title: Interactions of cytoplasmic granules with microtubules in human neutrophils.
abstract: Ultrastructural and functional studies of degranulation responses by human neutrophils have suggested that microtubules (MTs) have a role in the intracellular transport of neutrophil granules. We have found that granule-MT complexes can be isolated from disrupted taxol-treated (1.0 microM) neutrophils, visualized by electron microscopy, and quantified in terms of granules per MT length. After incubation of neutrophils with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), granule-MT complex formation was found to be increased two- to threefold. Enhanced binding of granules to MTs was detectable within 30 s of fMLP stimulation and was dependent on the concentration of fMLP. Incubation of cells with dibutyryl cAMP inhibited this fMLP-stimulated granule-MT complex formation in a dose-responsive fashion. These granule-MT interactions could be reproduced in a cell-free system with neutrophil granules isolated by density gradient centrifugation and MTs polymerized from phosphocellulose-purified tubulin. Furthermore, reconstituted granule-MT interactions were found to be modulated by ATPase inhibitors. Sodium orthovanadate increased granule-MT interactions in a concentration-dependent manner, while AMP-PNP, a nonhydrolyzable ATP analogue, and N-ethylmaleimide decreased or eliminated these interactions. In addition, we found that a MT-activated ATPase could be recovered from intact neutrophil granules by salt extraction, and that extracts enriched in this ATPase contained a polypeptide of between 115 and 120 kD which binds ATP and is immunologically related to kinesin. These studies demonstrate that cytoplasmic granules interact with MTs in human neutrophils in a regulated stimulus-responsive manner, and they suggest that such interactions may involve an MT-based, ATPase-dependent, vesicle translocation system as has been demonstrated in other types of cells.
authors.affiliation: Department of Hematology, Walter Reed Army Institute of Research, Washington, DC 20307-5100.
authors.forename: S W
authors.initials: SW
authors.lastname: Rothwell
authors.forename: J
authors.initials: J
authors.lastname: Nath
authors.forename: D G
authors.initials: DG
authors.lastname: Wright
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000502:physiology; D003994:Bucladesine / Q000494:pharmacology; D002458:Cell Fractionation; D002465:Cell Movement; D002474:Cell-Free System; D003594:Cytoplasmic Granules / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D016547:Kinesins; D008854:Microscopy, Electron; D008870:Microtubules / Q000502:physiology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000502:physiology; D009504:Neutrophils / Q000502:physiology* / Q000648:ultrastructure
chemical_list: D009419:Nerve Tissue Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D003994:Bucladesine; D000251:Adenosine Triphosphatases; D016547:Kinesins