Microtubule-associated Protein-dependent Binding of Phagosomes to Microtubules

doi:10.1074/jbc.271.7.3803

Microtubule-associated Protein-dependent Binding of Phagosomes to Microtubules (*)

From the Cell Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

^¶To whom correspondence should be addressed. Tel: 49 6221 387 267; Fax: 49 6221 387 306.

Abstract

In macrophages, phagosome movement is microtubule-dependent. Microtubules are a prerequisite for phagosome maturation because they facilitate interactions between phagosomes and organelles of the endocytic pathway. We have established an in vitro assay that measures the binding of purified phagosomes to microtubules. This binding depends on the presence of membrane proteins, most likely integral to the surface of phagosomes, and on macrophage cytosol. The cytosolic binding factor can interact with microtubules prior to the addition of phagosomes to the assay, suggesting that it is a microtubule-associated protein (MAP). Consistent with this, depletion of MAPs from the cytosol by microtubule affinity removes all binding activity. Microtubule motor proteins show no binding activity, whereas a crude MAP preparation is sufficient to support binding and to restore full binding activity to MAP-depleted cytosol. We show that the activating MAP factor is a heat-sensitive protein(s) that migrates at around 150 kDa by gel filtration.

Footnotes

↵^§ Supported by a European Molecular Biology long term fellowship.
↵** Supported by an HFSP fellowship.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Noc

nocodazole

AMP-PNP

5′-adenylylimidodiphosphate

ATPS

adenosine 5′-3-O-(thio)triphosphate

GTPS

guanosine 5′-3-O-(thio)triphosphate

CLIP

cytoplasmic linker protein

MAP

microtubule-associated protein

PI

protease inhibitor

MES

4-morpholineethanesulfonic acid.
↵²A. Blocker et al., manuscript in preparation.
↵³J. Olmsted, unpublished data.
↵⁴J. K. Burkhardt, unpublished data.
↵⁵A. Blocker, unpublished data.
- Received June 13, 1995.
- Revision received November 13, 1995.