TITLE: Detection of accessible cholesterol in primary cilia using purified His-ALOD4-mNeon in 3T3 Fibroblasts
AUTHORS: Sreeja V Nair, Ayan Adhikari, Suzanne R Pfeffer

[DESCRIPTION]
There exist at least three different pools of cholesterol in the plasma membrane: the essential pool, the sphingomyelin-sequestered pool, and the accessible pool (Radhakrishnan et al, 2020). Ciliary Hedgehog signaling is regulated by the accessible pool of cholesterol  (Kinnebrew et al, 2019), and His-mNeon-FLAG-ALOD4, a toxin-based probe, can be used to visualize this accessible pool. Here, we present a method for staining and measuring the amount of accessible cholesterol on cilia in 3T3 fibroblasts stably expressing Somatostatin Receptor (SSTR3)-mApple, a ciliary marker. The protocol described here is based upon previously established methods (Kinnebrew et al., 2019; Johnson and Radhakrishnan, 2021) and has also been used successfully to label cilia in RPE cells.


[GUIDELINES]
Purified ALOD4 is best used fresh and will lose activity if frozen; store at 4°C

[STEPS]
SECTION: His-mNeon ALOD4 purification
1. Make a 50 mL starter culture of BL21 DE3 Rosetta plysS cells expressing pRSET+ His-mNeon-FLAG-ALOD4, starting from a fresh colony picked from freshly transformed plates containing carbenicillin (100μg/ml) and chloramphenicol (34μg/ml). Grow   with shaking in an Erlenmeyer flask at 37 °C.

SECTION: His-mNeon ALOD4 purification
2. Day 2.  Using a 6 liter flask, prepare 2 X 2 liters LB by mixing 50 g LB powder with 2 L water.  Autoclave for 20 min using a liquid cycle.  Allow the media to cool to Room temperature

SECTION: His-mNeon ALOD4 purification
3. Add carbenicillin(100 µg)and chloramphenicol (34 µg) to each flask and             shake at 37 °C until an OD 0.5-0.6 is reached.  This usually takes ~2.5 hr.

SECTION: His-mNeon ALOD4 purification
4. Transfer flask to an 18 °C shaker and equilibrate temperature for 10-20 min.

SECTION: His-mNeon ALOD4 purification
5. Add IPTG to achieve a concentration of 0.3 millimolar (mM)

SECTION: His-mNeon ALOD4 purification
6. Induce expression overnight with shaking at    at 18 °C

SECTION: His-mNeon ALOD4 purification
7. Day 3.  Transfer bacterial culture to 4, 1 L centrifuge bottles.

SECTION: His-mNeon ALOD4 purification
8. Pellet bacteria by spinning at 4000 RPM for 20 min 4°C; discard supernatant.
SECTION: His-mNeon ALOD4 purification
9. Add 1 Roche protease inhibitor tablet to 50 mL of lysis buffer, rotate at 4 °C to              dissolve.

SECTION: His-mNeon ALOD4 purification
10. Set up a ~200 mL beaker with a stir bar at 4 °C in the cold room. 

SECTION: His-mNeon ALOD4 purification
11. Resuspend pelleted bacteria on ice or in cold room using ~10 mL of lysis buffer for each bottle. 

SECTION: His-mNeon ALOD4 purification
12. Vortex the bottles to help resuspend pellet; transfer resuspended pellets to the beaker with a stir bar. 
SECTION: His-mNeon ALOD4 purification
13. Take 40 mL of this suspension to the Emulsiflex cell breakage device and have ready, 4 x 65ml polycarbonate ultracentrifuge tubes on ice for the homogenate from the next step.  Pass 10 mL lysis buffer through the Emulsiflex once at 0-30K PSI to equilibrate the machine.  Pass suspension through the Emulsiflex once at ~50K PSI to lyse cells. Collect directly into polycarbonate tubes on ice.

SECTION: His-mNeon ALOD4 purification
14. Balance tubes and then spin at 40,000 RPM for 45 min in a 45Ti rotor at 4 °Cto pellet cell debris.  Collect supernatant and filter through a 500 mL, 0.45µm Millipore filter. Collect the filtered supernatant in a 100 mL glass beaker on ice.

SECTION: His-mNeon ALOD4 purification
15. Take a 1 mL HiTrap Talon column (Cytiva #28953766) and wash it with 10 column volumes (CV) of distilled, degassed water.  Equilibrate column with 10CV lysis buffer (50mM HEPES pH 8, 500 mM NaCl, 5mM MgCl2, 0.5mM TCEP, 10% glycerol, EDTA-free protease tablet)

SECTION: His-mNeon ALOD4 purification
16. Run the lysate through the column. Collect the flow through.
SECTION: His-mNeon ALOD4 purification
17. Wash the column with 30CV wash buffer (50mM HEPES pH 8, 500 mM NaCl, 5mM MgCl2, 0.5mM TCEP, 10% glycerol, 20mM imidazole). Collect the wash fractions.  Elute with an imidazole gradient of 100mM-500mM. Collect the elution fractions and analyze them by SDS PAGE.
SECTION: His-mNeon ALOD4 purification
18. Pool the fractions having the desired protein and buffer exchange the pool using a PD-10 desalting column (Cytiva #17085101) into the storage buffer (50mM HEPES pH 8, 150mM NaCl, 5mM MgCl2, 0.5mM TCEP, 10% glycerol).

 


SECTION: Cilia Staining
20. Seed 0.3 X 106 cells onto collagen-coated coverslips. Once attached, starve the cells in media without serum or in low serum for 1440 min to induce ciliogenesis.

SECTION: Cilia Staining
21. Set up an incubation chamber for ALOD4-mNeon staining: place a metal tray (covered with a layer of parafilm) on ice for 20-30 minutes before paraformaldehyde (PFA) fixation.

SECTION: Cilia Staining
22. Aspirate media and fix cells in 4% PFA in 1X PBS for 10 min at Room temperature.

SECTION: Cilia Staining
23. Wash the cells three times in 1X PBS; transfer the coverslips to the incubation chamber and incubate them for 60 min in 4μM ALOD4-mNeon diluted in 1% BSA 1X PBS, sterilized by passage through a 0.2 μm filter.

SECTION: Cilia Staining
24. Wash the cells twice in 1X PBS; fix the cells again in 2% PFA diluted in 1X PBS for 5 minutes on ice. Wash the cells again twice in 1X PBS.

SECTION: Cilia Staining
25. Optional: stain with DAPI (1:1000) for 4 minutes on ice.  Wash the cells twice in 1X PBS and mount them onto clean glass slides using 4 µl Mowiol. Air-dry the coverslips and proceed to imaging.

19.   

SECTION: Example of ciliary accessible cholesterol staining
26.