title: Late stages of the synchronized macrophage fusion in osteoclast formation depend on dynamin.
abstract: Macrophage fusion that leads to osteoclast formation is one of the most important examples of cell-cell fusion in development, tissue homoeostasis and immune response. Protein machinery that fuses macrophages remains to be identified. In the present study, we explored the fusion stage of osteoclast formation for RAW macrophage-like murine cells and for macrophages derived from human monocytes. To uncouple fusion from the preceding differentiation processes, we accumulated fusion-committed cells in the presence of LPC (lysophosphatidylcholine) that reversibly blocks membrane merger. After 16 h, we removed LPC and observed cell fusion events that would normally develop within 16 h develop instead within 30-90 min. Thus, whereas osteoclastogenesis, generally, takes several days, our approach allowed us to focus on an hour in which we observe robust fusion between the cells. Complementing syncytium formation assay with a novel membrane merger assay let us study the synchronized fusion events downstream of a local merger between two plasma membranes, but before expansion of nascent membrane connections and complete unification of the cells. We found that the expansion of membrane connections detected as a growth of multinucleated osteoclasts depends on dynamin activity. In contrast, a merger between the plasma membranes of the two cells was not affected by inhibitors of dynamin GTPase. Thus dynamin that was recently found to control late stages of myoblast fusion also controls late stages of macrophage fusion, revealing an intriguing conserved mechanistic motif shared by diverse cell-cell fusion processes.
authors.affiliation: *Section on Membrane Biology, Program of Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Building 10/Room 10D05, 10 Center Dr., Bethesda, MD 20892-1855, U.S.A.
authors.forename: Santosh K
authors.initials: SK
authors.lastname: Verma
authors.affiliation: *Section on Membrane Biology, Program of Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Building 10/Room 10D05, 10 Center Dr., Bethesda, MD 20892-1855, U.S.A.
authors.forename: Evgenia
authors.initials: E
authors.lastname: Leikina
authors.affiliation: *Section on Membrane Biology, Program of Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Building 10/Room 10D05, 10 Center Dr., Bethesda, MD 20892-1855, U.S.A.
authors.forename: Kamran
authors.initials: K
authors.lastname: Melikov
authors.affiliation: *Section on Membrane Biology, Program of Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Building 10/Room 10D05, 10 Center Dr., Bethesda, MD 20892-1855, U.S.A.
authors.forename: Leonid V
authors.initials: LV
authors.lastname: Chernomordik
mesh_terms: D000818:Animals; D002450:Cell Communication / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002459:Cell Fusion; D002478:Cells, Cultured; D034285:Dynamin II / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D010010:Osteoclasts / Q000187:drug effects / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D008244:Lysophosphatidylcholines; D034741:RNA, Small Interfering; D034285:Dynamin II