Reconstitution of ATP-dependent movement of endocytic vesicles along microtubules in vitro

An oscillatory bidirectional process

John W. Murray, Eustratios Bananis, Allan W. Wolkoff

Research output: Contribution to journalArticle

77 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)419-433
Number of pages15
JournalMolecular Biology of the Cell
Volume11
Issue number2
StatePublished - Feb 2000

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Transport Vesicles
Microtubules
Endosomes
Adenosine Triphosphate
Cytoplasmic Dyneins
Asialoglycoprotein Receptor
Ligands
In Vitro Techniques
Cytosol

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

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