Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Heddleston

et al. 2019

Preprint

Self Cite

Macrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating macrophage fusion is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase contrast video microscopy demonstrated that in the majority of events, short protrusions (3 ± 1 µm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (16 ± 7 µm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent actinbased protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP-and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data indicate that an actin-based protrusion formed at the leading edge initiates macrophage fusion.

Section: Phase-dense Protrusions At the Leading Edge Precede Macrophamentioning

confidence: 99%

Section: Lattice Light Sheet Microscopymentioning

confidence: 99%

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

Faust

Heddleston

et al. 2019

Preprint

Self Cite

“…Since PCTFE plastic is not amenable to most imaging techniques, we took advantage of recently developed optical-quality glass surfaces prepared by adsorption of long-chain hydrocarbons such as paraffin that promote high levels of macrophage fusion ( Faust et al. , 2017 , 2018 ).In this series of experiments, rather than macrophage cell lines, we used primary macrophages isolated from the inflamed mouse peritoneum ( Helming and Gordon, 2007a ; Podolnikova et al. , 2016 ; Faust et al.…”

Section: Resultsmentioning

confidence: 99%

“…, 2019 ). Briefly, peritoneal cells (5 × 10 6 cells/ml) in Hank’s balanced salt solution (HBSS; Cellgro, Manassas, VA) supplemented with 0.1% bovine serum albumin were applied to acid-cleaned or paraffin-coated glass coverslips (prepared as described by Faust et al., 2017 , 2018 ). Cells were incubated in 5% CO 2 at 37°C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

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

Podolnikova

Mursalimov

et al. 2020

MBoC

Self Cite

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]

“…To investigate the mechanism of ZLS formation, we established an in vitro system that allowed us to generate ZLSs reproducibly. Since PTFE plastic is not amenable to most imaging techniques, we took advantage of recently developed optical-quality glass surfaces prepared by adsorption of longchain hydrocarbons such as paraffin that promote high level of macrophage fusion (Faust et al, 2017;Faust et al, 2018). In this series of experiments, rather than macrophage cell lines, we used primary macrophages isolated from inflamed mouse peritonea (Helming and Gordon, 2007a;Podolnikova et al, 2016;Faust et al, 2019) to avoid the robust proliferation observed in the cultures of macrophage cell lines.…”

Section: Formation Of Zlss In Vitromentioning

confidence: 99%

Transition of Podosomes Into Zipper-Like Structures in Macrophage-Derived Multinucleated Giant Cells

Podolnikova

Mursalimov

et al. 2020

Preprint

Self Cite

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-4mediated 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.