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© The Rockefeller University Press, 0021-9525/1997//113 $5.00
The Journal of Cell Biology, Volume 137, Number 1, , 1997 113-129

Article

Molecular Requirements for Bi-directional Movement of Phagosomes Along Microtubules



Ariel Blocker*, Fedor F. Severin*, Janis K. Burkhardt*, James B. Bingham§, Hanry Yu*, Jean-Christophe Olivo{ddagger}, Trina A. Schroer§, Anthony A. Hyman*, and Gareth Griffiths*

* Cell Biology Programme, European Molecular Biology Laboratory, 69117 Heidelberg, Germany; {ddagger} Cell Biophysics Programme, European Molecular Biology Laboratory, 69117 Heidelberg, Germany; and § Department of Biology, The Johns Hopkins University, Baltimore, Maryland

Microtubules facilitate the maturation of phagosomes by favoring their interactions with endocytic compartments. Here, we show that phagosomes move within cells along tracks of several microns centrifugally and centripetally in a pH- and microtubuledependent manner. Phagosome movement was reconstituted in vitro and required energy, cytosol and membrane proteins of this organelle. The activity or presence of these phagosome proteins was regulated as the organelle matured, with "late" phagosomes moving threefold more frequently than "early" ones. The majority of moving phagosomes were minus-end directed; the remainder moved towards microtubule plus-ends and a small subset moved bi-directionally. Minus-end movement showed pharmacological characteristics expected for dyneins, was inhibited by immunodepletion of cytoplasmic dynein and could be restored by addition of cytoplasmic dynein. Plus-end movement displayed pharmacological properties of kinesin, was inhibited partially by immunodepletion of kinesin and fully by addition of an anti-kinesin IgG. Immunodepletion of dynactin, a dynein-activating complex, inhibited only minus-end directed motility. Evidence is provided for a dynactin-associated kinase required for dyneinmediated vesicle transport. Movement in both directions was inhibited by peptide fragments from kinectin (a putative kinesin membrane receptor), derived from the region to which a motility-blocking antibody binds. Polypeptide subunits from these microtubule-based motility factors were detected on phagosomes by immunoblotting or immunoelectron microscopy. This is the first study using a single in vitro system that describes the roles played by kinesin, kinectin, cytoplasmic dynein, and dynactin in the microtubule-mediated movement of a purified membrane organelle.

Abbreviations used in this paper: AMP-PNP, 5'-adenylylimidodiphosphate; DIC, 3,4-dichlorisocoumarin; CD, cytoplasmic dynein; FSG, fish skin gelatin; GTP, guanosine-5'-triphosphate; IM, internalization medium; MTOC, microtubule organizing center; NRK, normal rat kidney cells; PLO, poly-l-ornithine.

We are particularly indebted to Mike Sheetz for inspiring discussions and unpublished reagents against kinectin protein and to Anja Habermann for outstanding technical assistance throughout this work. We thank Steve Gill for kindly providing the unpublished antibody 150B. We thank Heinz Horstmann for initial electron microscopy, Frédérique Briquet-Laugier for help in developing the movement analysis programme, Alan Sawyer for advice on antibodies, Angel Nebreda for a guided tour of "kinase country," and Jacomine Krinjse-Locker for minimizing the radiation exposure of A. Blocker's unborn child. We are grateful to George Bloom and Eugeni Vaisberg for generous gifts of antibodies and discussions of work in progress. Finally, we thank George Bloom, Mike Sheetz, Kai Simons, Gert Vriend, and two anonymous reviewers for considerably improving the manuscript.

Please address all correspondence to A. Blocker, Unité de Pathogénie Microbienne Moléculaire, INSERM U389, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris cedex France. Tel.: 33 1 40 61 32 47. Fax: 33 1 45 68 89 53. E-Mail: ablocker{at}pasteur.fr


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