Reconstitution of ATP-dependent Movement of Endocytic Vesicles Along Microtubules In Vitro: An Oscillatory Bidirectional Process

Abstract

We have previously used the asialoglycoprotein receptor system to elucidate the pathway of hepatocytic processing of ligands such as asialoorosomucoid (ASOR). These studies suggested that endocytic vesicles bind to and travel along microtubules under the control of molecular motors such as cytoplasmic dynein. We now report reconstitution of this process in vitro with the use of a microscope assay to observe the interaction of early endocytic vesicles containing fluorescent ASOR with fluorescent microtubules. We find that ASOR-containing endosomes bind to microtubules and translocate along them in the presence of ATP. This represents the first time that mammalian endosomes containing a well-characterized ligand have been directly observed to translocate on microtubules in vitro. The endosome movement does not require cytosol or exogenous motor protein, is oscillatory, and is directed toward the plus and minus ends at equal frequencies. We also observe endosomes being stretched in opposite directions along microtubules, suggesting that microtubules could provide a mechanical basis for endocytic sorting events. The movement of endosomes in vitro is consistent with the hypothesis that microtubules actively participate in the sorting and distribution of endocytic contents.

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

“…This in vitro system has permitted identification of native endogenous proteins that are required for microtubulebased processing of endocytic vesicles. Using these tools, we found that plus-end motility of early endocytic vesicles from rat liver was mediated by the conventional kinesin Kif5B (standardized nomenclature kinesin-1) (Miki et al, 2003(Miki et al, , 2005Lawrence et al, 2004), and minus-end motility was mediated by Kifc2 (standardized nomenclature kinesin-14B) (Bananis et al, , 2003(Bananis et al, , 2004Murray et al, 2000). Whereas Kifc2 was initially described as a brain-specific minus-enddirected kinesin Saito et al, 1997), our studies showed that it was highly associated with these vesicles prepared from rat liver (Bananis et al, 2003).…”

“…Texas Red-labeled early endocytic vesicles were prepared from mouse or rat liver as described previously (Bananis et al, , 2003Murray et al, 2000). In brief, livers were harvested 5 min after portal venous injection of 50 g of Texas Red-labeled ASOR.…”

“…Previous studies indicated that this segregation event requires an intact microtubule cytoskeleton (Goltz et al, 1992;Novikoff et al, 1996;Murray et al, 2000;Bananis et al, 2003Bananis et al, , 2004. In more recent studies, microtubule-based endosome motility and segregation were reconstituted in vitro using fluorescent early endosomes prepared from rat liver 5 min after portal venous injection of Texas Red-labeled ASOR, a substrate for the hepatocytespecific asialoglycoprotein receptor (ASGPR) (Bananis et al, , 2003(Bananis et al, , 2004Murray et al, 2000;Murray and Wolkoff, 2003). On addition of ATP to the in vitro assay, these vesicles moved bidirectionally along microtubules.…”

“…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 .…”

CART: An Hrs/Actinin-4/BERP/Myosin V Protein Complex Required for Efficient Receptor Recycling

“…The complementary roles played by the actin cytoskeleton and microtubule network in the endocytic pathway (van Deurs et al, 1995;Durrbach et al, 1996;Maples et al, 1997;Murray et al, 2000) suggest that endosomes contain proteins allowing for movement on both types of cytoskeletal network. Small GTPases of the rab family have been suggested to be required for endosome motility and recycling.…”