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First published online 3 February 2004
doi: 10.1242/jcs.00935

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Endocytic trafficking in actively resorbing osteoclasts

Bone and Mineral Centre, University College London, 5 University Street, London WC1E 6JJ, UK

View larger version (48K): [in a new window]   Fig. 1. Substrate dependence of dextran endocytosis. Rabbit osteoclasts cultured overnight on glass (A-D) or dentine (E-H) were incubated for 5 minutes with either FITC-labelled dextran-3000 (FD3) (A,E) or FITC-labelled dextran-70,000 (FD70) (C,G). Cells plated on glass are non-resorbing and endocytose both markers to a similar extent (A,C), whereas resorbing osteoclast plated on dentine only endocytose low-molecular-weight dextran 3000 (E,G). F-actin was visualized with rhodamine-conjugated phalloidin (B,D,F,H). The typical ring-like actin structure of resorbing osteoclasts is only visible in cells plated on dentine (F,H, arrow). The outline of the cortical actin observed in B,D,F,H (triangle) is indicated in A,C,E,F with a white dashed line. Images shown are single CSLM sections taken close to the substrate surface, which is visible in E and G because of the binding of the negatively charged FITC-labelled dextran. Scale bar, 10 µm.

View larger version (11K): [in a new window]   Fig. 2. Endocytic activities of osteoclasts shift towards the ruffled border during resorption. Rabbit osteoclasts cultured overnight on dentine were incubated with vehicle control (A,C) or 10 µM cytochalasin D for 10 minutes (B,D) before the addition of FITC-BSA for 5 minutes and then either fixed directly (A,B) or washed and chased in medium without inhibitor and FITC-BSA for 30 minutes (C,D). FITC-BSA accumulates in the pit immediately below the cell only upon treatment with cytochalasin D (B, arrowheads). After the chase, endocytosis is only observed in cells that had been exposed to cytochalasin D (fluorescent structures inside of the cell are indicated with arrows in D), whereas vehicle-treated cells remained refractory to the label (C). The outline of the cells as delineated by vß3 antibodies is depicted by a dashed white line. Images shown are representative of 50 osteoclasts and lateral views of the cells taken with CSLM with a thickness of 1.008 µm. Scale bar, 10 µm.

View larger version (78K): [in a new window]   Fig. 3. Pathways of endocytosed fluid-phase marker and resorbed bone matrix converge. Osteoclasts were plated on fluorescently labelled dentine and incubated for 4 hours before the addition of TRITC-labelled dextran 3000 for 30 minutes. After fixation, the distribution of labelled bone matrix (A,C, green) and low-molecular-weight dextran (B,C, red) was analysed with CSLM. Most of the visible vesicular structures contain both labels (C, yellow); the few vesicular structures containing only dextran label are located towards the centre of the cell (C, arrowheads). The outline of the cells as delineated by vß3 integrin antibodies is depicted as a dashed white line. Scale bar, 10 µm.

View larger version (44K): [in a new window]   Fig. 4. Distribution of dextran in endocytic compartments. Osteoclasts cultured on dentine disks were incubated for 1 hour with TRITC-transferrin (A,B, red) before the addition of FITC-labelled dextran-3000 for 10 minutes (A,B, green). The cells were then fixed and processed for immunostaining with antibodies against the vß3 integrin (A-H, blue). Limited colocalization of both markers can be observed in the area of the ruffled border (A, yellow). The lateral view of the cell taken at the position indicated by the dotted white line in (A) confirms this observation because colocalization is seen only close to the pit (B, arrowhead). When the cells were labelled for 10 minutes with both markers together, the spatial separation between ruffled border and transferrin receptor became apparent. Transferrin is found mainly at the leading edge of the cell, whereas endocytosed dextran localized to the middle section of the osteoclasts (C,D, red and green, respectively). A similar picture was also observed when osteoclasts were prelabelled with DiI-LDL (E,F, red) for 30 minutes before the addition of FITC-labelled dextran for 10 minutes (E,F, green). LDL is found distributed throughout the osteoclasts and reaches the area close to the ruffled border, where colocalization with endocytosed dextran can be observed (E,F, yellow and arrowheads). When the cells were labelled for 5 minutes with both markers together and then chased for 25 minutes, the amount of colocalization was limited and localized to the cell periphery (G, yellow and arrowheads), whereas the area close to the ruffled border was devoid of colocalization because of the absence of dextran staining, which had moved towards the cell interior (H). Osteoclasts containing only endocytosed dextran but none of the other markers are indicated with a star in (A) and (E). The direction of movement of the cells is indicated with an open arrow in (B,D,F,H). The asterisks in (E) indicate the pits excavated by the osteoclasts and hence are devoid of surface label. Scale bar, 10 µm.

View larger version (38K): [in a new window]   Fig. 5. Kinetics of dextran endocytosis. For uptake kinetics, osteoclasts plated on dentine were incubated for 1 minute, 5 minutes, 10 minutes and 30 minutes with TRITC-labelled dextran-3000 before fixation and immunodecoration with antibodies against the vß3 integrin to identify the cells (A-C). To follow the trafficking of the label through the osteoclast, cells plated on dentine were labelled for 10 minutes with TRITC-labelled dextran-3000 before a chase period of 15 minutes, 30 minutes or 60 minutes in medium without label (D,E). Computer-assisted 3D reconstruction of x-y sections taken at a 0.5 µm step size was used to visualize the trafficking of the dextran through the osteoclasts (A,B,D, red indicates TRITC-labelled dextran and blue indicates vß3 integrin). At early time points, the dextran is exclusively found close to the bone surface and the pit (A,B) but, after 60 minutes of chase, the label is distributed throughout the cell and can be observed close to the basolateral membrane surface and outside the cell (D, arrowhead). The amount of fluorescence inside of the cells was determined using the Leica TCS-NT software package and expressed as arbitrary fluorescence units. Columns show mean fluorescence and s.e.m. (C,E).

View larger version (29K): [in a new window]   Fig. 6. Cytoskeletal requirements of dextran endocytosis and intracellular trafficking in osteoclasts. Osteoclasts settled overnight on dentine disks were incubated with vehicle (A), 10 µM cytochalasin D (B), 10 µM nocodazole (C) or 10 µM paclitaxel (D) for 30 minutes before the addition of TRITC-labelled dextran-3000 in the presence of the inhibitors. Representative lateral images of osteoclasts after 20 minutes of labelling are shown after immunodecoration with antibodies directed against the vß3 integrin (A-D, red indicates TRITC-labelled dextran and green indicates vß3 integrin); arrows indicate the position of the bone surface relative to the label. Scale bar, 10 µm. The number of osteoclasts with dextran endocytosis was counted and expressed as a percentage. Columns show mean percentage of osteoclasts with dextran endocytosis and s.e.m.

View larger version (56K): [in a new window]   Fig. 7. The pH dependence of dextran trafficking. Osteoclasts were plated on dentine disks and incubated for 16 hours before a 30 minute pre-incubation with 25 nM bafilomycin A1 or vehicle control. TRITC-labelled dextran-3000 was then added for 5 minutes before fixation and immunodecoration with antibodies against the vß3 integrin. Representative single CSLM sections for cells treated with the vehicle control (A) or bafilomycin A1 (B) are shown; the outline of the cells as delineated by vß3 antibodies is depicted by a dashed white line. Scale bar, 10 µm. The number of osteoclasts presenting endocytosed dextran was counted and expressed as the percentage of endocytosis inhibition ± s.e.m. (Table).

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