Nck1 and Grb2 localization patterns can distinguish invadopodia from podosomes

Invadopodia are matrix-degrading ventral cell surface structures formed in invasive carcinoma cells. Podosomes are matrix-degrading structures formed in normal cell types including macrophages, endothelial cells, and smooth muscle cells that are believed to be related to invadopodia in function. Both invadopodia and podosomes are enriched in proteins that regulate actin polymerization including proteins involved in N-WASp/WASp-dependent Arp2/3-complex activation. However, it is unclear whether invadopodia and podosomes use distinct mediators for N-WASp/WASp-dependent Arp2/3-complex activation. We investigated the localization patterns of the upstream N-WASp/WASp activators Nck1 and Grb2 in invadopodia of metastatic mammary carcinoma cells, podosomes formed in macrophages, and degradative structures formed in Src-transformed fibroblasts and PMA-stimulated endothelial cells. We provide evidence that Nck1 specifically localizes to invadopodia, but not to podosomes formed in macrophages or degradative structures formed in Src-transformed fibroblasts and PMA-stimulated endothelial cells. In contrast, Grb2 specifically localizes to degradative structures formed in Src-transformed fibroblasts and PMA-stimulated endothelial cells, but not invadopodia or podosomes formed in macrophages. These findings suggest that distinct upstream activators are responsible for N-WASp/WASp activation in invadopodia and podosomes, and that all these ventral cell surface degradative structures have distinguishing molecular as well as structural characteristics. These patterns of Nck1 and Grb2 localization, identified in our study, can be used to sub-classify ventral cell surface degradative structures.     

The matrix corroded: podosomes and invadopodia in extracellular matrix degradation

Podosomes and invadopodia are unique actin-rich adhesions that establish close contact to the substratum but can also degrade components of the extracellular matrix. Accordingly, matrix degradation localized at podosomes or invadopodia is thought to contribute to cellular invasiveness in physiological and pathological situations. Cell types that form podosomes include monocytic, endothelial and smooth muscle cells, whereas invadopodia have been mostly observed in carcinoma cells. This review highlights important new developments in the field, discusses the common and divergent features of podosomes and invadopodia and summarizes current knowledge about matrix-degrading proteinases at these structures.     

PtdIns(3,4)P2 instigates focal adhesions to generate podosomes

Cell-to-extracellular matrix (ECM) adhesion plays important roles in various biological events, such as proliferation, differentiation, and migration. Distinct from other types of adhesion structures (focal complexes, focal adhesions, and so on), podosomes and invadopodia are thought to have additional functions beyond attachment, possibly including invasion into the ECM. For podosomes and invadopodia to invade into the ECM, molecules involved in adhesion, actin polymerization, and ECM degradation must be recruited to sites of action. Our recent study demonstrated that podosomes form near newly formed focal adhesions via the minimally expressed phosphoinositide PtdIns(3,4)P2-mediated recruitment of the Tks5-Grb2 scaffold, followed by the accumulation of N-WASP. Although this study demonstrated details of molecular interplay during the transformation of focal adhesion, its regulation in the in vivo invasion process remains to be clarified. Here, we discuss the molecular bases of the transformation of focal adhesions to podosomes/invadopodia based on current understanding.     

PtdIns(3,4)P2 instigates focal adhesions to generate podosomes

Cell-to-extracellular matrix (ECM) adhesion plays important roles in various biological events, such as proliferation, differentiation and migration. Distinct from other types of adhesion structures (focal complexes, focal adhesions and so on), podosomes and invadopodia are thought to have additional functions beyond attachment, possibly including invasion into the ECM. For podosomes and invadopodia to invade into the ECM, molecules involved in adhesion, actin polymerization and ECM degradation must be recruited to sites of action. Our recent study demonstrated that podosomes form near newly formed focal adhesions via the minimally expressed phosphoinositide PtdIns(3,4) P2-mediated recruitment of the Tks5-Grb2 scaffold, followed by the accumulation of N-WASP. Although this study demonstrated details of molecular interplay during the transformation of focal adhesion, its regulation in the in vivo invasion process remains to be clarified. Here, we discuss the molecular bases of the transformation of focal adhesions to podosomes/invadopodia based on current understanding.Key words: podosome, invadopodium, focal adhesion, Tks5, PtdIns(3,4)P2, N-WASP     

Caldesmon suppresses cancer cell invasion by regulating podosome/invadopodium formation

The podosome and invadopodium are dynamic cell-adhesion structures that degrade the extracellular matrix (ECM) and promote cell invasion. We recently reported that the actin-binding protein caldesmon is a pivotal regulator of podosome formation. Here, we analyzed the caldesmon's involvement in podosome/invadopodium-mediated invasion by transformed and cancer cells. The ectopic expression of caldesmon reduced the number of podosomes/invadopodia and decreased the ECM degradation activity, resulting in the suppression of cell invasion. Conversely, the depletion of caldesmon facilitated the formation of podosomes/invadopodia and cell invasion. Taken together, our results indicate that caldesmon acts as a potent repressor of cancer cell invasion.     

Supervillin Reorganizes the Actin Cytoskeleton and Increases Invadopodial Efficiency

Tumor cells use actin-rich protrusions called invadopodia to degrade extracellular matrix (ECM) and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II, reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV induces redistribution of lamellipodial cortactin and lamellipodin/RAPH1/PREL1 away from the cell periphery to internal sites and concomitantly increases the numbers of F-actin punctae. Most punctae are highly dynamic and colocalize with the podosome/invadopodial proteins, cortactin, Tks5, and cdc42. Cortactin binds SV sequences in vitro and contributes to the formation of enhanced green fluorescent protein (EGFP)-SV induced punctae. SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases average numbers of ECM holes per cell; RNA interference-mediated knockdown of SV decreases these numbers. Although SV knockdown alone has no effect, simultaneous down-regulation of SV and the closely related protein gelsolin reduces invasion through ECM. Together, our results show that SV is a component of podosomes and invadopodia and that SV plays a role in invadopodial function, perhaps as a mediator of cortactin localization, activation state, and/or dynamics of metalloproteinases at the ventral cell surface.     

Extracellular matrix rigidity promotes invadopodia activity

Invadopodia are actin-rich subcellular protrusions with associated proteases used by cancer cells to degrade extracellular matrix (ECM) [1]. Molecular components of invadopodia include branched actin-assembly proteins, membrane trafficking proteins, signaling proteins, and transmembrane proteinases [1]. Similar structures exist in nontransformed cells, such as osteoclasts and dendritic cells, but are generally called podosomes and are thought to be more involved in cell-matrix adhesion than invadopodia [2-4]. Despite intimate contact with their ECM substrates, it is unknown whether physical or chemical ECM signals regulate invadopodia function. Here, we report that ECM rigidity directly increases both the number and activity of invadopodia. Transduction of ECM-rigidity signals depends on the cellular contractile apparatus [5-7], given that inhibition of nonmuscle myosin II, myosin light chain kinase, and Rho kinase all abrogate invadopodia-associated ECM degradation. Whereas myosin IIA, IIB, and phosphorylated myosin light chain do not localize to invadopodia puncta, active phosphorylated forms of the mechanosensing proteins p130Cas (Cas) and focal adhesion kinase (FAK) are present in actively degrading invadopodia, and the levels of phospho-Cas and phospho-FAK in invadopodia are sensitive to myosin inhibitors. Overexpression of Cas or FAK further enhances invadopodia activity in cells plated on rigid polyacrylamide substrates. Thus, in invasive cells, ECM-rigidity signals lead to increased matrix-degrading activity at invadopodia, via a myosin II-FAK/Cas pathway. These data suggest a potential mechanism, via invadopodia, for the reported correlation of tissue density with cancer aggressiveness.     

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.     

Coupling between acto-adhesive machinery and ECM degradation in invadosomes

Invadosomes have two main functions represented by their actin-rich and adhesive components and their polarized secretory pathways controlling the delivery of metalloproteases necessary to degrade extracellular matrix (ECM). Invadosomes include invadopodia and podosomes, which have subtle differences in molecular composition, dynamics, and structure. These differences could reflect different stages of invadosome maturation. This review will outline current knowledge on the coupling between the acto-adhesive machinery and the ECM degradation activity in invadosome diversity. Multiple works support that these two functions are not automatically linked but seem to be finely regulated to allow different functions of invadosomes. We will explore the paradigmatic aspect of invadosomes, which are able to interact with ECM to degrade it so as to better control their own dynamics. Understanding the fine-tuning between these two functions could serve to understand the link between the different types of invadosomes from invadopodia to podosomes.     

Coupling between acto-adhesive machinery and ECM degradation in invadosomes

Invadosomes have two main functions represented by their actin-rich and adhesive components and their polarized secretory pathways controlling the delivery of metalloproteases necessary to degrade extracellular matrix (ECM). Invadosomes include invadopodia and podosomes, which have subtle differences in molecular composition, dynamics, and structure. These differences could reflect different stages of invadosome maturation. This review will outline current knowledge on the coupling between the acto-adhesive machinery and the ECM degradation activity in invadosome diversity. Multiple works support that these two functions are not automatically linked but seem to be finely regulated to allow different functions of invadosomes. We will explore the paradigmatic aspect of invadosomes, which are able to interact with ECM to degrade it so as to better control their own dynamics. Understanding the fine-tuning between these two functions could serve to understand the link between the different types of invadosomes from invadopodia to podosomes.     

The interplay between the proteolytic,invasive, and adhesive domains of invadopodia and their roles in cancer invasion

Invadopodia are actin-based protrusions of the plasma membrane that penetrate into the extracellular matrix (ECM), and enzymatically degrade it. Invadopodia and podosomes, often referred to, collectively, as “invadosomes,” are actin-based membrane protrusions that facilitate matrix remodeling and cell invasion across tissues, processes that occur under specific physiological conditions such as bone remodeling, as well as under pathological states such as bone, immune disorders, and cancer metastasis. In this review, we specifically focus on the functional architecture of invadopodia in cancer cells; we discuss here three functional domains of invadopodia responsible for the metalloproteinase-based degradation of the ECM, the cytoskeleton-based mechanical penetration into the matrix, and the integrin adhesome-based adhesion to the ECM. We will describe the structural and molecular organization of each domain and the cross-talk between them during the invasion process.     

The interplay between the proteolytic,invasive, and adhesive domains of invadopodia and their roles in cancer invasion

Invadopodia are actin-based protrusions of the plasma membrane that penetrate into the extracellular matrix (ECM), and enzymatically degrade it. Invadopodia and podosomes, often referred to, collectively, as “invadosomes,” are actin-based membrane protrusions that facilitate matrix remodeling and cell invasion across tissues, processes that occur under specific physiological conditions such as bone remodeling, as well as under pathological states such as bone, immune disorders, and cancer metastasis. In this review, we specifically focus on the functional architecture of invadopodia in cancer cells; we discuss here three functional domains of invadopodia responsible for the metalloproteinase-based degradation of the ECM, the cytoskeleton-based mechanical penetration into the matrix, and the integrin adhesome-based adhesion to the ECM. We will describe the structural and molecular organization of each domain and the cross-talk between them during the invasion process.     

Invadopodia and matrix degradation, a new property of prostate cancer cells during migration and invasion

The present study demonstrated that invadopodia are associated with invasion by degradation of matrix in prostate cancer cells PC3. To find out the presence of invadopodia in PC3 cells, we performed a few comparative analyses with osteoclasts, which utilize podosomes for migration. Our investigations indeed demonstrated that invadopodia are comparable to podosomes in the localization of Wiskott-Aldrich syndrome protein (WASP)/matrix metalloproteinase-9 and the degradation of matrix. Invadopodia are different from podosomes in the localization of actin/vinculin, distribution during migration, and the mode of degradation of extracellular matrix. Invadopodia enable polarized invasion of PC3 cells into the gelatin matrix in a time-dependent manner. Gelatin degradation was confined within the periphery of the cell. Osteoclasts demonstrated directional migration with extensive degradation of matrix underneath and around the osteoclasts. A pathway of degradation of matrix representing a migratory track was observed due to the rearrangement of podosomes as rosettes or clusters at the leading edge. Reducing the matrix metalloproteinase-9 levels by RNA interference inhibited the degradation of matrix but not the formation of podosomes or invadopodia. Competition experiments with TAT-fused WASP peptides suggest that actin polymerization and formation of invadopodia involve the WASP-Arp2/3 complex pathway. Moreover, PC3 cells overexpressing osteopontin (OPN) displayed an increase in the number of invadopodia and gelatinolytic activity as compared with PC3 cells and PC3 cells expressing mutant OPN in integrin-binding domain and null for OPN. Thus, we conclude that OPN/integrin alphavbeta3 signaling participates in the process of migration and invasion of PC3 cells through regulating processes essential for the formation and function of invadopodia.     

Ubiquitous membrane-bound DNase activity in podosomes and invadopodia

Podosomes and invadopodia, collectively termed invadosomes, are adhesive and degradative membrane structures formed in many types of cells and are well known for recruiting various proteases. However, another major class of degradative enzymes, deoxyribonuclease (DNase), remains unconfirmed and not studied in invadosomes. Here, using surface-immobilized nuclease sensor (SNS), we demonstrated that invadosomes recruit DNase to their core regions, which degrade extracellular double-stranded DNA. We further identified the DNase as GPI-anchored membrane-bound DNase X. DNase recruitment is ubiquitous and consistent in invadosomes of all tested cell types. DNase activity exhibits within a minute after actin nucleation, functioning concomitantly with protease in podosomes but preceding it in invadopodia. We further showed that macrophages form DNase-active podosome rosettes surrounding bacteria or micropatterned antigen islets, and the podosomes directly degrade bacterial DNA on a surface, exhibiting an apparent immunological function. Overall, this work reports DNase in invadosomes for the first time, suggesting a richer arsenal of degradative enzymes in invadosomes than known before.     

Adhesions that mediate invasion

Infiltration of new tissue areas requires that a mammalian cell overcomes the physical and biochemical barrier of the surrounding extracellular matrix. Cell migration during embryonic development, and growth, invasion and dispersal of metastatic tumor cells depend to a large extent on the controlled degradation of extracellular matrix components. Localized degradation of the surrounding matrix is seen at defined adhesive (podosomes) and/or protrusive (invadopodia) locations in a variety of normal cells and aggressive carcinoma cells, suggesting that these membrane-associated cellular devices have a central role in mediating polarized migration in cells that cross-tissue boundaries. Here, we will discuss the recent advances and developments in this field, and provide our provisional outlook into the future understanding of the principles of focal extracellular matrix degradation by podosomes and invadopodia.     

Dense fibrillar collagen is a potent inducer of invadopodia via a specific signaling network

Cell interactions with the extracellular matrix (ECM) can regulate multiple cellular activities and the matrix itself in dynamic, bidirectional processes. One such process is local proteolytic modification of the ECM. Invadopodia of tumor cells are actin-rich proteolytic protrusions that locally degrade matrix molecules and mediate invasion. We report that a novel high-density fibrillar collagen (HDFC) matrix is a potent inducer of invadopodia, both in carcinoma cell lines and in primary human fibroblasts. In carcinoma cells, HDFC matrix induced formation of invadopodia via a specific integrin signaling pathway that did not require growth factors or even altered gene and protein expression. In contrast, phosphoproteomics identified major changes in a complex phosphosignaling network with kindlin2 serine phosphorylation as a key regulatory element. This kindlin2-dependent signal transduction network was required for efficient induction of invadopodia on dense fibrillar collagen and for local degradation of collagen. This novel phosphosignaling mechanism regulates cell surface invadopodia via kindlin2 for local proteolytic remodeling of the ECM.     

Formation of atypical podosomes in extravillous trophoblasts regulates extracellular matrix degradation

Throughout pregnancy the cytotrophoblast, the stem cell of the placenta, gives rise to the differentiated forms of trophoblasts. The two main cell lineages are the syncytiotrophoblast and the invading extravillous trophoblast. A successful pregnancy requires extravillous trophoblasts to migrate and invade through the decidua and then remodel the maternal spiral arteries. Many invasive cells use specialised cellular structures called invadopodia or podosomes in order to degrade extracellular matrix. Despite being highly invasive cells, the presence of invadapodia or podosomes has not previously been investigated in trophoblasts. In this study these structures have been identified and characterised in extravillous trophoblasts. The role of specialised invasive structures in trophoblasts in the degradation of the extracellular matrix was compared with well characterised podosomes and invadopodia in other invasive cells and the trophoblast specific structures were characterised by using a sensitive matrix degradation assay which enabled visualisation of the structures and their dynamics. We show trophoblasts form actin rich protrusive structures which have the ability to degrade the extracellular matrix during invasion. The degradation ability and dynamics of the structures closely resemble podosomes, but have unique characteristics that have not previously been described in other cell types. The composition of these structures does not conform to the classic podosome structure, with no distinct ring of plaque proteins such as paxillin or vinculin. In addition, trophoblast podosomes protrude more deeply into the extracellular matrix than established podosomes, resembling invadopodia in this regard. We also show several significant pathways such as Src kinase, MAPK kinase and PKC along with MMP-2 and 9 as key regulators of extracellular matrix degradation activity in trophoblasts, while podosome activity was regulated by the rigidity of the extracellular matrix.     

Laminin-111 derived peptides AG73 and C16 regulate invadopodia activity of a human adenoid cystic carcinoma cell line

Adenoid cystic carcinoma is a frequently occurring malignant salivary gland neoplasm with high level of recurrence and distant metastasis long time after treatment. Metastatic tumor cells that actively migrate and invade surrounding tissues rely on invadopodia to degrade extracellular matrix (ECM) barriers. Invadopodia are actin-rich membrane protrusions that localize enzymes required for ECM degradation. Breakdown of ECM macromolecules releases fragments and bioactive peptides. We have already demonstrated that laminin-111 and its derived peptides regulate migration, invasion and protease activity of adenocarcinoma cells. Here we addressed the role of laminin-111 peptides AG73 and C16 in invadopodia activity of cells (CAC2) derived from human adenoid cystic carcinoma. CAC2 cells were treated by AG73 and C16, and subjected to fluorescent gelatin substrate degradation assay. In this assay invadopodia activity areas appear as black dots in a fluorescent background. Both peptides significantly increased invadopodia formation and activity compared to controls. We analyzed putative receptors and signaling pathways related to peptide effects. β1 integrin silencing by siRNA decreased AG73- and C16-induced invadopodia. Furthermore inhibition of Rac1 and ERK signaling pathways decreased both C16- and AG73-related invadopodia activities. We propose that laminin-111 peptides AG73 and C16 increase invadopodia activity in CAC2 cells through β1 integrin. Rac1 and ERK1/2 signaling pathways would transduce signals generated by both peptides.     

p53 in cell invasion,podosomes, and invadopodia

Cell invasion of the extracellular matrix is prerequisite to cross tissue migration of tumor cells in cancer metastasis, and vascular smooth muscle cells in atherosclerosis. The tumor suppressor p53, better known for its roles in the regulation of cell cycle and apoptosis, has ignited much interest in its function as a suppressor of cell migration and invasion. How p53 and its gain-of-function mutants regulate cell invasion remains a puzzle and a challenge for future studies. In recent years, podosomes and invadopodia have also gained center stage status as veritable apparatus specialized in cell invasion. It is not clear, however, whether p53 regulates cell invasion through podosomes and invadopodia. In this review, evidence supporting a negative role of p53 in podosomes formation in vascular smooth muscle cells will be surveyed, and signaling nodes that may mediate this regulation in other cell types will be explored.