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.     

Cdc42 and Tks5: A minimal and universal molecular signature for functional invadosomes

Invadosomes are actin-based structures involved in extracellular-matrix degradation. Invadosomes, either known as podosomes or invadopodia, are found in an increasing number of cell types. Moreover, their overall organization and molecular composition may vary from one cell type to the other. Some are constitutive such as podosomes in hematopoietic cells whereas others are inducible. However, they share the same feature, their ability to interact and to degrade the extracellular matrix. Based on the literature and our own experiments, the aim of this study was to establish a minimal molecular definition of active invadosomes. We first highlighted that Cdc42 is the key RhoGTPase involved in invadosome formation in all described models. Using different cellular models, such as NIH-3T3, HeLa, and endothelial cells, we demonstrated that overexpression of an active form of Cdc42 is sufficient to form invadosome actin cores. Therefore, active Cdc42 must be considered not only as an inducer of filopodia, but also as an inducer of invadosomes. Depending on the expression level of Tks5, these Cdc42-dependent actin cores were endowed or not with a proteolytic activity. In fact, Tks5 overexpression rescued this activity in Tks5 low expressing cells. We thus described the adaptor protein Tks5 as a major actor of the invadosome degradation function. Surprisingly, we found that Src kinases are not always required for invadosome formation and function. These data suggest that even if Src family members are the principal kinases involved in the majority of invadosomes, it cannot be considered as a common element for all invadosome structures. We thus define a minimal and universal molecular signature of invadosome that includes Cdc42 activity and Tks5 presence in order to drive the actin machinery and the proteolytic activity of these invasive structures.     

Physiological type I collagen organization induces the formation of a novel class of linear invadosomes

Invadosomes are F-actin structures capable of degrading the matrix through the activation of matrix metalloproteases. As fibrillar type I collagen promotes pro-matrix metalloproteinase 2 activation by membrane type 1 matrix metalloproteinase, we aimed at investigating the functional relationships between collagen I organization and invadosome induction. We found that fibrillar collagen I induced linear F-actin structures, distributed along the fibrils, on endothelial cells, macrophages, fibroblasts, and tumor cells. These structures share features with conventional invadosomes, as they express cortactin and N-WASP and accumulate the scaffold protein Tks5, which proved essential for their formation. On the basis of their ability to degrade extracellular matrix elements and their original architecture, we named these structures "linear invadosomes." Interestingly, podosomes or invadopodia were replaced by linear invadosomes upon contact of the cells with fibrillar collagen I. However, linear invadosomes clearly differ from classical invadosomes, as they do not contain paxillin, vinculin, and β1/β3 integrins. Using knockout mouse embryonic fibroblasts and RGD peptide, we demonstrate that linear invadosome formation and activity are independent of β1 and β3 integrins. Finally, linear invadosomes also formed in a three-dimensional collagen matrix. This study demonstrates that fibrillar collagen I is the physiological inducer of a novel class of invadosomes.     

Invadosomes: Intriguing structures with promise

Podosomes and invadopodia are highly dynamic, actin-rich adhesion structures and represent the two founding members of the invadosome family. Podosomes form spontaneously in cells of the myelomonocytic lineage but a plethora of other cells are endowed with this capacity, under appropriate stimulation, such as a soluble factor, matrix receptor, or cell stress. Related structures called invadopodia are detected in some cancer cells or appear on cells upon oncogenic transformation. In contrast to other cell adhesion devices, invadosomes harbour metalloproteases which degrade components of the extracellular matrix. Because of this distinctive feature, invadosomes have been systematically linked with invasion processes. However, it now appears that these intriguing structures are endowed with other functions and are therefore expected to contribute to a wider range of biological processes. The invadosome field has been progressing for thirty years, expanding exponentially during the last decade, where tremendous advances have been made regarding the molecular mechanism underlying their formation, dynamics and function. Invadosomes are involved in human diseases but the causative link remains to be established. To this end, 3D analysis of invadosomes is now being actively developed in ex vivo and in vivo models to demonstrate their occurrence and establish their role in pathological and physiological processes.     

Invadosome regulation by adhesion signaling

Invadosomes are adhesive mechanosensory modules composed of a dense F-actin core surrounded by a ring of adhesion molecules and able to infiltrate compact tissue environment in physiological and pathological conditions. These structures comprise podosomes that are found in a variety of cells under physiological conditions and invadopodia in transformed or cancer cells. Invadosomes are regulated by extracellular matrix signals and are endowed with degradative machinery for extracellular matrix. The ability of extracellular matrix signals to orchestrate the building, dynamics, and function of invadosomes is based on mechano-chemical integrin outside-in signaling and requires integrin cross-talk. This review highlights recent findings that place Src as an inducer and PKC as an amplifier in the assembly of integrin stimulated invadosome through mechanotransduction and polarized endo/exocytic trafficking pathways for key proteolytic and enzymatic activities in a temporally and spatially confined manner.     

αII-Spectrin Regulates Invadosome Stability and Extracellular Matrix Degradation

Invadosomes are actin-rich adhesion structures involved in tissue invasion and extracellular matrix (ECM) remodelling. αII-Spectrin, an ubiquitous scaffolding component of the membrane skeleton and a partner of actin regulators (ABI1, VASP and WASL), accumulates highly and specifically in the invadosomes of multiple cell types, such as mouse embryonic fibroblasts (MEFs) expressing SrcY527F, the constitutively active form of Src or activated HMEC-1 endothelial cells. FRAP and live-imaging analysis revealed that αII-spectrin is a highly dynamic component of invadosomes as actin present in the structures core. Knockdown of αII-spectrin expression destabilizes invadosomes and reduces the ability of the remaining invadosomes to digest the ECM and to promote invasion. The ECM degradation defect observed in spectrin-depleted-cells is associated with highly dynamic and unstable invadosome rings. Moreover, FRAP measurement showed the specific involvement of αII-spectrin in the regulation of the mobile/immobile β3-integrin ratio in invadosomes. Our findings suggest that spectrin could regulate invadosome function and maturation by modulating integrin mobility in the membrane, allowing the normal processes of adhesion, invasion and matrix degradation. Altogether, these data highlight a new function for spectrins in the stability of invadosomes and the coupling between actin regulation and ECM degradation.     

Essential Function of Dynamin in the Invasive Properties and Actin Architecture of v-Src Induced Podosomes/Invadosomes

The large GTPase dynamin plays a key role in endocytosis but is also localized at numerous actin rich sites. We investigated dynamin functions at podosomes/invadosomes, actin-based cellular adhesion structures implicated in tissue invasion. Podosomes/invadosomes are constituted of long F-actin bundles perpendicular to the substratum (actin cores), connected to randomly arranged F-actin fibers parallel to the substratum (actin cloud). We show here that dynamin depletion in v-Src-transformed fibroblasts triggers a massive disorganization of podosomes/invadosomes (isolated or in rosettes), with a corresponding inhibition of their invasive properties. The action of dynamin at podosomes/invadosomes requires a functional full-length protein, suggesting that the effects of dynamin at these sites and in membrane remodelling during endocytosis are mediated by similar mechanisms. In order to determine direct effect of dynamin depletion on invadosome, an optogenetic approach based on the photosensitizer KillerRed was developed. Acute dynamin photo-inactivation leads to a very rapid disorganization of invadosome without affecting focal adhesions. Dynamin therefore is a key regulator of the architecture of actin in podosomes/invadosomes.     

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.     

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.     

β1A integrin is a master regulator of invadosome organization and function

Invadosomes are adhesion structures involved in tissue invasion that are characterized by an intense actin polymerization–depolymerization associated with β1 and β3 integrins and coupled to extracellular matrix (ECM) degradation activity. We induced the formation of invadosomes by expressing the constitutive active form of Src, SrcYF, in different cell types. Use of ECM surfaces micropatterned at the subcellular scale clearly showed that in mesenchymal cells, integrin signaling controls invadosome activity. Using β1^−/− or β3^−/− cells, it seemed that β1A but not β3 integrins are essential for initiation of invadosome formation. Protein kinase C activity was shown to regulate autoassembly of invadosomes into a ring-like metastructure (rosette), probably by phosphorylation of Ser785 on the β1A tail. Moreover, our study clearly showed that β1A links actin dynamics and ECM degradation in invadosomes. Finally, a new strategy based on fusion of the photosensitizer KillerRed to the β1A cytoplasmic domain allowed specific and immediate loss of function of β1A, resulting in disorganization and disassembly of invadosomes and formation of focal adhesions.     

Invadopodia and podosomes in tumor invasion

Cell migration through the extracellular matrix (ECM) is necessary for cancer cells to invade adjacent tissues and metastasize to an organ distant from primary tumors. Highly invasive carcinoma cells form ECM-degrading membrane protrusions called invadopodia. Tumor-associated macrophages have been shown to promote the migratory phenotypes of carcinoma cells, and macrophages are known to form podosomes, similar structures to invadopodia. However, the role of invadopodia and podosomes in vivo remains to be determined. In this paper, we propose a model for possible functions and interactions of invadopodia and podosomes in tumor invasion, based on observations that macrophage podosomes degrade ECM and that podosome formation is regulated by colony-stimulating factor-1 signaling.     

Discoidin domain receptor 1 controls linear invadosome formation via a Cdc42–Tuba pathway

Accumulation of type I collagen fibrils in tumors is associated with an increased risk of metastasis. Invadosomes are F-actin structures able to degrade the extracellular matrix. We previously found that collagen I fibrils induced the formation of peculiar linear invadosomes in an unexpected integrin-independent manner. Here, we show that Discoidin Domain Receptor 1 (DDR1), a collagen receptor overexpressed in cancer, colocalizes with linear invadosomes in tumor cells and is required for their formation and matrix degradation ability. Unexpectedly, DDR1 kinase activity is not required for invadosome formation or activity, nor is Src tyrosine kinase. We show that the RhoGTPase Cdc42 is activated on collagen in a DDR1-dependent manner. Cdc42 and its specific guanine nucleotide-exchange factor (GEF), Tuba, localize to linear invadosomes, and both are required for linear invadosome formation. Finally, DDR1 depletion blocked cell invasion in a collagen gel. Altogether, our data uncover an important role for DDR1, acting through Tuba and Cdc42, in proteolysis-based cell invasion in a collagen-rich environment.     

ECM Cross-Linking Regulates Invadopodia Dynamics

Invadopodia are membrane protrusions dynamically assembled by invasive cancer cells in contact with the extracellular matrix (ECM). Invadopodia are enriched by the structural proteins actin and cortactin as well as metalloproteases such as MT1-MMP, whose function is to degrade the surrounding ECM. During metastasis, invadopodia are necessary for cancer cell intravasation and extravasation. Although signaling pathways involved in the assembly and function of invadopodia are well studied, few studies address invadopodia dynamics and how the cell-ECM interactions contribute to cell invasion. Using iterative analysis based on time-lapse microscopy and mathematical modeling of invasive cancer cells, we found that cells oscillate between invadopodia presence and cell stasis—termed the “invadopodia state”—and invadopodia absence during cell translocation—termed the “migration state.” Our data suggest that β1-integrin-ECM binding and ECM cross-linking control the duration of each of the two states. By changing the concentration of cross-linkers in two-dimensional and three-dimensional cultures, we generate an ECM in which 0–0.92 of total lysine residues are cross-linked. Using an ECM with a range of cross-linking degrees, we demonstrate that the dynamics of invadopodia-related functions have a biphasic relationship to ECM cross-linking. At intermediate levels of ECM cross-linking (0.39), cells exhibit rapid invadopodia protrusion-retraction cycles and rapid calcium spikes, which lead to more frequent MT1-MMP delivery, causing maximal invadopodia-mediated ECM degradation. In contrast, both extremely high or low levels of cross-linking lead to slower invadopodia-related dynamics and lower ECM degradation. Additionally, β1-integrin inhibition modifies the dynamics of invadopodia-related functions as well as the length of time cells spend in either of the states. Collectively, these data suggest that β1-integrin-ECM binding nonlinearly translates small physical differences in the extracellular environment to differences in the dynamics of cancer cell behaviors. Understanding the conditions under which invadopodia can be reduced by subtle environment-targeting treatments may lead to combination therapies for preventing metastatic spread.     

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.     

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.     

Rapid Remodeling of Invadosomes by Gi-coupled Receptors: DISSECTING THE ROLE OF Rho GTPases*

Invadosomes are actin-rich membrane protrusions that degrade the extracellular matrix to drive tumor cell invasion. Key players in invadosome formation are c-Src and Rho family GTPases. Invadosomes can reassemble into circular rosette-like superstructures, but the underlying signaling mechanisms remain obscure. Here we show that Src-induced invadosomes in human melanoma cells (A375M and MDA-MB-435) undergo rapid remodeling into dynamic extracellular matrix-degrading rosettes by distinct G protein-coupled receptor agonists, notably lysophosphatidic acid (LPA; acting through the LPA₁ receptor) and endothelin. Agonist-induced rosette formation is blocked by pertussis toxin, dependent on PI3K activity and accompanied by localized production of phosphatidylinositol 3,4,5-trisphosphate, whereas MAPK and Ca²⁺ signaling are dispensable. Using FRET-based biosensors, we show that LPA and endothelin transiently activate Cdc42 through G_i, concurrent with a biphasic decrease in Rac activity and differential effects on RhoA. Cdc42 activity is essential for rosette formation, whereas G_12/13-mediated RhoA-ROCK signaling suppresses the remodeling process. Our results reveal a G_i-mediated Cdc42 signaling axis by which G protein-coupled receptors trigger invadosome remodeling, the degree of which is dictated by the Cdc42-RhoA activity balance.     

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.     

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.