Biochemical procedures

2.2.3.1 Preparation of whole cell lysates from adherent cells

Whole cell lysates were prepared by resuspending previously harvested adherent cell pellets on ice in 50–150µL of lysis buffer (1.5% N-dodecyl β-D-maltoside in PBS supplemented with protease and phosphatase inhibitor), depending on pellet size. The cell suspension was transferred to 1.5ml Eppendorf tubes and lysed by rotation at 4°C for 30 minutes. The cell homogenate was then centrifuged at 16000rpm, 4°C for 20 minutes. The whole cell lysate (supernatant) was collected into new 1.5mL Eppendorf tubes and the protein concentration was determined by the Bradford Assay. In order to avoid freeze-thaw cycles, lysates were either used for further determinations or aliquoted and stored at -80°C.

2.2.3.2 Preparation of whole tissue lysates

For tissue lysates, previously cryoground tissue powder in 1.5mL Eppendorf tubes were resuspended in 250–400µL of RIPA buffer supplemented with protease and phosphatase inhibitor (depending on tissue size), vortexed briefly and lysed by rotation at 4°C for 1 hour.

Following lysis, the tissue homogenate was centrifuged at 16000rpm, 4°C for 20 minutes. The tissue lysate (supernatant) was collected into new 1.5mL Eppendorf tubes and the protein concentration was determined by the Pierce Assay. In order to avoid freeze-thaw cycles, lysates were used for further determinations or aliquoted, snap frozen in liquid nitrogen and stored at -80°C.

2.2.3.3 Protein concentration determination using Bradford assay

Protein concentrations of whole cell lysates were determined using Protein Assay Dye Reagent Concentrate (Bio-Rad) following manufacturer’s protocol. Along with standards using bovine serum albumin (BSA), the protein concentration of cell lysates was determined by mixing, in duplicates, 800µL of ddH2O, 1µL of sample or standard and 200µL of protein assay dye reagent concentrate in a glass tube and vortexed briefly. The tubes containing mixtures were incubated at RT in the dark for 5 minutes and transferred into plastic cuvettes. The absorbance at 595 nm was measured using a GeneQuant 1300 spectrophotometer. The protein concentration of the cell lysates was calculated based on the standard calibration curve using Microsoft Excel 2013.

2.2.3.4 Protein concentration determination using Pierce BCA assay

Protein concentrations of tissue lysates and purified tissue mitochondria were determined using Pierce BCA Protein Assay Kit (Bio-Rad) according to manufacturer’s instructions. Along with albumin standards, the protein concentration of tissue lysates or purified mitochondria was determined by mixing, in duplicates, 1mL of Pierce BCA working reagent (reagents A and B, 50:1) and 50µL of standards or 1:40 diluted samples in a glass tube and vortexed briefly. The tubes containing mixture were incubated at 37°C for 30 minutes in the dark and transferred into plastic cuvettes. The absorbance at 562 nm was measured using a GeneQuant 1300 spectrophotometer. The protein concentration of the cell lysates was calculated based on the standard calibration curve while taking into consideration the sample dilution factor using Microsoft Excel 2013.

2.2.3.5 SDS-PAGE

SDS-PAGE is a widely used technique to separate denatured molecules according to their weight. Briefly, using the Bio-Rad Gel preparation system, 1mm thick 12% resolving gels were made with the composition described previously in section 2.1.3, poured between the gel glass plates and covered with isopropanol to prevent air bubbles. After the gels were set, the excess isopropanol was drained and the gels were rinsed with ddH2O. The excess water was also drained and the 4% stacking gel (composition previously described) was poured on top of the polymerized resolving gel. A desired multi-well comb was quickly inserted into the stacking gel and allowed to set. The polymerized gel was used immediately or stored in a container filled

with 1X running buffer at 4°C. To run the SDS-PAGE, the Mini-Protean Tetra System (Bio-Rad) was set up with gels without the combs and filled with 1X running buffer. An equal amount of protein from cell or tissue lysates was mixed with 4µL of 6X SDS loading buffer, made up to 24µL with lysis buffer, boiled at 95°C for 5 minutes and loaded per well in a 10-well gel along with a molecular weight standard, the PageRuler plus Prestained ladder. For 15-well gels, an equal amount of protein and 3µL of 6X SDS made up to 18µL with lysis buffer instead was loaded per well after boiling at 95°C for 5 minutes. Within the stacking gel, the electrophoresis was run at an initial constant voltage of 80V and switched to constant 180V when samples were in the resolving gel and run until samples reached the bottom of the gel.

2.2.3.6 Immunoblotting

Following SDS-PAGE and in order to detect specific target proteins, Western blot was carried according to protocols described by (Gallagher et al 2011). Briefly, polyvinylidene fluoride (PVDF) membranes were rinsed for 15 sec in methanol to activate them and washed with ddH2O for 3 minutes. Previously run gels were disassembled from the gel glass plates, stacking gels were cut off and the gels along with activated PVDF membranes were equilibrated at RT in 1X transfer buffer (contains 20% methanol and cold at all times). The transfer sponges and sheets of Whatman paper were also soaked in 1X transfer buffer. Using the tank transfer system, the transfer sandwich was assembled on a plastic transfer cassette completely immersed in 1X transfer buffer to avoid trapping air bubbles. The order of the transfer assembly was as follows:

place a soaked sponge on the bottom half of the plastic transfer cassette followed by two sheets of wet Whatman paper, place gel on top of the papers and remove any air bubbles by gently rolling a roller over the gel surface, place wet PVDF membrane on top of the gel and roll over membrane surface to remove any air bubbles and place again two sheets of wet Whatman paper followed by one wet transfer sponge. The top half of the transfer cassette was locked in place and the transfer sandwich was placed in the transfer apparatus in a tank filled with 1X transfer buffer while ensuring the correct orientation (bottom half towards cathode). A cold ice pack was also placed in the tank to avoid excessive heating of the transfer buffer. The proteins were transferred from gel to PVDF membrane at constant 100V for 1.5 hours in a cold room.

2.2.3.7 Immunodetection

After complete electrotransfer of proteins onto PVDF membranes, the transfer sandwich was disassembled and the PVDF membranes were blocked in blocking buffer with mild shaking for 1 hour at RT. The membranes were then washed three times in 1X TBST for 5 minutes and labeled with primary antibodies (diluted in blocking buffer) by overnight incubation at 4 °C. The membranes were washed three times in 1X TBST for 10 minutes and incubated with appropriate HRP-conjugated secondary antibodies diluted in blocking buffer at RT for 1 hour and washed three more times in 1X TBST for 10 minutes. Signals representing proteins by chemiluminescence were detected by draining excess 1X TBST on membranes, incubating membranes in Luminata Western HRP substrate for 3 minutes and developing signals on medical X-ray films using the AGFA Curix 60 processor. The films were scanned with the Epson Perfection V850 Pro Scanner and band densities were quantified with Image J software.

2.2.3.8 BN-PAGE and respiratory chain complex activity staining

BN-PAGE, unlike SDS-PAGE, resolves non-denatured proteins thereby also preserving their physiological interactions with other proteins. As a result, the electrophoretic mobility depends not only on the charge-to-mass ratio but also on the size and shape of proteins. As this technique was developed to separate mitochondrial proteins and complexes, under low detergent conditions, respiratory chain supercomplexes are preserved, allowing for further in-gel staining of their activities (Schägger and Pfeiffer, 2000; Schägger and von Jagow, 1991).

We took advantage of this technique to characterize the role of lysosomal malfunction in mitochondrial complex assembly and function.

2.2.3.8.1 BN-PAGE: sample preparation

50µg each of previously isolated mitochondria from mouse cortices was centrifuged at 14000rpm, 2°C for 5 minutes. The supernatant was removed and the mitochondrial pellet was resuspended in 50µL of ice-cold digitonin solubilization buffer. The mitochondria were solubilized by pipetting up and down (10 times) without generating foam. The mitochondria were mixed on a shaker at RT for 10 seconds and put back on ice for 15 minutes to complete solubilization. To remove insoluble material, the samples were centrifuged at 14000rpm, 2°C for 5 minutes and the supernatant was transferred into new pre-cooled Eppendorf tubes. 5µL of 10X BN–loading dye was added to 45µL of the solubilized mitochondria and cleared by

spinning at 14000rpm, 2°C for 2 minutes. Each 50µL sample was loaded per well in a 6–16.5%

gradient gel along with a molecular weight standard.

2.2.3.8.2 BN-PAGE: casting gels and electrophoresis

For native separation of mitochondrial protein complexes by BN-PAGE, the protocol described in Dekker et al. (1997) was used. The gels were cast and run using the SE600 Ruby system.

Briefly, resolving gels were made from equal volumes of 6% and 16.5% acrylamide (stock: a mixture of 48% acrylamide and 1.5% bisacrylamide) in 1X BN–gel buffer supplemented with 0.44% APS and TEMED and cast using a gradient gel maker with pump. The stacking gel contained 4% acrylamide solution in 1X BN-gel buffer, 0.08% APS and 0.133% TEMED.

Following loading of samples in the gels, the cathode buffer was gently overlaid on the samples.

The gel was then run in a cooled gel-chamber at 15mA per gel at 100V overnight at 4°C. The cathode buffer was replaced with the anode buffer when the electrophoresis was half-way through the gel. The electrophoresis was completed just prior to the blue loading dye running out of the gel into the anode buffer.

2.2.3.8.3 BN-PAGE: enzyme activity staining

To assess the activity of complex IV of the respiratory chain, we took advantage of the COX-catalyzed reaction, which is coupled to the oxidation of 2, 2-diaminobenzidine (DAB). Briefly, electrophoresed gels were equilibrated in 50mM Potassium phosphate buffer (pH 7.4) for at least 15 minutes at RT. The gels were then incubated in 10mL of complex IV activity staining buffer (see section 2.1.3) at 30°C until the brown bands of oxidized DAB were visible. The gels were then rinsed briefly in equilibration buffer and scanned using the Epson Perfection V850 Pro Scanner. The intensity of DAB stains was quantified using Image J software.