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 ∼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 μM 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.

Kif5B and Kifc1 Interact and Are Required for Motility and Fission of Early Endocytic Vesicles in Mouse Liver

“…Specifically, there was no inhibition of vesicle motility in the presence of 5 M vanadate (Figure 4), a treatment that inhibits dynein-mediated motility Sarkar et al, 2006), and there was no immunocolocalization of dynein with fluorescent ASOR-containing vesicles ( Figure 6A). Rather, as was found in studies of early endocytic vesicles from the rat, all motility in both wild-type and Kifc2 knockout mouse vesicles was inhibited by 1 mM AMP-PNP, an inhibitor of kinesin based motility (Vale et al, 1992;Bananis et al, 2000;Murray et al, 2000). A recent study in which HeLa cells were loaded with fluorescent epidermal growth factor for 2-3 min and followed for as long as 30 min was interpreted as showing a role for dynein in processing of early endocytic vesicles (Driskell et al, 2007), in contrast to other studies (Nielsen et al, 1999;Bananis et al, 2003Bananis et al, .…”

“…When the vesicles were preincubated with 5 M vanadate, an inhibitor of the minus-end-directed motor dynein (Kobayashi et al, 1978;Bananis et al, 2000Bananis et al, , 2004Murray et al, 2000), motility and fission were essentially unchanged (Figure 4, A and B). However, pretreatment with 1 mM AMP-PNP, a kinesin inhibitor (Vale et al, 1992;Bananis et al, 2000Bananis et al, , 2004Murray et al, 2000), before ATP addition inhibited vesicle motility and fission completely (Figure 4, A and B). These results suggest that microtubule-based motility of mouse early endocytic vesicles is mediated by kinesins and not dynein, similar to results obtained with rat early endocytic vesicles .…”

Caspases rule the intracellular trafficking cartel

“…Intra-Golgi trafficking involves motor kinesin movement toward the plus end of microtubules. Moreover, motility of early endocytic vesicles is also performed by kinesin [58]. As both traffickings to and from the Golgi as well as the integrity of the Golgi itself are dependent on microtubule motors [59][60][61][62], it is not surprising that components of minus and plus end-directed microtubule motors are targeted by caspases during apoptosis.…”

Cellular Mechanisms for Dopamine D4 Receptor-induced Homeostatic Regulation of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors

“…To examine the involvement of kinesin motors directly, we dialyzed a monoclonal antibody against KIF5, which is known to bind to kinesin and block the motor function (30), and examined the effect of PD168077 on AMPAR-EPSC in bicucullinepretreated slices. As shown in Fig.…”