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 processes4,5,6,7,8,9. 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 trafficking7,11,12. Of interest, various research groups have illustrated the importance of ARF6 in endocytic recycling13,14,15,16,17. 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
- 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. - The next day, transfect the cells with Lipofectamine according to the manufacturer's instructions.
2. Cell harvest
- Discard the culture medium 48 h post-transfection.
- Wash the cells with ice-cold PBS (10 mM sodium phosphates, 2.68 mM potassium chloride, 140 mM sodium chloride) twice.
- Add 1 mL of ice-cold PBS+ (PBS supplemented with 0.5x protease inhibitor cocktail and 0.5x phosphatase inhibitor cocktail) to each dish.
- Collect the cells with a cell scraper and transfer the cell suspension to a 15 mL centrifuge tube.
- Pellet the cells by centrifugation using a swing bucket rotor at 400 x g for 5 min.
- 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).
- Collect the cells by centrifugation at 1,300 x g for 10 min.
- Resuspend the cell pellet in 1 mL of HB.
- 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. - 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. - Add 0.7 mL of HB to the homogenate.
- Spin the diluted homogenate at 2,000 x g for 10 min at 4 °C.
NOTE: The pellet contains nuclei and unbroken cells. - Collect 1.5 mL of the supernatant and repeat step 2.12 once.
- Collect 1.4 mL of the supernatant and label it as post-nuclear supernatant (PNS).
3. Density gradient column preparation
- Transfer 1.2 mL of PNS to an ultracentrifuge tube.
- 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. - 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.
- 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. - 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
- Centrifuge the column at 210,000 x g for 3 h at 4 °C.
- 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. - Dilute all the fractions with 1 mL of dilution buffer (3 mM imidazole at pH 7.4, 1 mM EDTA).
- Centrifuge the diluted sample at 100,000 x g for 1 h at 4 °C.
- Aspirate the supernatant and add 50 µL of 1x sample buffer to harvest the fractions.
- Analyze the protein contents in the fractions by western blotting.
Representative 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 subcellular markers, including β-COP, COX IV, GAPDH, EEA1, Rab7, and Lamp1, were also probed. A positive EEA1 signal is also detected in fraction 7 as well. The band intensities of GAPDH, COX IV, and Rab11 in both fraction 7 and PNS were measured with ImageLab software (Bio-Rad). The intensity ratios of the markers in fraction 7 to PNS were calculated and expressed in a bar chart ± SD.
The previous studies suggest that ARF6 and ELMO1 interact with FE65 to promote Rac1-mediated neurite outgrowth21,22. Since ARF6 is a regulator of endocytic recycling and ELMO1 plasma membrane targeting is critical for the subsequent Rac1 activation, it is hypothesized that FE65 connects ARF6 and ELMO1 to mediate the trafficking of ELMO1 to the plasma membrane. Therefore, this method was employed to investigate the ELMO1 level in the isolated recycling endosomes. HEK293 cells were transfected with either ELMO1, ELMO1 + ARF6 Q67L, or ELMO1 + ARF6 Q67L + FE65. No changes were found in ELMO1 distribution with ARF6 Q67L and FE65 overexpression (Figure 2A). Next, the ELMO1 level in fraction 7 (Rab11-positive) were compared between different transfections. The amount of ELMO1 in the fraction is found to elevate after the co-transfection of ARF6 Q67L. Further increase in ELMO1 level is observed when both ARF6 Q67L and FE65 are co-expressed (Figure 2B). Conversely, knockout of FE65 diminished the ARF6-mediated ELMO1 enrichment in fraction 7 (Figure 2C).
Figure 1: Recycling endosomes are found in fraction 7 after density gradient ultracentrifugation. Untransfected HEK293 cells were fractionated and the protein contents in the obtained fractions were analyzed with western blotting. Rab11 is detected in fraction 7 with an anti-Rab11 antibody (1:500). Other subcellular markers were detected with their specific antibodies, including β-COP (1:1000), COX IV (1:1000), GAPDH (1:10,000), EEA1 (1:1000), Rab7 (1:500), and Lamp1 (1:1000). Fraction 1 is the less dense top fraction, while fraction 12 is the denser bottom fraction. The bar chart shows the ratio of GAPDH, COX IV, and Rab11 in fraction 7 to PNS ± SD. This figure has been modified from Chan, W. W. R. et al.22. Please click here to view a larger version of this figure.
Figure 2: Expression of FE65 promotes ARF6-mediated endocytic recycling of ELMO1. (A) The cells transfected with either ELMO1, ELMO1 + ARF6 Q67L, or ELMO1 + ARF6 Q67L + FE65 were fractionated using density gradient ultracentrifugation. All the fractions were subjected to western blotting for analyzing the distributions of ELMO1, ARF6 Q67L, and FE65. Fraction 1 is the less dense top fraction, while fraction 12 is the denser bottom fraction. (B) The Rab11-positive fraction 7 from different transfections was analyzed with Western blotting to evaluate the levels of ELMO1 and ARF6 Q67L. The amount of ELMO1 in fraction 7 was elevated when the cells were co-transfecting with ARF6 Q67L. Co-expression of ARF6 Q67L and FE65 further increases the amount of ELMO1 in the fraction. (C) Wildtype HEK293 was transfected with ELMO1 or ELMO1 + ARF6 Q67L, whereas FE65 KO HEK293 was transfected with ELMO1 + ARF6 Q67L. The ARF6-mediated ELMO1 enrichment in fraction 7 significantly diminished in FE65 KO cells. (B–C) Recycling endosome marker Rab11 (1:500) and cytosol marker GAPDH (1:10,000) were probed. ELMO1, FE65, and ARF6 Q67L were detected with anti-ELMO1 B-7 (1:1000), anti-FE65 E-20 (1:1000), and anti-myc 9B11 (1:5000), respectively. The relative level of ELMO1 in fraction 7 was expressed as the densitometric ratio of the ELMO1 in fraction 7/total ELMO1. Data for the bar chart were obtained from three independent experiments. One-way ANOVA with Bonferroni post hoc test was employed for statistical analysis. *p < 0.001. Results are mean fold change ± SD22. This figure has been modified from Chan, W. W. R. et al.22. Please click here to view a larger version of this figure.
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 is recommended to validate the refractive indexes of the solutions with a refractometer. The refractive index of the 62%, 35%, and 25% sucrose solutions at room temperature are 1.44, 1.39, and 1.37, respectively. Also, air bubbles should be avoided from the gradient. The presence of bubbles in the column may disrupt the continuity of the gradient. Detergents should be avoided in the homogenization process since they damage the membrane of organelles. This leads to the releasing of proteins from membrane-bound organelles and could result in severe contamination. Also, all homogenizing tools should be pre-cooled before use to avoid protein degradation during homogenization. Once the gradient is prepared, it should be used as soon as possible. Although the prepared gradient could temporarily be stored at 4 °C (1-2 h), prolonged storage may interfere with the density gradient due to diffusion.
Sucrose is a widely used gradient medium because of its easy availability. In fact, there are many other alternatives, including Percoll and Ficoll-40023,24. These media have different physical properties when compared with sucrose. For instance, Percoll has lower osmolarity and viscosity than sucrose. These allow rapid banding of particles using lower centrifugal forces. Ficoll-400 has a lower permeability toward membranes than sucrose because of its high molecular weight and low content of dialyzable material. Therefore, changing the gradient medium may achieve a higher endosome isolation efficiency.
Apart from density gradient ultracentrifugation, other methods can be used for cell fractionation, including free-flow electrophoresis (FFE)25, fluorescence-activated organelle sorting (FAOS)26, and immunoisolation27. FFE is a liquid phase separation method. The sample flows through the separation buffer under the influence of an electric field perpendicular to the flow direction. Deflection levels of different organelles vary based on their surface charges25. FAOS usually uses a fluorescent tag or antibody to label specific organelle and then followed by flow cytometry for the isolation28,29. Immunoisolation relies on detecting specific antigens on the surface of the targeted organelle and subsequent precipitation by antibodies30.
When comparing with these alternatives, density gradient ultracentrifugation has its own advantages. First of all, a distribution profile of the interested protein can be obtained by performing Western blotting with the isolated fractions (Figure 2A). Any changes in protein subcellular localization could be easily detected. Also, ultracentrifuge is a standard instrument in most institutes, and the technical requirement for operating the centrifuge is low. In contrast, a flow cytometer and a specific electrophoresis system are required for the isolation process of FAOS and FFE, respectively. There is no specific equipment required for immunoisolation. However, it is mainly used for isolating endosomes from a small number of cells. The preparation scale of ultracentrifugation is larger than that of immunoisolation. Besides, an antibody with high specificity is necessary for immunoisolation31. Furthermore, detergent- and high salt-containing buffer cannot be used in the washing steps to ensure the integrity of the endosomes. This may lead to high background and reduce the purity of the isolated organelle31.
Since density gradient ultracentrifugation separates organelles based on density, its most significant limitation is that of resolving power toward organelles with similar density. As shown in Figure 1, both Rab11 and EEA1 are detected in fraction 7 because of the similar physical properties of recycling and early endosomes. Further assays are needed to confirm the changes in the level of the targeted protein in the recycling endosome. In the previous study, co-immunostaining on ELMO1 and Rab11 was performed in cells22. In addition to performing other assays, some measures can be adopted to overcome this problem. A continuous density gradient can resolve organelles with minor density differences32. However, the yield in a continuous gradient is significantly lower than that of a discontinuous gradient. It is possible to manipulate the density of the endosome by pre-treating the cells with latex beads33. The beads are internalized by the cells via endocytosis. The density of the beads containing endosomes is significantly reduced and can be separated from other organelles. Usually, adding another level of isolation could significantly improve the isolation of organelles with similar densities. For example, perform immunoisolation from the isolated fraction34. By using a specific antibody, recycling endosomes can be separated from the contaminants. Fluorescent-labeled antibodies and probes, combined with flow cytometry analysis, could also be used for precise endosome isolation28. FFE is also applicable for separating organelles with similar densities based on their surface charge25.
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by funds from the Research Grants Council Hong Kong, CUHK direct grant scheme, United College endowment fund, and the TUYF Charitable Trust. The figures in this work were adapted from our previous publication, "ARF6-Rac1 signaling-mediated neurite outgrowth is potentiated by the neuronal adaptor FE65 through orchestrating ARF6 and ELMO1" published in the FASEB Journal in October 2020.
Materials
| 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 |
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