Proactive for invasion: Reuse of matrix metalloproteinase for structural memory

Migratory cells translocate membrane type-1 matrix metalloproteinase (MT1-MMP) to podosomes or invadosomes to break extracellular matrix barriers. In this issue, El Azzouzi et al. (2016. J. Cell. Biol. http://dx.doi.org/10.1083/jcb.201510043) describe an unexpected function for the MT1-MMP cytoplasmic domain in imprinting spatial memory for podosome reformation via assembly in membrane islets.Invasion of most normal and cancer cells across basement membranes and collagen-rich interstitial tissues involves degradation of the ECM by membrane type-1 matrix metalloproteinase (MT1-MMP/MMP14; Willis et al., 2013). To fulfill this activity, MT1-MMP is transported to podosomes, the specialized ECM-degrading membrane protrusions found in highly migratory cells such as activated macrophages, osteoclasts, endothelial cells, and smooth muscle cells (Murphy and Courtneidge, 2011). In cancer cells, MT1-MMP is transported to ECM-degrading invasive structures called invadopodia (Poincloux et al., 2009). Both these membrane protrusions, collectively called invadosomes, are composed of an actin-rich core surrounded by scaffold and adhesion proteins, and numerous mechanisms of invadosome assembly, maturation, and dynamics have been identified (Poincloux et al., 2009; Murphy and Courtneidge, 2011). MT1-MMP activity is regulated at multiple levels to achieve targeted ECM degradation, cell surface protein processing, and protease activation (Sato et al., 1994; Osenkowski et al., 2004; Sugiyama et al., 2013; Willis et al., 2013; Itoh, 2015). Potential regulatory functions of MT1-MMP toward the cytoskeleton have, however, remained unclear. In this issue, El Azzouzi et al. describe an unexpected and novel function for MT1-MMP that goes beyond its traditional proteolytic activity: they show that MT1-MMP accumulates in membrane islets that provide macrophages with spatial information, or memory, in sites of podosome dissolution so as to enable efficient podosome reassembly.El Azzouzi et al. (2016) first used total internal reflection fluorescence microscopy and a pH-sensitive version of MT1-MMP devised to fluoresce only when the MT1-MMP ectodomain is exposed to the extracellular environment’s pH. With this approach, they show that, on the ventral surface of cultured human macrophages, MT1-MMP is localized at two different membrane compartments: underneath the podosome core, as previously suggested based on matrix degradation and colocalization with podosome markers, and in distinct islets devoid of other podosome components, CD44, or integrin-mediated adhesion to the ECM (Fig. 1; Osiak et al., 2005). MT1-MMP islets were dependent on intact cortical actin, but became more apparent and persisted after podosome disruption by pharmacological perturbation of key components of podosome assembly and maturation, such as integrin adhesion, Src kinase activity, and the Arp2/3 complex essential for actin nucleation and branched actin cytoskeleton. Podosomes often reemerge at sites of previous podosome localization, and El Azzouzi et al. (2016) hypothesized that MT1-MMP islets might mark sites of podosome formation. They treated cells with an Arp2/3 inhibitor to disrupt podosomes and induce the appearance of MT1-MMP membrane clusters, and used time-lapse imaging to track what happens upon washout and podosome reformation. Interestingly, they show that these novel MT1-MMP structures serve as remarkably immobile cell membrane anchors capable of rerecruiting the podosomal actin cores/scaffolds to the same islets.Open in a separate windowFigure 1.MT1-MMP islets as memory sites for podosome reformation. Migratory cells translocate MT1-MMP (red) to podosomes or invadosomes to degrade the ECM (green fibers). These membrane structures are composed of an actin-rich core (brown) surrounded by adhesion and scaffold proteins (beige) such as integrins (blue). El Azzouzi et al. (2016) show a function for MT1-MMP accumulation in membrane ”islets” (1), where they imprint spatial memory for podosome reemergence after podosome disassembly (2). Unlike dynamic mature podosomes (3), MT1-MMP assembles in stable islets via anchorage to cortical actin. Future work in the fields of inflammation, cancer, and angiogenesis will need to address the nature of the cytoskeletal dynamics mediating islet formation, the involvement of microtubules in islet formation, the exact islet protein composition, and the relevance of these memory sites to 2D or 3D environments and to other cell types beyond macrophages, including endothelial cells and invasive cancer cells.Further, by expressing mutant MT1-MMP proteins in cells silenced for the endogenous proteinase and using a podosome reformation assay (based on pharmacological dissolution of podosomes via Src inhibition, followed by podosome reformation after washout), El Azzouzi et al. (2016) pinpointed the region of MT1-MMP critical for islet formation, the LLY-sequence in its cytoplasmic domain. Moreover, when attached to the membrane by the MT1-MMP transmembrane domain, the 20–amino acid cytoplasmic tail appeared necessary and sufficient to form the islets. Considering the LLY sequence–dependent actin-binding ability of MT1-MMP (Yu et al., 2012) coupled with the observed necessity of cortical actin for islet appearance and podosome reformation, the direct interaction with unbranched cortical actin was suggested by the authors as a likely decisive mechanism for the remarkable MT1-MMP islet stabilization in podosome-free areas, although a possible indirect interaction was not ruled out. Actin binding through the MT1-MMP cytosolic tail was likewise suggested as a potential means for podosome rerecruitment by MT1-MMP memory islets.Although cortical actin is instrumental for the emergence of the spatially and temporally stable MT1-MMP islets upon podosome dissolution in macrophages and direct actin–MT1-MMP interaction has been proven in vitro and suggested as a means for retaining MT1-MMP in invadopodia, a Src-regulated interaction between MT1-MMP’s cytoplasmic domain and the actin-binding scaffold protein palladin has also been shown to regulate MT1-MMP targeting into invadopodia (Yu et al., 2012; von Nandelstadh et al., 2014). Moreover, the LLY sequence in MT1-MMP’s cytoplasmic tail harbors a Src substrate sequence and mediates an interaction between MT1-MMP and AP-2 that is important for MT1-MMP internalization and dynamics in cell migration and invasion (Uekita et al., 2001; Nyalendo et al., 2007). Intriguingly, El Azzouzi et al. (2016) did not find evidence of involvement of dynamin-dependent membrane trafficking events in the ability of MT1-MMP islets to function as memory sites. However, their results after treatment with the microtubule inhibitor nocodazole indicated that although the islets themselves remained intact, podosome reappearance was mislocalized, suggesting that microtubules contribute by as yet undefined mechanisms to the ability of MT1-MMP islets to provide spatial memory and to facilitate podosome reassembly. Therefore, further identification of drivers and specific regulatory mechanisms of the actin–MT1-MMP interaction dynamics in podosomes, of the stable actin–MT1-MMP interaction and structures in podosome-free areas, and of microtubule-dependent podosome reassembly will be of interest.A striking observation of this study is that MT1-MMP islets do not display degradative activity in matrix degradation assays. In addition, inhibition of the proteolytic activity of MT1-MMP through pharmacological agents or via an inactivating mutation did not impact islet appearance or podosome reemergence at sites of MT1-MMP clustering. Overall, on the extracellular side of the plasma membrane, the apparent lack of contact and degradation of the ECM as well as the relatively minor impact of the N-terminal MT1-MMP ectodomain on islet formation and podosome reemergence are peculiar features of the MT1-MMP islets. However, El Azzouzi et al. (2016) show evidence for somewhat impaired islet formation in cells expressing an MT1-MMP mutant lacking the entire ectodomain, and they demonstrate that endogenous MT1-MMP must be silenced for the LLY MT1-MMP mutant to disrupt islet localization. Based on these results, the authors suggest the possible influence of MT1-MMP oligomerization and of MT1-MMP–ECM binding on islet recruitment and stabilization. Nevertheless, these observations altogether indicate that the adhesive and degradative activities of MT1-MMP memory islets toward the ECM are minor and, intriguingly, do not influence the structure or function of these islets as currently characterized in 2D cultures.Furthermore, the aforementioned results raise questions about the possible contribution of the different molecular forms of MT1-MMP (e.g., cleaved or uncleaved and inhibitor bound or not) to the stabilization and podosome reassembly function of MT1-MMP islets. In cells and conditions in which MT1-MMP activity is high, MT1-MMP turnover is typically fast via autocatalytic cleavage or shedding of the N-terminal catalytic domain (Lehti et al., 1998; Yana and Weiss, 2000; Itoh et al., 2001; Osenkowski et al., 2004). After interaction with inhibitors such as tissue inhibitors of metalloproteinases, active endocytosed MT1-MMP may dissociate from the bound inhibitor to be recycled to the plasma membrane (Jiang et al., 2001; Remacle et al., 2003). However, in the absence of interaction with a protease inhibitor or collagen/matrix substrate, MT1-MMP oligomerization facilitates MT1-MMP turnover via autocatalytic inactivating cleavage (Itoh et al., 2001; Lehti et al., 2002; Osenkowski et al., 2004). In the current study, El Azzouzi et al. (2016) used MT1-MMP proteins with a pH sensor inserted N-terminally to the transmembrane domain, so that the probe is located extracellularly on the surface-exposed protease. The fluorescence signal from these constructs is not expected to be affected by proteolytic processing or shedding of the catalytic domain, so it is unclear whether the MT1-MMP proteins clustered in islets are cleaved or not. However, FRAP results showed that the turnover of MT1-MMP molecules within the islets is relatively slow. It thus remains to be clarified if and how the proteolytically active or possibly processed or protease inhibitor–bound inactive forms of MT1-MMP are stabilized in these MT1-MMP islets.As posodomes are highly dynamic protrusions, their rapid turnover implicates a constant disassembly at the rear and formation at the front of migrating macrophages. Assembly and dissassembly are known to depend on Arp2/3-mediated actin nucleation and fission of preexisting podosomes, respectively (Linder et al., 2000). Both of these mechanisms may contribute to podosome reassembly at MT1-MMP memory sites. Considering that these islets are laterally immobile and overall stable in at least unpolarized cells, it is unclear how migrating cells coordinate their actin and microtubule cytoskeletons for podosome reassembly at the front using islets formed upon podosome dissolution at the rear of the cell (Fig. 1). Moreover, the structural and functional features of MT1-MMP islets in the scenario of 3D cell–ECM microenvironments is intriguing and will need to be investigated at high resolution, as cytoskeletal dynamics, cell polarity, and matrix stiffness greatly differ in 3D tissues and matrices from the simple 2D setting of cultured cells, and all are known to influence cell behavior. Although the transient nature of these MT1-MMP islets in bridging podosome disassembly and reassembly exemplifies and reflects the efficiency of podosome reusage, probing the protein composition of these islets as well as the dynamics of podosome reassembly will likely be challenging. Future studies comparing MT1-MMP state, dynamics, reuse, and turnover in different types of invadosomes, islets, and other subcellular compartments will be instrumental to better understand how cells integrate the different types of MT1-MMP membrane structures and cell–ECM communication with other cellular signals and with drivers of cytoskeletal dynamics.The identification of the molecular mechanisms of structural and functional podosome memory are not only relevant to the fields of macrophage biology and inflammation but also more broadly to those of tissue invasion and matrix remodeling. For instance, endothelial cells, smooth muscle cells, and cancer cells are also known to target MT1-MMP to podosomes or related invadosomes. Examining MT1-MMP memory in such specialized subcellular compartments will be interesting beyond the podosome field, as the podosome counterparts in cancer cells may display and use MT1-MMP or other metalloproteinases in a similar manner. By shedding light on the mechanisms of dynamic protrusion formation and function, this paper not only opens new avenues of investigation into the cellular structures marking protrusion sites as “memory devices” but also brings about a new concept to the fields of cell invasion, angiogenesis, and cancer.     

TLR ligand-induced podosome disassembly in dendritic cells is ADAM17 dependent

Toll-like receptor (TLR) signaling induces a rapid reorganization of the actin cytoskeleton in cultured mouse dendritic cells (DC), leading to enhanced antigen endocytosis and a concomitant loss of filamentous actin–rich podosomes. We show that as podosomes are lost, TLR signaling induces prominent focal contacts and a transient reduction in DC migratory capacity in vitro. We further show that podosomes in mouse DC are foci of pronounced gelatinase activity, dependent on the enzyme membrane type I matrix metalloprotease (MT1-MMP), and that DC transiently lose the ability to degrade the extracellular matrix after TLR signaling. Surprisingly, MMP inhibitors block TLR signaling–induced podosome disassembly, although stimulated endocytosis is unaffected, which demonstrates that the two phenomena are not obligatorily coupled. Podosome disassembly caused by TLR signaling occurs normally in DC lacking MT1-MMP, and instead requires the tumor necrosis factor α–converting enzyme ADAM17 (a disintegrin and metalloprotease 17), which demonstrates a novel role for this “sheddase” in regulating an actin-based structure.     

Transforming growth factor beta induces rosettes of podosomes in primary aortic endothelial cells

Cytoskeletal rearrangements are central to endothelial cell physiology and are controlled by soluble factors, matrix proteins, cell-cell interactions, and mechanical forces. We previously reported that aortic endothelial cells can rearrange their cytoskeletons into complex actin-based structures called podosomes when a constitutively active mutant of Cdc42 is expressed. We now report that transforming growth factor beta (TGF-beta) promotes podosome formation in primary aortic endothelial cells. TGF-beta-induced podosomes assembled together into large ring- or crescent-shaped structures. Their formation was dependent on protein synthesis and required functional Src, phosphatidylinositide 3-kinase, Cdc42, RhoA, and Smad signaling. MT1-MMP and metalloprotease 9 (MMP9), both upregulated by TGF-beta, were detected at sites of podosome formation, and MT1-MMP was found to be involved in the local degradation of extracellular matrix proteins beneath the podosomes and required for the invasion of collagen gels by endothelial cells. We propose that TGF-beta plays an important role in endothelial cell physiology by inducing the formation of podosomal structures endowed with metalloprotease activity that may contribute to arterial remodeling.     

The Novel Adaptor Protein Tks4 (SH3PXD2B) Is Required for Functional Podosome Formation

Metastatic cancer cells have the ability to both degrade and migrate through the extracellular matrix (ECM). Invasiveness can be correlated with the presence of dynamic actin-rich membrane structures called podosomes or invadopodia. We showed previously that the adaptor protein tyrosine kinase substrate with five Src homology 3 domains (Tks5)/Fish is required for podosome/invadopodia formation, degradation of ECM, and cancer cell invasion in vivo and in vitro. Here, we describe Tks4, a novel protein that is closely related to Tks5. This protein contains an amino-terminal Phox homology domain, four SH3 domains, and several proline-rich motifs. In Src-transformed fibroblasts, Tks4 is tyrosine phosphorylated and predominantly localized to rosettes of podosomes. We used both short hairpin RNA knockdown and mouse embryo fibroblasts lacking Tks4 to investigate its role in podosome formation. We found that lack of Tks4 resulted in incomplete podosome formation and inhibited ECM degradation. Both phenotypes were rescued by reintroduction of Tks4, whereas only podosome formation, but not ECM degradation, was rescued by overexpression of Tks5. The tyrosine phosphorylation sites of Tks4 were required for efficient rescue. Furthermore, in the absence of Tks4, membrane type-1 matrix metalloproteinase (MT1-MMP) was not recruited to the incomplete podosomes. These findings suggest that Tks4 and Tks5 have overlapping, but not identical, functions, and implicate Tks4 in MT1-MMP recruitment and ECM degradation.     

Microtubule Dynamic Instability Controls Podosome Patterning in Osteoclasts through EB1, Cortactin,and Src

In osteoclasts (OCs) podosomes are organized in a belt, a feature critical for bone resorption. Although microtubules (MTs) promote the formation and stability of the belt, the MT and/or podosome molecules that mediate the interaction of the two systems are not identified. Because the growing “plus” ends of MTs point toward the podosome belt, plus-end tracking proteins (+TIPs) might regulate podosome patterning. Among the +TIPs, EB1 increased as OCs matured and was enriched in the podosome belt, and EB1-positive MTs targeted podosomes. Suppression of MT dynamic instability, displacement of EB1 from MT ends, or EB1 depletion resulted in the loss of the podosome belt. We identified cortactin as an Src-dependent interacting partner of EB1. Cortactin-deficient OCs presented a defective MT targeting to, and patterning of, podosomes and reduced bone resorption. Suppression of MT dynamic instability or EB1 depletion increased cortactin phosphorylation, decreasing its acetylation and affecting its interaction with EB1. Thus, dynamic MTs and podosomes interact to control bone resorption.     

The non-redundant role of N-WASP in podosome-mediated matrix degradation in macrophages

Wiskott-Aldrich Syndrome Protein (WASP) is a hematopoietic cell-specific regulator of Arp2/3-dependent actin polymerization. Despite the presence of the highly homologous N-WASP (neural-WASP), macrophages from WAS patients are devoid of podosomes, adhesion structures in cells of the monocytic lineage capable of matrix degradation via matrix metalloproteases (MMPs), suggesting that WASP and N-WASP play unique roles in macrophages. To determine whether N-WASP also plays a unique role in macrophage function, N-WASP expression was reduced using silencing RNA in a sub-line of RAW 264.7 macrophages (RAW/LR5). Similar to reduction in WASP levels, cells with reduced N-WASP levels were rounder and less polarized. Interestingly, podosomes still formed when N-WASP was reduced but they were unable to perform matrix degradation. This defect was rescued by re-expression of N-WASP, but not by over-expression of WASP, indicating that these proteins play distinct roles in podosome function. Additionally, reducing N-WASP levels mistargets the metalloprotease MT1-MMP and it no longer localizes to podosomes. However, N-WASP was only found to co-localize with MT1-MMP positive vesicles at podosomes, suggesting that N-WASP may play a role on the targeting or fusion of MMP-containing vesicles to podosomes in macrophage-like cells.     

The protein kinase C cascade regulates recruitment of matrix metalloprotease 9 to podosomes and its release and activation

Podosomes are transient cell surface structures essential for degradation of extracellular matrix during cell invasion. Protein kinase C (PKC) is involved in the regulation of podosome formation; however, the roles of individual PKC isoforms in podosome formation and proteolytic function are largely unknown. Recently, we reported that PDBu, a PKC activator, induced podosome formation in normal human bronchial epithelial cells. Here, we demonstrate that phorbol-12,13-dibutyrate (PDBu)-induced podosome formation is mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes. Interestingly, although blocking atypical PKCζ did not affect PDBu-induced podosome formation, it significantly reduced matrix degradation at podosomes. Inhibition of PKCζ reduced recruitment of matrix metalloprotease 9 (MMP-9) to podosomes and its release and activation. Downregulation of MMP-9 by small interfering RNA (siRNA) or neutralization antibody also significantly reduced matrix degradation. The regulatory effects of PKCζ on matrix degradation and recruitment of MMP-9 to podosomes were PKCζ kinase activity dependent. PDBu-induced recruitment of PKCζ and MMP-9 to podosomes was blocked by inhibition of novel PKC with rottlerin or PKCδ siRNA. Our data suggest that multiple PKC isozymes form a signaling cascade that controls podosome formation and dynamics and MMP-9 recruitment, release, and activation in a coordinated fashion.     

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.     

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.     

Macrophage podosomes go 3D

Macrophage tissue infiltration is a critical step in the immune response against microorganisms and is also associated with disease progression in chronic inflammation and cancer. Macrophages are constitutively equipped with specialized structures called podosomes dedicated to extracellular matrix (ECM) degradation. We recently reported that these structures play a critical role in trans-matrix mesenchymal migration mode, a protease-dependent mechanism. Podosome molecular components and their ECM-degrading activity have been extensively studied in two dimensions (2D), but yet very little is known about their fate in three-dimensional (3D) environments. Therefore, localization of podosome markers and proteolytic activity were carefully examined in human macrophages performing mesenchymal migration. Using our gelled collagen I 3D matrix model to obligate human macrophages to perform mesenchymal migration, classical podosome markers including talin, paxillin, vinculin, gelsolin, cortactin were found to accumulate at the tip of F-actin-rich cell protrusions together with β1 integrin and CD44 but not β2 integrin. Macrophage proteolytic activity was observed at podosome-like protrusion sites using confocal fluorescence microscopy and electron microscopy. The formation of migration tunnels by macrophages inside the matrix was accomplished by degradation, engulfment and mechanic compaction of the matrix. In addition, videomicroscopy revealed that 3D F-actin-rich protrusions of migrating macrophages were as dynamic as their 2D counterparts. Overall, the specifications of 3D podosomes resembled those of 2D podosome rosettes rather than those of individual podosomes. This observation was further supported by the aspect of 3D podosomes in fibroblasts expressing Hck, a master regulator of podosome rosettes in macrophages. In conclusion, human macrophage podosomes go 3D and take the shape of spherical podosome rosettes when the cells perform mesenchymal migration. This work sets the scene for future studies of molecular and cellular processes regulating macrophage trans-migration.     

Biophysical Induction of Vascular Smooth Muscle Cell Podosomes

Vascular smooth muscle cell (VSMC) migration and matrix degradation occurs with intimal hyperplasia associated with atherosclerosis, vascular injury, and restenosis. One proposed mechanism by which VSMCs degrade matrix is through the use of podosomes, transient actin-based structures that are thought to play a role in extracellular matrix degradation by creating localized sites of matrix metalloproteinase (MMP) secretion. To date, podosomes in VSMCs have largely been studied by stimulating cells with phorbol esters, such as phorbol 12,13-dibutyrate (PDBu), however little is known about the physiological cues that drive podosome formation. We present the first evidence that physiological, physical stimuli mimicking cues present within the microenvironment of diseased arteries can induce podosome formation in VSMCs. Both microtopographical cues and imposed pressure mimicking stage II hypertension induce podosome formation in A7R5 rat aortic smooth muscle cells. Moreover, wounding using a scratch assay induces podosomes at the leading edge of VSMCs. Notably the effect of each of these biophysical stimuli on podosome stimulation can be inhibited using a Src inhibitor. Together, these data indicate that physical cues can induce podosome formation in VSMCs.     

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.     

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.     

Invasive matrix degradation at focal adhesions occurs via protease recruitment by a FAK-p130Cas complex

Tumor cell migration and the concomitant degradation of extracellular matrix (ECM) are two essential steps in the metastatic process. It is well established that focal adhesions (FAs) play an important role in regulating migration; however, whether these structures contribute to matrix degradation is not clear. In this study, we report that multiple cancer cell lines display degradation of ECM at FA sites that requires the targeted action of MT1-MMP. Importantly, we have found that this MT1-MMP targeting is dependent on an association with a FAK-p130Cas complex situated at FAs and is regulated by Src-mediated phosphorylation of Tyr 573 at the cytoplasmic tail of MT1. Disrupting the FAK-p130Cas-MT1 complex significantly impairs FA-mediated degradation and tumor cell invasion yet does not appear to affect invadopodia formation or function. These findings demonstrate a novel function for FAs and also provide molecular insights into MT1-MMP targeting and function.     

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.     

MT-LOOP-dependent Localization of Membrane Type I Matrix Metalloproteinase (MT1-MMP) to the Cell Adhesion Complexes Promotes Cancer Cell Invasion

Localization of membrane type I matrix metalloproteinase (MT1-MMP) to the leading edge is thought to be a crucial step during cancer cell invasion. However, its mechanisms and functional impact on cellular invasion have not been clearly defined. In this report, we have identified the MT-LOOP, a loop region in the catalytic domain of MT1-MMP (¹⁶³PYAYIREG¹⁷⁰), as an essential region for MT1-MMP to promote cellular invasion. Deletion of the MT-LOOP effectively inhibited functions of MT1-MMP on the cell surface, including proMMP-2 activation, degradation of gelatin and collagen films, and cellular invasion into a collagen matrix. This is not due to loss of the catalytic function of MT1-MMP but due to inefficient localization of the enzyme to β1-integrin-rich cell adhesion complexes at the plasma membrane. We also found that an antibody that specifically recognizes the MT-LOOP region of MT1-MMP (LOOP_Ab) inhibited MT1-MMP functions, fully mimicking the phenotype of the MT-LOOP deletion mutant. We therefore propose that the MT-LOOP region is an interface for molecular interactions that mediate enzyme localization to cell adhesion complexes and regulate MT1-MMP functions. Our findings have revealed a novel mechanism regulating MT1-MMP during cellular invasion and have identified the MT-LOOP as a potential exosite target region to develop selective MT1-MMP inhibitors.     

L-Plastin Nanobodies Perturb Matrix Degradation,Podosome Formation,Stability and Lifetime in THP-1 Macrophages

Podosomes are cellular structures acting as degradation ‘hot-spots’ in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein. The presence of the capping protein CapG in podosomes has not yet been investigated. We used an innovative approach to investigate the role of these proteins in macrophage podosomes by means of nanobodies or Camelid single domain antibodies. Nanobodies directed against distinct domains of gelsolin, L-plastin or CapG were stably expressed in macrophage-like THP-1 cells. CapG was not enriched in podosomes. Gelsolin nanobodies had no effect on podosome formation or function but proved very effective in tracing distinct gelsolin populations. One gelsolin nanobody specifically targets actin-bound gelsolin and was effectively enriched in podosomes. A gelsolin nanobody that blocks gelsolin-G-actin interaction was not enriched in podosomes demonstrating that the calcium-activated and actin-bound conformation of gelsolin is a constituent of podosomes. THP-1 cells expressing inhibitory L-plastin nanobodies were hampered in their ability to form stable podosomes. Nanobodies did not perturb Ser5 phosphorylation of L-plastin although phosphorylated L-plastin was highly enriched in podosomes. Furthermore, nanobody-induced inhibition of L-plastin function gave rise to an irregular and unstable actin turnover of podosomes, resulting in diminished degradation of the underlying matrix. Altogether these results indicate that L-plastin is indispensable for podosome formation and function in macrophages.     

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).     

The cortactin-binding domain of WIP is essential for podosome formation and extracellular matrix degradation by murine dendritic cells

In immature dendritic cells (DCs) podosomes form and turn over behind the leading edge of migrating cells. The Arp2/3 complex activator Wiskott-Aldrich Syndrome Protein (WASP) localises to the actin core of forming podosomes together with WASP-Interacting Protein (WIP). A second weaker Arp2/3 activator, cortactin, is also found at podosomes where it has been proposed to participate in matrix metalloproteinase (MMP) secretion. We have previously shown that WIP(-/-) DCs are unable to make podosomes. WIP binds to cortactin and in this report we address whether WIP regulates cortactin-mediated MMP activity. Using DCs derived from splenic murine precursors, we found that wild-type cells were able to localise MMPs at podosomes where matrix degradation takes place. In contrast, WIP(-/-) DCs remain able to synthesise MMPs but do not degrade the extracellular matrix. Infection of WIP KO DCs with lentivirus expressing WIP restored both podosome formation and their ability to degrade the extracellular matrix, implicating WIP-induced podosomes as foci of functional MMP location. When WIP KO DCs were infected with a mutant form of WIP lacking the cortactin-binding domain (WIPΔ110-170) DCs were only able to elaborate disorganised podosomes that were unable to support MMP-mediated matrix degradation. Taken together, these results suggest a role for WIP not only in WASP-mediated actin polymerisation and podosome formation, but also in cortactin-mediated extracellular matrix degradation by MMPs.