Transition of podosomes into zipper-like structures in macrophage-derived multinucleated giant cells

Abstract

Macrophage fusion resulting in the formation of multinucleated giant cells (MGCs) is a multistage process that requires many adhesion-dependent steps and involves the rearrangement of the actin cytoskeleton. The diversity of actin-based structures and their role in macrophage fusion is poorly understood. In this study, we revealed hitherto unrecognized actin-based zipper-like structures (ZLSs) that arise between MGCs formed on the surface of implanted biomaterials. We established an in vitro model for the induction of these structures in mouse macrophages undergoing IL-4–mediated fusion. Using this model, we show that over time MGCs develop cell-cell contacts containing ZLSs. Live-cell imaging using macrophages isolated from mRFP- or GFP-Lifeact mice demonstrated that ZLSs are dynamic formations undergoing continuous assembly and disassembly and that podosomes are precursors of these structures. Immunostaining experiments showed that vinculin, talin, integrin αMβ2, and other components of podosomes are present in ZLSs. Macrophages deficient in WASp or Cdc42, two key molecules involved in actin core organization in podosomes, as well as cells treated with the inhibitors of the Arp2/3 complex failed to form ZLSs. Furthermore, E-cadherin and nectin-2 were found between adjoining membranes, suggesting that the transition of podosomes into ZLSs is induced by bridging plasma membranes by junctional proteins. [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Multinucleated Giant Cells: Current Insights in Phenotype, Biological Activities, and Mechanism of Formation

“…Moreover, E-cadherin neutralizing antibodies impair IL-4 induced macrophage fusion, especially in early stages of differentiation ( Moreno et al, 2007 ), and fusion is diminished in macrophages derived from E-cadherin deficient mice ( Van de Bossche et al, 2009 ; Van de Bossche et al, 2012 ). Fusion-efficient macrophages are characterized by membrane extension, called podosomes, which form zipper-like structures at cell fusion sites ( Balabiyev et al, 2020 ). E-cadherin is probably implicated in the generation of these zipper-like structures as neutralizing antibodies disrupt formation of these structures and subsequent fusion ( Balabiyev et al, 2020 ).…”

Cellular and molecular actors of myeloid cell fusion: podosomes and tunneling nanotubes call the tune

“…It is induced in a STAT-6 dependent manner consequently to IL-4 or IL-13 treatments [ 44 , 105 , 132 , 133 ]. In IL-4 stimulated macrophages, E-cadherin forms complexes with catenins that accumulate at the sites of cell contact [ 133 , 134 ]. Different approaches using treatment with specific antibodies and E-cadherin-deficient macrophages show that this protein participates through homotypic interaction in IL-4-induced MGC formation [ 44 , 133 ].…”

“…A recent and elegant study demonstrated the existence of zipper-like structures in FBGCs. Indeed, this study revealed zipper-like structures that arise between mouse MGCs induced by IL-4 stimulation in vitro and following biomaterial implantation in vivo [ 134 ]. Using live imaging and super-resolution microscopy, the group of Ugarova showed that podosomes are the precursors of these zipper-like structures.…”

“…As a consequence, and similarly to OC zipper-like structures described above, these zipper-like structures contain many podosome proteins, and their assembly and stability are dependent on Arp2/3, Wasp, and Cdc42. Furthermore, they found that the junctional protein E-cadherin and SIRPα localize at the intercellular space between adjacent cell membranes within zipper-like structures[ 134 ]. Interestingly, these two proteins have been linked to macrophage fusion (see “ Molecular actors involved in cell-to-cell fusion ”).…”

“…Although the in vitro models, generally limited to a single cell type cultured at high density, provide valuable information for the identification of critical actors in macrophage fusion, they could bias the choice of the F-actin structures involved. For example, at an advanced stage of OC differentiation, the proximity of MGCs will favor the formation of zipper-like structures with the emergence of fine TNT-type structures that induce the fusion process [ 29 , 95 , 134 ]; the presence of these structures still needs to be demonstrated on a relevant substrate, such as bone matrix for OCs, and in more complex three-dimensional settings. In addition, the existence of TNTs in vivo is still a matter of debate in the field, and has never been described in the bone environment.…”