Nanoscale architecture and coordination of actin cores within the sealing zone of human osteoclasts.
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ABSTRACT:
SUBMITTER: Portes M
PROVIDER: S-EPMC9255968 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive super-structure known as the sealing zone (SZ). The SZ is a dynamic structure, consisting of a condensed array of podosomes, the elementary adhesion-mediating structures of osteoclasts, interconnected by F-actin filaments. The molecular composition and structure of the SZ were extensively investigated, yet despite its major importance for bone formation and remodelling, the mechanisms underlying its assembly and dynamics are still poorly understood. Here we determine the relations between matrix adhesiveness and the formation, stability and expansion of the SZ. By growing differentiated osteoclasts on micro-patterned glass substrates, where adhesive areas are separated by non-adhesive PLL-g-PEG barriers, we show that SZ growth and fusion strictly depend on the continuity of substrate adhesiveness, at the micrometer scale. We present a possible model for the role of mechanical forces in SZ formation and reorganization, inspired by the current data.
| S-EPMC3230604 | biostudies-literature
Project description:Bone resorption by osteoclasts (OCs) depends on the formation and stability of the sealing zone (SZ), a peripheral belt of actin and integrin-based podosomes. Recent studies demonstrated that the SZ is a highly dynamic structure, undergoing cycles of assembly and disassembly. In this study, we explored the mechanisms underlying the regulation of SZ stability and reorganization in OCs cultured on glass slides, and forming an SZ-like podosome belt (SZL). By monitoring this belt in cultured RAW264.7 cells expressing GFP-tagged actin, we show here that SZL stability is usually locally regulated, and its dissociation, occurring mostly in concave segments, is manifested in the loss of both podosome coherence, and actin belt continuity. Double labeling of cells for actin and tubulin indicated that microtubules (MTs) are mostly confined by the inner aspect of the stable SZL-associated actin belt. However, in unstable regions of the SZL, MTs tend to extend radially, across the SZL, toward the cell edge. Disruption of MTs by nocodazole induces SZ disassembly, without affecting individual podosome stability. Inspection of the MT network indicates that it is enriched along stable SZL regions, while bypassing disorganized regions. These results suggest that the SZL is stabilized by MTs flanking its inner aspect, while disruption or misalignment of MTs leads to SZL destabilization. We further demonstrate that the MT-associated protein dynamin2 is involved in the regulation of SZL stability, and dynamin2 knockdown or inactivation cause SZL destabilization.
| S-EPMC5347972 | biostudies-literature
Project description:BackgroundOsteoclasts are bone-degrading cells, which play a central role in physiological bone remodeling. Unbalanced osteoclast activity is largely responsible for pathological conditions such as osteoporosis. Osteoclasts develop specialized adhesion structures, the so-called podosomes, which subsequently undergo dramatic reorganization into sealing zones. These ring-like adhesion structures, which delimit the resorption site, effectively seal the cell to the substrate forming a diffusion barrier. The structural integrity of the sealing zone is essential for the cell ability to degrade bone, yet its structural organization is poorly understood.Principal findingsCombining high-resolution scanning electron microscopy with fluorescence microscopy performed on the same sample, we mapped the molecular architecture of the osteoclast resorptive apparatus from individual podosomes to the sealing zone, at an unprecedented resolution. Podosomes are composed of an actin-bundle core, flanked by a ring containing adhesion proteins connected to the core via dome-like radial actin fibers. The sealing zone, hallmark of bone-resorbing osteoclasts, consists of a dense array of podosomes communicating through a network of actin filaments, parallel to the substrate and anchored to the adhesive plaque domain via radial actin fibers.SignificanceThe sealing zone of osteoclasts cultured on bone is made of structural units clearly related to individual podosomes. It differs from individual or clustered podosomes in the higher density and degree of inter-connectivity of its building blocks, thus forming a unique continuous functional structure connecting the cell to its extracellular milieu. Through this continuous structure, signals reporting on the substrate condition may be transmitted to the whole cell, modulating the cell response under physiological and pathological conditions.
| S-EPMC1779809 | biostudies-literature
Project description:Bone homeostasis is continuously regulated by the coordinated action of bone-resorbing osteoclasts and bone-forming osteoblasts. Imbalance between these two cell populations leads to pathological bone diseases such as osteoporosis and osteopetrosis. Osteoclast functionality relies on the formation of sealing zone (SZ) rings that define the resorption lacuna. It is commonly assumed that the structure and dynamic properties of the SZ depend on the physical and chemical properties of the substrate. Considering the unique complex structure of native bone, elucidation of the relevant parameters affecting SZ formation and stability is challenging. In this study, we examined in detail the dynamic response of the SZ to the microtopography of devitalized bone surfaces, taken from the same area in cattle femur. We show that there is a significant enrichment in large and stable SZs (diameter larger than 14 µm; lifespan of hours) in cells cultured on rough bone surfaces, compared with small and fast turning over SZ rings (diameter below 7 µm; lifespan approx. 7 min) formed on smooth bone surfaces. Based on these results, we propose that the surface roughness of the physiologically relevant substrate of osteoclasts, namely bone, affects primarily the local stability of growing SZs.
| S-EPMC5332580 | biostudies-literature
Project description:Osteoclasts use actin-rich attachment structures in place of focal adhesions for adherence to bone and non-bone substrates. On glass, osteoclasts generate podosomes, foot-like processes containing a core of F-actin and regulatory proteins that undergo high turnover. To facilitate bone resorption, osteoclasts generate an actin-rich sealing zone composed of densely packed podosome-like units. Patterning of both podosomes and sealing zones is dependent upon an intact microtubule system. A role for unconventional myosin X (Myo10), which can bind actin, microtubules, and integrins, was examined in osteoclasts. Immunolocalization showed Myo10 to be associated with the outer edges of immature podosome rings and sealing zones, suggesting a possible role in podosome and sealing zone positioning. Further, complexes containing both Myo10 and beta-tubulin were readily precipitated from osteoclasts lysates. RNAi-mediated suppression of Myo10 led to decreased cell and sealing zone perimeter, along with decreased motility and resorptive capacity. Further, siRNA-treated cells could not properly position podosomes following microtubule disruption. Osteoclasts overexpressing dominant negative Myo10 microtubule binding domains (MyTH4) showed a similar phenotype. Conversely, overexpression of full-length Myo10 led to increased formation of podosome belts along with larger sealing zones and enhanced bone resorptive capacity. These studies suggest that Myo10 plays a role in osteoclast attachment and podosome positioning by direct linkage of actin to the microtubule network.
| S-EPMC2843201 | biostudies-literature
Project description:Osteoclasts are multinucleated bone-resorbing cells with a dynamic actin cytoskeleton. Osteoclasts are derived from circulating mononuclear precursors. Confocal and stimulated emission depletion (STED) super-resolution microscopy was used to investigate peripheral blood-derived human osteoclasts cultured on glass surfaces. STED and confocal microscopy demonstrated that the actin was curved and branched, for instance, in the vicinity of membrane ruffles. The overall architecture of the curved actin network extended from the podosomes to the top of the cell. The other novel finding was that a micrometer-level tube containing actin bridged the osteoclasts well above the level of the culture glass. The actin filaments of the tubes originated from the network of curved actin often surrounding a group of nuclei. Furthermore, nuclei were occasionally located inside the tubes. Our findings demonstrated the accumulation of c-Src, cortactin, cofilin, and actin around nuclei suggesting their role in nuclear processes such as the locomotion of nuclei. ARP2/3 labeling was abundant at the substratum level of osteoclasts and in the branched actin network, where it localized to the branching points. We speculate that the actin-containing tubes of osteoclasts may provide a means of transportation of nuclei, e.g., during the fusion of osteoclasts. These novel findings can pave the way for future studies aiming at the elucidation of the differentiation of multinucleated osteoclasts.
| S-EPMC5532409 | biostudies-literature
Project description:The process of sealing ring formation requires major actin filament reorganization. We previously demonstrated that an actin-bundling protein L-plastin has a role in the cross-linking of actin filaments into tight bundles and forms actin aggregates (denoted as nascent sealing zones). These nascent sealing zones mature into fully functional sealing rings. We have shown here that TNF-alpha signaling regulates the phosphorylation of serine-5 and -7 in L-plastin which increases the actin bundling capacity of L-plastin and hence the formation of nascent sealing zones in mouse osteoclasts. Using the TAT-mediated transduction method, we confirmed the role of L-plastin in nascent sealing zones formation at the early phase of the sealing ring assembly. Transduction of TAT-fused full-length L-plastin peptide significantly increases the number of nascent sealing zones and therefore sealing rings. But, transduction of amino-terminal L-plastin peptides consisting of the serine-5 and -7 reduces the formation of both nascent sealing zones and sealing rings. Therefore, bone resorption in vitro was reduced considerably. The decrease was associated with the selective inhibition of cellular L-plastin phosphorylation by the transduced peptides. Neither the formation of podosomes nor the migration was affected in these osteoclasts. Phosphorylation of L- plastin on serine 5 and -7 residues increases the F-actin bundling capacity. The significance of our studies stands on laying the groundwork for a better understanding of L-plastin as a potential regulator at the early phase of sealing ring formation and could be a new therapeutic target to treat bone loss.
| S-EPMC6181588 | biostudies-literature
Project description:The aim of this study is to identify the exact mechanism(s) by which cytoskeletal structures are modulated during bone resorption. In this study, we have shown the possible role of different actin-binding and signaling proteins in the regulation of sealing ring formation. Our analyses have demonstrated a significant increase in cortactin and a corresponding decrease in L-plastin protein levels in osteoclasts subjected to bone resorption for 18 h in the presence of RANKL, M-CSF, and native bone particles. Time-dependent changes in the localization of L-plastin (in actin aggregates) and cortactin (in the sealing ring) suggest that these proteins may be involved in the initial and maturation phases of sealing ring formation, respectively. siRNA to cortactin inhibits this maturation process but not the formation of actin aggregates. Osteoclasts treated as above but with TNF-α demonstrated very similar effects as observed with RANKL. Osteoclasts treated with a neutralizing antibody to TNF-α displayed podosome-like structures in the entire subsurface and at the periphery of osteoclast. It is possible that TNF-α and RANKL-mediated signaling may play a role in the early phase of sealing ring configuration (i.e. either in the disassembly of podosomes or formation of actin aggregates). Furthermore, osteoclasts treated with alendronate or αv reduced the formation of the sealing ring but not actin aggregates. The present study demonstrates a novel mechanistic link between L-plastin and cortactin in sealing ring formation. These results suggest that actin aggregates formed by L-plastin independent of integrin signaling function as a core in assembling signaling molecules (integrin αvβ3, Src, cortactin, etc.) involved in the maturation process.
| S-EPMC2943304 | biostudies-literature
Project description:The actin cytoskeleton is essential for osteoclasts main function, bone resorption. Two different organizations of actin have been described in osteoclasts, the podosomes belt corresponding to numerous F-actin columns arranged at the cell periphery, and the sealing zone defined as a unique large band of actin. To compare the role of these two different actin organizations, we imaged osteoclasts on various substrata: glass, dentin, and apatite. Using primary osteoclasts expressing GFP-actin, we found that podosome belts and sealing zones, both very dynamic actin structures, were present in mature osteoclasts; podosome belts were observed only in spread osteoclasts adhering onto glass, whereas sealing zone were seen in apico-basal polarized osteoclasts adherent on mineralized matrix. Dynamic observations of several resorption cycles of osteoclasts seeded on apatite revealed that 1) podosomes do not fuse together to form the sealing zone; 2) osteoclasts alternate successive stationary polarized resorption phases with a sealing zone and migration, nonresorption phases without any specific actin structure; and 3) apatite itself promotes sealing zone formation though c-src and Rho signaling. Finally, our work suggests that apatite-mediated sealing zone formation is dependent on both c-src and Rho whereas apico-basal polarization requires only Rho.
| S-EPMC532006 | biostudies-literature
Project description:Basic principles of statistical mechanics require that proteins sample an ensemble of conformations at any nonzero temperature. However, it is still common to treat the crystallographic structure of a protein as the structure of its native state, largely because high-resolution structural characterization of protein flexibility remains a profound challenge. To assess the typical degree of conformational heterogeneity within folded proteins, we construct Markov state models describing the thermodynamics and kinetics of proteins ranging from 72 to 263 residues in length. Each of these models is built from hundreds of microseconds of atomically detailed molecular dynamics simulations. Examination of the side-chain degrees of freedom reveals that almost every residue visits at least two rotameric states over this time frame, with rotamer transition rates spanning a wide range of time scales (from nanoseconds to tens of microseconds). We also report substantial backbone dynamics on time scales longer than are typically addressed by experimental measures of protein flexibility, such as NMR order parameters. Finally, we demonstrate that these extensive rearrangements are consistent with NMR and crystallographic data, which supports the validity of our models. Altogether, these results depict the interior of proteins not as well-ordered solids, as is often imagined, but instead as dense fluids, which undergo substantial structural fluctuations despite their high packing fraction.
| S-EPMC4065209 | biostudies-literature