title: Role of actin filaments in fusopod formation and osteoclastogenesis.
abstract: Cell fusion process is a critical, rate-limiting step in osteoclastogenesis but the mechanisms that regulate fusopod formation are not defined. We characterized fusopod generation in cultured pre-osteoclasts derived from cells stably transfected with a plasmid that expressed a short, actin filament binding peptide (Lifeact) fused to mEGFP that enables localization of actin filaments in living cells. Fusion was initiated at fusopods, which are cell extensions of width >2 μm and that are immunostained for myosin-X at the extension tips. Fusopods formed at the leading edge of larger migrating cells and from the tail of adjacent smaller cells, both of which migrated in the same direction. Staining for DC-STAMP was circumferential and did not localize to cell-cell fusion sites. Compared with wild-type cells, monocytes null for Rac1 exhibited 6-fold fewer fusopods and formed 4-fold fewer multinucleated osteoclasts. From time-lapse images we found that fusion was temporally related to the formation of coherent and spatially isolated bands of actin filaments that originated in cell bodies and extended into the fusopods. These bands of actin filaments were involved in cell fusion after approaching cells formed initial contacts. We conclude that the formation of fusopods is regulated by Rac1 to initiate intercellular contact during osteoclastogenesis. This step is followed by the tightly regulated assembly of bands of actin filaments in fusopods, which lead to closure of the intercellular gap and finally, cell fusion. These novel, actin-dependent processes are important for fusion processes in osteoclastogenesis.
authors: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada. Electronic address: yongqiang.wang@utoronto.ca.
authors: Yongqiang
authors: Y
authors: Wang
authors: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
authors: Patricia Joyce
authors: PJ
authors: Brooks
authors: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
authors: Janet Jinyoung
authors: JJ
authors: Jang
authors: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
authors: Alexandra Shade
authors: AS
authors: Silver
authors: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
authors: Pamma D
authors: PD
authors: Arora
authors: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
authors: Christopher A
authors: CA
authors: McCulloch
authors: Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada.
authors: Michael
authors: M
authors: Glogauer
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques; D002459:Cell Fusion; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010012:Osteogenesis / Q000187:drug effects; D053245:RANK Ligand / Q000494:pharmacology; D012995:Solubility; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: D000199:Actins; D053245:RANK Ligand; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D020830:rac1 GTP-Binding Protein
keywords: Actin filaments; Fusopods; Osteoclast fusion; Rac1