Microtubule and motor-dependent endocytic vesicle sorting in vitro

Endocytic vesicles undergo fission to sort ligand from receptor. Using quan titative immunofluorescence and video imaging, we provide the first in vitr o reconstitution of receptor-ligand sorting in early endocytic vesicles der ived from rat liver. We show that to undergo fission, presegregation vesicl es 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 daught er vesicle! whereas ligand is enriched by similar to 300% with respect to r eceptor in the other daughter vesicle. Although studies performed on polari ty marked MTs showed approximately equal plus and minus end-directed motili ty, immunofluorescence microscopy revealed that kinesins, but not dynein, w ere associated with these vesicles. Motility and fission were prevented by addition of 1 mM 5'-adenylylimido-diphosphate (AMP-PNP, an inhibitor of kin esins) or incubation with kinesin antibodies, but were unaffected by additi on of 5 mu M vanadate (a dynein inhibitor) or dynein antibodies. These stud ies indicate an essential role of kinesin-based MT motility in endocytic ve sicle sorting, providing a system in which factors required for endocytic v esicle processing can be identified and characterized.