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STAR Protoc
STAR Protoc
STAR Protocols
2666-1667
Elsevier

S2666-1667(24)00418-0
10.1016/j.xpro.2024.103253
103253
Protocol
Protocol for the isolation and purification of endoplasmic reticulum-plasma membrane junctions from the mouse brain
Weesner Jason A. 13
van de Vlekkert Diantha 1
Fremuth Leigh Ellen 1
d’Azzo Alessandra sandra.dazzo@stjude.org
124∗
1 St. Jude Children’s Research Hospital, Department of Genetics, Memphis, TN 38105, USA
2 University of Tennessee Health Science Center, Department of Anatomy and Physiology, Memphis, TN 38163, USA
∗ Corresponding author sandra.dazzo@stjude.org
3 Technical contact

4 Lead contact

10 8 2024
20 9 2024
10 8 2024
5 3 103253© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Dynamic communication between intracellular organelles often takes place at specialized membrane contact sites that form between their membranes. Here we detail a procedure for the purification of endoplasmic reticulum-plasma membrane (ER-PM) junctions from the mouse brain. We describe steps for homogenizing isolated brain hemispheres and sequential centrifugation to remove the nuclear fraction from the other membrane fractions. We then detail procedures for separating the resulting crude membrane fractions by sucrose density gradients and purifying into their respective pellets.

For complete details on the use and execution of this protocol, please refer to Weesner et al.1

Graphical abstract

Highlights

• Mouse brain isolation, tissue homogenization, and subcellular fractionation

• Purification of ER, PM, and ER-PM junctions from membrane fractions

• Analysis of protein and lipid contents from purified ER-PM junctions

• Protocol applicable to GM1-gangliosidosis and other neurodegenerative diseases

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Dynamic communication between intracellular organelles often takes place at specialized membrane contact sites that form between their membranes. Here we detail a procedure for the purification of endoplasmic reticulum-plasma membrane (ER-PM) junctions from the mouse brain. We describe steps for homogenizing isolated brain hemispheres and sequential centrifugation to remove the nuclear fraction from the other membrane fractions. We then detail procedures for separating the resulting crude membrane fractions by sucrose density gradients and purifying into their respective pellets.

Subject areas

Cell Membrane
Cell separation/fractionation
Neuroscience
==== Body
pmcBefore you begin

Glycosphingolipids (GSLs) are integral components of membranes and play a pivotal role in the formation of MCSs.2 Changes in GSL concentration due their improper processing and degradation, affects the composition and functions of MCSs.2 To study how GSL content affects the composition and function of MCSs, we have developed a protocol for the isolation of ER-PM junctions from the mouse brain. The protocol is an updated version of the one developed in 1962 for the isolation of synaptosomes from the mouse brain3 and includes a series of additional homogenization and fractionization steps to dismantle the organellar membranes prior to separation on the sucrose gradient. The protocol is adapted from our previously published protocol for isolation of mitochondria-associated ER membranes4 and optimized for the purification of ER-PM junctions. We have used this protocol to study the role of the GSL, GM1-ganglioside (GM1) at neuronal ER-PM junctions under both physiological and neuropathological conditions in wild-type and β-Gal−/− mice.1,5 Although optimized for mouse brain tissue, this reproducible protocol can be adapted for the isolation of these MCSs from any tissue or cell types. Most importantly, purified preparations of ER-PM junctions can be subjected to proteomic analysis and various biochemical assays. This purification protocol provides the means to gain insights into the lipid and protein composition of these MCSs, and in turn, inform on how alterations in any of these constituents can elicit neuropathological processes, leading to neurodegeneration.

The day prior to brain dissection and ER-PM junction isolation:1. Sterilize equipment, including Dounce homogenizers, tweezers, and scissors.a. Sterilize Dounce tissue grinder, pestles, glassware, tweezers, and scissors by autoclave.

b. Equipment that cannot be autoclaved (i.e., dissection plate), must be cleaned with 70% ethanol.

2. Label all tubes appropriately prior to beginning the procedure.

3. Adjust all working reagents/solutions to the appropriate pH on ice, as detailed below, and store them at 4°C.

4. Prepare Isolation Buffer on ice and titrate with 500 mM HCL to pH 6. Store at 4°C for up to 6 weeks (for recipe see materials and equipment).

Institutional permissions

All experiments described have been conducted in accordance with the Policies on animal protocols approved by the St. Jude Children’s Research Hospital Institutional Animal Care and Use Committee (IACUC) and the NIH guidelines. In brief, Animals were housed in a fully AAALAC (Assessment and Accreditation of Laboratory Animal Care)-accredited animal facility with controlled temperature (22°C), humidity, and lighting (alternating 12 h light/dark cycles). Food and water were provided ad libitum. The β-Gal−/− mice are a model of the lysosomal storage disease GM1 gangliosidosis and were generated to lack a functional β-Gal gene.5 Wild-type (WT) and β-Gal−/− mice (C57BL/6J background) of 6 months of age were used in this protocol; however, ER-PM junction isolation can likely be applied to brain tissue from mice of various age and genetic background, depending on the scope of the experiments. Both male and female mice were used in all experiments without any apparent sex differences. Users are reminded that they should acquire the necessary permissions from the relevant Institutions.

Preparation of solutions and reagents

Timing: 1 h

All solutions should be prepared by the day prior to the isolation and kept at 4°C. At this time, dissection tools should be autoclaved, and tubes should be labeled.5. Prepare solution A on ice and titrate with 500 mM HCL to pH 7.4. Store at 4°C for up to 24 h (for recipe see materials and equipment).

Note: Add fresh protease and phosphatase inhibitors to the solution up to 24 h prior to isolation

6. Prepare 11%, 38%, 43%, and 53% weight to volume sucrose solution in Isolation Buffer (see before you begin). Store at 4°C for up to 1 week (for recipe see materials and equipment).

7. Prepare Washing Buffer and store at 4°C for up to 6 weeks (for recipe see materials and equipment).

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Antibodies	
	
Rabbit polyclonal anti-calnexin antibody, 1:1,000	Novus Biologicals	Cat #: NB100-1965; RRID: AB_10002123	
Rabbit anti-Laminin A/C antibody, 1:1,000	Cell Signaling Technology	Cat #: 2032; RRID: AB_2136278	
Goat anti-LDH antibody, 1:1,000	Chemicon International	Cat #: AB1222; RRID: AB_90491	
Mouse anti-N-Cadherin antibody, 1:1,000	BD Biosciences	Cat #: 610921; RRID: 398236	
Mouse monoclonal anti-TOMM20 antibody, 1:1,000	Santa Cruz	Cat #: sc-11415; RRID: AB_2207533	
	
Chemicals, peptides, and recombinant proteins	
	
Bis-Tris	bioWORLD	Cat #: 40220016-1	
Calcium chloride	Fisher Scientific	Cat #: C77-500	
cOmplete EDTA-free protease inhibitors	Roche	Cat #: 05056489001	
D-mannitol	Millipore Sigma	Cat #: M9546-1KG	
EDTA disodium salt dehydrate	Fisher Scientific	Cat #: BP120-500	
EGTA	Millipore Sigma	Cat #: E4378-250G	
HEPES	Millipore Sigma	Cat #: 54457	
Hydrochloric acid	Fisher Scientific	Cat #: A144S-500	
Magnesium chloride	Millipore Sigma	Cat #: M-8266	
PhosSTOP phosphatase inhibitors	Roche	Cat #: 04906837001	
Sodium dodecyl sulfate (SDS)	Millipore Sigma	Cat #: L3771-100G	
Sodium bicarbonate	Fisher Scientific	Cat #: S233-500	
Sucrose	Fisher Scientific	Cat #: S5-500	
Tris	Bio-Rad	Cat #: 1610716	
Triton X-100	Millipore Sigma	Cat #: X100-500ML	
	
Experimental models: Organisms/strains	
	
Wild-type and B6.129P2-Glb1tm1Adz/J (β-Gal−/−) murine model, ages between 1–4 months, males and females	Jackson Laboratory	Strain #: 037063	
	
Other	
	
5430 R tabletop centrifuge	Eppendorf	Cat #: 022620667	
6″ micro dissecting forceps	Roboz	Cat #: RS-5359	
Corex 30 mL round-bottom glass tubes or Nalgene high-speed round-bottom 38 mL tubes	Corex
Thermo Fisher Scientific	Cat #: 8445
Cat #: 3110-0380	
Fiberlite F20-12 × 50 LEX rotor	Thermo Fisher Scientific	Cat #: 096-124375	
KIMBLE Dounce tissue grinder set (homogenizer, pestle A, and pestle B)	Millipore Sigma	Cat #: D8938-1SET	
Optima XPN-90 ultracentrifuge	Beckman Coulter	Cat #: A94468	
Rubber adapter sleeve for Kimax high strength centrifuge tube	Millipore Sigma	Cat #: Z514942	
Sharp blunt 27 mm scissors	Roboz	Cat #: RS-5960	
Sharp blunt 45 mm scissors	Roboz	Cat #: RS-6780	
Single edge carbon steel razor	Electron Microscopy Sciences	Cat #: 71964	
Sorvall LYNX 6000 centrifuge	Thermo Fisher Scientific	Cat #: 75006590	
SW 32 Ti rotor	Beckman Coulter	Cat #: 369650	
SW 41 Ti rotor	Beckman Coulter	Cat #: 331362	

Materials and equipment

Recipes of stock solutions and reagents

• Sucrose stock solution (1.5 M): Dissolve 102.69 g of sucrose in a total volume of 200 mL using ddH2O. Store at 4°C for up to 6 weeks.

• Sodium bicarbonate stock solution (100 mM): Dissolve 1.68 g of NaHCO3 in a total volume of 200 mL ddH2O. Store at 4°C, solution is stable for long term.

• Magnesium Chloride stock solution (100 mM): Dissolve 1.90 g of MgCl2 in a total volume of 200 mL ddH2O. Store at 4°C, solution is stable for long term.

• Calcium Chloride stock solution (50 mM): Dissolve 1.11 g of CaCl2 in a total volume of 200 mL ddH2O. Store at 4°C, solution is stable for long term.

• Mannitol stock solution (500 mM): Dissolve 91.07 g of Mannitol in a total volume of 1 L ddH2O. Store at 4°C for up to 6 weeks.

• Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate stock solution (100 mM): Dissolve 7.45 g of EDTA in a total volume of 200 mL ddH2O and pH to 7.4. Store at 4°C, solution is stable for long term.

• Ethylene glycol tetraacetic acid (EGTA) stock solution (100 mM): Add 7.45 g of EGTA in a total volume of 200 mL ddH2O and pH to 8 to dissolve. Once dissolved, pH stock solution to 7.4. Store at 4°C, solution is stable for long term.

• 11% w/v Sucrose solution: Dissolve 1.1 g of sucrose in a total volume of 10 mL Isolation Buffer (see below for recipe). Store at 4°C for up to 1 week.

• 38% w/v Sucrose solution: Dissolve 7.6 g of sucrose in a total volume of 20 mL Isolation Buffer (see below for recipe). Store at 4°C for up to 1 week.

• 43% w/v Sucrose solution: Dissolve 8.6 g of sucrose in a total volume of 20 mL Isolation Buffer (see below for recipe). Store at 4°C for up to 1 week.

• 53% w/v Sucrose solution: Dissolve 5.3 g of sucrose in a total volume of 10 mL Isolation Buffer (see below for recipe). Store at 4°C for up to 1 week.

• Tris stock solution, pH 7.4 (1 M): Dissolve 6.06 g of tris in a total volume of 50 mL ddH2O and pH to 7.4 using 500 mM HCL. Store at 4°C, solution is stable for long term.

• Tris stock solution, pH 8.8 (1 M): Dissolve 6.06 g of tris in a total volume of 50 mL ddH2O and pH to 7.4 using 500 mM HCL. Store at 4°C, solution is stable for long term.

• Hydrochloric acid titrating solution (500 mM): Dilute 4.17 mL of 12.1 N HCl in a total volume of 100 mL ddH2O. Store at RT, solution is stable for long term.

• HEPES, pH 7.4 (1 M): Dissolve 47.66 g of HEPES in a total volume of 200 mL ddH2O and pH to 7.4 using 500 mM HCL. Store at 4°C, solution is stable for long term.

• Sodium Chloride (1 M): Dissolve 11.71 g of NaCl in a total volume of 200 mL ddH2O. Store at 4°C, solution is stable for long term.

Solution A

Solution A∗	Final concentration	Amount	
Sucrose (1.5 M)	320 mM	10.67 mL	
NaHCO3 (100 mM)	1 mM	500 μL	
MgCl2 (100 mM)	1 mM	500 μL	
CaCl2 (50 mM)	500 μM	500 μL	
ddH2O	N/A	Up to 50 mL	
Total	N/A	50 mL	
∗Protease and phosphatase inhibitors should be added prior to isolation; solution A can be stored at 4°C for up to 24 h prior to isolation.

CRITICAL: pH should be adjusted to 7.4 using 500 mM HCL on ice after the addition of inhibitors.

Isolation Buffer

Isolation buffer∗	Final concentration	Amount	
Bis-tris (Powder)	5 mM	525 mg	
EDTA:2H2O (100 mM)	200 μM	1 mL	
ddH2O	N/A	Up to 500 mL	
Total	N/A	500 mL	
∗pH should be adjusted to 6 using 500 mM HCL on ice; Isolation Buffer can be stored at 4°C for up to 1 week.

Washing Buffer

Washing buffer∗	Final concentration	Amount	
Mannitol (500 mM)	225 mM	90 mL	
Sucrose (1.5 M)	75 mM	10 mL	
EGTA (100 mM)	100 μM	200 μL	
Tris, pH 7.4 (1 M)	5 mM	1 mL	
ddH2O	N/A	Up to 200 mL	
Total	N/A	200 mL	
∗Solution can be stored at 4°C for up to 6 weeks.

Washing (−) Buffer

Washing (−) buffer∗	Final concentration	Amount	
EGTA (100 mM)	100 μM	200 μL	
Tris, pH 7.4 (1 M)	5 mM	1 mL	
ddH2O	N/A	Up to 200 mL	
Total	N/A	200 mL	
∗Solution can be stored at 4°C for up to 6 weeks.

GEM Extraction Buffer

GEM extraction buffer∗	Final concentration	Amount	
HEPES, pH 7.4 (1 M)	25 mM	1.25 mL	
NaCl (1 M)	150 mM	7.5 mL	
Triton X-100	1%	500 μL	
ddH2O	N/A	Up to 50 mL	
Total	N/A	50 mL	
∗Solution can be stored at 4°C for up to 6 weeks.

GEM Solubilization Buffer

GEM solubilization buffer∗	Final concentration	Amount	
Tris, pH 8.8 (1 M)	50 mM	2.5 mL	
EDTA:2H2O (100 mM)	5 mM	2.5 mL	
SDS	1%	0.5 g	
ddH2O	N/A	Up to 50 mL	
Total	N/A	50 mL	
∗Solution can be stored at 4°C for up to 6 weeks.

∗Solution must be thawed between 20°C–25°C for 30 min prior to use to resuspend precipitated SDS.

Equipment setup

• Centrifuges, rotors, and tube holders should be precooled to 4 °C at the start of the isolation.

• Dissection tools should be autoclaved, and dissection surfaces should be cleaned thoroughly with 70% Ethanol.

Step-by-step method details

Mouse brain isolation

Timing: 30 min

At this step, all buffers should be ready and kept on ice. Dissections can be performed outside the hood, provided that all surfaces are cleaned with 70% ethanol, and the proper PPE for handling animals is used. It is important that brain dissection is performed as quickly as possible and that the isolated brains are kept on ice to avoid tissue breakdown and preserve integrity. Timing for all future steps is set for the isolation of two mouse brains. If additional brains are needed, timing should be adjusted.1. Euthanize and isolate brain from mouse.a. Euthanize mouse in a CO2 chamber, according to institutional and regional regulations (Figure 1A).Figure 1 Isolate the mouse brain

Isolate the brain in Step 1 a-f by (A) euthanizing the mouse, (B) removing the skull, (C and D) cutting open the skull and peeling the scalp from the brain, (E) removing the brain, and (F) cutting the brain in half.

b. Immediately remove the head using surgical scissors and remove all fur and connective tissue around the skull (Figure 1B).

c. Remove the snout and cut open the skull down the middle starting from between the eyes (Figures 1C and 1D).

d. Carefully remove the skull from the brain over each hemisphere.

e. Isolate the intact brain using forceps (Figure 1E).

f. With a scalpel, separate the brain into two hemispheres, and place each half in a 2 mL tube on ice (Figure 1F).

Note: Be sure to collect the brain hemispheres quickly and keep them on ice to avoid tissue breakdown.

2. Weigh each brain hemisphere to determine the appropriate volume of Solution A to be used in the following steps.

Note: Each brain half is processed separately during the steps below.

Manual dissociation of mouse brain and removal of nuclei, intact cells and cellular debris

Timing: 45 min

This major step is to perform subcellular fractionation of mouse brain tissue to remove the nuclear fraction and cellular debris. At this step, each brain hemisphere is weighed, and the volume of solution A is calculated based on 10 times w/v.3. Homogenize the brain hemispheres.a. Place one hemisphere in a pre-chilled 2 mL glass Dounce tissue grinder containing 1 mL cold Solution A (Figure 2A).Figure 2 Homogenize the mouse brain in Dounce Homogenizer

After weighing brain, (A) place brain in a Dounce homogenizer and (B) homogenize with Pestle A. Step 3.

b. Homogenize the tissue using a large clearance pestle (pestle A) with a total of 15 strokes to dissociate the brain sample and initiate the lysis of cells (Figure 2B).

Note: Pre-cool all glass tubes and homogenizers in ice.

CRITICAL: Be sure that all brain hemispheres are homogenized with the same number of pestle strokes.

4. Transfer the 1–1.5 mL lysate to a 15 mL Falcon tube, on ice.a. Add additional Solution A to bring to volume up to a total of 10 volumes (w/v). (For example, a 225 mg brain should be lysed in 1 mL and 1.25 mL of Solution A should be added to reach a total volume of 2.25 mL).

b. Mix the lysate by pipetting up and down ∼10 times with a p1250 pipette (Methods video S1).

Note: Be sure to thoroughly mix the homogenates by pipetting the samples up and down an equal number of times.

5. Centrifuge the sample at 1400 × g for 10 min at 4°C and place it back on ice.

6. Carefully transfer the supernatant to a 30 mL round-bottom glass centrifuge tube and set aside on ice (to be used in Step 12) (Figure 3).Figure 3 Remove nucleus and cell debris from lysate

Representative image of pellet and supernatant obtained in Step 6 from WT and β-Gal−/− mice.

Note: Alternatively, if a 30 mL Corex tube is unavailable, you can use a Nalgene High-Speed Round-Bottom 38 mL Tubes.

Note: Be careful to not disturb the pellet while collecting the supernatants.

7. Resuspend the pellet in the same volume of Solution A as shown in Step 4. (Following the same example, a 225 mg brain should be resuspended in 2.25 mL of Solution A) (Figure 4).Figure 4 Resuspend the pellet in Solution A

Add solution A to the pellet in Step 7 and pipette up and down.

8. Homogenize the resuspended pellet (1.5 mL at a time) in the same grinder using a small clearance pestle (Pestle B), 6 strokes in total, to ensure full lysis of the cells (Methods video S2).

9. Transfer the homogenate to a fresh 15 mL Falcon tube (Figure 5).Figure 5 Homogenize the pellet

Representative image of pellet homogenate collected in Step 9 from WT and β-Gal−/− mice.

10. Centrifuge at 710 × g for 10 min at 4°C. This results in a pellet of nuclei and cell debris (Figures 6A and 6B).Figure 6 Nuclear removal

(A) Representative image of pellet (containing the nucleus) and supernatant collected in Step 10 from WT and β-Gal−/− mice. (B) Collect supernatant and transfer to a 30 mL Corex glass tube in Step 11.

Note: The pellet can be discarded or can be used as a crude nuclear fraction that can be further purified as needed.

11. Carefully remove this supernatant and combine it with the supernatant saved in Step 6 on ice (Figures 6A and 6B).

Methods video S1. Mixing homogenized lysate into Solution A, related to Step 4

Methods video S2. Homogenize the resuspended pellet

Resuspend the pellet obtained in Step 8 and homogenize using 6 strokes of Pestle B.

Isolation of crude membrane fractions

Timing: 1.5 h

The purpose of this major step is to remove unbound ER by spinning down the sample collected in Step 11. The resulting supernatant is enriched in unbound ER and the cytosolic fraction, while the pellet contains PM, ER-PM junctions, and mitochondria membranes.12. Insert the 30 mL round-bottom glass tubes containing the pooled supernatants in adaptor sleeves and place the samples in a Sorvall centrifuge (Figure 7).Figure 7 Pellet the supernatant containing membrane fractions

Insert the Corex tubes from Step 12 and place them in a Sorvall Centrifuge.

13. Centrifuge at 13,800 × g for 10 min at 4°C.

14. Transfer supernatant, containing the cytosol and unbound ER fractions, to a 13.2 mL ultra-clear Beckman centrifuge tube, and save on ice (to be used in Step 27) (Figures 8A–8C).Figure 8 Separate the unbound ER and cytosolic fraction from the crude membrane fraction

(A) Representative image of pellet (containing the membranes) and supernatant (containing unbound ER and cytosol) collected in Step 14 from WT and β-Gal−/− mice.

(B) Carefully remove the supernatant and (C) transfer the supernatant to a 13.2 mL ultra-clear Beckman centrifuge tube.

Note: The supernatant collected in this step will be combined with additional supernatants obtained in Step 19.

15. Resuspend pellet in the same volume of Solution A used in Step 4 and transfer to a 15 mL Falcon tube on ice.

16. Homogenize the resuspended pellet using a small clearance pestle (pestle B) with 6 strokes, 1.5 mL at a time and transfer the lysate to the 30 mL round-bottom glass centrifuge tubes used in Step 12.

17. Centrifuge the lysate at 13,800 × g for 10 min at 4°C.

18. Repeat Steps 14–16 once.

19. Carefully remove the supernatants (cytosol and ER) and combine them with the supernatants collected in Step 14; cover with parafilm and keep on ice.

Note: The supernatants will be further processed in Step 27.

20. Resuspend the resulting pellet (containing membranes and mitochondria) in 2 mL of the 11% sucrose solution and transfer to a fresh grinder.

21. Homogenize with 10 strokes, using a small clearance pestle (pestle B).

Fraction separation by sucrose gradient

Timing: 2.5 h

The purpose of this major step is to separate the membrane fractions (PM, ER-PM junctions, and mitochondria membranes) using a discontinuous sucrose gradient. After spinning the gradient, each fraction appears aa a milky band at the interface of the sucrose layer (see Step 24).22. Prepare a discontinuous sucrose gradient in a 13.2 mL ultra-clear Beckman centrifuge tube by overlaying the following solutions:a. Add 2 mL of 53% sucrose to the bottom of the tube (Figure 9A).Figure 9 Layer the sucrose gradient

(A) Add 2 mL of 53% sucrose in a 13.2 mL Beckman centrifuge tube.

(B–D) Carefully layer (B) 43% and (C) 38% sucrose on top. (D) Layer the homogenized pellet obtained in Step 21 on top of the gradient. Related to Step 22a-d.

b. Carefully layer 3.5 mL of 43% sucrose solution (Figure 9B).

c. Carefully layer 3.5 mL of 38% sucrose solution (Figure 9C).

d. Carefully overlay the resuspended pellet (∼2 mL) from Step 21 to the top of gradient (Figure 9D) (Methods video S3).

Note: Carefully layer the solutions avoiding the formation of bubbles in the gradient. It is recommended to use a 10 mL serological pipette at the lowest setting to apply the sucrose layers by gravitational force. To provide additional control to the flow rate, it is recommended to attach a p200 pipette tip to the end of the 10 mL stereological pipette.

23. Balance the tubes using the 11% sucrose solution.

24. Centrifuge at 100,000 × g for 2.5 h at 4°C in an ultracentrifuge using a SW 41 Ti Beckman rotor. After centrifugation, the resulting gradient will appear as three defined bands consisting of ER-PM junctions, crude mitochondria, and PM (Figure 10).a. Top band: ER-PM junctions (11%–38% interface).

b. Middle band: crude mitochondria (38%–43% interface).Note: The crude mitochondria can be further processed into a pure mitochondrial fraction (See Annunziata et al., 2021, section 3.1.3).

c. Bottom band: PM (43%–53% interface). Troubleshooting 1 and 2.CRITICAL: It is important to remove the three bands from the sucrose gradient starting from the top one that will be further processed in the next section.

Figure 10 Separate the membrane fractions on a sucrose gradient

After 2.5 h spin in Step 24, there are 3 bands containing the ER-PM junctions, crude mitochondria, and plasma membrane fractions.

Methods video S3. Layering sample on sucrose gradient, related to Step 22

Purification of ER, PM, and ER-PM junctions

Timing: 2.5 h

For this major step, the bands from Step 24 are further processed into pure fractions. The resulting pellets can be used for multiple analyses, including immunoblots, co-immunoprecipitations, proteomics and lipid analysis using HP-TLC. For the following steps the samples obtained from the two brain hemispheres can be combined into a single sample.25. Isolation of ER-PM junctions. Troubleshooting 5.a. Collect the top band containing the ER-PM junctions using a P1000 pipette tip and place it into a fresh 30 mL round bottom glass tube on ice (Methods video S4).

Methods video S4. Removal of ER-PM junctions from sucrose gradient, related to Step 25

b. Combine the ER-PM junction bands from each brain hemisphere and measure their volume (∼4 mL total).

c. Add 2 x volume of Washing Buffer (∼8 mL) and mix thoroughly by pipetting the solution up and down.

d. Centrifuge at 10,000 × g for 10 min at 4°C to remove mitochondria contaminants from ER-PM junctions. Troubleshooting 3.Optional: Transfer the resulting supernatant (from Step 25d) to a fresh 30 mL round bottom glass tube, adjust the volume and centrifuge again at 10,000 × g for 10 min at 4°C to remove any remaining mitochondria.

e. Transfer supernatant to a 13.2 mL ultra-clear centrifuge tube and centrifuge at 100,000 × g for 45 min at 4°C.

f. Decant the supernatant and carefully remove any remaining liquid using a P200 pipette.

g. Dry the resulting ER-PM junction pellet for ∼5 min between 20°C–25°C by placing the tube upside down on a paper towel (Figure 11). Troubleshooting 6.Note: The pellet can be resuspended in the appropriate buffer. Alternatively, the pellet can be flash frozen on dry ice, covered with parafilm and stored for a short period at −20°C.

Optional: The middle band of the gradient contains the crude mitochondrial fraction. This band can be further purified into a pure mitochondrial fraction.

Figure 11 Purify the ER-PM junctions

Representative image of pellet containing purified ER-PM junctions obtained in Step 25g from WT and β-Gal−/− mice.

26. Isolation of PM.a. Collect and combine the bottom bands, containing the PM fractions from the two brain hemispheres, using a glass Pasteur pipette into a 30 mL round bottom glass tube on ice, and add 5x volume of Washing Buffer.

b. Centrifuge at 10,000 × g for 10 min at 4°C to remove mitochondria contaminants from the PM.

c. Transfer the supernatant to a clean 30 mL round bottom glass tube, and centrifuge again at 10,000 × g for 10 min at 4°C to remove any remaining mitochondria. Troubleshooting 3.Optional: Transfer the resulting supernatant (from Step 26c) to a fresh 30 mL round bottom glass tube, adjust the volume and centrifuge again at 10,000 × g for 10 min at 4°C to remove any remaining mitochondria.

d. Transfer supernatant to an ultra-clear centrifuge tube and centrifuge at 48,000 × g for 20 min at 4°C.

e. Decant the supernatant and carefully remove any remaining liquid using a P200 pipette.

f. Dry the resulting PM pellet for ∼5 min between 20°C–25°C by placing the tube upside down on a paper towel (Figure 12). Troubleshooting 6.Note: The pellet can be resuspended in the appropriate buffer. Alternatively, the pellet can be flash frozen on dry ice, covered with parafilm and stored for a short period at −20°C.

Figure 12 Purify the plasma membrane fraction

Representative image of pellet containing purified plasma membrane fraction obtained in Step 26f from WT and β-Gal−/− mice.

27. Isolation of ER.a. Take the supernatant saved in Step 14, and centrifuge it at 100,000 × g for 1 h at 4°C.

b. Remove the supernatant containing the cytosolic fraction and place 1 mL in a 1.7 mL Eppendorf tube.

c. Carefully remove any remaining liquid from the pellet using a P200 pipette.

d. Dry the resulting ER pellet for ∼5 min between 20°C–25°C by placing the tube upside down on a paper towel. (Figure 13). Troubleshooting 6.Figure 13 Purify the plasma membrane fraction

Representative image of pellet containing purified ER fraction obtained in Step 27d from WT and β-Gal−/− mice.

Note: The pellet can be resuspended in the appropriate buffer. Alternatively, the pellet can be flash frozen on dry ice, covered with parafilm and stored for a short period at −20°C.

Optional: Isolation of GEMs from ER, PM, and ER-PM junctions

Timing: 45 min

For this optional step, you can isolate the glycosphingolipid enriched microdomain (GEM) fraction from the pellets of the collected fractions (ER, PM, ER-PM junctions).28. Resuspend the pellets obtained in Steps 25g, 26f, and 27d in the following amount of GEM Extraction Buffer.a. Resuspend ER-PM junction pellet (Step 25g) in 600 μL GEM Extraction Buffer.

b. Resuspend PM Pellet (Step 26f) in 300 μL GEM Extraction Buffer.

c. Resuspend ER Pellet (Step 27d) in 300 μL GEM Extraction Buffer.

29. Transfer each resuspended pellet to a 1.7 mL Eppendorf tube on ice for 20 min, vortex every 5 min.

30. Centrifuge at 14,000 × g for 5 min at 4°C.

31. Transfer each supernatant into a new 1.7 mL Eppendorf tube and centrifuge at 14,000 × g for 5 min at 4°C. Keep the pellets on ice.

32. Carefully remove the supernatant from each sample and resuspend the resulting pellets with 100 μL GEM Solubilization Buffer. Use these 100 μL slurries to resuspend and combine the individual pellets from each sample saved in Step 31.

Expected outcomes

The enrichment of specific organellar markers recovered in each fraction after the “isolation of crude membrane fractions” can be tested by immunoblot analysis with specific antibodies. As described in Weesner et al. 2024, the nuclear removal step (Step 10) should result in a pellet enriched in the nuclear marker, lamin A/C, and in a supernatant containing cytosolic (LDH), PM (N-Cadherin), ER (calnexin), and mitochondria (TOMM20) markers (Figure 14).1 The pooled supernatants collected at Step 21 are centrifuged and the resulting pellet should contain ER, PM, and mitochondria markers, while the resulting supernatant should contain the unbound ER and cytosolic fractions (Figure 14). After ER, PM, and ER-PM junction purification (Steps 25–27), you should expect an enrichment in their corresponding purity markers, see Weesner et al. 2024, Figure 3A, for an example1 troubleshooting 4. If the optional purification of the GEM fractions is performed (Steps 28–32), the purity of these fractions can be tested using flotillin-1, as demonstrated in Weesner et al. 2024, Figure S3D.1Figure 14 Expected purity marker for first 2 major steps

Schematic representation of “Manual dissociation of mouse brain” and “Isolation of crude membrane fractions” major steps. Western blot analyses demonstrate purity markers for each fraction described in the expected outcomes. (Adapted from Weesner et al. 20241).

The amount of sample yielded by this protocol is dependent on the starting weight of the mouse brain. For a 6-month-old animal, the resulting PM (Step 26) and ER-PM junction pellets (Step 25) should contain a 200–600 μg total protein content, while the unbound ER pellet (Step 27) should contain 1.5–2.5 mg of proteins. Pellets should be visible by the naked eye and can be resuspended in the appropriate volume of buffer needed for experimentation (Table 1). Alternatively, pellets can be flash frozen in dry ice and stored short term at −20°C.Table 1 Volume of buffers to resuspend the ER, PM, and ER-PM junction pellets based on the experiment performed

	Unbound ER	Plasma membrane	ER-PM junctions	
Immunoblot/co-IP analysis	500 μL buffer	150 μL buffer	150 μL buffer	
High-performance thin layer chromatography	300 μL ddH2O	100 μL ddH2O	100 μL ddH2O	
Proteomic analysis	500 μL buffer	150 μL buffer	150 μL buffer	
Related to expected outcomes.

When isolating ER-PM junctions from WT and β-Gal−/− mice, the protein content of β-Gal−/− ER-PM junctions was higher even though the brains had similar weights as demonstrated in Weesner et al. 2024, Figure S3C.1

Limitations

Overall, we found this procedure reproducible and consistent. The protocol has been optimized for the isolation of ER-PM junctions from the mouse brain; however, it can also be adapted to isolate these contact sites from other tissues or cultured cells. The isolation of ER-PM junctions from cultured cells would require a considerable number of cells as starting material to yield sufficient amounts of ER-PM junctions for downstream analyses. A major challenge arises when using primary cultured neurons, as these do not proliferate, and it is difficult to obtain enough cells as starting material for the isolation of a sufficient amount of ER-PM junctions. In addition, the initial manual dissociation using the Dounce tissue grinder would require optimization based on the cell type to ensure complete lysis of the cells and dissociation of their membranes.

The timing of this protocol is based on the use of two mouse brains (4 hemispheres). If more than 2 brains are used (e.g., up to 6 total), the timing of each section would increase accordingly. In addition, for more than 6 hemispheres, the users would require a second Beckman ultracentrifuge to spin down multiple sucrose gradients simultaneously or perform the isolations and centrifugation on a second day, as the protocol takes over 8 h. We do not recommend isolating more than 6 brains at a time.

During this protocol, the integrity of the mitochondria and ER is disrupted which can cause the release of their luminal contents. For this we recommend the use of membrane-bound proteins to serve as markers for individual organelles.

Troubleshooting

Problem 1

The sucrose gradient separates into two bands, with no band present at 43%–53% sucrose interfaces (Step 24) (Figure 15).Figure 15 Improper separation of plasma membrane and mitochondria bands

Example of improper separation of plasma membrane and mitochondria band from sucrose gradient described in problem 1.

Potential solution

Careful layering of the sucrose gradient, to prevent mixing of the layers, is critical to ensure proper separation. We found that the PM band at the 43%–53% sucrose interface is the most difficult to separate and can be mis-localized to the 38%–43% sucrose interface along with the mitochondrial fraction. This gives the appearance of two bands at the 38%–43% sucrose interface: one band on top that is less dense and appears fuzzy (mitochondrial fraction) and a second band directly underneath that appears dense and lipid rich (PM fraction) (Figure 15).

To successfully purify the PM band, we recommend removing the PM fraction first using a p1000 pipette. The high lipid content of the PM allows the band to be removed in a single motion (Methods video S5). We recommend performing the optional spin in Step 26c to ensure complete removal of the mitochondria contamination from the PM fraction.

Methods video S5. Removal of band containing the plasma membrane fraction aberrantly located at the 38%–43% sucrose interface, rather than the 43%–53% sucrose interface, related to problem 1

Problem 2

The sucrose gradient separates into two distinct bands, and an additional diffuse band spanning between the two interfaces (Step 24) (Figure 16).Figure 16 Diffuse plasma membrane band

Examples of improper separation of plasma membrane band from sucrose gradient described in problem 2.

Potential solution

It is recommended to layer the resuspended pellet in Step 22d on top of the sucrose gradient as quickly as possible to prevent the layers from diffusing. If you find that one layer diffuses between the interfaces after the 2.5 h spin in Step 24 (Figure 16), you should collect the entire sucrose layer containing the diffuse band to ensure that the fraction is fully recovered in the pellet.

Problem 3

There is a large amount of mitochondria contamination in the ER-PM junctions and PM fractions (Steps 25d and 26c) (Figure 17).Figure 17 Removal of mitochondria contamination from fractions

Examples of (A) low mitochondria contamination and (B) too much mitochondria contamination as described in problem 3.

Potential solution

To remove mitochondria contamination, it is recommended to perform a second 10,000 × g spin after Steps 25d or 26c. If mitochondria continue to contaminate your PM and ER-PM junction fractions, you may increase the optional spin to 13,000 × g, which should enable further removal of these contaminants.

Problem 4

Purity markers for each organelle are found in all the fractions (Expected Outcomes).

Potential solution

Complete lysis of the cells in Steps 3–4 is essential to ensure proper organellar separation during the subsequent steps. For tissues, we find that the use of Dounce homogenizers is sufficient to ensure a complete lysis. If specific organellar markers spread over fractions that should not contain them, you will need to adjust the number of pestle strokes in Steps 3–4 to obtain a more homogeneous lysis of the tissue. For cells, it may be difficult to obtain a homogenous lysate using only a Dounce homogenizer; complete lysis of the cells may require additional use of a syringe needle. To do this, prior to Step 3, you can pass the lysate through an 18-gauge needle ∼20 times.

Problem 5

Low ER-PM junction yield following purification (Step 25) (Figure 18).Figure 18 Low ER-PM junction yield

Examples of low ER-PM junction recovery as described in problem 5.

Potential solution

We found that this protocol gives an adequate yield of ER, PM and ER-PM junction fractions from mice aged one month or older, if the brain hemispheres are combined in Steps 25–27. When using pups younger than one month, it may be necessary to combine 2 or more brains to obtain a workable pellet at Step 3. If 2 brains weighting <250 mg are combined, you may first lyse the whole brain in Step 3 rather than separating the brain hemispheres. Then, at Steps 25–27, the two brain lysates can be combined for subsequent purification of the individual fractions.

Problem 6

The ER-PM junction fraction contains sucrose contamination that interferes with analyses (Expected Outcomes) (Figure 19).Figure 19 Example of a high-performance thin-layer chromatography plate with sucrose contamination as described in problem 6

Potential solution

When performing ganglioside analysis using high-performance thin-layer chromatography (HP-TLC), we found that the mannitol and sucrose contained in the Washing Buffer react with the resolving HP-TLC spray, giving an extra contaminant band (figure). To minimize the amount of sucrose and mannitol contained in these pellets, it is recommended that one additional washing step is applied. This can be achieved by resuspending the pellets collected in Steps 25–27 with 10 mL of a Washing (−) Buffer without sucrose or mannitol and performing the separation spin again (Steps 25e, 26d, 27a) to pellet the fractions (Figure 20).Figure 20 Example of a high-performance thin-layer chromatography with low sucrose contamination after wash with Washing (−) Buffer as described in Solution 6

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Alessandra d’Azzo, at Sandra.Dazzo@stjude.org.

Technical contact

Further information for technical concerns should be directed to the technical contact, Dr. Jason Andrew Weesner, at Jason.Weesner@stjude.org.

Materials availability

This study did not generate new unique reagents.

Data and code availability

• All relevant data are available within the article or from the lead contact upon reasonable request.

• This paper does not report original code.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

A.d’A. holds the Jewelers for Children Endowed Chair in Genetics and Gene Therapy. This work was supported in part by NIH grants, DK052025 and CA021764 , and grants from the Assisi Foundation of Memphis, the National Tay-Sachs and Allied Diseases Association. (NTSAD), and the American Lebanese Syrian Associated Charities (ALSAC). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author contributions

Conceptualization, J.A.W. and A.d’A.; methodology, J.A.W. and L.E.F.; validation, J.A.W. and D.v.d.V.; formal analysis, J.A.W. and D.v.d.V.; investigation, J.A.W. and A.d’A.; data curation, J.A.W. and D.v.d.V.; writing – original draft, J.A.W.; writing – review and editing, J.A.W. and A.d’A.; visualization, J.A.W. and A.d’A.; supervision, A.d’A.; project administration, A.d’A.; funding acquisition, A.d’A.

Declaration of interests

The authors declare no competing interests.

Supplemental information can be found online at https://doi.org/10.1016/j.xpro.2024.103253.
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