Podosomes at a glance (2005)

Cells can contact the extracellular matrix (ECM) through a variety of specialized structures. These cell-matrix contacts contain membrane proteins such as integrins, which bind both matrix components and cellular proteins, thus bridging the substratum and the cytoskeleton (reviewed in Adams, 2001). Matrix contacts provide the physical linkage that enables cells to adhere to and migrate on (or through) the matrix. Moreover, they can also act as localized `relay stations' for important signaling pathways. Well-known cell-matrix contacts include focal complexes and focal adhesions, whereas others, such as fibrillar adhesions, podosomes and invadopodia are just beginning to attract widespread attention.FIG1Focal complexes have a dash-like appearance, a length of ∼0.5 μm and constitute some of the earliest matrix contacts formed by a protruding cell. They are integrin-based and enriched in talin, paxillin and vinculin (Zamir and Geiger, 2001). Focal complexes can mature into focal adhesions through the application of force – for example, during the retraction of cellular protrusions. This transformation is accompanied by an increase in size, the formation of actin bundles (stress fibres), that contact the structure, and the recruitment of zyxin (Zaidel-Bar et al., 2004). Focal adhesions are formed on rigid substrates and contain high levels of phosphotyrosine but low amounts of the actin-binding protein tensin. On soft matrices, however, focal adhesions can give rise to a third type of contact, fibrillar adhesions. In contrast to focal adhesions, fibrillar adhesions display high levels of tensin but contain little phosphotyrosine (Zamir et al., 1999). In sum, these matrix contacts not only impart close adhesion to the substratum but also act as mechanosensors, and their ultimate form (focal adhesion vs fibrillar adhesion) depends strongly on matrix rigidity (Zamir and Geiger, 2001).Podosomes and invadopodia constitute two forms of dot-like matrix contact that differ from those described above structurally and functionally (reviewed in Linder and Aepfelbacher, 2003; Buccione et al., 2004). Structurally, their most distinguishing feature is their two-part architecture: they have a core of F-actin and actin-associated proteins that is surrounded by a ring structure consisting of plaque proteins such as talin or vinculin. This actin-rich core is not present in other cell-matrix contacts. Consequently, actin regulatory pathways exert a major additional influence on podosome-type contacts. Functionally, the ability of podosomes and invadopodia to engage in matrix degradation clearly sets them apart from other cell-matrix contacts.Podosomes are typically formed in cells of the monocytic lineage, such as macrophages (Lehto et al., 1982; Linder et al., 1999), osteoclasts (Marchisio et al., 1984) or (immature) dendritic cells (Burns et al., 2001). However, they can also be found or induced in a variety of other cell types, including smooth muscle cells (Gimona et al., 2003) and endothelial cells (Moreau et al., 2003; Osiak et al., 2005).By contrast, invadopodia are found in fibroblasts transformed with viral oncogenes encoding protein tyrosine kinases (Tarone et al., 1985) and in some malignant cell types (Buccione et al., 2004). Indeed, the first observation of podosome-type adhesions 25 years ago described such virally induced invadopodia (David-Pfeuty and Singer, 1980).Podosomes and invadopodia differ in their respective sizes and numbers. Typically, a cell forms dozens of podosomes, but there are only a few invadopodia per cell. However, what invadopodia lack in numbers, they make up for in size: Podosomes have a diameter of 0.5-1 μm and a depth of 0.2-0.4 μm, whereas invadopodia can correspond to an array of membrane invaginations that have a diameter of ∼8 μm and also can form root-like extensions into the matrix that are several micrometers deep (Buccione et al., 2004; McNiven et al., 2004).Despite these differences, basic similarities between the two structures, both in composition and architecture, are apparent. Indeed, it has been proposed that invadopodia might develop from podosomal precursors (Linder and Aepfelbacher, 2003; Buccione et al., 2004). It is therefore an attractive speculation that podosomes and invadopodia represent a continuum of specialized matrix contacts comparable to the succession of focal complexes, focal adhesions and fibrillar adhesions described above.The growing list of podosome-containing cells may point to a widespread ability of cells to form this type of actin-rich structure. Furthermore, the fact that podosomes are also formed on soft substrates, such as endothelial monolayers (Linder and Aepfelbacher, 2003), adds to the notion that podosomes are physiological structures that are also formed in the context of tissues.The main functions of podosomes appear to be adhesion and matrix degradation. Moreover, podosomes have also been implicated in cell migration and invasion. However, much of the evidence for these functions is still circumstantial and needs to be rigorously tested.It is very possible that podosomes have a role in adhesion, because they establish close contact to the substratum, which can be shown by total internal reflection (TIRF) microscopy, are enriched in adhesion-mediating integrins (Linder and Aepfelbacher, 2003) and form only at the substrate-attached cell side.The aptly named invadopodia were defined through their ability to perform matrix degradation (Buccione et al., 2004; McNiven et al., 2004). For podosomes, the case has been less clear cut: podosomes in osteoclasts are enriched in matrix metalloproteases (Sato et al., 1997; Delaissé et al., 2000), but it was not shown until recently that, in various cell systems, podosomes overlap regions of matrix degradation (Burgstaller and Gimona, 2005; Osiak et al., 2005). It is therefore very possible that podosomes, like invadopodia, have an inherent ability to lyse the ECM.Podosomes may also play an accessory role in cell migration. They might help to establish localized anchorage, thus stabilizing sites of cell protrusion and ultimately enabling productive directional movement. The observation that podosomes are recruited to sites of cell protrusion, especially to the leading edge, appears to be in line with such a concept.Podosome-type adhesions are mainly formed in cells that have to cross tissue boundaries such as monocytes, immature dendritic cells or some types of cancer cell (invadopodia in this case). Podosome-localized matrix degradation at the leading edge may therefore also confer invasive potential to cells.Finally, podosomes are a prominent part of the actin cytoskeleton in osteoclasts, where they form continuous belts at the cell periphery (Pfaff and Jurdic, 1999). Their probable roles in adhesion and matrix degradation suggest an important role in bone homeostasis. Moreover, their fusion was believed to form the bone-remodeling organelle of osteoclasts, the so-called sealing zone, a tightly adherent structure surrounding the space into which lytic enzymes and protons are secreted (Lakkakorpi and Väänänen, 1996). However, recent data suggest that both types of organelle are formed independently (Saltel et al., 2004). Furthermore, on mineralized bone, their physiological substrate, osteoclasts form only sealing zones, but not podosomes (Saltel et al., 2004). The proposed role for podosomes – as opposed to sealing zones – in bone homeostasis is therefore again open for debate.Podosomes comprise a core of F-actin and actin-associated proteins embedded in a ring structure of integrins and integrin-associated proteins (see poster, middle and right). Ring and core are probably linked by bridging molecules such as α-actinin, and the whole structure is surrounded by a cloud of mostly monomeric actin molecules (Destaing et al., 2003). Many podosome components show a distinct localization to either the core or ring structure. Typical core components are F-actin, actin regulators such as members of the Wiskott-Aldrich Syndrome protein (WASP) family, the Arp2/3 complex, gelsolin and cortactin, whereas adhesion mediators such as paxillin, vinculin or talin, and kinases such as PI3K or Pyk2/FAK preferentially associate with the ring structure (Linder and Aepfelbacher, 2003; Buccione et al., 2004).Podosomes are anchored in the extracellular matrix through integrins. These are distributed over the whole outer surface of the podosome, but show an isotype-specific localization: β1 integrins localize mostly to the core, whereas β2 and β3 integrins localize to the ring.Podosomes are influenced by a variety of cellular signaling pathways. Major modes of podosome regulation include signaling by Rho family GTPases, actin regulatory pathways, protein tyrosine phosphorylation, and the influence of the microtubule system.The RhoGTPases RhoA, Rac1 and CDC42 have all been shown to regulate podosome turnover in various cell types. Their influence on podosomes is undisputed; however, their particular mode of action may depend on the cell type. For example, both dominant active and inactive mutants of these GTPases can interfere with podosome formation or localization in dendritic cells (Burns et al., 2001), while expression of dominant active CDC42 leads to podosome formation in aortic endothelial cells (Moreau et al., 2003). In any case, podosome turnover appears to need subcellular fine tuning of the GTP/GDP cycles of these cellular master switches (Linder and Aepfelbacher, 2003). Accordingly, active Rho has been localized to invadopodia in transformed fibroblasts (Berdeaux et al., 2004).The most prominent feature of podosomes is their F-actin-rich core, which is also necessary for the stability of the whole structure (Lehto et al., 1982). Members of the WASP family, as well as actin-nucleating Arp2/3 complex are both strongly enriched in the podosome core. Absence of these components, either induced artificially (Linder et al., 2000a) or in disease (Linder et al., 1999; Burns et al., 2001) (reviewed in Calle et al., 2004), results in disruption of podosomes. Another prominent podosome component involved in actin regulation is gelsolin. It probably contributes to actin turnover through its ability to both sever and cap actin filaments (Chellaiah et al., 1998).It was noted early on that podosome-type adhesions can be induced by transformation of cells with viruses whose oncogenes encode tyrosine kinases such as v-Src (Tarone et al., 1985; Marchisio et al., 1987). Indeed, one of the best ways to visualize podosomes is by staining phosphorylated tyrosine (Linder and Apfelbacher, 2003), which is highly enriched in some types of adhesive structure. Not surprisingly, cellular tyrosine kinases such as Src and Csk play major roles in podosome regulation (Howell and Cooper, 1994), and tools for podosome manipulation include Src kinase inhibitors, which disrupt podosomes (Linder et al., 2000b), and vanadate, a phosphotyrosine phosphatase inhibitor, which is able to induce podosome formation (Marchisio et al., 1988).Microtubules are closely associated with podosomes. Moreover, they have been shown to stabilize podosome patterns such as the marginal belts in osteoclasts (Babb et al., 1997), to influence the fusion and fission rates of podosome precursors in murine macrophages (Evans et al., 2003) and to regulate podosome formation in human macrophages (Linder et al., 2000b).Analysis of podosome-inducing pathways has been performed in a variety of cell types. From these data we can propose the following simplified model for podosome formation: The key signal for initiation is attachment of the cell to the substrate, because podosomes are only observed in adherent cells. This leads to clustering and activation of integrins and signaling by receptor tyrosine kinases. One of the most upstream signals is probably PKC activity. This is underscored by the fact that podosome formation can be induced by PKC-activating agents, such as phorbol esters (Gimona et al., 2003). An important subsequent switch is activation of Src. Downstream, a variety of pathways are initiated, most notably PI3K signaling (Chellaiah et al., 1998), which leads to the formation of the phosphatidylinositols PtdIns(3,4)P2 and PtdIns(3,4,5)P3, and activation of focal adhesion kinase (FAK) or its haematopoietic relative Pyk2. 3′ phosphoinositide signaling is also influenced by the RhoGTPases Rho and (probably) Rac (Chellaiah et al., 2001; Sechi and Wehland, 2000).CDC42, another RhoGTPase, is crucial for the local initiation of actin filament formation, which gives rise to the formation of the actin-rich core structure. This is achieved by releasing the autoinhibition of N-WASP or its relative WASP, which in turn activates actin-nucleating Arp2/3 complex (Linder et al., 1999; Linder et al., 2000a; Burns et al., 2001). Turnover of core-localized actin filaments is probably facilitated by gelsolin (Chellaiah et al., 1998) and the GTPase dynamin (McNiven et al., 2004; Buccione et al., 2004). Additionally, dynamin may also play a role in podosome-localized matrix degradation by facilitating matrix metalloprotease release.It is unclear how core- and ring-formation are coordinated. However, similarly to core molecules, central components of the ring structure, such as talin and vinculin, are also influenced by phosphoinositides (Sechi and Wehland, 2000), and activation of paxillin through integrin-related signaling (Pfaff and Jurdic, 2001) may constitute one of the earliest signals in ring formation.Podosomes are highly dynamic organelles with a half-life of 2-12 minutes. Moreover, their inner dynamic is even faster: F-actin in the core turnes over 2-3 times during the life span of a podosome (Destaing et al., 2003). Individual podosomes are motile within a certain radius, but movement of larger podosome groups is achieved through assembly at the front and disassembly at the rear. This is especially visible in migratory cells where podosomes are recruited to the leading edge.Podosomes can assemble de novo or by branching off precursor clusters, which undergo constant fusion and fission (Evans et al., 2003) (see poster, lower right). `Regular' podosomes are mostly found in the inner regions of the cell, while the larger precursor clusters localize to the cell periphery or the leading lamella of migrating cells.Podosomes are assembled from molecules that have multiple functions in the cell, such as actin and Src kinases. Inhibitors of podosome assembly therefore also have profound effects on other parts of the cytoskeleton. Similarly, the absence of podosomes in diseases based on defects in a podosome component(s) may only constitute a side effect. The podosome aficionado should therefore take care to ascertain whether a lack of podosomes causes or is simply symptomatic of a particular phenotype.Podosome-associated diseases include Wiskott-Aldrich Syndrome (WAS) and chronic myeloid leukemia (CML). WAS-patient macrophages (Linder et al., 1999) and dendritic cells from CML patients (Dong et al., 2003) both display pronounced defects in podosome formation and chemotaxis.We thank Bettina Ebbing for help with TIRF microscopy, Alexander Bershadsky for the gift of YFP-vinculin, Peter C. Weber and Jürgen Heesemann for continuous support and Barbara Böhlig for expert technical assistance. Work from our lab is supported by the Deutsche Forschungsgemeinschaft (GRK 438, SFB 413), the Friedrich Baur Stiftung and the August Lenz Stiftung. We apologize to all whose work was not mentioned owing to space limitations.