TITLE: Isolation of lysosomes using the Tagless LysoIP method in PBMCs
AUTHORS: Daniel Saarela, Esther Sammler, Dario R Alessi, Francesca Tonelli

[DESCRIPTION]
Molecular homeostasis in cells is regulated in part by protein degradation, which is facilitated by the proteasome and lysosomal proteolysis. Lysosomes are membrane bound organelles involved in the turnover of proteins, metabolites and lipids. Recent literature implicates lysosomal dysfunction to be a feature of many a disease, including neurodegenerative diseases. Focused investigation of lysosomal content (proteome/lipidome/metabolome) in disease states could lead to the discovery of novel therapeutics and disease mechanisms. Here we describe our method to isolate peripheral blood mononuclear cells (PBMCs) and perform rapid isolation of intact, tag-less lysosomes from PBMC homogenates using ball-bearing cell breakers and anti-TMEM192 -antibody coupled to magnetic beads. First, cells are broken with physical sheering force as the cell suspension passes through a narrow gap within the cell-breaker, leading to plasma membrane rupture but due to their small size, lysosomes remain intact (Figure 1). Then, the cell homogenate is incubated with the antibody-coupled magnetic beads to allow for rapid immunopurification of lysosomes by binding to the transmembrane protein of lysosomes TMEM192 (Figure 3). The purified lysosomes can be processed and analysed with a variety of techniques including immunoblotting analysis, proteomic/lipidomic/metabolomic tools, fluorescence-activated cell sorting and GCase activity assay. The immunopurification protocol is very fast, less than 15 minutes from start of the incubation with the beads to washed, pure lysosomes. The same protocol can also be used to immunopurify lysosomes from commonly cultured cells such as mouse embryonic fibroblast, HEK293 and A549 cells.


[GUIDELINES]
Supporting figures

Figure 1

 
Graphical overview of the workflow of PBMC isolation and homogenisation. Note that images are not to scale, and the homogenising is performed with a different instrument, as outlined in Figure 2.


 
Figure 2.
 
(A) Isobiotec cell-breaker with one side panel open and a 10 μm-clearance metal ball with its container.
(B) Example of assembled cell-breaker being used to homogenise cells on ice.  
  
Figure 3.
 


 
(A) Graphic describing the design of anti-TMEM192 antibody-bound beads attached to a lysosome.
(B) Beads are incubated with cell homogenate containing lysosomes for 5 min. After immunoprecipitation, the beads are immobilised with magnets and washed with KPBS. Lysosomes attached to the beads are lysed and the supernatant containing lysosomal contents is collected for analysis.

Figure 4.
 
Immunoblot analysis confirming enrichment of lysosomes with Tagless Lyso-IP in PBMCs. PBMCs were treated with 0.5 mM diisopropylfluorophosphate (DIFP) to prevent rapid degradation of proteins. Whole-cell lysates (2 µg) as well as the resuspended immunoprecipitates (IPs) (2 µg) were subjected to immunoblotting with the lysosomal (LAMP1, TMEM55B, LAMTOR1), Golgi (GM130), cytosolic (α-tubulin, GAPDH) and mitochondrial (HSP60) markers. The data shown is from 3 healthy male donors.  
 
 





[STEPS]
SECTION: Methods
1. 
 
Prepare a 0.5 Molarity (M) stock solution of DIFP by diluting in isopropanol under a fume hood. Stock solution can be stored in -80 °C until needed.
 

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2. Collect blood into a BD Vacutainer sodium heparin 10-mL tubes. 
 

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3. Add density gradient medium to the SepMate tube using a 20-mL syringe with a large bore needle (at least 21G) by carefully dispensing 15 mL through the central hole of the SepMate insert. 
 

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4. Transfer the blood from the BD Sodium heparin tubes into a 50-mL Falcon tubes.
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5. Dilute the blood by adding an equivalent volume of PBS containing 2% (v/v) FBS to achieve a 1:1 ratio. Mix gently by inversion.
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6. Gently pipette the blood into the SepMate tube containing the density gradient medium.
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7. Centrifuge for 1200 x g, 10 min, 4 °C.

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8. Decant the top layer (containing PBMCs) to a new 50-ml Falcon tube, leaving the red bottom layer in the tube. 
 

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9. Top up the Falcon tube from Step 8 with PBS up to 50 mL and centrifuge the cells at 450 x g, 5 min, 4 °C. Discard the supernatant.
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10. Repeat Step 9: Top up the Falcon tube from Step 8 with PBS up to 50 mL and centrifuge the cells at 450 x g, 5 min, 4 °C. Discard the supernatant.
 

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11. After last wash with PBS, resuspend the pellet in 1 mL KPBS and transfer to a low-binding Eppendorf tube.

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12. 
 
Aliquot 100 µL of pre-prepared TMEM192-beads per IP into a fresh Eppendorf tube. 
 

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13. Immobilize the beads by placing the tubes from Step 12 into a Dyna-Mag tube holder for 30 s.

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14. Remove the supernatant using a pipette and gently resuspend the beads in 1 mL of KPBS.

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15. Repeat Step 13: Immobilize the beads by placing the tubes from Step 12 into a Dyna-Mag tube holder for 30 s.
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16. Remove the supernatant using a pipette.
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17. Gently resuspend the beads in n x 100 µL of KPBS, where n = number of samples to be subjected to immunoprecipitation.

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18. Aliquot the beads from Step 17 into fresh low-binding Eppendorf tubes (100 µL of slurry each). Leave the tubes on ice until use (Step 27).

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19. Centrifuge the cells from Step 11 at 1500 x g, 2 min, 4 °C to compare total cell pellets between samples. Note down the size and colouring of the pellets. 
 

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20. Resuspend the cells in 800 µL KPBS and add DIFP to a final concentration of 0.5 millimolar (mM) (1:1000 dilution of stock solution). Leave the tubes on ice.
 


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21. Transfer 50 µL of whole cell sample (WC) from Step 20 to a fresh Eppendorf tube and place on ice.

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22. To prepare the Isobiotec cell-breaker, assemble it by inserting the ball with the size of your choosing inside the machine and screw the lids on tightly. For PBMCs 12 μm gap is effective. Place on aluminium foil on ice and push 3 mL of KPBS through the machine to wash it. Carefully tap dry. 
 

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23. Transfer the PBMCs from Step 20 into a KPBS rinsed, ice-cold Isobiotec cell-breaker with gap-size of 12 μm using a 1-ml syringe and 21G needle. Homogenise the cells with 15-20 passes through the cell breaker using two 1-ml syringes.
 

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24. Collect the homogenate from the cell breaker into a fresh Eppendorf tube using a 1-ml syringe. 
 

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25. Centrifuge at 1500 x g, 2 min, 4 °C.
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26. Transfer the supernatant to a fresh Eppendorf tube on ice. 
 

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27. Add the supernatant from Step 26 to 100 µL of the prewashed beads (from Step 18). Mix by pipetting gently three times, then place on a belly-dancer orbiter for 5 min at 4 °C. 
 

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28. 
 
Place the IPs from Step 27 on a Dyna-Mag tube holder for 30 s to immobilise the beads. Discard the supernatant or collect as flowthrough sample. 
 

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29. Resuspend the beads from Step 28 in 1 mL of KBPS and immobilise the beads by placing the tubes in a Dyna-Mag tube holder for 30 s. Discard the supernatant.

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30. Repeat Step 29: Resuspend the beads in 1 mL of KBPS and immobilise the beads by placing the tubes in a Dyna-Mag tube holder for 30 s. Discard the supernatant.
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31. Resuspend the beads in 1 mL of KPBS and transfer to a new Eppendorf tube on ice.

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32. Place the tubes in a Dyna-Mag tube holder for 30 s. Discard the supernatant.

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33. The lysosomes attached to the dry beads from Step 32 can now be either eluted off the beads with SDS or Triton-X100 based lysis buffer or stored as they are at -80 °C for later use. Alternatively, they can be immediately processed for:
Immunoblotting analysis
Lipidomic analysis
Metabolomic analysis
Proteomic analysis

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34. 
 
Open the cell-breaker from one side.
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35. Take the metal ball out and rinse with MillIQ-water.
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36. Flush the cell breaker thoroughly with MilliQ-water. 
 

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37. Reassemble the cell-breaker by re-inserting the metal ball into the instrument and close the side panel tightly using the screws.
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38. Flush the cell breaker through the syringe inlets with 5 mL of KPBS for both inlets using 5-ml syringes. 
 


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39. Proceed to homogenise the next sample.
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40. Once done using the homogeniser for the experiment, repeat Step 36.
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41. Take all pieces apart (both side panels, panel screws and the metal ball).
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42. Clean each part with generous amount of 70% (v/v) ethanol in MilliQ-water.
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43. Wipe all parts dry and leave pieces apart to air-dry 30 s.