Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

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.