Microtubule and motor-dependent endocytic vesicle sorting in vitro

Abstract

Endocytic vesicles undergo fission to sort ligand from receptor. Using quantitative immunofluorescence and video imaging, we provide the first in vitro reconstitution of receptor-ligand sorting in early endocytic vesicles derived from rat liver. We show that to undergo fission, presegregation vesicles must bind to microtubules (MTs) and move upon addition of ATP. Over 13% of motile vesicles elongate and are capable of fission. After fission, one vesicle continues to move, whereas the other remains stationary, resulting in their separation. On average, almost 90% receptor is found in one daughter vesicle, whereas ligand is enriched by approximately 300% with respect to receptor in the other daughter vesicle. Although studies performed on polarity marked MTs showed approximately equal plus and minus end-directed motility, immunofluorescence microscopy revealed that kinesins, but not dynein, were associated with these vesicles. Motility and fission were prevented by addition of 1 mM 5'-adenylylimido-diphosphate (AMP-PNP, an inhibitor of kinesins) or incubation with kinesin antibodies, but were unaffected by addition of 5 microM vanadate (a dynein inhibitor) or dynein antibodies. These studies indicate an essential role of kinesin-based MT motility in endocytic vesicle sorting, providing a system in which factors required for endocytic vesicle processing can be identified and characterized.

Figure 1

Immunolocalization of ASGPR and ASOR in isolated endocytic vesicles. A preparation enriched in endocytic vesicles was prepared from rat liver that was obtained 5 min after i.v. injection of Texas red–ASOR, as described in Materials and Methods. Vesicles were incubated at 4°C for 1 min in anti-ASGPR IgG. Cy2-labeled goat anti–rabbit IgG was added and incubation was continued for an additional 6 min. Vesicles were hydrostatically attached to a glass slide and examined by immunofluorescence microscopy. Many receptor containing vesicles were observed (a). There were fewer ligand-containing vesicles (b). A merged view (c) shows that although there were many receptor-containing vesicles devoid of ligand, virtually all ligand-containing vesicles also contained receptor, indicating that they represent a population of early endocytic vesicles.

Figure 2

MT-based motility and fission of endocytic vesicles. Fluorescent early endocytic vesicles were prepared after injection of a rat with Texas red–labeled ASOR. Endocytic vesicles were flowed into a 3-μl microscopy chamber in which taxol-stabilized MTs had been attached to the glass surface. Vesicle motility was examined after addition of 50 μM ATP. In some experiments, vesicles were preincubated with rabbit anti–rat ASGPR IgG directed against the receptor cytoplasmic tail, nonimmune mouse IgG, or mouse mAbs IgG or IgM against kinesin or cytoplasmic dynein. Distribution of antibody was visualized after addition of Cy2-labeled goat anti–rabbit IgG, Cy2-labeled goat anti–mouse IgG, or FITC-labeled anti–mouse IgM, as appropriate. The bars in the left graph indicate the percentage of MT-bound vesicles that moved upon ATP addition. The total number of MT-bound vesicles that were examined in each experiment is in parentheses. The bars in the right graph indicate the percentage of moving vesicles that underwent fission.

Figure 2

MT-based motility and fission of endocytic vesicles. Fluorescent early endocytic vesicles were prepared after injection of a rat with Texas red–labeled ASOR. Endocytic vesicles were flowed into a 3-μl microscopy chamber in which taxol-stabilized MTs had been attached to the glass surface. Vesicle motility was examined after addition of 50 μM ATP. In some experiments, vesicles were preincubated with rabbit anti–rat ASGPR IgG directed against the receptor cytoplasmic tail, nonimmune mouse IgG, or mouse mAbs IgG or IgM against kinesin or cytoplasmic dynein. Distribution of antibody was visualized after addition of Cy2-labeled goat anti–rabbit IgG, Cy2-labeled goat anti–mouse IgG, or FITC-labeled anti–mouse IgM, as appropriate. The bars in the left graph indicate the percentage of MT-bound vesicles that moved upon ATP addition. The total number of MT-bound vesicles that were examined in each experiment is in parentheses. The bars in the right graph indicate the percentage of moving vesicles that underwent fission.

Figure 3

Fission of an endocytic vesicle on an MT after addition of ATP. Texas red–ASOR and rhodamine-labeled MTs are visualized in the left panels, whereas rabbit IgG against the cytoplasmic tail of the ASGPR after incubation with Cy2-labeled secondary antibody is visualized in the right panels. Time in seconds after the addition of 50 μM ATP is shown in the upper left. Before addition of ATP (0 s), the arrowhead points to a single vesicle that is attached to an MT and contains ligand and receptor. In subsequent panels, the arrow indicates the nonmotile daughter vesicle that contains only ∼3% of the original receptor fluorescence. This study corresponds to event 14 in Table . Bar, 10 μm.

Figure 4

Effect of vesicle dilution on motility and fission. Vesicles were diluted by 50% before binding to MTs and addition of 50 μM ATP, to test the possibility that under more concentrated conditions, movement of two overlapping vesicles could produce a spurious fission. Black bars indicate results for 1,093 vesicles that were bound to MTs when concentrated; white bars indicate results for 169 vesicles that were bound to MTs when diluted. Motility and fission were determined as described in Materials and Methods.

Figure 5

Colocalization of early endocytic vesicles with kinesin, but not with cytoplasmic dynein. Two representative studies are shown in this figure in which Texas red–ASOR and rhodamine-labeled MTs are visualized (a and d). Mouse mAbs (IgM) against the kinesin heavy chain (b) and the dynein intermediate chain (e) were incubated with vesicles and visualized after addition of FITC-labeled goat anti–mouse IgM. Merged images reveal that the majority of ligand-containing vesicles are associated with kinesin (c) but not with dynein (f).

Figure 6

Effects of vanadate and AMP-PNP on vesicle motility and fission. MTs were bound to the glass chamber via DEAE-dextran. Studies were performed at ATP concentrations of 50 μM (black bars) or 4 mM (cross-hatched bars) in the absence of a regenerating system. The bars in the left graph indicate the percentage of MT-bound vesicles that moved upon ATP addition in the presence or absence of varied concentrations of vanadate or 1 mM AMP-PNP. The total number of MT-bound vesicles that were examined in each experiment is in parentheses. The bars in the right graph indicate the percentage of moving vesicles that underwent fission.

Figure 6

Effects of vanadate and AMP-PNP on vesicle motility and fission. MTs were bound to the glass chamber via DEAE-dextran. Studies were performed at ATP concentrations of 50 μM (black bars) or 4 mM (cross-hatched bars) in the absence of a regenerating system. The bars in the left graph indicate the percentage of MT-bound vesicles that moved upon ATP addition in the presence or absence of varied concentrations of vanadate or 1 mM AMP-PNP. The total number of MT-bound vesicles that were examined in each experiment is in parentheses. The bars in the right graph indicate the percentage of moving vesicles that underwent fission.