Quantifying Drp1-Mediated Mitochondrial Fission by Immunostaining in Fixed Cells

Abstract

Dynamin-like protein 1 (Drp1) is the master regulator of mitochondrial fission. Drp1 translocates from the cytosol to the mitochondrial outer membrane to execute the scission process. Here we describe an immunofluorescence-based method to measure the mitochondrial translocation of Drp1 and quantify Drp1-related mitochondrial fission by labeling the mitochondrial import receptor subunit TOM20 in fixed cell culture.

1. Introduction

Mitochondrial fission is executed by dynamin-related protein 1 (Drp1) along with its adaptor proteins on the mitochondrial outer membrane [1, 2]. Under normal conditions, Drp1 is mainly distributed in cytosol [3]. Various cues including posttranslational modifications (e.g., phosphorylation) and protein-protein interactions, however, induce the mitochondrial translocation of Drp1 to sever mitochondrial filaments at pre-marked constriction sites [3], resulting in the generation of small and rounded mitochondrial fragments. Drp1-mediated mitochondrial fission can be examined by directly visualizing mitochondrial morphology as well as the subcellular localization of Drp1. Here we describe an immunofluorescence method for imaging mitochondria, by targeting the mitochondrial import receptor subunit TOM20, which is widely distributed on mitochondrial outer membrane. Mitochondrial translocation of Drp1 is then analyzed by immunolabeling.

2. Materials

Prepare all solutions using double-distilled water and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Follow waste disposal regulations when disposing waste materials.

2.1. Drp1 KO Mouse Embryonic Fibroblast Cell Culture

1. Culture medium: DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% (v/v) heat inactivated FBS and 1% (v/v) penicillin/streptomycin.

2. DPBS (Fisher scientific, Cat. No. SH30028FS).

3. Trypsin (Fisher scientific, Cat. No. SH303236.01).

4. Cell culture plates: 10 cm, 12- and 24-well.

2.2. Coating

1. 12 mm microscope cover glass (Fisher scientific, Cat. No. 12-545-82).

2. 2% Gelatin (Sigma, Cat. No. G1393).

2.3. Transfection

1. Opti-MEM®|reduced-serum medium (Thermo fisher scientific, Cat. No. #31985070).

2. Transfection reagent T2020 (Mirus, Cat. No. MIR5406) (see Note 1).

2.4. Immunofluorescence Staining of Fixed Cell

1. Paraformaldehyde 16% (TED PELLA, Cat. No. 18505).

2. PBS (see Note 2).

3. Triton X-100 (Fisher scientific, Cat. No. BP-151-500).

4. Normal goat serum (Invitrogen, Cat. No. 10000C).

5. Hoechst 33342 (Invitrogen, Cat. No. H21492).

6. Microscope slides (Fisher scientific, Cat. No. 12-544-2).

2.5. Examine the Expression of Myc-Drp1 by Western Blot

1. Total cell lysis buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail.

2. 5% stacking gel.

3. 10% resolving gel.

4. SDS-PAGE running buffer.

5. 2× sample loading buffer (reducing): Add 5 mL 1 M Tris (pH 7), 25 mL 20% SDS, 20 mL glycerol, 2 mg bromophenol blue, 50 μL β-mercaptoethanol, and water up to 100 mL.

6. Nitrocellulose membranes.

7. Transfer buffer.

8. Tris-buffered saline (TBS).

9. TBS containing 0.05% Tween-20 (Anatrace, Cat. No. T1003) (TBST).

10. Blocking solution: 5% milk in TBST. Store at 4 °C.

11. ECL solution: Add 12.2 μL H₂O₂ (10 M), 200 μL luminol (250 mM), 80 μL coumaric acid (90 mM), 4 mL Tris-HCl (1 M, pH 8.5), and water up to 40 mL. Store at 4 °C (see Note 3).

2.6. Antibodies

1. Mouse anti-c-Myc antibody (Santa Cruz, Cat. No. sc-40).

2. Rabbit anti-Tom20 antibody (Santa Cruz, Cat. No. 11415).

3. Anti-β-Actin antibody (Sigma-Aldrich, Cat. No. A1978).

4. Goat anti-mouse HRP (Thermo Scientific/Fisher, Cat. No. 31430).

5. Alexa 488, goat anti-rabbit IgG (H + L) (Invitrogen, A11034).

3. Methods

Carry out all procedures at room temperature unless otherwise specified. Carry out all of the cell culture studies in cell culture hood. All cells mentioned below are maintained in an incubator at 37 °C in 5% CO₂.

3.1. Restore Drp1 KO Mouse Embryonic Fibroblast

1. Restore a vial of Drp1 knock out (KO) mouse embryonic fibroblast (MEF) [4] from liquid nitrogen or −80 °C.

2. After completely thawing, transfer the cells to a 15 mL tube. Add 10 mL culture medium and gently pipette several times (see Note 4).

3. Spin down the cells under 1000 rpm (or 400 × g) for 5 min.

4. Aspirate the medium and resuspend the cell pellet in 1 mL fresh culture medium. Transfer the cell to a 10 cm cell culture plate with a total of 10 mL medium (see Note 4) and incubate for 24 h at 37 °C in 5% CO₂.

5. Change the culture medium after 24 h.

6. Split the cells once it reaches 80% confluence.

3.2. Splitting Cells

1. Aspirate the culture medium and wash the cells once with warmed DPBS.

2. Add 1 mL trypsin to the plate and incubate for 3–5 min until most of the cells are detached.

3. Add 5 mL culture medium to the plate and gently pipette down the cells. Transfer the cells to a 15 mL tube (see Note 4).

4. Spin down the cells at 1000 rpm (or 400 × g) for 5 min.

5. Aspirate the medium above the cell pellet in the tube and resuspend the cell pellet in 1 mL culture medium (see Note 4).

6. Transfer the cells into a new culture plate.

3.3. Coating

1. Put the glass coverslips in 24-well plate (see Note 5).

2. Sterilize the plate with coverslips under UV inside the cell culture hood for 30 min.

3. Add 500 μL coating solution made of 0.1% gelatin in water in each well to fully cover/merge the glass coverslips.

4. Incubate the 24-well plate under 37 °C for 1 h or under 4 °C overnight.

5. Aspirate out the coating solution and wash with PBS three times before plating the cells on coverslips.

6. Split the Drp1 KO MEF cells and plate 40,000 cells in each well.

3.4. Transfection

1. Transfect cells with empty control vector or Myc-Drp1 vector [5] on the second day following split as detailed below (see Note 1).

2. For transfection of cells in each well of the 24-well plate, add 50 μL Opti-MEM®|reduced-serum medium to a sterile 1.5 mL Eppendorf tube.

3. Add 0.5 μg of plasmid DNA to the tube.

4. Add 1.5 μL transfection reagent T2020 to the tube and mix by pipetting.

5. Let the tube incubate in the cell culture hood for 25 min.

6. Transfer the medium in the Eppendorf tube to a well (see Note 6).

7. Cells are ready for immunofluorescence staining 48 h after transfection.

3.5. Immunofluorescence Staining of Mitochondria in Fixed Cell

1. Wash the cells with PBS once.

2. Fix the cell with 4% paraformaldehyde diluted in PBS for 20 min.

3. Wash the cells with PBS three times.

4. Incubate the fixed cells with 0.1% Triton-X-100 diluted in PBS for 5 min.

5. Take out the 0.1% Triton-X-100 and incubate the cells in blocking buffer (2% normal goat serum and 0.05% Triton X-100 in PBS) for 1 h.

6. Take out the blocking buffer and add the primary antibodies, anti-Tom20 and anti-Myc antibodies, diluted in blocking buffer (1:1000). Incubate overnight at 4 °C or 2 h at room temperature.

7. Wash the cell with PBS three times.

8. Add the secondary antibody, Alexa 488 anti-rabbit IgG and Alexa 568 anti-mouse IgG, diluted in blocking buffer (1:1000). Incubate for 2 h in dark.

9. Wash the cell with PBS three times.

10. Add Hoechst for nuclei staining for 10 min.

11. Wash the cell with PBS three times.

12. Add one droplet of mounting solution on a cover slide. Use tweezers to put the coverslip upside down on the mounting solution, and avoid any bubbles if possible.

13. Dry out the slides overnight at 4 °C in the dark.

14. Use 40× magnification on a fluorescence microscope (e.g., Olympus FV1000 confocal microscope) to observe mitochondrial morphology and Myc-Drp1 distribution the next day as illustrated in Figs. 1 and 2.

Fig. 1.

Treatment with small peptide inhibitor P110 prevented mitochondrial fragmentation in mouse striatal HdhQ111 cells (HdhQ7-control cell, HdhQ111-mutant cell). Tom20 in green. (Reproduced from Guo X et al., Inhibition of mitochondrial fragmentation diminishes Huntington’s disease-associated neurodegeneration, J Clin Invest, 123, 5371–5388 (2013))

Fig. 2.

Specific small peptide inhibitor P110 prevents the mitochondrial translocation of Drp1 and mitochondrial fragmentation in fibroblasts derived from Huntington’s disease (HD) patients (Con-control subject, HD1/2-HD patients). Drp1 in red and Tom20 in green. (Reproduced from Guo X et al., Inhibition of mitochondrial fragmentation diminishes Huntington’s disease-associated neurodegeneration, J Clin Invest, 123, 5371–5388 (2013))

3.6. Quantification of Mitochondrial Fragmentation

1. Count the total number of cells immune-positive for anti-Myc antibody, which indicate the Myc-Drp1 expressing cells.

2. Count the total number of Myc-positive cells with fragmented mitochondria among the cells counted in step 1 as below.

3. Count at least 100 cells. The percentage of Myc-positive cells with fragmented mitochondrial can be calculated as number of Myc-positive cells with fragmented mitochondria over total number of Myc-positive cells (see Note 7).

3.7. Examine the Expression Level of Myc-Drp1 in Cells by Western Blot

1. Split the Drp1 KO MEFs into a 12-well plate with 200,000 cells per well.

2. On the second day, transfect the cell with 1 μg Myc-Drp1 vector using 3 μL transfection reagent following the procedures as indicated in Subheading 3.4.

3. After 48 h transfection, gently wash the cells with cold PBS twice.

4. Incubate cells on ice for 30 min in total cell lysis buffer.

5. Collect the cells and spin for 10 min at 12,000 rpm (or 13,523 × g) in a table top centrifuge set at 4 °C.

6. Keep the supernatant as total cell lysates.

7. Measure the protein concentration of the total cell lysates and prepare equal amount of proteins for load in each group (see Note 8).

8. Prepare the sample using 1 × reducing sample loading buffer and boil at 100 °C for 10 min.

9. Run SDS-PAGE.

10. Transfer the protein onto nitrocellulose membranes using standard western blotting procedures.

11. Rinse the membrane once with TBST.

12. Block the membrane with blocking solution for 1 h.

13. Cut the membrane at 50 kDa. Incubate the top half of the membrane with anti-c-Myc antibody diluted in blocking solution (1:1000) and bottom half of the membrane with anti-actin antibody at 4 °C overnight.

14. Wash the membranes with TBST for 5 min each for a total of four times.

15. Incubate the membranes with the goat anti-mouse antibody diluted in blocking solution (1:5000) for 1 h.

16. Wash the membrane with TBST for 5 min each for a total of four times.

17. Incubate the membrane with ECL solution for 2 min.

18. Develop the membrane.

19. The band shown around 75 kDa is Myc-Drp1 and the band shown around 37 kDa is actin. The relative level of Drp1 is determined as the ratio of Myc-Drp1 density over actin density.