TITLE: Generation of stable LysoTag expressing cell lines and LysoTag immunoprecipitation of lysosomes
AUTHORS: Daniel Saarela, Dario Alessi

[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 how to produce stable LysoTag expressing cell lines and how to perform rapid isolation of lysosomes in cultured cells with immunoprecipitation of the LysoTag using HA-coupled magnetic beads. The immunoprecipitation protocol is very fast, less than 15min from start of the incubation with the beads. The protocol can be used to immunoprecipitate lysosomes from commonly cultured cells such as mouse embryonic fibroblast, HEK293 and A549 cells expressing the LysoTag.

[STEPS]
SECTION: Packaging LysoTag and HA-Empty (Mock) construct
3. Prepare a transfection mix to generate LysoTag expressing lentivirus in 1.5ml Eppendorf tube containing:
a. 3.8 µg pGag/Pol plasmid
b. 2.2 µg pVSVG plasmid
c. 6 µg pLJC5 TMEM192 3XHA plasmid
d. 300 µL OptiMem
 

SECTION: Packaging LysoTag and HA-Empty (Mock) construct
4. Prepare a transfection mix to generate MockTag expressing lentivirus in 1.5ml Eppendorf tube containing:
a. 3.8 µg pGag/Pol plasmid
b. 2.2 µg pVSVG plasmid
c. 6 µg pLJC5 short kozak 3HA plasmid
d. 300 µL OptiMem

SECTION: Packaging LysoTag and HA-Empty (Mock) construct
5. Prepare two PEI mixtures in 1.5ml Eppendorf tube (one per transfection mix).

SECTION: Packaging LysoTag and HA-Empty (Mock) construct
1. 
 

SECTION: Packaging LysoTag and HA-Empty (Mock) construct
6. Incubate each mixture separately for 5 min at Room temperature.

SECTION: Packaging LysoTag and HA-Empty (Mock) construct
7. Add the PEI mixture (Step 5) to the transfection mix (Steps 3 or 4) and repeat for the MockTag.
SECTION: Packaging LysoTag and HA-Empty (Mock) construct
8. Mix by gently  ting up and down and incubate for 20 min at Room temperature.

SECTION: Packaging LysoTag and HA-Empty (Mock) construct
9. Add each mixture dropwise using a P1000 sterile pipette into a 10cm HEK293FT containing Petri dish per transfection.
SECTION: Packaging LysoTag and HA-Empty (Mock) construct
10. Incubate cells at 37 °C for 1440 min.

SECTION: Packaging LysoTag and HA-Empty (Mock) construct
11. Replace the growth media with fresh Growth Media and incubate cells at 37 °C for further 1440 min .
SECTION: Packaging LysoTag and HA-Empty (Mock) construct
12. Collect the media that contains the lentivirus and pass through 0.45µm syringe filter. This is now the lentivirus infection media. This could be used immediately or snap frozen in liquid nitrogen and stored at -80 °C .


SECTION: Infecting cells to stably express Lyso/MockTag
13. 
 
Grow HEK293 cells in a 10cm Petri dish to 60% confluency.

SECTION: Infecting cells to stably express Lyso/MockTag
14. Mix 5 mL of infection media (Step 12) with 5 mL of fresh growth media of your cells you are intending to infect in a 15ml Falcon tube.

SECTION: Packaging LysoTag and HA-Empty (Mock) construct
2. Grow one 10cm Petri dish of HEK293FT cells to 60% confluency per transfection (2)
SECTION: Infecting cells to stably express Lyso/MockTag
15. Add Polybrene reagent (10 µg/ml) dissolved in MilliQ water) to the infection mix (Step 14) to a final concentration 10 Mass Percent Polybrene. 

SECTION: Infecting cells to stably express Lyso/MockTag
16. Remove growth media from your HEK293 cells (Step 13).
SECTION: Infecting cells to stably express Lyso/MockTag
17. Gently add the mix (Step 14) to the HEK293 cells (Step16).

 

SECTION: Infecting cells to stably express Lyso/MockTag
18. Incubate at 37 °C for 1440 min.

SECTION: Infecting cells to stably express Lyso/MockTag
19. Replace the infection media with fresh normal growth media and further incubate at 37 °C for 1440 min .

SECTION: Infecting cells to stably express Lyso/MockTag
20. Select for cells expressing your Lyso/Mock Tag by replacing the growth media with selection media for 1440 min .

SECTION: Infecting cells to stably express Lyso/MockTag
21. Remove and replenish the selection media every 1440 min for 4320 min.

 

SECTION: Infecting cells to stably express Lyso/MockTag
22. Once the transfected cells reach confluency in transfection media, expand and/or freezestore cells for use for LysoTag-immunoprecipitation experiments.
SECTION: Pre-clearing of anti-HA beads
23. 
 
Pipette 100 µL anti-HA bead slurry into 1.5ml Eppendorf tube.
SECTION: Pre-clearing of anti-HA beads
24. Place the tube containing the bead slurry onto a tube magnet for 30 s. 

SECTION: Pre-clearing of anti-HA beads
25. Remove supernatant and resuspend with 100 µL of ice cold KPBS off magnet, making sure to disperse clumps from the slurry.

SECTION: Pre-clearing of anti-HA beads
26. Repeat steps 24 and 25, 3 more times.
SECTION: Pre-clearing of anti-HA beads
27. Store on ice for later use.

 

SECTION: Preparation of Isobiotec cell breaker
28. To prepare the Isobiotec cell-breaker, assemble it by inserting the ball inside the machine and screw the lids on tightly. Place on aluminium foil on ice and push 3 mL of KPBS through the machine to wash it. Carefully tap dry.

 


SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
29. 
 
Grow cells to a confluency of 80-90% in 15cm Petri dishes.
SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
30. Place cells on aluminium covered ice and remove media.
SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
31. Add 5 mL ice cold PBS and swirl it to cover all of the plate.

SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
32. Remove the PBS and add another 5 mL ice cold PBS and swirl the plate.

SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
33. Remove the PBS and add 800 µL ice cold KPBS to the top of the plate.

SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
34. Scrape off the cells in the KPBS with a cell lifter.
SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
35. Transfer the cell/KPBS mixture to a 1.5ml Eppendorf tube using a P1000 pipette.
SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
36. Pellet the cells at 1500 x g, 4 °C, 2 min .

SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
37. Discard the supernatant carefully to not disturb the pellet.
SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
38. Resuspend the pellet in 800 µL ice cold KPBS using a P1000 pipette.

SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
39. Take 50 µL of the cell suspension aside as your whole cell sample (WC)

 

SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
40. Using a 1ml syringe and 21 gauge needle, aspirate the cell suspension(Step 38) into the
syringe and discard the needle.
SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
41. Transfer the cell suspension into a KPBS rinsed, ice-cold Isobiotec cell-breaker with gapsize of 10 μm. Homogenise the cells with 15 passes through the cell breaker using two 1ml syringes.
SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
42. Collect the homogenate from the cell breaker into a fresh 1.5ml Eppendorf.
 


SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
43. Pellet at 1000 x g for 2 min at 4 °C .

 

SECTION: Homogenisation of Lyso/MockTag expressing HEK293 cells
44. Take 50 µL of cell homogenate and place it in a fresh Eppendorf on ice. This is your input sample.

SECTION: LysoTag and MockTag immunoprecipitation
45. Transfer the supernatant to 100 µL of the prewashed beads (step 27). 

 

46. Mix by pipetting gently three times, then place on a belly-dancer orbiter for 5 min at
4 °C. 
 



47. Place the IPs on a tube magnet for 30 s to immobilise the beads out of the supernatant.
 


48. Resuspend in 1 mL of KBPS and immobilise the beads using the magnet for 30 s. Discard the supernatant. 

49. Repeat Step 48.
50. Resuspend the beads in 1 mL of KPBS and transfer to a new tube. 

51. Place the tube on the magnet and after 30 s discard the supernatant.

52. You are now left with beads attached to lysosomes. The sample can be used for:
a. Store in -80 °C
b. Immunoblot
c. Prepared for lipidomics
d. Prepared for metabolomics
e. Prepared for proteomics

SECTION: Packaging LysoTag and HA-Empty (Mock) construct
5.1. Dissolve 20 µL 1 mg/ml PEI Max 40K in distilled water.

SECTION: Packaging LysoTag and HA-Empty (Mock) construct
5.2. 300 µLOptiMem

SECTION: Pre-clearing of anti-HA beads
26.1. Place the tube containing the bead slurry onto a tube magnet for 30 s. 
Remove supernatant and resuspend with 100 µL of ice cold KPBS off magnet, making sure to disperse clumps from the slurry. (1/3)
SECTION: Pre-clearing of anti-HA beads
26.2. Place the tube containing the bead slurry onto a tube magnet for 30 s. 
Remove supernatant and resuspend with 100 µL of ice cold KPBS off magnet, making sure to disperse clumps from the slurry. (2/3)
SECTION: Pre-clearing of anti-HA beads
26.3. Place the tube containing the bead slurry onto a tube magnet for 30 s. 
Remove supernatant and resuspend with 100 µL of ice cold KPBS off magnet, making sure to disperse clumps from the slurry. (3/3)
49.1. Resuspend in 1 mL of KBPS and immobilise the beads using the magnet for 30 s. Discard the supernatant.