The circle of life: Phases of podosome formation,turnover and reemergence

Podosomes are highly dynamic actin-rich structures in a variety of cell types, especially monocytic cells. They fulfill multiple functions such as adhesion, mechanosensing, or extracellular matrix degradation, thus allowing cells to detect and respond to a changing environment. These abilities are based on an intricate architecture that enables podosomes to sense mechanical properties of their substratum and to transduce them intracellularly in order to generate an appropriate cellular response. These processes are enabled through the tightly orchestrated interplay of more than 300 different components that are dynamically recruited during podosome formation and turnover. In this review, we discuss the different phases of the podosome life cycle and the current knowledge on regulatory factors that impact on the genesis, activity, dissolution and reemergence of podosomes. We also highlight mechanoregulatory processes that become important during these different stages, on the level of individual podosomes, and also at podosome sub- and superstructures.     

Cofilin Activation during Podosome Belt Formation in Osteoclasts

Podosomes are dynamic actin-based structures found constitutively in cells of monocytic origin such as macrophages, dendritic cells and osteoclasts. They have been involved in osteoclast cell adhesion, motility and matrix degradation, and all these functions rely on the ability of podosomes to form supra-molecular structures called podosome belts or sealing zones on mineralized substrates. Podosomes contain two distinct domains, an actin-rich core enriched in actin polymerization regulators, surrounded by a ring of signaling and plaque molecules. The organization of podosome arrays into belts is linked to actin dynamics. Cofilin is an actin-severing protein that is known to regulate cytoskeleton architecture and cell migration. Cofilin is present in lamellipodia and invadopodia where it regulates actin polymerization. In this report, we show that cofilin is a novel component of the podosome belt, the mature osteoclast adhesion structure. Time-course analysis demonstrated that cofilin is activated during primary osteoclast differentiation, at the time of podosome belt assembly. Immunofluorescence studies reveal a localization of active cofilin in the podosome core structure, whereas phosphorylated, inactive cofilin is concentrated in the podosome cloud. Pharmacological studies unraveled the role of a specific cofilin phosphatase to achieve cofilin activation during osteoclast differentiation. We ruled out the implication of PP1/PP2A and PTEN in this process, and rather provided evidence for the involvement of SSH1. In summary, our data involve cofilin as a regulator of podosome organization that is activated during osteoclast differentiation by a RANKL-mediated signaling pathway targeting the SSH1 phosphatase.     

Combined AFM and super-resolution localisation microscopy: Investigating the structure and dynamics of podosomes

Podosomes are mechanosensitive attachment/invasion structures that form on the matrix-adhesion interface of cells and protrude into the extracellular matrix to probe and remodel. Despite their central role in many cellular processes, their exact molecular structure and function remain only partially understood. We review recent progress in molecular scale imaging of podosome architecture, including our newly developed localisation microscopy technique termed HAWK which enables artefact-free live-cell super-resolution microscopy of podosome ring proteins, and report new results on combining fluorescence localisation microscopy (STORM/PALM) and atomic force microscopy (AFM) on one setup, where localisation microscopy provides the location and dynamics of fluorescently labelled podosome components, while the spatial variation of stiffness is mapped with AFM. For two-colour localisation microscopy we combine iFluor-647, which has previously been shown to eliminate the need to change buffer between imaging modes, with the photoswitchable protein mEOS3.2, which also enables live cell imaging.     

Podosomes: Multipurpose organelles?

Thirty years of research have accumulated ample evidence that podosome clusters qualify as genuine cellular organelles that are being found in more and more cell types. A podosome is a dynamic actin-based and membrane-bound microdomain and the organelle consists in an interconnected network of such basic units, forming a cytoskeletal superstructure linked to the plasma membrane. At this strategic location, podosomes are privileged sites of interactions with the pericellular environment that regulates their formation, density, lifetime, distribution, architecture and functioning. Actin polymerization is the driving force behind most podosome characteristics. In contrast to classical organelles, podosomes are not vital at the cell level but rather serve diverse and often intricate functions of which adhesion, matrix degradation and substrate sensing are the most established. These capabilities involve specific molecules, depend on podosome organization and may vary according to the cell type in which they form. Podosome-associated diseases manifest by loss or gain of podosome functions and include genetic diseases affecting podosome components and various cancers where tumor cells ectopically express podosome equivalents (invadopodia).     

VAMP3 regulates podosome organisation in macrophages and together with Stx4/SNAP23 mediates adhesion, cell spreading and persistent migration

The ability of cells to adhere, spread and migrate is essential to many physiological processes, particularly in the immune system where cells must traffic to sites of inflammation and injury. By altering the levels of individual components of the VAMP3/Stx4/SNAP23 complex we show here that this SNARE complex regulates efficient macrophage adhesion, spreading and migration on fibronectin. During cell spreading this complex mediates the polarised exocytosis of VAMP3-positive recycling endosome membrane into areas of membrane expansion, where VAMP3's surface partner Q-SNARE complex Stx4/SNAP23 was found to accumulate. Lowering the levels of VAMP3 in spreading cells resulted in a more rounded cell morphology and most cells were found to be devoid of the typical ring-like podosome superstructures seen normally in spreading cells. In migrating cells lowering VAMP3 levels disrupted the polarised localisation of podosome clusters. The reduced trafficking of recycling endosome membrane to sites of cell spreading and the disorganised podosome localisation in migrating macrophages greatly reduced their ability to persistently migrate on fibronectin. Thus, this important SNARE complex facilitates macrophage adhesion, spreading, and persistent macrophage migration on fibronectin through the delivery of VAMP3-positive membrane with its cargo to expand the plasma membrane and to participate in organising adhesive podosome structures.     

Regulatory signals for endothelial podosome formation

Podosomes are punctate actin-rich adhesion structures which spontaneously form in cells of the myelomonocytic lineage. Their formation is dependent on Src and RhoGTPases. Recently, podosomes have also been described in vascular cells. These podosomes differ from the former by the fact that they are inducible. In endothelial cells, such a signal can be provided by either constitutively active Cdc42, the PKC activator PMA or TGFbeta, depending on the model. Consequently, other regulatory pathways have been reported to contribute to podosome formation. To get more insight into the mechanisms by which podosomes form in endothelial cells, we have explored the respective contribution of signal transducers such as Cdc42-related GTPases, Smads and PKCs in three endothelial cell models. Results presented demonstrate that, in addition to Cdc42, TC10 and TCL GTPases can also promote podosome formation in endothelial cells. We also show that PKCalpha can be either necessary or entirely dispensable, depending on the cell model. In contrast, PKCdelta is essential for podosome formation in endothelial cells but not smooth muscle cells. Finally, although podosomes vary very little in their molecular composition, the signalling pathways involved in their assembly appear very diverse.     

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.     

Osteoprotegerin induces podosome disassembly in osteoclasts through calcium,ERK, and p38 MAPK signaling pathways

Osteoclasts are critical for bone resorption and use podosomes to attach to bone matrix. Osteoprotegerin (OPG) is a negative regulator of osteoclast function that can affect the formation and function of podosomes. However, the signaling pathways that link OPG to podosome function have not been well characterized. Therefore, this study examined the roles of intracellular calcium and MAPKs in OPG-induced podosome disassembly in osteoclasts. We assessed the effects of the intracellular calcium chelator Bapta-AM, ERK inhibitor U0126, and p38 inhibitor SB202190 on OPG-treated osteoclast differentiation, adhesion structures, intracellular free Ca²⁺ concentration and the phosphorylation state of podosome associated proteins (Pyk2 and Src). Mouse monocytic RAW 264.7 cells were differentiated to osteoclasts using RANKL (30 ng/mL) and M-CSF (25 ng/mL). The cells were pretreated with Bapta-AM (5 μM), U0126 (5 μM), or SB202190 (10 μM) for 30 min, followed by 40 ng/mL OPG for 3 h. Osteoclastogenesis, adhesion structure, viability and morphology, intracellular free Ca²⁺ concentration and the phosphorylation state of Pyk2 and Src were measured by TRAP staining, scanning electron microscopy, real-time cell analyzer, flow cytometry and western blotting, respectively. OPG significantly inhibited osteoclastogenesis, the formation of adhesion structures, and reduced the amount of phosphorylated Pyk2 and Src-pY527, but increased phosphorylation of Src-pY416. Bapta-AM, U0126, and SB202190 partially restored osteoclast differentiation and adhesion structures. Both Bapta-AM and U0126, but not SB202190, restored the levels of intracellular free Ca²⁺ concentration, phosphorylated Pyk2 and Src-pY527. All three inhibitors blocked OPG-induced phosphorylation at Src-pY416. These results suggest OPG disrupts the attachment structures of osteoclasts and activates Src as an adaptor protein that competes for the reduced amount of phosphorylated Pyk2 through calcium- and ERK-dependent signaling pathways. p38 MAPK signaling may have a different role in OPG-induced osteoclast retraction. Our findings potentially offer novel insights into the signaling mechanisms downstream of OPG that affect osteoclast attachment to the extracellular matrix.     

Lasp-1 regulates podosome function

Eukaryotic cells form a variety of adhesive structures to connect with their environment and to regulate cell motility. In contrast to classical focal adhesions, podosomes, highly dynamic structures of different cell types, are actively engaged in matrix remodelling and degradation. Podosomes are composed of an actin-rich core region surrounded by a ring-like structure containing signalling molecules, motor proteins as well as cytoskeleton-associated proteins. Lasp-1 is a ubiquitously expressed, actin-binding protein that is known to regulate cytoskeleton architecture and cell migration. This multidomain protein is predominantely present at focal adhesions, however, a second pool of Lasp-1 molecules is also found at lamellipodia and vesicle-like microdomains in the cytosol.In this report, we show that Lasp-1 is a novel component and regulator of podosomes. Immunofluorescence studies reveal a localization of Lasp-1 in the podosome ring structure, where it colocalizes with zyxin and vinculin. Life cell imaging experiments demonstrate that Lasp-1 is recruited in early steps of podosome assembly. A siRNA-mediated Lasp-1 knockdown in human macrophages affects podosome dynamics as well as their matrix degradation capacity. In summary, our data indicate that Lasp-1 is a novel component of podosomes and is involved in the regulation of podosomal function.     

Sequential signals toward podosome formation in NIH-src cells

Podosomes (also termed invadopodia in cancer cells) are actin-rich adhesion structures with matrix degradation activity that develop in various cell types. Despite their significant physiological importance, the molecular mechanism of podosome formation is largely unknown. In this study, we investigated the molecular mechanisms of podosome formation. The expression of various phosphoinositide-binding domains revealed that the podosomes in Src-transformed NIH3T3 (NIH-src) cells are enriched with PtdIns(3,4)P2, suggesting an important role of this phosphoinositide in podosome formation. Live-cell imaging analysis revealed that Src-expression stimulated podosome formation at focal adhesions of NIH3T3 cells after PtdIns(3,4)P2 accumulation. The adaptor protein Tks5/FISH, which is essential for podosome formation, was found to form a complex with Grb2 at adhesion sites in an Src-dependent manner. Further, it was found that N-WASP bound all SH3 domains of Tks5/FISH, which facilitated circular podosome formation. These results indicate that augmentation of the N-WASP-Arp2/3 signal was accomplished on the platform of Tks5/FISH-Grb2 complex at focal adhesions, which is stabilized by PtdIns(3,4)P2.     

Adhesion structures and their cytoskeleton-membrane interactions at podosomes of osteoclasts in culture

The organization of the cytoskeleton in the podosomes of osteoclasts was studied by use of cell shearing, rotary replication, and fluorescence cytochemical techniques. After shearing, clathrin plaques and particles associated with the cytoskeleton were left behind on the exposed cytoplasmic side of the membrane. The cytoskeleton of the podosomes was characterized by two types of actin filaments: relatively long filaments in the portion surrounding the podosome core, and highly branched short filaments in the core. Individual actin filaments radiating from the podosomes interacted with several membrane particles along the length of the filaments. Many lateral contacts with the membrane surface by the particles were made along the length of individual actin filaments. The polarity of actin filaments in podosomes became oriented such that their barbed ends were directed toward the core of podosomes. The actin cytoskeletons terminated or branched at the podosomes, where the membrane tightly adhered to the substratum. Microtubules were not usually present in the podosome structures; however, certain microtubules appeared to be morphologically in direct contact with the podosome core. Most of the larger clathrin plaques consisted of flat sheets of clathrin lattices that interconnected neighboring clathrin lattices to form an extensive clathrin area. However, the small deeply invaginated clathrin plaques and the podosomal cytoskeleton were located close together. Thus, the clathrin plaques on the ventral membrane of osteoclasts might be involved in both cell adhesion and the formation of receptor-ligand complexes, i.e., endocytosis. This work was supported by the following grants to T.A.: Grants-in-Aid for Scientific Research (C) (18592020) from the Ministry of Education, Science, and Culture of Japan and the Miyata Research Fund of Asahi University.     

Hematopoietic cell kinase (Hck) isoforms and phagocyte duties – From signaling and actin reorganization to migration and phagocytosis

The activity of hematopoietic cell kinase (Hck), a member of the Src family kinases, is modulated by regulatory mechanisms leading to distinct protein conformations with gradual levels of activity. Hck is mostly expressed in phagocytes as two isoforms, p59Hck and p61Hck, which show distinct subcellular localizations and trigger distinct phenotypes when expressed ectopically in fibroblasts. Hck has been reported to be involved in phagocytosis, adhesion and migration, and to regulate formation of membrane protrusions, lysosome exocytosis, podosome formation, and actin polymerization. The present review focuses on the mechanisms regulating Hck activity as well as on the functions of Hck isoforms in phagocytes, and presents selected examples of Hck substrates and/or adaptors shown to interact with the kinase in myeloid cells. Deciphering Hck signaling pathways is a challenge to progress in the understanding of innate immune responses and pathologies involving phagocytes such as inflammatory diseases, leukemia, and human immunodeficiency virus-1 (HIV-1) infection.     

Rho GTPases in osteoclasts: orchestrators of podosome arrangement

Cells from the myeloid lineage, namely macrophages, dendritic cells and osteoclasts, develop podosomes instead of stress fibers and focal adhesions to adhere and migrate. Podosomes share many components with focal adhesions but differ in their molecular organization, with a dense core of polymerized actin surrounded by scaffolding proteins, kinases and integrins. Podosomes are found either isolated both in macrophages and dendritic cells or arranged into superstructures in osteoclasts. When osteoclasts resorb bone, they form an F-actin rich sealing zone, which is a dense array of connected podosomes that firmly anchors osteoclasts to bone. It delineates a compartment in which protons and proteases are secreted to dissolve and degrade the mineralized matrix. Since Rho GTPases have been shown to control F-actin stress fibers and focal adhesions in mesenchymal cells, the question of whether they could also control podosome formation and arrangement in cells from the myeloid lineage, and particularly in osteoclasts, rapidly emerged. This article considers recent advances made in our understanding of podosome arrangements in osteoclasts and how Rho GTPases may control it.     

Actin cytoskeletal organisation in osteoclasts: a model to decipher transmigration and matrix degradation

Osteoclasts are large monocyte-derived multinucleated cells whose function is to resorb bone, i.e. a mineralised extracellular matrix. They exhibit two different actin cytoskeleton organisations according to their substratum. On non-mineralised substrates they form canonical podosomes, but on mineralised extracellular matrices they form a sealing zone. Podosomes consist of two functionally different actin subdomains: a podosome core, probably made of branched actin organised through a CD44 transmembrane receptor, and an actin cloud of actin cables organised around alphavbeta3 integrin. During osteoclast differentiation, podosome patterning is highly dynamic, and we propose that it ends up in a sealing zone in mature bone-resorbing osteoclasts after a complete reorganisation of the two subdomains. In addition to matrix degradation, osteoclasts share with tumour cells the ability to transmigrate through cell layers and-for that purpose-can arrange their cytoskeleton in long protrusions reminiscent of invadopodia. In this review, we discuss the relationships between podosomes and sealing zone, comparing their structures, their molecular composition and their abilities to degrade extracellular matrices. The dynamic actin remodelling in osteoclasts appears then as a major factor to understand their unusual abilities reminiscent of metastatic tumour cells.     

Podosomes: adhesion hot-spots of invasive cells

Podosomes are highly dynamic, actin-rich adhesion structures of monocyte-derived cells, certain transformed fibroblasts and carcinoma cells and have recently also been discovered in an increasing number of other cell types. Because they are found mainly in motile cells and control the activity of matrix metalloproteases, podosomes are thought to contribute to tissue invasion and matrix remodeling. Importantly, podosomes are physiologically relevant organelles because they can be found in ex vivo models of invasive cells. Regulators of podosome turnover include tyrosine kinases, RhoGTPases, actin regulators and the microtubule system. Podosomes might also serve as an attractive model to study how integration of various signaling pathways controls actin dynamics. Here, we summarize and discuss the known structural, regulatory and functional features of podosomes, our aim being to stimulate further research into these unique structures.     

Re-arrangements of podosome structures are observed when Hck is activated in myeloid cells

Podosomes are adhesion structures with an extracellular matrix-degrading capacity mostly found in monocyte-derived cells. We have previously shown that the protein tyrosine kinase Hck, a member of the Src family, triggers the de novo formation of podosome rosettes in a lysosome-dependent manner when expressed in its constitutively active form. Hck is specifically expressed in myeloid cells. In human monocyte-derived macrophages (MDMs) it is present at podosomes. Here we addressed whether its activation by lipopolysaccharide and interferon-gamma has an effect on podosome organization in MDMs. Several structures were observed evolving from individual podosomes to clusters, aggregates and rosettes. In chronic myeloid leukemia cells, Hck is constitutively activated by the fusion protein Bcr-Abl and podosome-like structures were present. Finally, in monocyte-derived osteoclasts, Hck was found to accumulate at podosome belts. In conclusion, in monocyte-derived cells, it is likely that Hck could play a role in podosome re-arrangements.     

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.     

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.     

A Functional Interplay between the Small GTPase Rab11a and Mitochondria-shaping Proteins Regulates Mitochondrial Positioning and Polarization of the Actin Cytoskeleton Downstream of Src Family Kinases

It is believed that mitochondrial dynamics is coordinated with endosomal traffic rates during cytoskeletal remodeling, but the mechanisms involved are largely unknown. The adenovirus early region 4 ORF4 protein (E4orf4) subverts signaling by Src family kinases (SFK) to perturb cellular morphology, membrane traffic, and organellar dynamics and to trigger cell death. Using E4orf4 as a model, we uncovered a functional connection between mitochondria-shaping proteins and the small GTPase Rab11a, a key regulator of polarized transport via recycling endosomes. We found that E4orf4 induced dramatic changes in the morphology of mitochondria along with their mobilization at the vicinity of a polarized actin network typifying E4orf4 action, in a manner controlled by SFK and Rab11a. Mitochondrial remodeling was associated with increased proximity between Rab11a and mitochondrial membranes, changes in fusion-fission dynamics, and mitochondrial relocalization of the fission factor dynamin-related protein 1 (Drp1), which was regulated by the Rab11a effector protein FIP1/RCP. Knockdown of FIP1/RCP or inhibition of Drp1 markedly impaired mitochondrial remodeling and actin assembly, involving Rab11a-mediated mitochondrial dynamics in E4orf4-induced signaling. A similar mobilization of mitochondria near actin-rich structures was mediated by Rab11 and Drp1 in viral Src-transformed cells and contributed to the biogenesis of podosome rosettes. These findings suggest a role for Rab11a in the trafficking of Drp1 to mitochondria upon SFK activation and unravel a novel functional interplay between Rab11a and mitochondria during reshaping of the cell cytoskeleton, which would facilitate mitochondria redistribution near energy-requiring actin-rich structures.     

Cortactin regulates podosome formation: roles of the protein interaction domains

Cortactin, a multi-domain scaffolding protein involved in actin polymerization, is enriched in podosomes induced by phorbol ester in vascular smooth muscle cells. We generated several functional and truncation mutants of cortactin to probe the roles of various protein interaction domains in the regulation of the dynamics of podosome formation. At the onset of podosome genesis, cortactin clustered near the ends of stress fibers that appeared to act as nucleation platforms onto which the actin polymerization machinery assembled. Translocation of cortactin to these pre-podosome clusters required the intact N-WASp-binding SH3 domain. Overexpression of the C-terminal third of cortactin containing the intact SH3 domain inhibited podosome formation presumably by sequestering of N-WASp and prevented cortactin clustering. Subsequent assembly of the actin-rich core of podosomes required translocation of additional cortactin to the actin core, a process that required the actin-binding repeats, but not the Arp2/3-binding N-terminal acidic region nor the SH3 domain. These results suggest that the SH3 domain and the actin-binding repeat region are involved, respectively, in the early and late stages of podosome formation process.