Fig. 4. ^emGFP-Slc37a2⁺ tubules orientate towards the ruffled border and fuse with the bone-apposed plasma membrane.

a and b Multilevel confocal images of a mouse osteoclast cultured on bone expressing ^emGFP-Slc37a2 isoform 2 and stained with rhodamine-phalloidin to indicate the F-actin ring and underlying ruffled border. Representative XY serial confocal sections depict ^emGFP-Slc37a2 distribution at the basolateral, nuclear (blue), and ruffled border level(s) (n = 3). XZ denotes the side view corresponding to the white horizontal line in the merged panel. Dashed lines indicate ruffled border regions within sealing zones (yellow), resorptive pit (blue) and cell border (white). Bars 10 µm. c Multilevel XY and XZ confocal images of a live osteoclast cultured on bone expressing ^emGFP-Slc37a2 isoform 2 and labeled with LysoTracker Red. Dashed lines denote the plasma membrane (PM, white), sealing zone (yellow), and bone surface (blue). Bars, 10 µm. (n = 3). d Bottom-up view of the ruffled border level with the sealing zone denoted by the yellow dashed line. Bar, 10 µm. (n = 3). e and f Magnified regions of a time-lapse series illustrating extension, membrane contact, transient fusion, and retraction of an ^emGFP-Slc37a2⁺ tubule (blue arrow) with the bone-lining plasma membrane (yellow dashed line), with time points, indicated. White arrows highlight content release/exocytosis indicative of tubule fusion with the PM as confirmed by corresponding signal intensities in fluorescence line scans intensities (arbitrary units). Bar, 2 µm. (n = 2). Osteoclast cartoons depicted in panels a and c are adapted from ref. ⁷². See also related Supplementary Movies 4, 5.