A net-like structure with pores is observed during cell fusion induced by the receptor FGFRL1

Abstract

FGFRL1 is the fifth member of the fibroblast growth factor receptor (FGFR) family. Similar to the other members, it harbors three Ig loops in its extracellular domain, but in contrast to the other receptors, it lacks the intracellular protein tyrosine kinase domain that would be required for signaling by transphosphorylation. FGFRL1 is mainly found in the musculoskeletal system, where it appears to inhibit cell proliferation but to induce cell adhesion and differentiation. Mice with a targeted disruption of the FGFRL1 gene die during birth due to a malformed diaphragm muscle, which is not strong enough to inflate the lungs after birth. Expression of FGFRL1 is highly upregulated during the differentiation of myoblasts to multinucleated myotubes, suggesting an important role for FGFRL1 in cell-cell fusion. Recently we showed that FGFRL1 does indeed induce fusion of cultured cells into large syncytia. A reporter gene assay demonstrated that the third Ig domain and the transmembrane domain of FGFRL1 are both necessary and sufficient to fuse CHO cells into syncytia comprising several hundred nuclei. At the contact site, the fusing cells reveal a peculiar net-like structure with pores of about 1 µm diameter. It is possible that these structures represent membrane areas with fusion pores that set in motion the cell-cell fusion process. FGFRL1 is the first mammalian protein that is capable of triggering cell-cell fusion in vitro.

Keywords: CD9; FGFRL1; cell-cell fusion; fibroblast growth factor (FGF); fibroblast growth factor receptor (FGFR); fusion pores; muscle formation.

Figure 1

A net-like structure is observed during fusion of HEK-TetOn cells with CHO cells. (A and B) HEK-TetOn cells that express FGFRL1 on their cell surface were seeded together with CHO cells that had been transfected with a GFP construct, which is under the control of the Tet transactivator protein. Photographs of two syncytial cells were taken at different magnifications. FGFRL1 at the surface of the syncytia was stained with a monoclonal antibody, followed by a Cy3 labeled secondary antibody (red). GFP (green) is expressed after the Tet transactivator protein has diffused from the HEK-TetOn cells to the CHO cells. At higher magnification, a net-like structure with pores (arrow) becomes visible. (C and D) The net-like structure is also observed when the HEK-TetOn cells were cultivated without the CHO cells. In this case, FGFRL1 was stained with the monoclonal antibody, followed by a Cy2-labeled secondary antibody (green). When the HEK-TetOn cells were seeded at higher density, the net-like structures are found to be distributed around the entire cells.

Figure 2

The net-like structures on the surface of HEK-TetOn cells show pores with a diameter of 1 ?m. (A and B) The net-like structures are preferentially observed at membrane regions where two cells touch each other (arrow). (C and D) The diameter of the pores is approximately 1 µm as determined at higher magnification.