Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein - PubMed (original) (raw)
Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein
Olivier Destaing et al. Mol Biol Cell. 2003 Feb.
Abstract
Podosomes, small actin-based adhesion structures, differ from focal adhesions in two aspects: their core structure and their ability to organize into large patterns in osteoclasts. To address the mechanisms underlying these features, we imaged live preosteoclasts expressing green fluorescent protein-actin during their differentiation. We observe that podosomes always form inside or close to podosome groups, which are surrounded by an actin cloud. Fluorescence recovery after photobleaching shows that actin turns over in individual podosomes in contrast to cortactin, suggesting a continuous actin polymerization in the podosome core. The observation of podosome assemblies during osteoclast differentiation reveals that they evolve from simple clusters into rings that expand by the continuous formation of new podosomes at their outer ridge and inhibition of podosome formation inside the rings. This self-organization of podosomes into dynamic rings is the mechanism that drives podosomes at the periphery of the cell in large circular patterns. We also show that an additional step of differentiation, requiring microtubule integrity, stabilizes the podosome circles at the cell periphery to form the characteristic podosome belt pattern of mature osteoclasts. These results therefore provide a mechanism for the patterning of podosomes in osteoclasts and reveal a turnover of actin inside the podosome.
Figures
Figure 1
Actin-GFP–expressing RAW cells differentiate into osteoclasts. Actin-GFP RAW cells were stimulated daily with RANK-L/M-CSF to induce differentiation. (A) After 8 d, fully differentiated, multinucleated osteoclasts are revealed by TRAP activity (purple stain). (B) Resorption pits (dark purple) formed after 12 d of culture on dentine slices. (C) A single osteoclast is shown with a podosome belt; colocalization of actin-GFP (green) with phalloidin-rhodamine-isothiocyanate (red) is revealed by yellow/orange color.
Figure 2
Podosome clusters move with a play of podosome assembly and disassembly. A murine primary osteoclast intranuclearly microinjected with an actin-GFP expression plasmid was observed by confocal time-lapse microscopy. GFP fluorescence images were inverted for better visualization and significant frames were extracted. (A) Overview of the cell that shows the different clusters. (B–E) Zoom on a cluster delineated by a dark line and composed of podosomes (black dots) surrounded by an actin cloud (gray). Although the whole structure (delimited by the dark line in B and E) has completely translated in 9 min, specific podosomes did not move as exemplified by the circled podosome.
Figure 3
FRAP analysis indicates actin turnover in podosomes. Osteoclasts derived from actin-GFP and cortactin-RFP RAW cells were imaged using fluorescein-type and rhodamine-type filter set, respectively. Actin and cortactin (A and A′), respectively, before photobleaching; after photobleaching in the white box (B and B′); and first image showing complete recovery of photobleaching (C and C′). (D and D′) Overlay of A, B, and C (A′, B′, and C′, respectively) white dots result from podosomes present in all three images; yellow signals podosomes present in A and C (A′ and C′, respectively). The lack of “yellow” podosomes in D′ compared with D illustrates that cortactin does not turnover in individual podosomes, whereas actin does. (E) Actin-GFP fluorescence over time in podosomes and in the cloud quantified using masks (see MATERIALS AND METHODS) after photobleaching. Both experimental data sets fit an exponential law with a similar characteristic time of 30 s (dashed lines). (E′) Cortactin-RFP fluorescence recovery measured in the whole area photobleached, the data set fits an exponential law (dashed line) with a characteristic time of 3 min. Experiment representative from five independent measures. (F) Plot of the characteristic time of FRAP in podosome (τpodo) vs. the characteristic time of the cloud (τcloud) in 11 independent experiments. Although the characteristic time varies from 20 to 45 s, τcloud and τpodo remain tightly correlated. Bar, 5 μm.
Figure 4
Podosomes are organized after three distinct patterns during osteoclast differentiation. Mouse spleen cells seeded on glass coverslips were induced into osteoclast differentiation with RANK-L and M-CSF for up to 8 d, fixed every day, and double stained for actin with phalloidin-rhodamine-isothiocyanate (red) and for vinculin (green). The first osteoclasts (≥3 nuclei/cells) with podosomes were visible at day 5. A single osteoclast is shown in each picture, podosomes look like red dots circled with green. They are grouped (arrowhead) either in clusters (A), rings (B), or belts (C). The ratio of cells displaying these structures to the total number of osteoclasts counted for each day (n) is shown in D. Rings appeared as a transient structure during the differentiation process.
Figure 5
Expanding podosome rings are formed inside podosome clusters and grow larger as differentiation progresses. (A) Actin-GFP RAW osteoclasts at early stage of ring formation (a) overview of the cell with a mix of rings (black arrow) and clusters (white arrowhead). (b–e) Zoomed area. Inside a cluster (b), a ring formed and expanded (c). No podosome formation was observed inside the area delimited by the ring. (d) The ring eventually stopped and collapsed back to a cluster. (B) Osteoclast at later stage of ring formation. Three different rings formed asynchronously from different areas of the cell (a); thus, rings expanded (b) and eventually fused (c). Resultant ring progressed to the periphery (d).
Figure 6
The belt is rapidly destabilized by a nocodazole treatment and is reformed after washout. Actin-GFP RAW osteoclasts at late stages of maturation were imaged by time-lapse confocal fluorescence microscopy. (A) Podosome belts in two different cells (arrowheads 1) were stable before the treatment. (B) After 30 min of a 2 μM nocodazole treatment, the belts have disappeared and replaced by podosomes rings and clusters (arrowheads 2 and 3). (C) After nocodazole washout more rings were formed and they were more stable. (D) After fusion and stabilization at the periphery, rings reformed two belts. Bar, 50 μm.
Figure 7
Models of podosome internal dynamics and podosome self-patterning. The integrins and associated proteins such as vinculin and paxillin surround the actin core and presumably induce the formation of a patch of cortactin/WASP-Arp2/3 that controls the actin polymerization. The actin core of the podosome is produced by the combined action of actin polymerization and actin severing. During osteoclastogenesis, rings start to from inside clusters and expand. The ring dynamics constantly induce the formation of new podosomes at its outer ridge while inhibiting new podosomes to form inside. This mechanism drives progressively the formation of podosomes at the cell periphery. A functional microtubule network is needed to stabilize the podosome belt at the cell periphery.
References
- Akisaka T, Yoshida H, Inoue S, Shimizu K. Organization of cytoskeletal F-actin, G-actin, and gelsolin in the adhesion structures in cultured osteoclast. J Bone Miner Res. 2001;16:1248–1255. - PubMed
- Arnett TR, Dempster DW. A comparative study of disaggregated chick and rat osteoclasts in vitro: effects of calcitonin and prostaglandins. Endocrinology. 1987;120:602–608. - PubMed
- Babb SG, Matsudaira P, Sato M, Correia I, Lim SS. Fimbrin in podosomes of monocyte-derived osteoclasts. Cell Motil Cytoskeleton. 1997;37:308–325. - PubMed
- Ballestrem C, Wehrle-Haller B, Imhof BA. Actin dynamics in living mammalian cells. J Cell Sci. 1998;111:1649–1658. - PubMed
- Bourette RP, Mouchiroud G, Ouazana R, Morle F, Godet J, Blanchet JP. Expression of human colony-stimulating factor-1 (CSF-1) receptor in murine pluripotent hematopoietic NFS-60 cells induces long-term proliferation in response to CSF-1 without loss of erythroid differentiation potential. Blood. 1993;81:2511–2520. - PubMed
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