Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Summary

This paper aims to present a protocol for preparing recycling endosomes from mammalian cells using sucrose density gradient ultracentrifugation.

Abstract

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma membrane and then transported to the early endosomes. The internalized proteins could be transited to the lysosome for degradation or recycled back to the plasma membrane. A robust endocytic recycling pathway is required to balance the removal of membrane materials from endocytosis. Various proteins are reported to regulate the pathway, including ADP-ribosylation factor 6 (ARF6). Density gradient ultracentrifugation is a classical method for cell fractionation. After the centrifugation, organelles are sedimented at their isopycnic surface. The fractions are collected and used for other downstream applications. Described here is a protocol to obtain a recycling endosome-containing fraction from transfected mammalian cells using density gradient ultracentrifugation. The isolated fractions were subjected to standard Western blotting for analyzing their protein contents. By employing this method, we identified that the plasma membrane targeting of engulfment and cell motility 1 (ELMO1), a Ras-related C3 botulinum toxin substrate 1 (Rac1) guanine nucleotide exchange factor, is through ARF6-mediated endocytic recycling.

Introduction

Endosomal trafficking is an essential physiological process that implicates various biological events¹, for example, the transportation of signaling receptors, ion channels, and adhesion molecules. Proteins localized at the plasma membrane are internalized by endocytosis². The internalized proteins are then sorted by the early endosome³. Some of the proteins are targeted to lysosomes for degradation⁴. However, a significant amount of proteins are recycled back to the cell surface by fast recycling and slow recycling processes. In fast recycling, proteins leave the early endosomes and directly return to the plasma membrane. Conversely, in slow recycling, proteins are first sorted to the endocytic recycling compartment and then transported back to the plasma membrane. Various cargo proteins, for example, clathrin, retromer complex, retriever complex and Wiskott-Aldrich syndrome protein, and SCAR Homologue (WASH) complex, participate in such membrane recycling processes^4,5,6,7,8,9. The balance of the endocytosis and recycling event is crucial for cell survival and contributes to various cellular events¹⁰, for instance, cell adhesion, cell migration, cell polarity, and signal transduction.

ARF6, a small GTPase, is a reported regulator of endocytic trafficking^7,11,12. Of interest, various research groups have illustrated the importance of ARF6 in endocytic recycling^{13,14,15,16,17}. The study aims to investigate the relationship between ARF6-mediated neurite outgrowth and endocytic recycling. The previous report suggests that the activation of ARF6 is upstream to Rac1 activity through acting on ELMO1-dedicator of cytokinesis 1 (DOCK180) complex¹⁸. However, how ARF6 triggers ELMO1-DOCK180 mediated Rac1 signaling remains unclear. Density gradient ultracentrifugation was employed to investigate the role of ARF6-mediated endocytic recycling in such process. By using that, the recycling endosome-containing fraction was obtained from cell lysates¹⁹. The fraction was subjected to Western blotting for protein content analysis. The immunoblot results revealed that under the presence of FE65, a brain-enriched adaptor protein, active ARF6 substantially increased the level of ELMO1 in the recycling endosome-containing fraction. The following protocol includes the procedures for (1) transfecting mammalian cells; (2) preparing the samples and density gradient columns; and (3) obtaining the recycling endosome-containing fraction.

Protocol

1. Mammalian cell culture and transfection

1. Plate 2 x 10⁶ cells in a 100 mm culture dish. Use four dishes for each transfection.
   NOTE: The number of cells required may vary for different cell lines. Optimization may be necessary before proceeding to the isolation step.
2. The next day, transfect the cells with Lipofectamine according to the manufacturer's instructions.

2. Cell harvest

1. Discard the culture medium 48 h post-transfection.
2. Wash the cells with ice-cold PBS (10 mM sodium phosphates, 2.68 mM potassium chloride, 140 mM sodium chloride) twice.
3. Add 1 mL of ice-cold PBS⁺ (PBS supplemented with 0.5x protease inhibitor cocktail and 0.5x phosphatase inhibitor cocktail) to each dish.
4. Collect the cells with a cell scraper and transfer the cell suspension to a 15 mL centrifuge tube.
5. Pellet the cells by centrifugation using a swing bucket rotor at 400 x g for 5 min.
6. Discard the supernatant and resuspend the cell pellet gently in 5 mL of homogenization buffer (HB; 250 mM sucrose, 3 mM imidazole at pH 7.4, 1 mM EDTA supplemented with 0.03 mM cycloheximide, 1x protease inhibitor cocktail, and 1x phosphatase inhibitor cocktail).
7. Collect the cells by centrifugation at 1,300 x g for 10 min.
8. Resuspend the cell pellet in 1 mL of HB.
9. Homogenize the cells with a Dounce homogenizer for 15-20 strokes.
   NOTE: Other homogenization methods, for example, passing the sample through a syringe, could be used. The homogenization efficiency could be revealed by observing the homogenate under a phase-contrast microscope.
10. Transfer the homogenate to a 2 mL centrifugation tube.
    NOTE: Harvest 50 µL of homogenate with 12.5 µL of 5x sample buffer and label it as total lysate.
11. Add 0.7 mL of HB to the homogenate.
12. Spin the diluted homogenate at 2,000 x g for 10 min at 4 °C.
    NOTE: The pellet contains nuclei and unbroken cells.
13. Collect 1.5 mL of the supernatant and repeat step 2.12 once.
14. Collect 1.4 mL of the supernatant and label it as post-nuclear supernatant (PNS).

3. Density gradient column preparation

1. Transfer 1.2 mL of PNS to an ultracentrifuge tube.
2. Add 1 mL of 62% sucrose solution (2.351 M sucrose, 3 mM imidazole at pH 7.4) to the sample and mix well by gentle pipetting.
   NOTE: The resultant solution is a 40.6% sucrose solution.
3. Add 3.3 mL of 35% sucrose solution (1.177 M sucrose, 3 mM imidazole at pH 7.4) carefully on top of the sample.
4. Add 2.2 mL of 25% sucrose solution (0.806 M sucrose, 3 mM imidazole at pH 7.4) carefully on top of the 35% sucrose solution.
   NOTE: The refractive index of the 62%, 40.6%, 35%, and 25% sucrose solutions at room temperature are 1.44, 1.40, 1.39, and 1.37, respectively. The refractive indexes of the sucrose solutions could be checked with a refractometer to ensure the precision and consistency of the experiment.
5. Fill up the ultracentrifugation tube with HB.
   ​NOTE: Temporarily store the prepared density gradient column at 4 °C.

4. Fractionation and recovery of recycling endosome-containing fraction

1. Centrifuge the column at 210,000 x g for 3 h at 4 °C.
2. Collect 12 fractions (1 mL each) carefully, starting from the top of the gradient.
   NOTE: The recycling endosomes should be found at the interface between 35% and 25% sucrose solutions. The collected fractions can be snap-frozen in liquid nitrogen and stored at -80 °C.
3. Dilute all the fractions with 1 mL of dilution buffer (3 mM imidazole at pH 7.4, 1 mM EDTA).
4. Centrifuge the diluted sample at 100,000 x g for 1 h at 4 °C.
5. Aspirate the supernatant and add 50 µL of 1x sample buffer to harvest the fractions.
6. Analyze the protein contents in the fractions by western blotting.

Results

After fractionating the untransfected HEK293 cells by density gradient ultracentrifugation, 12 fractions were collected starting from the top of the gradient. The harvested fractions were diluted with the dilution buffer in a 1:1 ratio and subjected to a second round of centrifugation. The samples were then subjected to western blotting for analyzing their protein contents. As shown in Figure 1, the recycling endosome marker Rab11 is detected in fraction 7²⁰. Other su...

Discussion

The above protocol outlines the procedures for isolating recycling endosomes from cultured cells by ultracentrifugation. The reliability of this method has been demonstrated by the latest publication²², proving that recycling endosomes are successfully isolated from other organelles (Figure 1), such as the Golgi apparatus and mitochondria. Some critical steps need to be paid attention to for obtaining a good separation result. While preparing the sucrose solutions, it...

Materials

Name	Company	Catalog Number	Comments
1 mL, Open-Top Thickwall Polypropylene Tube, 11 x 34 mm	Beckman Coulter	347287
100 mm tissue culture dish	SPL	20100
13.2 mL, Certified Free Open-Top Thinwall Ultra-Clear Tube, 14 x 89 mm	Beckman Coulter	C14277
5x Sample Buffer	GenScript	MB01015
cOmplete, EDTA-free Protease Inhibitor Cocktail	Roche	11873580001
COX IV (3E11) Rabbit mAb	Cell Signaling Technology	4850S	Rabbit monoclonal antibody for detecting COX IV.
Cycloheximide	Sigma-Aldrich	C1988
Dounce Tissue Grinder, 7 mL	DWK Life Sciences	357542
Dulbecco's Modified Eagle Medium (DMEM) with low glucose	HyClone	SH30021.01
ELMO1 antibody (B-7)	Santa Cruz Biotechnology	SC-271519	Mouse monoclonal antibody for detecting ELMO1.
EndoFree Plasmid Maxi Kit	QIAGEN	12362
FE65 antibody (E-20)	Santa Cruz Biotechnology	SC-19751	Goat polyclonal antibody for detecting FE65.
Fetal Bovine Serum, Research Grade	HyClone	SV30160.03
GAPDH Monoclonal Antibody (6C5)	Ambion	AM4300	Mouse monoclonal antibody for detecting GAPDH.
ImageLab Software	Bio-Rad		Measurement of band intensity
Imidazole	Sigma-Aldrich	I2399
Lipofectamine 2000 Transfection Reagent	Invitrogen	11668019
Monoclonal Anti-β-COP antibody	Sigma	G6160	Mouse monoclonal antibody for detecting β-COP.
Myc-tag (9B11) mouse mAb	Cell Signaling Technology	2276S	Mouse monoclonal antibody for detecting myc tagged proteins.
OmniPur EDTA, Disodium Salt, Dihydrate	Calbiochem	4010-OP
Optima L-100 XP	Beckman Coulter	392050
Optima MAX-TL	Beckman Coulter	A95761
Opti-MEM I Reduced Serum Media	Gibco	31985070
PBS Tablets	Gibco	18912014
PhosSTOP	Roche	4906845001
RAB11A-Specific Polyclonal antibody	Proteintech	20229-1-AP	Rabbit polyclonal antibody for detecting Rab11.
Sucrose	Affymetrix	AAJ21931A4
SW 41 Ti Swinging-Bucket Rotor	Beckman Coulter	331362
TLA-120.2 Fixed-Angle Rotor	Beckman Coulter	362046
Trypsin-EDTA (0.05%), phenol red	Gibco	25300062