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.<...

<ol>
	<li>Elkin, S. R., Lakoduk, A. M., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytic+pathways+and+endosomal+trafficking:+a+primer.">Endocytic pathways and endosomal trafficking: a primer.</a> Wiener Medizinische Wochenschrift. 166 (7-8), 196-204 (2016).</li><li>Kumari, S., Mg, S., Mayor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+unplugged:+multiple+ways+to+enter+the+cell.">Endocytosis unplugged: multiple ways to enter the cell.</a> Cell Research. 20 (3), 256-275 (2010).</li><li>Naslavsky, N., Caplan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enigmatic+endosome+-+sorting+the+ins+and+outs+of+endocytic+trafficking.">The enigmatic endosome - sorting the ins and outs of endocytic trafficking.</a> Journal of Cell Science. 131 (13), (2018).</li><li>Cullen, P. J., Steinberg, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+degrade+or+not+to+degrade:+mechanisms+and+significance+of+endocytic+recycling.">To degrade or not to degrade: mechanisms and significance of endocytic recycling.</a> Nature Reviews. Molecular Cell Biology. 19, 679-696 (2018).</li><li>Weeratunga, S., Paul, B., Collins, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognising+the+signals+for+endosomal+trafficking.">Recognising the signals for endosomal trafficking.</a> Current Opinion in Cell Biology. 65, 17-27 (2020).</li><li>Khan, I., Steeg, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis:+a+pivotal+pathway+for+regulating+metastasis.">Endocytosis: a pivotal pathway for regulating metastasis.</a> British Journal of Cancer. 124 (1), 66-75 (2021).</li><li>Grant, B. D., Donaldson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+and+mechanisms+of+endocytic+recycling.">Pathways and mechanisms of endocytic recycling.</a> Nature Reviews. Molecular Cell Biology. 10 (9), 597-608 (2009).</li><li>Maxfield, F. R., McGraw, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytic+recycling.">Endocytic recycling.</a> Nature Reviews. Molecular Cell Biology. 5 (2), 121-132 (2004).</li><li>McDonald, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explosion+in+the+complexity+of+membrane+protein+recycling.">Explosion in the complexity of membrane protein recycling.</a> American Journal of Physiology. Cell Physiology. 320 (4), 483-494 (2021).</li><li>O'Sullivan, M. J., Lindsay, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Endosomal+Recycling+pathway-at+the+crossroads+of+the+cell.">The Endosomal Recycling pathway-at the crossroads of the cell.</a> International Journal of Molecular Sciences. 21 (17), 6074 (2020).</li><li>D'Souza-Schorey, C., Li, G., Colombo, M. I., Stahl, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+role+for+ARF6+in+receptor-mediated+endocytosis.">A regulatory role for ARF6 in receptor-mediated endocytosis.</a> Science. 267 (5201), 1175-1178 (1995).</li><li>Schweitzer, J. K., Sedgwick, A. E., D'Souza-Schorey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARF6-mediated+endocytic+recycling+impacts+cell+movement,+cell+division+and+lipid+homeostasis.">ARF6-mediated endocytic recycling impacts cell movement, cell division and lipid homeostasis.</a> Seminars in Cell and Developmental Biology. 22 (1), 39-47 (2011).</li><li>Finicle, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingolipids+inhibit+endosomal+recycling+of+nutrient+transporters+by+inactivating+ARF6.">Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6.</a> Journal of Cell Science. 131 (12), (2018).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APE1+upregulates+MMP-14+via+redox-sensitive+ARF6-mediated+recycling+to+promote+cell+invasion+of+esophageal+adenocarcinoma.">APE1 upregulates MMP-14 via redox-sensitive ARF6-mediated recycling to promote cell invasion of esophageal adenocarcinoma.</a> Cancer Research. 79 (17), 4426-4438 (2019).</li><li>Qi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arf6-driven+endocytic+recycling+of+CD147+determines+HCC+malignant+phenotypes.">Arf6-driven endocytic recycling of CD147 determines HCC malignant phenotypes.</a> Journal of Experimental and Clinical Cancer Research. 38 (1), 471 (2019).</li><li>Crupi, M. J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GGA3-mediated+recycling+of+the+RET+receptor+tyrosine+kinase+contributes+to+cell+migration+and+invasion.">GGA3-mediated recycling of the RET receptor tyrosine kinase contributes to cell migration and invasion.</a> Oncogene. 39 (6), 1361-1377 (2020).</li><li>Gamara, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Arf6+deletion+in+PLB-985+differentiated+in+neutrophil-like+cells+and+in+mouse+neutrophils:+impact+on+adhesion+and+migration.">Assessment of Arf6 deletion in PLB-985 differentiated in neutrophil-like cells and in mouse neutrophils: impact on adhesion and migration.</a> Mediators of Inflammation. 2020, 2713074 (2020).</li><li>Santy, L. C., Ravichandran, K. S., Casanova, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DOCK180/Elmo+complex+couples+ARNO-mediated+Arf6+activation+to+the+downstream+activation+of+Rac1.">The DOCK180/Elmo complex couples ARNO-mediated Arf6 activation to the downstream activation of Rac1.</a> Current Biology. 15 (19), 1749-1754 (2005).</li><li>Wibo, M., Dumont, J. E., Brown, B. L., Marshall, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fractionation+by+centrifugation+methods.">Cell fractionation by centrifugation methods.</a> Eukaryotic Cell Function and Growth: Regulation by Intracellular Cyclic Nucleotides. , 1-17 (1976).</li><li>Kelly, E. E., Horgan, C. P., McCaffrey, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab11+proteins+in+health+and+disease.">Rab11 proteins in health and disease.</a> Biochemical Society Transactions. 40 (6), 1360-1367 (2012).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+adaptor+FE65+stimulates+Rac1-mediated+neurite+outgrowth+by+recruiting+and+activating+ELMO1.">Neuronal adaptor FE65 stimulates Rac1-mediated neurite outgrowth by recruiting and activating ELMO1.</a> The Journal of Biological Chemistry. 293 (20), 7674-7688 (2018).</li><li>Chan, W. W. R., Li, W., Chang, R. C. C., Lau, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARF6-Rac1+signaling-mediated+neurite+outgrowth+is+potentiated+by+the+neuronal+adaptor+FE65+through+orchestrating+ARF6+and+ELMO1.">ARF6-Rac1 signaling-mediated neurite outgrowth is potentiated by the neuronal adaptor FE65 through orchestrating ARF6 and ELMO1.</a> FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology. 34 (12), 16397-16413 (2020).</li><li>Huber, L. A., Pfaller, K., Vietor, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+proteomics:+implications+for+subcellular+fractionation+in+proteomics.">Organelle proteomics: implications for subcellular fractionation in proteomics.</a> Circulation Research. 92 (9), 962-968 (2003).</li><li>Fleischer, S., Kervina, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+fractionation+of+rat+liver.">Subcellular fractionation of rat liver.</a> Methods in Enzymology. 31, 6-41 (1974).</li><li>Marsh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosome+and+lysosome+purification+by+free-flow+electrophoresis.">Endosome and lysosome purification by free-flow electrophoresis.</a> Methods in Cell Biology. 31, 319-334 (1989).</li><li>Stasyk, T., Huber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zooming+in:+fractionation+strategies+in+proteomics.">Zooming in: fractionation strategies in proteomics.</a> Proteomics. 4 (12), 3704-3716 (2004).</li><li>Iordachescu, A., Hulley, P., Grover, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+collection+of+nanoscopic+vesicles+from+an+organotypic+culture+model.">A novel method for the collection of nanoscopic vesicles from an organotypic culture model.</a> RSC Advances. 8 (14), 7622-7632 (2018).</li><li>Chavrier, P., vander Sluijs, P., Mishal, Z., Nagelkerken, B., Gorvel, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+endosome+membrane+dynamics+characterized+by+flow+cytometry.">Early endosome membrane dynamics characterized by flow cytometry.</a> Cytometry. 29 (1), 41-49 (1997).</li><li>Chasan, A. I., Beyer, M., Kurts, C., Burgdorf, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+specialized,+antigen-loaded+early+endosomal+subpopulation+by+flow+cytometry.">Isolation of a specialized, antigen-loaded early endosomal subpopulation by flow cytometry.</a> Methods in Molecular Biology. 960, 379-388 (2013).</li><li>Thapa, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-3-OH+kinase+signaling+is+spatially+organized+at+endosomal+compartments+by+microtubule-associated+protein+4.">Phosphatidylinositol-3-OH kinase signaling is spatially organized at endosomal compartments by microtubule-associated protein 4.</a> Nature Cell Biology. 22 (11), 1357-1370 (2020).</li><li>Guimaraes de Araujo, M. E., Fialka, I., Huber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Endocytic Organelles: Methods For Preparation And Analysis. In eLS. , (2001).</li><li>Rickwood, D., Graham, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Centrifugation Techniques. , (2015).</li><li>Lamberti, G., de Araujo, M. E., Huber, L. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;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-&#946;-COP antibody</td>
			<td>Sigma</td>
			<td>G6160</td>
			<td>Mouse monoclonal antibody for detecting &#946;-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

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...

Watch this Scientific Journal Video about Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells at JoVE.com

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

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 ...

This protocol helps in isolating the recycling endosomes containing fraction from transfected mammalian cells to facilitate the investigation of protein trafficking via endocytic trafficking. This technique is easy to handle and applicable to both tissue and cell samples. Demonstrating the procedure will be Miss Zhai Yu Qi, a PhD student from Dr.Lao's Laboratory.To begin, plate 2 times 10 to the 6 cells in a 100 millimeter culture dish. On the next day, transfect the cells with Lipofectamine according to the manufacturer's instructions. To harvest the cells, discard the culture medium 48 hours post-transfection and wash the cells twice with ice cold PBS.Then add one milliliter of ice cold PBS supplemented with 0.5X protease inhibitor cocktail in 0.5X phosphatase inhibitor cocktail to each dish. Next, using a cell scraper, collect the cells and transfer the cell suspension to a 15 milliliter centrifuge tube. the cells by centrifugation using a swing bucket rotor.Discard the supernatant and resuspend the cell pellet gently in five milliliters of homogenization buffer. Collect the cells again by centrifugation and resuspend the cell pellet in one milliliter of homogenization buffer. Homogenize the cells with a Dounce Homogenizer using 15 to 20 strokes.Then transfer the homogonate to a two milliliter centrifugation tube. Add 700 microliters of homogenization buffer to the homogonate. Then spin the diluted homogonate and collect 1.5 milliliters of the supernatant.Spin the collected supernatant again, then collect 1.4 milliliters of the supernatant and label it as post-nuclear supernatant. To prepare the density gradient column, transfer 1.2 milliliters of the post-nuclear supernatant to an ultracentrifuge tube. Then add one milliliter of 62%sucrose solution to the sample and mix well by gentle pipetting.Next, carefully add 3.3 milliliters of 35%sucrose solution on top of the sample, followed by 2.2 milliliters of 25%sucrose solution on top of the 35%sucrose solution. Finally, fill the ultracentrifuge tube to the top with homogenization buffer. Centrifuge the density gradient column and carefully collect 12 fractions of 1 milliliter each, starting from the top of the gradient.Next, dilute all the fractions with one milliliter of dilution buffer and centrifuge the diluted samples. After centrifugation, aspirate the supernatant and add 50 microliters of 1X sample buffer to harvest the fractions. The recycling endosome marker Rab11 was detected in Fraction seven.Other subcellular markers, including beta COP, COX IV, GAPDH, EEA1, Rab 7, and Lamp1 were also probed. A positive EEA1 signal was also detected in Fraction seven. The measured band intensities of GAPDH, Cox IV, and Rab11 in both Fraction seven and the post-nuclear supernatant are shown here.After transecting HEC293 cells with either ELMO1, ELMO1 ARF6Q67L, or ELMO1 ARF6Q67L FE65, no changes were found in ELMO1 distribution with ARF6Q67L and FE65 overexpression. The ELMO1 level in Fraction seven was compared between different transfections and found to be elevated after cotransfection of ARF6Q67L. A further increase was observed when both ARF6Q67L and FE65 were co-expressed.Conversely, knocking out FE65 diminished the ARF6 mediated Elmo1 enrichment in Fraction seven. The fraction obtained could be used for subsequent analysis. For example, western blotting to visualize protein level changes in the fraction.

density-gradient-ultracentrifugation-for-investigating-endocytic

Research

JoVE Journal

Biochemistry

3.8K Views.  The Chinese University of Hong Kong. Questo protocollo aiuta a isolare gli endosomi di riciclaggio contenenti frazione da cellule di mammifero trasfettate per facilitare lo studio del traffico proteico attraverso il traffico endocitico. Questa tecnica è facile da maneggiare e applicabile sia ai campioni di tessuto che a quelli cellulari. A dimostrare la procedura sarà miss Zhai Yu Qi, dottoranda del Dr.Lao's Laboratory.Per iniziare, piastra 2 volte 10 alle 6 celle in un piatto di coltura da 100 millimetri. Il giorno suc...