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.

An actin-based protrusion originating from a podosome-enriched region initiates macrophage fusion

“…Less frequently, filopodium-like protrusions that formed after a transient expansion of podosome-like protrusions were observed. Wang et al have also observed finger-like extensions (~ 2.5 µm) in RAW264.7 cultures, which they termed fusopods (Wang et al, 2015). In that investigation, fusopods were the predominant structures, although some cells reportedly fused at contacts between broad surfaces.…”

“…Recently, a number of studies have reported the involvement of membrane extensions during osteoclastogenesis (Oikawa et al, 2012;Soe et al, 2015;Wang et al, 2015). Using RAW264.7 macrophages differentiated into osteoclasts by M-CSF and RANKL, Oikawa et al demonstrated that cell-cell fusion was mediated by short membrane protrusions that emerged from the sealing belt, a structure formed in large multinucleated osteoclasts from a ring of "circumferential" podosomes (Oikawa et al, 2012).…”

Differential intensity‐dependent effects of pulsed electromagnetic fields on RANKL‐induced osteoclast formation, apoptosis, and bone resorbing ability in RAW264.7 cells

“…Podosomes are organized in clusters at the beginning of differentiation, followed by evolvement into dynamic rings and final stabilization at the cell edge to form a podosome belt [Song et al, ; Ti et al, ]. The cell periphery of immature osteoclasts shows dendritic filopodial extensions, while mature osteoclasts exhibit smooth plasma membranes [Wang et al, ]. Mature osteoclasts can form a resorptive microenvironment consisting of a refuled border and an actin ring on the bone‐cell interface.…”

Osteoclast Fusion: Time‐Lapse Reveals Involvement of CD47 and Syncytin‐1 at Different Stages of Nuclearity

“…These analyses are powerful, but are not able to elucidate on the details of individual fusion events leading to this outcome. However, recently analyses of OC fusion in real‐time have been used to identify novel details with regard to the individual fusion partners, their molecular‐ and fusion‐characteristics (Takito and Nakamura, 2012; Takito et al, 2012; Levaot et al, 2015; Soe et al, 2015; Wang et al, 2015; Fiorino and Harrison, 2016). We have recently published a study (Soe et al, 2015), in which we analyzed a large quantity of time‐lapse recordings made over a period of 4 days.…”