Leupeptin

Measurement of Bulk Autophagy by a Cargo Sequestration Assay

Nikolai Engedal, Morten Luhr, Paula Szalai, and Per O. Seglen

Abstract
The nonselective bulk sequestration of cytoplasm and its subsequent delivery to lysosomes for degradation was originally defined as autophagy or macroautophagy. However, both terms are now increasingly being applied in a generic sense to encompass the many recently described mechanisms for selective sequestration and degradation of individual cellular elements. We will therefore use the term bulk autophagy to denote the non-exclusive and largely nonselective process.
Bulk autophagy can be measured directly by a cargo sequestration assay, using a cargo marker represen- tative of total cytoplasm. The assay described here measures the sequestration and accumulation of the ubiquitous cytosolic protein lactate dehydrogenase (LDH) in the sedimentable autophagic vacuoles of cells incubated with an inhibitor of intravacuolar degradation such as bafilomycin or leupeptin. Electrodisrup- tion of the plasma membrane followed by centrifugal sedimentation of the “cell corpses” (which retain their organelles in an intact state, bound to the cytoskeleton) is a convenient, efficient, and reproducible way to separate the small fraction of sequestered, sedimentable LDH from the major pool of cytosolic LDH.

1 Introduction
Cargo assays of actual bulk-autophagic activity were introduced in the early 1980s [1–4], based on the sequestration of soluble cargo markers, initially distributed throughout the cytoplasm, into sedimentable autophagic vacuoles. The first markers were membrane-impermeant radiolabeled sugars such as [14C]sucrose and [3H]raffinose, which were introduced into isolated rat hepato- cytes by reversible electropermeabilization of the plasma membrane prior to the autophagy measurements. Later, the ubiquitous cyto- solic enzyme lactate dehydrogenase (LDH) was used as a cargo probe [5, 6], obviating the need for electropermeabilization, but necessitating treatment of the cells with an inhibitor of intravacuo- lar degradation such as leupeptin [5, 6] or bafilomycin A1 [7–9] to prevent LDH degradation during the sequestration period (Fig. 1). The largely nonselective nature of bulk autophagy during amino acid starvation has been well documented by the identical seques- tration rates measured for cytosolic enzymes with widely different half-lives [5] as well as by the identical subcellular compositions of purified autophagosomes and total cytoplasm [10].
As a rapid way of separating the small fraction of autophagically sequestered probe from the total cytoplasmic pool, the method of electrodisruption has been found to be convenient [1, 5]. By sub- mitting cells suspended in a nonionic medium (isotonic sucrose) to a strong electric pulse, the plasma membrane can be completely disrupted, while intracellular organelles (including autophagic vacuoles) remain intact (Fig. 1). The organelles are enmeshed by the cytoskeleton and can be cleanly separated from the leaked-out cytosol by centrifugal sedimentation of the coherent “cell corpses” [5–9].
For measurements of bulk autophagy in isolated rat hepato- cytes by the LDH cargo assay, leupeptin has routinely been used to prevent LDH degradation [5, 6], apparently acting both as a direct protease inhibitor and as a suppressant of the fusion between lyso- somes and prelysosomal amphisomes [11, 12]. The hepatocyte assay, which is still recommended for the study of bulk autophagy in this cell type, has been described in detail previously [5, 6], and will, therefore, not be further considered here.
However, in a variety of cultured cell lines investigated, we have found leupeptin to be relatively inefficient as an inhibitor of intra- vacuolar LDH degradation, possibly reflecting its poor cellular uptake. In contrast, bafilomycin A1, a proton pump inhibitor [13], prevents the degradation of sequestered LDH in all cell lines examined [7–9, 14]. In the routine cargo assay for bulk autophagy measurements in cultured cells described below, we have, accordingly, replaced leupeptin with bafilomycin as an inhibi- tor of LDH degradation. Other inhibitors of intravacuolar degra- dation such as concanamycin A or ammonia (10 mM NH4Cl) may also be used.

2 Materials
2.1 Chemicals
1. Accumax (Innovative Cell Technologies).
2. Bafilomycin A1.
3. Imidazole.
4. Pyruvate.
5. NADH.

2.2 Buffers and Solutions
1. Earle’s balanced salt solution (EBSS, with 0.1% glucose).
2. Phosphate-buffered saline (PBS).
3. Fetal bovine serum (10% FBS).
4. Bovine serum albumin (BSA).
5. Triton X-405.
6. Tween-20.
7. Phosphate-buffered sucrose: 100 mM sodium monopho- sphate, 2 mM EDTA, 2 mM DTT, 1.75% sucrose, pH 7.5.
8. Resuspension buffer: 50 mM sodium monophosphate, 1 mM EDTA, 1 mM DTT.
9. Pyruvate-imidazole buffer: 0.75 mM pyruvate, 65 mM imid- azole, pH 7.5.
10. NADH-imidazole buffer: 1.8 mM NADH, 65 mM imidazole, pH 7.5.

2.3 Labware
1. 12-well plates.
2. 0.4 cm electroporation cuvette.
3. 1.5 and 2 mL microcentrifuge tubes.

2.4 Instruments
1. Electroporator (e.g., BTX Harvard EMC 630).
2. Multianalyzer (MaxMat PL-II, Erba Diagnostics).

3 Method
1. Seed cells onto plates of any format and let them grow to the desired confluency in the medium chosen (see Note 1).
2. For experimental incubations, aspirate the medium, rinse the cells once with the medium or buffer to be used in the experi- ment, and add fresh medium/buffer with the additives required in the experiment (see Note 2).
3. Incubate the cells (triplicate samples for each condition tested is recommended) for the desired length of time (e.g., 3–4 h) in the presence of bafilomycin A1 (100–200 nM) (see Note 3). Always include 0-h samples (in fresh medium/buffer only) to define the background of sedimented LDH.
4. At the end of the experiment, aspirate the medium, rinse the cells once with 500 μL PBS, and incubate them at 37 ◦C with 200 μL Accumax until adherent cells have detached. Add 800 μL PBS/5% BSA and gently pipette the cells to obtain a near single-cell suspension, which is transferred to a microcentrifuge tube and kept on ice (see Note 4).
5. Centrifuge the cells at ~500 × g for 5 min at 4 ◦C.
6. Aspirate the supernatant, add 400 μL ice-cold 10% sucrose to the pellet, and gently resuspend the cells to a near single-cell suspension (see Note 5).
7. Transfer the cell suspension to a 0.4-cm electroporation cuvette and discharge a single electric pulse of ~8 ms duration (800 V, 25 μF, and 400 Ω settings in the BTX electroporator). This electric shock will cause a uniform and selective plasma membrane electrodisruption, which converts the cells to perme- able cell corpses from which cytosolic LDH leaks out (Fig. 1). However, autophagic vacuoles with sequestered LDH remain intact in situ and thus sediment readily along with the cell corpses (see Note 6).
8. Transfer the cell disruptate (400 μL) to a microcentrifuge tube containing 400 μL ice-cold phosphate-buffered sucrose. Mix by pipetting and store on ice.
9. Repeat steps 7 and 8 with each sample (one at a time).
10. To obtain a measure of the total cellular LDH content (“total LDH”) in the sample, pipette 100 μL of the diluted disruptate into a microcentrifuge tube and freeze-thaw it once at —80 ◦C (storage at —80 ◦C overnight may be convenient).
11. To measure sedimentable LDH, transfer 500 μL of the diluted disruptate to a microcentrifuge tube containing 900 μL ice-cold “resuspension buffer” with 0.5% BSA and 0.01% Tween-20. Mix briefly by pipetting and store on ice.
12. Centrifuge the mixture from the previous step at 18,000g for 45 min at 4 ◦C (see Note 7).
13. Aspirate the supernatant and freeze-thaw the pellet (“sedimen- ted LDH”) at —80 ◦C (see Note 8).
14. Thaw all frozen-stored samples on ice.
15. Dilute the “total LDH” sample (from step 10) with 300 μL ice-cold resuspension buffer containing 1.33% Triton X-405 and resuspend until the solution is homogeneous.
16. Add 400 μL ice-cold resuspension buffer containing 1% Triton X-405 to the “sedimented LDH” pellet (from step 13) and resuspend to homogeneity.
17. Centrifuge all samples at 18,000 g for 5 min at 4 ◦C (to sedi- ment debris).
18. Measure LDH activity in the “sedimented LDH” and “total LDH” samples spectrophotometrically as the decline in NADH absorbance at 340 nm (see Note 9).
19. Calculate LDH values back to the total cell sample, taking dilutions and sampling into account. Subtract the 0-h “sedi- mentable LDH” values from the final “sedimentable LDH” values and divide by each sample’s “total LDH”/100 to obtain the percentage of cellular LDH rendered sedimentable (i.e., sequestered) during the incubation period. Bulk-autophagic sequestration rates can be conveniently expressed as %/h (see Note 10).

4 Notes
1. 12-well plates with a growth surface of ~3.8 cm2 per well (and harvested at ~70% confluency) will usually yield enough cells for the cargo assay. The culture medium should be chosen to suit the cell line and experimental conditions used.
2. To measure maximal bulk-autophagic activity (i.e., capacity), the cells may be washed and incubated in an amino acid-free buffer such as EBSS (with glucose). Make sure to use bicarbonate-buffered solutions when cells are incubated in a CO2 incubator, otherwise not.
3. Bafilomycin addition defines the start of the experiment, when intravacuolar LDH degradation is stopped and autophagically sequestered LDH begins to accumulate.
4. Accumax contains DNase, which degrades “sticky” DNA and helps to maintain single-cell suspensions.
5. The supernatant should be aspirated thoroughly by suction to leave the pellet as dry as possible. Most cultured cells will form a well-packed pellet with little carry-over of medium and mini- mal cell damage, thus with insignificant transfer of ions to the subsequently added nonionic sucrose solution. However, if the cell disruption (see step 7) is incomplete (<99%), the cells should be washed once in ice-cold 10% sucrose. 6. While standard homogenization and subcellular fractionation methods can, in principle, be used to separate cytosol from the formed elements of the cell, electrodisruption is both an extremely simple and an exceptionally mild procedure which preserves the structure of intracellular organelles perfectly [5]. 7. This step may need to be adjusted to suit the cell type or cell line used. Too harsh Leupeptin centrifugation may result in too packed pellets that are difficult too solubilize, whereas too weak centri- fugation may cause incomplete cell corpse sedimentation or loose pellets with excessive carry-over of cytosolic LDH. Cen- trifugation at 18,000 g for 45 min at 4 ◦C has been shown to be suitable for most cell lines tested with the current protocol.
8. The supernatant should be aspirated thoroughly by suction to leave the pellet as dry as possible.
9. In principle, quantified immunoblots can also be used in this assay, but enzyme activity measurements are more accurate. An autoanalyzer/multianalyzer is very convenient for this pur- pose; we routinely use the MaxMat instrument. We previously used the accompanying LDH assay kit (RM LADH0126V from Erba Diagnostics), but we now make our own assay solution by mixing four parts of cold pyruvate-imidazole buffer with one part of cold NADH-imidazole buffer to obtain an imidazole working solution containing 0.6 mM pyruvate and 0.36 mM NADH. We found this NADH concentration (twice as high as in the Erba kit) to be necessary for adequate LDH measurements.
10. Bulk-autophagic sequestration rates, measured under amino acid starvation conditions, vary widely between different cell types, with approximate values of, e.g., Huh7, 0.2; U2OS, 0.6; PC3, 0.9; LNCaP, 1.7; and primary rat hepatocytes, 3–5%/h.