Figure 1.
Figure 1. Polarized membrane extensions mediate osteoclast fusion. (A) Schematic representation of the mRNA encoding the reporter constructs used for detection of PIs. IRES, internal ribosome entry site. (B) Live imaging of a mononuclear cell (top) and a multinuclear osteoclast (bottom) in RANKL-stimulated cultures of RAW264.7 cells expressing the reporter constructs in A. Cells were exposed to DAPI at 1 µg/ml (blue) for 1 h before observation to visualize nuclei. AktPH showed a polarized distribution, whereas PLCδ1PH was localized uniformly along the plasma membrane. (C) Live imaging of cells as in B during osteoclastogenesis. Arrows indicate a podosome-related protrusion in a multinucleated osteoclast. Closed and open arrowheads indicate a transient membrane expansion and filopodium-like protrusions, respectively. Pixel intensities on the traversing dashed lines were measured using LAS AF software and shown in the boxed areas (arbitrary units). See also Videos 1, 2, 3, and 4. (D, top) Immunoblot analysis of lysates of RAW264.7 macrophages stimulated with RANKL for the indicated times with antibodies to Ser473-phosphorylated (p) or total forms of Akt. (bottom) The pAkt/Akt signal intensity ratio was determined. Means ± SD from three independent experiments are indicated. *, P < 0.05 versus 0 h (Student’s t test). (E, left) RAW264.7 macrophages stimulated with RANKL for the indicated times were stained with rhodamine-phalloidin (red) to visualize F-actin and with DAPI (green) to visualize nuclei. (right) The distribution of the number of nuclei per cell was determined, with >100 cells counted at each time point. (F) RAW264.7 macrophages were stimulated with RANKL for 72 h, with the addition of 10 µM LY294002 or 0.1 µg/ml Latrunculin B (Lat B) for the last 16 h. DMSO was used as a vehicle control. Cell lysates were then subjected to immunoblot analysis with antibodies to NFATc1 or to Ser473-phosphorylated or total forms of Akt. (G, left) RAW264.7 macrophages treated with RANKL and inhibitors as in F were stained for TRAP activity. (right) The number of TRAP-positive cells with the indicated diameters was also determined. Data are means ± SD from three independent experiments. Bars: (B and C) 25 µm; (E) 250 µm; (G) 200 µm.

Polarized membrane extensions mediate osteoclast fusion. (A) Schematic representation of the mRNA encoding the reporter constructs used for detection of PIs. IRES, internal ribosome entry site. (B) Live imaging of a mononuclear cell (top) and a multinuclear osteoclast (bottom) in RANKL-stimulated cultures of RAW264.7 cells expressing the reporter constructs in A. Cells were exposed to DAPI at 1 µg/ml (blue) for 1 h before observation to visualize nuclei. AktPH showed a polarized distribution, whereas PLCδ1PH was localized uniformly along the plasma membrane. (C) Live imaging of cells as in B during osteoclastogenesis. Arrows indicate a podosome-related protrusion in a multinucleated osteoclast. Closed and open arrowheads indicate a transient membrane expansion and filopodium-like protrusions, respectively. Pixel intensities on the traversing dashed lines were measured using LAS AF software and shown in the boxed areas (arbitrary units). See also Videos 1, 2, 3, and 4. (D, top) Immunoblot analysis of lysates of RAW264.7 macrophages stimulated with RANKL for the indicated times with antibodies to Ser473-phosphorylated (p) or total forms of Akt. (bottom) The pAkt/Akt signal intensity ratio was determined. Means ± SD from three independent experiments are indicated. *, P < 0.05 versus 0 h (Student’s t test). (E, left) RAW264.7 macrophages stimulated with RANKL for the indicated times were stained with rhodamine-phalloidin (red) to visualize F-actin and with DAPI (green) to visualize nuclei. (right) The distribution of the number of nuclei per cell was determined, with >100 cells counted at each time point. (F) RAW264.7 macrophages were stimulated with RANKL for 72 h, with the addition of 10 µM LY294002 or 0.1 µg/ml Latrunculin B (Lat B) for the last 16 h. DMSO was used as a vehicle control. Cell lysates were then subjected to immunoblot analysis with antibodies to NFATc1 or to Ser473-phosphorylated or total forms of Akt. (G, left) RAW264.7 macrophages treated with RANKL and inhibitors as in F were stained for TRAP activity. (right) The number of TRAP-positive cells with the indicated diameters was also determined. Data are means ± SD from three independent experiments. Bars: (B and C) 25 µm; (E) 250 µm; (G) 200 µm.

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