study the mechanisms of HS biosynthesis, HS–protein interactions, and the impact of GAG structural modifications on signaling events. Thus, this novel in vitro system nicely complements Drosophila genetics, making the Drosophila model a highly unique and powerful platform to understand the structure–function relationship of HS.

2. Materials

2.1. Fly Strains and Egg Collection

1. Drosophila strains: actin-Gal4 (Bloomington stock number: 25374, 3954).

2. UAS-Ras^V12 (Bloomington stock number: 64196, 64195).

3. Incubator set at 25 °C.

2.2. Cell Culture

1. M3 insect medium (Sigma).

2. Grape juice agar plates (see Note 1).

3. Embryo collection cages (see Note 2).

4. Nylon mesh filter assembly (egg basket).

5. Egg rinsing solution (0.7% NaCl, 0.02% w/v Triton-X 100).

6. Bleach (diluted to 50% before use).

7. 70% ethanol.

8. 5-ml glass homogenizer.

9. 50-ml T-flask (Falcon 3014).

2.3. Immunostaining

1. PBS (phosphate-buffered saline).

2. PBT (0.05% w/v Triton X-100 in PBS).

3. 4% paraformaldehyde (freshly prepared).

4. Normal goat serum.

5. Appropriate primary antibodies (e.g., rabbit anti-dMef2 (a gift from B. Paterson), a mesoderm marker; rat anti-E-cadherin (Developmental Studies Hybridoma Bank), an epithelial marker; mouse 22C10/anti-Futsch (Developmental Studies Hybridoma Bank), a neuronal marker).

6. Phalloidin-Alexa Fluor 633 (Thermo Fisher Scientific).

7. Appropriate secondary antibodies (e.g., the AlexaFluor series, Thermo Fisher Scientific).

8. Moist chamber (60 mm plastic dish with wet filter paper).

9. Coverslips (12 mm Round, Warner Instruments), washed with HCl.

10. Vectashield mounting medium with DAPI (Vector Laboratory).

11. Fluorescence microscope or confocal microscope.

2.4. Radio-Labeling and Glycosaminoglycan Structural Analyses

1. A custom-ordered, M3 insect medium without MgSO₄ (Life Technologies).

2. 50 μCi/ml Na₂³⁵SO₄ (specific activity, 1500 Ci/mmol; Perki-nElmer Life Sciences).

3. Extraction buffer (50 mM Tris–HCl, pH 7.4, 1% Triton X-100).

4. Complete protease inhibitor cocktail (Roche).

5. DEAE-Sephacel column (2 ml, GE Healthcare).

6. DEAE washing buffer (50 mM NaAc, pH 4.5, 0.25 M NaCl).

7. DEAE elution buffer (50 mM NaAc, pH 4.5, 1.5 M NaCl).

8. PD-10 column (GE Healthcare).

9. 0.5 N NaOH.

10. HNO₂ at pH 1.5.

2.5. Dpp Signaling Assay

1. Drosophila S2 cells.

2. Dpp-expression construct (pAW-HA-dpp) [12, 14].

3. Recombinant Dpp peptide (R&D systems).

4. Rabbit anti-pMad antibody (EP823Y, 1:2000, Epitomics).

5. Mouse anti-α-tubulin antibody (DM1A, Sigma).

6. Secondary antibodies (anti-rabbit or mouse IgG conjugated with horseradish peroxidase, Sigma).

7. ECL-Plus (GE Healthcare Life Sciences).

3. Methods

3.1. Preparation of Parental Fly Strains

1. Generate two recombinant chromosomes: one with a mutation of your interest (*) and an actin-Gal4 transgene, and another with this mutation (*) and a UAS-Ras^V12 transgene. These two (* actin-Gal4 and * UAS-Ras^V12) should be on the same chromosome (X, second, or third), and maintained with an appropriate balancer chromosome.

2. Set up a cross between these lines to obtain embryos with four different genotypes (Fig. 1a).

Fig. 1.

Establishment of HS mutant cell lines. (a) Genetic crosses to obtain a mutant cell line. The mutation is shown by the asterisk (*). A cross between * Act-Gal4 and * UAS-Ras^V12 will yield a mixture of embryos with four genotypes. Among these, only homozygous mutant embryos bear both Act-Gal4 and UAS-Ras^V12 transgenes (boxed), thus expressing Ras^V12. After embryos with mixed genotypes are homogenized and primary cultures are plated, only homozygous mutant cells survive during early passages. (b) A light microscope image of wild-type cells. (c) A confocal image showing anti-dMef2 antibody staining of wild-type cells. The cells show spindle-shaped morphology and a high level of nuclear dMef2 staining (green). Nucleus and cell cortex are stained with DAPI (blue) and Phalloidin (red), respectively. (This figure is modified from [7])

3.2. Establishment of Cell Lines

Establishment of cell lines from the Drosophila embryos is described in the original protocol [4-6]. We made some minor modifications.

1. Collect embryos overnight from the cross using embryo collection cages with grape juice agar plates. If there are hatched larvae, remove all with forceps. The larvae are the cause of yeast contamination in the primary culture.

2. Add some distilled water in the agar plate and brush the embryos gently into the suspension. Pour the water containing the embryos into the egg basket. Wash with copious amounts of distilled water using a squirt bottle to get rid of the yeast paste.

3. Transfer the embryos into ice-cold egg rinsing solution in a petri dish. Wash the embryos by pipetting with 1 ml Pipetman. Check again to confirm that there is no hatched larva under dissection microscope (see Note 3).

4. Transfer the embryos into a 15-ml conical tube. After the embryos sink to the bottom, remove the supernatant. Add 5 ml of ice-cold egg rinsing solution.

5. Bring the tube inside the clean bench and remove the supernatant. Add 5 ml of 50% commercial bleach solution. Cap the tube and gently invert the tube three times.

6. Incubate 3 min. This treatment removes the chorion (egg-shell). Do not overexpose embryos to bleach, or they will be damaged.

7. Remove the supernatant and wash the embryos with egg rinsing solution two times.

8. Sterilize the surface of the dechlorinated embryos by rinsing with 70% ethanol. Wash the embryos with egg rinsing solution two times.

9. Take approximately 50–100 μl of the packed volume of the embryos into a 5-ml glass homogenizer. Wash the embryo once with water and once with M3 medium supplemented with 10% FBS. Add 3 ml of the medium and homogenize them with three gentle strokes.

10. Centrifuge the homogenate at 1400 × g for 2 min at room temperature.

11. Wash the pellet with the medium two times.

12. Suspend the washed embryos in 3 ml of the medium and culture in a T-flask at 25 °C.

13. Change the medium after 10 days for the first time, and thereafter split the cells in half when the culture reaches 70% confluency (Fig. 1B).

14. After a few passages (approximately 4 weeks of culture), confirm the establishment of null mutant cell lines by PCR. Use primer sets that specifically detect wild-type alleles for the genomic region of each gene, but not mutant alleles.

3.3. Immunostaining of Cell Lines

To identify the cellular origin of the established mutant cell lines, the cells can be stained with a panel of antibodies. The following antibodies are commonly used:anti-dMef2 antibody (a mesodermal marker), anti-E-cadherin (an epithelial marker), and 22C10 (anti-Futsch, a neuronal marker).

1. Wash coverslips (12 mm round) by incubating in HCl overnight.

2. Seed cells on the coverslips placed in a 35-mm culture dish and incubated at 25 °C overnight.

3. After the cells were washed once with PBS, fix the cells in 4% paraformaldehyde for 25 min.

4. After washes with PBT (0.05% Triton X-100 in PBS), block the fixed cells in 5% normal goat serum in PBT for 1 h at room temperature.

5. Apply primary antibodies (e.g., anti-dMef2 antibody) in a 20 μl solution onto a coverslip and incubate for 2 h at 25 °C in a moist chamber.

6. Wash the cells with PBT.

7. Incubate with secondary antibodies (1:500) and Phalloidin-Alexa Fluor 633 (ThermoFisher Scientific; 1:500) for 1 h.

8. After extensive washes with PBT, mount the cells using Vectashield mounting medium with DAPI.

9. Observe and image the samples under fluorescence or confocal microscope (Fig. 1c).

3.4. Radiolabeling and Structural Analyses of Glycosaminoglycan Chains

Metabolic ³⁵S-labeling and characterization of glycosaminoglycan chains are described in previous publications [15-17].

1. Culture the cells in M3 medium without sulfate supplemented with 10% FBS and 0.15 mM MgSO₄ to 95% confluency.

2. Add 50 μCi/ml Na₂³⁵SO₄ (specific activity, 1500 Ci/mmol) and incubate the cells for 6–24 h.

3. Separate the cells from the medium.

4. After washing the cells, solubilize the cells in extraction buffer with protease inhibitor cocktail at 4 °C for 1 h under gentle shaking.

5. Purify ³⁵S-labeled macromolecules in medium and cell lysate fractions by DEAE-ion exchange chromatography. Following extensive washing with DEAE washing buffer, elute bound materials with DEAE elution buffer. Desalt eluates on a PD-10 column and lyophilize. Dissolve samples in 50 mM Tris-HCl, pH 8.0, 30 μM Na-acetate, 0.1 mg/ml BSA.

6. To release the GAG chains, treat the cell lysate and medium fractions with 0.5 N NaOH on ice overnight.

7. To identify HS fraction in the ³⁵S-labeled macromolecules, treat the cell lysate and medium fractions with HNO₂ at pH 1.5. HS is susceptible to treatment with nitrous acid.

8. Separate the samples by gel chromatography on Superose 6 before or after treatment with alkali and nitrous acid (Fig. 2).

Fig. 2.

Gel chromatography on Superose 6 of ³⁵S-labeled PGs/GAGs from wild-type cells. Samples obtained from medium (top) or cell lysate (bottom) were analyzed directly (open circle), after alkali treatment (open square), or after treatment with both alkali and nitrous acid at pH 1.5 (filled triangle). Fractions of 0.5 ml were collected and analyzed by scintillation counting. More than 90% of the ³⁵S-labeled macromolecules isolated from cell fraction were susceptible to treatment with nitrous acid, demonstrating their HS nature. (This figure is modified from [7])

3.5. Dpp Signaling Assay

Cell-based Dpp signaling assay using Drosophila S2 cells has been previously described [11, 12]. This method detects phosphorylation of Mad protein, as a direct readout of Dpp signaling (Fig. 3). Unlike S2 cells, Ras-RMCE cells endogenously express Mad [7]. Therefore, there is no need to transfect a Mad-expressing construct.

Fig. 3.

Dpp signaling assay using Hs6st mutant cell line. (a) Wild-type and Hs6st mutant cells were treated with Dpp-containing conditioned medium for 1 h at 25 °C (+). Control cells (−) were treated with conditioned medium of S2 cells without a Dpp transgene. Dpp signaling was measured by immunoblot analysis of the cell lysate using anti-pMad antibody (pMad). α-Tubulin staining was used as an internal control (Tub). (This figure is modified from [7])

1. Prepare Dpp-containing conditioned medium from Drosophila S2 cells expressing Dpp (transfected with pAW-HA-dpp). Control-conditioned medium should be prepared from S2 cells without pAW-HA-dpp transfection. Alternatively, a recombinant Dpp peptide (commercially available, R&D systems) can also be used. Addition of relatively low levels of Dpp (approximately 2 × 10⁻⁹ M) can stimulate phosphorylation of Mad in wild-type cells [7].

2. Incubate wild-type and mutant cells in the Dpp-containing conditioned medium at 25 °C for 1 h.

3. Spin down the cells and lyse in SDS sample buffer.

4. Assay Dpp signaling by immunoblot analysis using rabbit anti-pMad antibody. Mouse anti-α-tubulin antibody can be used for a loading control.

5. Incubate the blot with secondary antibodies (anti-rabbit or mouse IgG conjugated with horseradish peroxidase).

6. Detect signals using ECL-Plus (Fig. 3).

4. Notes

1. To make grape juice agar plates (76 plates), prepare the following solutions.

   Solution A: 12.5 g glucose, 125 ml grape juice.

   Solution B: 3.5 g agar, 125 ml water (heat with stirring until dissolved).

   Tegosept solution: 2.25 g Tegosept, 9.4 ml ethanol.

   1. Add Solution A to Solution B and cool it down until 60 °C with stirring.
   2. Add Tegosept solution and stir.
   3. Pour it into 60 mm × 5 mm petri dishes.

2. Homemade embryo collection cages are made from 100-ml plastic beaker. A hundred of holes were made with a needle in the bottom and the wall of the plastic beaker.

3. The complete removal of hatched larvae is essential to avoid yeast contamination.