Reconstitution of Phagosome-Lysosome Fusion in StreptolysinO-permeabilized Cells*
- From the ‡Animal and Cellular Systems Lab, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-01, Japan, §Universidad Nacional de Cuyo, Facultad de Ciencias Medican, Instituto de Histologia y Embiologia, Casilla de Correo 56, 5500 Mendoza, Argentina, the **Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India, and the ‖Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
Abstract
We have reconstituted fusion between phagosomes and lysosomes in streptolysin O-permeabilized J774-E macrophages. Fusion was assessed by measuring the delivery of avidin-conjugated horseradish peroxidase pre-internalized into lysosomes to phagosomes containing biotinylated β-glucuronidase-conjugated paramagnetic beads (1–2 μm). Fusion was dependent on energy and exogenously supplied cytosol. Phagosome-lysosome fusion was greatly inhibited when microtubules were depolymerized by nocodazole treatment, suggesting that fusion occurs via microtubule-dependent transport. Furthermore, fusion was inhibited by GTPγS and Rab GDP dissociation inhibitor. These results suggest that rab proteins are involved in the regulation of fusion. Lastly, anti-NEM-sensitive factor (NSF) antibodies inhibited fusion, and addition of recombinant NSF wild type partially restored the fusogenic activity, indicating that NSF is required for fusion between phagosomes and lysosomes.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants (to P. D. S.) and by the Japan Society for Promotion of Science (JSPS) (to K. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ Recipient of a CONICET Fellowship, Argentina.
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↵‡ Recipient of a DBT Associateship from the Government of India.
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↵§§ To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 South Ave., St. Louis, MO 63110. Tel.: 314-362-6952; Fax: 314-362-7463.
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↵1 The abbreviations used are: NEM,N-ethylmaleimide; SL-O, streptolysin O; HRP, horseradish peroxidase; GTPγS, guanosine 5′-3-O-(thio)triphosphate; Rab-GDI, Rab GDP dissociation inhibitor; ATPγS, adenosine 5′-O-(thio)triphosphate; NSF, NEM-sensitive factor; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; wt, wild type; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; TES,N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; PBS, phosphate-buffered saline; BSA, bovine serum albumin; HBSA, Hanks’ balanced salt solution with 0.1% BSA; MES, 4-morpholineethanesulfonic acid.
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- Received February 6, 1997.
- Revision received April 28, 1997.
