Self-organized podosomes are dynamic mechanosensors

Podosomes are self-organized, dynamic, actin-containing structures that adhere to the extracellular matrix via integrins [1-5]. Yet, it is not clear what regulates podosome dynamics and whether podosomes can function as direct mechanosensors, like focal adhesions [6-9]. We show here that myosin-II proteins form circular structures outside and at the podosome actin ring to regulate podosome dynamics. Inhibiting myosin-II-dependent tension dissipated podosome actin rings before dissipating the myosin-ring structure. As podosome rings changed size or shape, tractions underneath the podosomes were exerted onto the substrate and were abolished when myosin-light-chain activity was inhibited. The magnitudes of tractions were comparable to those generated underneath focal adhesions, and they increased with substrate stiffness. The dynamics of podosomes and of focal adhesions were different. Torsional tractions underneath the podosome rings were generated with rotations of podosome rings in a nonmotile, nonrotating cell, suggesting a unique feature of these circular structures. Stresses applied via integrins at the apical surface directly displaced podosomes near the basal surface. Stress-induced podosome displacements increased nonlinearly with applied stresses. Our results suggest that podosomes are dynamic mechanosensors in which interactions of myosin tension and actin dynamics are crucial for regulating these self-organized structures in living cells.     

Sodium fluoride induces podosome formation in endothelial cells

Background information. Fluoride is a well‐known G‐protein activator. Exposure of cultured cells to its derivatives results in actin cytoskeleton remodelling. Podosomes are actin‐based structures endowed with adhesion and matrix‐degradation functions. This study investigates actin cytoskeleton reorganization induced by fluoride in endothelial cells. Results. Treatment of cultured endothelial cells with sodium fluoride (NaF) results in a rapid and potent stimulation of podosome formation. Furthermore, we show that Cdc42 (cell‐division cycle 42), Rac1 and RhoA activities are stimulated in NaF‐treated cells. However, podosome assembly is dependent on Cdc42 and Rac1, but not RhoA. Although the sole activation of Cdc42 is sufficient to induce individual podosomes, a balance between RhoGTPase activities regulates podosome formation in response to NaF, which in this case are often found in groups or rosettes. As in other models, podosome formation in endothelial cells exposed to NaF also involves Src. Finally, we demonstrate that NaF‐induced podosomes are fully competent for matrix protein degradation. Conclusions. Taken together, our findings establish NaF as a novel inducer of podosomes in endothelial cells in vitro.     

Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein

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.     

Effects of tyrosine phosphorylation of cortactin on podosome formation in A7r5 vascular smooth muscle cells

Cortactin, a predominant substrate of Src family kinases, plays an important role in Arp2/3-dependent actin polymerization in lamellipodia and membrane ruffles and was recently shown to be enriched in podosomes induced by either c-Src or phorbol ester. However, the mechanisms by which cortactin regulates podosome formation have not been determined. In this study, we showed that cortactin is required for podosome formation, using siRNA knockdown of cortactin expression in smooth muscle A7r5 cells. Treatment with phorbol ester or expression of constitutively active c-Src induced genesis of cortactin-containing podosomes as well as increase in phosphorylation of cortactin at Y421 and Y466, the Src phosphorylation sites on cortactin. The Src kinase inhibitor SU-6656 significantly inhibited formation of podosomes induced by phorbol ester and phosphorylation of cortactin, whereas PKC inhibitor did not affect podosome formation in c-Src-transfected cells. Unexpectedly, expression of cortactin mutants containing Y421F, Y421D, Y466F, or Y466D mutated sites did not affect podosome formation or cortactin translocation to podosomes, although endogenous tyrosine-phosphorylated cortactin at Y421 and Y466 was present in podosomes. Our data indicate that 1) PKC acts upstream of Src in phosphorylation of cortactin and podosome formation in smooth muscle cells; 2) expression of cortactin is essential for genesis of podosomes; 3) phosphorylation at Y421 and Y466 is not required for translocation of cortactin to podosomes, although phosphorylation at these sites appears to be enriched in podosomes; and 4) tyrosine phosphorylation of cortactin may be involved in regulation of stability and turnover of podosomes, rather than targeting this protein to the site of podosome formation. actin cytoskeleton; Src; protein kinase C     

Macrophage podosomes assemble at the leading lamella by growth and fragmentation

Podosomes are actin- and fimbrin-containing adhesions at the leading edge of macrophages. In cells transfected with beta-actin-ECFP and L-fimbrin-EYFP, quantitative four-dimensional microscopy of podosome assembly shows that new adhesions arise at the cell periphery by one of two mechanisms; de novo podosome assembly, or fission of a precursor podosome into daughter podosomes. The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, fimbrin, integrin, and is in close apposition to the substratum. Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures. Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.     

Changes in the balance between caldesmon regulated by p21-activated kinases and the Arp2/3 complex govern podosome formation

Podosomes are dynamic cell adhesion structures that degrade the extracellular matrix, permitting extracellular matrix remodeling. Accumulating evidence suggests that actin and its associated proteins play a crucial role in podosome dynamics. Caldesmon is localized to the podosomes, and its expression is down-regulated in transformed and cancer cells. Here we studied the regulatory mode of caldesmon in podosome formation in Rous sarcoma virus-transformed fibroblasts. Exogenous expression analyses revealed that caldesmon represses podosome formation triggered by the N-WASP-Arp2/3 pathway. Conversely, depletion of caldesmon by RNA interference induces numerous small-sized podosomes with high dynamics. Caldesmon competes with the Arp2/3 complex for actin binding and thereby inhibits podosome formation. p21-activated kinases (PAK)1 and 2 are also repressors of podosome formation via phosphorylation of caldesmon. Consequently, phosphorylation of caldesmon by PAK1/2 enhances this regulatory mode of caldesmon. Taken together, we conclude that in Rous sarcoma virus-transformed cells, changes in the balance between PAK1/2-regulated caldesmon and the Arp2/3 complex govern the formation of podosomes.     

Phagocytosis is coupled to the formation of phagosome-associated podosomes and a transient disruption of podosomes in human macrophages

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.     

Plectin deposition at podosome rings requires myosin contractility

Metalloproteinase-dependent tissue invasion requires the formation of podosomes and invadopodia for localized matrix degradation. Actin cytoskeleton remodeling via Arp2/3-mediated actin polymerization is essential for podosome formation, and dynamic microtubules have an important role in maintaining podosome turnover in macrophages and osteoclasts. Little is known, however, about the involvement of the intermediate filament cytoskeleton in formation, stabilization, and turnover of podosomes. Here we show that vimentin intermediate filaments colocalize with the early sites of podosome formation at the stress fiber - focal adhesion interface in cultured vascular smooth muscle cells, but do not directly contribute to podosome formation, or stabilization. In unstimulated A7r5 cells the cytolinker protein plectin poorly colocalized with vimentin and the microdomains, but following induction by phorbol ester accumulated in the rings that surround the podosomes. In plectin-deficient A7r5 cells actin stress fiber remodelling is reduced in response to PDBu, and small podosomes remain localized at stable actin stress fibres. Pharmacological inhibition of actomyosin contractility by blebbistatin leads to an aberrant localization of podosomes away from the cell periphery and induces failure of plectin to surround the outer perimeter of these invasive adhesions. Taken together, we conclude that plectin is involved in growth and maturation of podosomes by reducing focal adhesion and stress fiber turnover, and that actomyosin-dependent contractility is required for the peripheral localization and specific deposition of plectin at the podosome rings.     

Podosome and sealing zone: specificity of the osteoclast model

The bone resorption function of osteoclasts is dependent on the integrity of the actin cytoskeleton. Depending on the substratum upon which the osteoclasts are spread, there are two different structures of actin known as podosomes and the sealing zone. To understand the specific properties and relationship of podosomes and the sealing zone, we used live-cell imaging of cultured osteoclasts. When cultured on extracellular matrix components, podosomes in these cells are organized in higher-ordered structures. These are clustered podosomes that will arrange later into dynamic short-lived rings which finally expand to the cell periphery to form a stable long-lived podosome belt in fully differentiated cells. In osteoclasts, this specific podosome patterning is under the control of microtubules (MTs). Indeed, nocodazole treatment does not affect podosome formation but only the transition between clusters/rings and belts. During this transition, MTs accumulate a specific post-translational modification of tubulin by acetylation. This process is repressed by an inhibitory pathway involving the GTPase Rho, its effector mDIA2 and the recently discovered tubulin deacetylase HDAC6. The specific function of this acetylation is still unknown but is also observed in active osteoclasts forming a sealing zone which is also MT dependent. Thus, it appears that the podosome belt is reminiscent of the sealing zone. Indeed, podosome belts and sealing zones are characterized by their overall stability. Despite their similar behavior, a sealing zone is not formed by fusion of podosomes. The formation of a podosome belt or a sealing zone is controlled by the external environment. Indeed, only the bone mineral fraction, known as apatite crystal, is able to induce sealing zone formation in mature osteoclasts. Contact of osteoclasts with apatite stimulates the non-receptor tyrosine kinase c-Src and the GTPase Rho in order to form the sealing zone. As we will discuss in this review, it appears that podosomes and the sealing zone are strikingly linked.     

Spatiotemporal organization and mechanosensory function of podosomes

Podosomes are small, circular adhesions formed by cells such as osteoclasts, macrophages, dendritic cells, and endothelial cells. They comprise a protrusive actin core module and an adhesive ring module composed of integrins and cytoskeletal adaptor proteins such as vinculin and talin. Furthermore, podosomes are associated with an actin network and often organize into large clusters. Recent results from our laboratory and others have shed new light on podosome structure and dynamics, suggesting a revision of the classical “core-ring” model. Also, these studies demonstrate that the adhesive and protrusive module are functionally linked by the actin network likely facilitating mechanotransduction as well as providing feedback between these two modules. In this commentary, we briefly summarize these recent advances with respect to the knowledge on podosome structure and discuss force distribution mechanisms within podosomes and their emerging role in mechanotransduction.     

The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts

Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src^−/− OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src^−/− OCLs. Videomicroscopy and fluorescence recovery after photobleaching analysis revealed that the life span of Src^−/− podosomes was increased fourfold and that the rate of actin flux in the core was decreased by 40%. Thus, Src regulates the formation, structure, life span, and rate of actin polymerization in podosomes and in the actin cloud. Rescue of Src^−/− OCLs with Src mutants showed that both the kinase activity and either the SH2 or the SH3 binding domain are required for Src to restore normal podosome organization and dynamics. Moreover, inhibition of Src family kinase activities in Src^−/− OCLs by Src inhibitors or by expressing dominant-negative Src^K295M induced the formation of abnormal podosomes. Thus, Src is an essential regulator of podosome structure, dynamics and organization.     

Podosome organization drives osteoclast-mediated bone resorption

Osteoclasts are the cells responsible for physiological bone resorption. A specific organization of their most prominent cytoskeletal structures, podosomes, is crucial for the degradation of mineralized bone matrix. Each podosome is constituted of an F-actin-enriched central core surrounded by a loose F-actin network, called the podosome cloud. In addition to intrinsic actin dynamics, podosomes are defined by their adhesion to the extracellular matrix, mainly via core-linking CD44 and cloud-linking integrins. These properties allow podosomes to collectively evolve into different patterns implicated in migration and bone resorption. Indeed, to resorb bone, osteoclasts polarize, actively secrete protons, and proteases into the resorption pit where these molecules are confined by a podosome-containing sealing zone. Here, we review recent advancements on podosome structure and regulatory pathways in osteoclasts. We also discuss the distinct functions of different podosome patterns during the lifespan of a single osteoclast.     

Spatiotemporal organization and mechanosensory function of podosomes

Podosomes are small, circular adhesions formed by cells such as osteoclasts, macrophages, dendritic cells, and endothelial cells. They comprise a protrusive actin core module and an adhesive ring module composed of integrins and cytoskeletal adaptor proteins such as vinculin and talin. Furthermore, podosomes are associated with an actin network and often organize into large clusters. Recent results from our laboratory and others have shed new light on podosome structure and dynamics, suggesting a revision of the classical “core-ring” model. Also, these studies demonstrate that the adhesive and protrusive module are functionally linked by the actin network likely facilitating mechanotransduction as well as providing feedback between these two modules. In this commentary, we briefly summarize these recent advances with respect to the knowledge on podosome structure and discuss force distribution mechanisms within podosomes and their emerging role in mechanotransduction.     

Podosome rings generate forces that drive saltatory osteoclast migration

Podosomes are dynamic, actin-containing adhesion structures that collectively self-organize as rings. In this study, we first show by observing osteoclasts plated on bead-seeded soft substrates that podosome assemblies, such as rings, are involved in tension forces. During the expansion of a podosome ring, substrate displacement is oriented outward, suggesting that podosomal structures push the substrate away. To further elucidate the function of forces generated by podosomes, we analyze osteoclast migration. Determining the centers of mass of the whole cell (G) and of actin (P), we demonstrate that osteoclasts migrate by "jumps" and that the trajectories of G and P are strongly correlated. The velocity of the center of mass as a function of time reveals that osteoclasts rapidly catch up with podosomal structures in a periodic pattern. We conclude that actin dynamics inside the cell are not only correlated with cell migration, but drive it.     

Cortactin has an essential and specific role in osteoclast actin assembly

Osteoclasts are essential for bone dynamics and calcium homeostasis. The cells form a tight seal on the bone surface, onto which they secrete acid and proteases to resorb bone. The seal is associated with a ring of actin filaments. Cortactin, a c-Src substrate known to promote Arp2/3-mediated actin assembly in vitro, is expressed in osteoclasts and localizes to the sealing ring. To address the role of cortactin and actin assembly in osteoclasts, we depleted cortactin by RNA interference. Cortactin-depleted osteoclasts displayed a complete loss of bone resorption with no formation of sealing zones. On nonosteoid surfaces, osteoclasts flatten with a dynamic, actin-rich peripheral edge that contains podosomes, filopodia, and lamellipodia. Cortactin depletion led to a specific loss of podosomes, revealing a tight spatial compartmentalization of actin assembly. Podosome formation was restored in cortactin-depleted cells by expression of wild-type cortactin or a Src homology 3 point mutant of cortactin. In contrast, expression of a cortactin mutant lacking tyrosine residues phosphorylated by Src did not restore podosome formation. Cortactin was found to be an early component of the nascent podosome belt, along with dynamin, supporting a role for cortactin in actin assembly.     

Gelsolin-independent podosome formation in dendritic cells

Podosomes, important structures for adhesion and extracellular matrix degradation, are claimed to be involved in cell migration. In addition, podosomes are also reported to be of importance in tissue remodelling, e.g., in osteoclast-mediated bone resorption. Podosomes are highly dynamic actin-filament scaffolds onto which proteins important for their function, such as matrix metallo-proteases and integrins, attach. The dynamics of the podosomes require the action of many proteins regulating actin assembly and disassembly. One such protein, gelsolin, which associates to podosomes, has been reported to be important for podosome formation and function in osteoclasts. However, podosome-like structures have been reported in gelsolin-deficient dendritic cells, but the identity of these structures was not confirmed, and their dynamics and function was not investigated. Like many other cells, dendritic cells of the immune system also form matrix degrading podosomes. In the present study, we show that dendritic cells form podosomes independently of gelsolin, that there are no major alterations in their dynamics of formation and disassembly, and that they exhibit matrix-degrading function. Furthermore, we found that gelsolin is not required for TLR4-induced podosome disassembly. Thus, the actin cytoskeleton of podosomes involved in dendritic cell extracellular matrix degradation appears to be regulated differently than the cytoskeleton in podosomes of osteoclasts mediating bone resorption.     

The kinesin KIF1C and microtubule plus ends regulate podosome dynamics in macrophages

Microtubules are important for the turnover of podosomes, dynamic, actin-rich adhesions implicated in migration and invasion of monocytic cells. The molecular basis for this functional dependency, however, remained unclear. Here, we show that contact by microtubule plus ends critically influences the cellular fate of podosomes in primary human macrophages. In particular, we identify the kinesin KIF1C, a member of the Kinesin-3 family, as a plus-end-enriched motor that targets regions of podosome turnover. Expression of mutation constructs or small interfering RNA-/short hairpin RNA-based depletion of KIF1C resulted in decreased podosome dynamics and ultimately in podosome deficiency. Importantly, protein interaction studies showed that KIF1C binds to nonmuscle myosin IIA via its PTPD-binding domain, thus providing an interface between the actin and tubulin cytoskeletons, which may facilitate the subcellular targeting of podosomes by microtubules. This is the first report to implicate a kinesin in podosome regulation and also the first to describe a function for KIF1C in human cells.     

Polyphosphoinositides-dependent regulation of the osteoclast actin cytoskeleton and bone resorption

Background  

Gelsolin, an actin capping protein of osteoclast podosomes, has a unique function in regulating assembly and disassembly of the podosome actin filament. Previously, we have reported that osteopontin (OPN) binding to integrin α_vβ₃ increased the levels of gelsolin-associated polyphosphoinositides, podosome assembly/disassembly, and actin filament formation. The present study was undertaken to identify the possible role of polyphosphoinositides and phosphoinositides binding domains (PBDs) of gelsolin in the osteoclast cytoskeletal structural organization and osteoclast function.