Name: Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells (Video) | JoVE Journal
Uploaded: 2021-06-30T13:15:00.000+00:00
Duration: 5 min 13 s
Description: Learn density gradient ultracentrifugation for isolating recycling endosomes in mammalian cells. This method enables investigation of protein trafficking via endocytic pathways using transfected cell samples.

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5 minutes

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, &#34;ARF6-Rac1 signaling-mediated neurite outgrowth is potentiated by the neuronal adaptor FE65 through orchestrating ARF6 and ELMO1&#34; published in the FASEB Journal in October 2020.

1. Mammalian cell culture and transfection
<ol>
	<li>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.</li>
	<li>The next day, transfect the cells with Lipofectamine according to the manufacturer&#39;s instructions.</li>
</ol>
2. Cell harvest
<ol>
	<li>Discard the culture medium 48 h post-transfection.</li>
	<li>Wash the cells with ice-cold PBS (10 mM sodium phosphates, 2.68 mM potassium chloride, 140 mM sodium chloride) twice.</li>
	<li>Add 1 mL of ice-cold PBS+ (PBS supplemented with 0.5x protease inhibitor cocktail and 0.5x phosphatase inhibitor cocktail) to each dish.</li>
	<li>Collect the cells with a cell scraper and transfer the cell suspension to a 15 mL centrifuge tube.</li>
	<li>Pellet the cells by centrifugation using a swing bucket rotor at 400 x g for 5 min.</li>
	<li>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).</li>
	<li>Collect the cells by centrifugation at 1,300 x g for 10 min.</li>
	<li>Resuspend the cell pellet in 1 mL of HB.</li>
	<li>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.</li>
	<li>Transfer the homogenate to a 2 mL centrifugation tube. 
	NOTE: Harvest 50 &#181;L of homogenate with 12.5 &#181;L of 5x sample buffer and label it as total lysate.</li>
	<li>Add 0.7 mL of HB to the homogenate.</li>
	<li>Spin the diluted homogenate at 2,000 x g for 10 min at 4 &#176;C. 
	NOTE: The pellet contains nuclei and unbroken cells.</li>
	<li>Collect 1.5 mL of the supernatant and repeat step 2.12 once.</li>
	<li>Collect 1.4 mL of the supernatant and label it as post-nuclear supernatant (PNS).</li>
</ol>
3. Density gradient column preparation
<ol>
	<li>Transfer 1.2 mL of PNS to an ultracentrifuge tube.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#160;could be checked with a refractometer to ensure the precision and consistency of the experiment.</li>
	<li>Fill up the ultracentrifugation tube with HB. 
	&#8203;NOTE: Temporarily store the prepared density gradient column at 4 &#176;C.</li>
</ol>
4. Fractionation and recovery of recycling endosome-containing fraction
<ol>
	<li>Centrifuge the column at 210,000 x g for 3 h at 4 &#176;C.</li>
	<li>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 &#176;C.</li>
	<li>Dilute all the fractions with 1 mL of dilution buffer (3 mM imidazole at pH 7.4, 1 mM EDTA).</li>
	<li>Centrifuge the diluted sample at 100,000 x g for 1 h at 4 &#176;C.</li>
	<li>Aspirate the supernatant and add 50 &#181;L of 1x sample buffer to harvest the fractions.</li>
	<li>Analyze the protein contents in the fractions by western blotting.</li>
</ol>

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</ol>

The authors declare that they have no conflicts of interest with the contents of this article.

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...

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.

<table><tbody><tr><td>1 mL, Open-Top Thickwall Polypropylene Tube, 11 x 34 mm</td><td>Beckman Coulter</td><td>347287</td><td></td></tr><tr><td>100 mm tissue culture dish</td><td>SPL</td><td>20100</td><td></td></tr><tr><td>13.2 mL, Certified Free Open-Top Thinwall Ultra-Clear Tube, 14 x 89 mm</td><td>Beckman Coulter</td><td>C14277</td><td></td></tr><tr><td>5x Sample Buffer</td><td>GenScript</td><td>MB01015</td><td></td></tr><tr><td>cOmplete, EDTA-free Protease Inhibitor Cocktail</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>COX IV (3E11) Rabbit mAb</td><td>Cell Signaling Technology</td><td>4850S</td><td>Rabbit monoclonal antibody for detecting COX IV.</td></tr><tr><td>Cycloheximide</td><td>Sigma-Aldrich</td><td>C1988</td><td></td></tr><tr><td>Dounce Tissue Grinder, 7 mL</td><td>DWK Life Sciences</td><td>357542</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM) with low glucose</td><td>HyClone</td><td>SH30021.01</td><td></td></tr><tr><td>ELMO1 antibody (B-7)</td><td>Santa Cruz Biotechnology</td><td>SC-271519</td><td>Mouse monoclonal antibody for detecting ELMO1.</td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>QIAGEN</td><td>12362</td><td></td></tr><tr><td>FE65 antibody (E-20)</td><td>Santa Cruz Biotechnology</td><td>SC-19751</td><td>Goat polyclonal antibody for detecting FE65.</td></tr><tr><td>Fetal Bovine Serum, Research Grade</td><td>HyClone</td><td>SV30160.03</td><td></td></tr><tr><td>GAPDH Monoclonal Antibody (6C5)</td><td>Ambion</td><td>AM4300</td><td>Mouse monoclonal antibody for detecting GAPDH.</td></tr><tr><td>ImageLab Software</td><td>Bio-Rad</td><td></td><td>Measurement of band intensity</td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I2399</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Invitrogen</td><td>11668019</td><td></td></tr><tr><td>Monoclonal Anti-&amp;beta;-COP antibody</td><td>Sigma</td><td>G6160</td><td>Mouse monoclonal antibody for detecting &amp;beta;-COP.</td></tr><tr><td>Myc-tag (9B11) mouse mAb</td><td>Cell Signaling Technology</td><td>2276S</td><td>Mouse monoclonal antibody for detecting myc tagged proteins.</td></tr><tr><td>OmniPur EDTA, Disodium Salt, Dihydrate</td><td>Calbiochem</td><td>4010-OP</td><td></td></tr><tr><td>Optima L-100 XP</td><td>Beckman Coulter</td><td>392050</td><td></td></tr><tr><td>Optima MAX-TL</td><td>Beckman Coulter</td><td>A95761</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Media</td><td>Gibco</td><td>31985070</td><td></td></tr><tr><td>PBS Tablets</td><td>Gibco</td><td>18912014</td><td></td></tr><tr><td>PhosSTOP</td><td>Roche</td><td>4906845001</td><td></td></tr><tr><td>RAB11A-Specific Polyclonal antibody</td><td>Proteintech</td><td>20229-1-AP</td><td>Rabbit polyclonal antibody for detecting Rab11.</td></tr><tr><td>Sucrose</td><td>Affymetrix</td><td>AAJ21931A4</td><td></td></tr><tr><td>SW 41 Ti Swinging-Bucket Rotor</td><td>Beckman Coulter</td><td>331362</td><td></td></tr><tr><td>TLA-120.2 Fixed-Angle Rotor</td><td>Beckman Coulter</td><td>362046</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Gibco</td><td>25300062</td><td></td></tr></tbody></table>

density gradient ultracentrifugation for investigating endocytic recycling in mammalian cells

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

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.

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...

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

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma ...

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Research

JoVE Journal

Biochemistry

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Learn density gradient ultracentrifugation for isolating recycling endosomes in mammalian cells. This method enables investigation of protein trafficking via endocytic pathways using transfected cell samples.

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

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