Ventral Cell Surface Research Articles

Fibroblasts secrete fibronectin under lamellipodia in a microtubule- and myosin II-dependent fashion.

Fibronectin (FN) is an essential structural and regulatory component of the extracellular matrix (ECM), and its binding to integrin receptors supports cell adhesion, migration, and signaling. Here, using live-cell microscopy of fibroblasts expressing FN tagged with a pH-sensitive fluorophore, we show that FN is secreted predominantly at the ventral surface of cells in an integrin-independent manner. Locally secreted FN then undergoes β1 integrin-dependent fibrillogenesis. We find that the site of FN secretion is regulated by cell polarization, which occurs in bursts under stabilized lamellipodia at the leading edge. Moreover, analysis of FN secretion and focal adhesion dynamics suggest that focal adhesion formation precedes FN deposition and that deposition continues during focal adhesion disassembly. Lastly, we show that the polarized FN deposition in spreading and migrating cells requires both intact microtubules and myosin II-mediated contractility. Thus, while FN secretion does not require integrin binding, the site of exocytosis is regulated by membrane and cytoskeletal dynamics with secretion occurring after new adhesion formation.

Invadopodia: clearing the way for cancer cell invasion.

The invasive nature of many cancer cells involves the formation of F-actin-based, lipid-raft-enriched membrane protrusions known as invadopodia or, more broadly, invadosomes. Invadopodia are specialized adhesive structures arising from ventral cell surface within cell-extracellular matrix (ECM) contacts and concentrate high proteolytic activities that allow cells to overcome the dense scaffold of local microenvironment, comprising a natural barrier to cell spreading. This degradative activity distinguishes invadopodia from other adhesive structures like focal adhesions, lamellipodia or filopodia, and is believed to drive cancer progression.

Cell Behavior within Nanogrooved Sandwich Culture Systems.

Grooved topography and inherent cell contact guidance has shown promising results regarding cell proliferation, morphology, and lineage-specific differentiation. Yet these approaches are limited to 2D applications. Sandwich-culture conditions are developed to bridge the gap between 2D and 3D culture, enabling both ventral and dorsal cell surface stimulation. The effect of grooved surface topography is accessed on cell orientation and elongation in a highly controlled manner, with simultaneous and independent stimuli on two cell sides. Nanogrooved and non-nanogrooved substrates are assembled into quasi-3D systems with variable relative orientations. A plethora of sandwich-culture conditions are created by seeding cells on lower, upper, or both substrates. Software image analysis demonstrates that F-actin of cells acquires the orientation of the substrate on which cells are initially seeded, independently from the orientation of the second top substrate. Contrasting cell morphologies are observed, with a higher elongation for nanogrooved 2D substrates than nanogrooved sandwich-culture conditions. Correlated with an increased pFAK activity and vinculin staining for sandwich-culture conditions, these results point to an enhanced cell surface stimulation versus control conditions. The pivotal role of initial cell-biomaterial contact on cellular alignment is highlighted, providing important insights for tissue engineering strategies aiming to guide cellular response through mechanotransduction approaches.

Keratin-binding ability of the N-terminal Solo domain of Solo is critical for its function in cellular mechanotransduction.

Solo (ARHGEF40) is a RhoA-targeting guanine nucleotide exchange factor that regulates tensional force-induced cytoskeletal reorganization. Solo binds to keratin 8/keratin 18 (K8/K18) filaments through multiple sites, but the roles of these interactions in the localization and mechanotransduction-regulating function of Solo remain unclear. Here, we constructed two Solo mutants (L14R/L17R and L49R/L52R) with leucine-to-arginine replacements in the N-terminal conserved region (which we termed the Solo domain) and analyzed their K18-binding activities. These mutations markedly decreased the K18-binding ability of the N-terminal fragment (residues 1-329) of Solo but had no apparent effect on the K18-binding ability of full-length (FL) Solo. When expressed in cultured cells, wild-type Solo-FL showed a unique punctate localization near the ventral surface of cells and caused the reinforcement of actin filaments. In contrast, despite retaining the K18-binding ability, the L14R/L17R and L49R/L52R mutants of Solo-FL were diffusely distributed in the cytoplasm and barely induced actin cytoskeletal reinforcement. Furthermore, wild-type Solo-FL promoted traction force generation against extracellular matrices and tensional force-induced stress fiber reinforcement, but its L14R/L17R and L49R/L52R mutants did not. These results suggest that the K18-binding ability of the N-terminal Solo domain is critical for the ventral localization of Solo and its function in regulating mechanotransduction.

Subcellular topography modulates actin dynamics and signaling in B-cells.

B-cell signaling activation is most effectively triggered by the binding of B-cell receptors (BCRs) to membrane-bound antigens. In vivo, B-cells encounter antigen on antigen-presenting cells (APC), which possess complex surfaces with convoluted topographies, a fluid membrane and deformable cell bodies. However, whether and how the physical properties of antigen presentation affect B-cell activation is not well understood. Here we use nanotopographic surfaces that allow systematic variation of geometric parameters to show that surface features on a subcellular scale influence B-cell signaling and actin dynamics. Parallel nanoridges with spacings of 3 microns or greater induce actin intensity oscillations on the ventral cell surface. Nanotopography-induced actin dynamics requires BCR signaling, actin polymerization, and myosin contractility. The topography of the stimulatory surface also modulates the distribution of BCR clusters in activated B-cells. Finally, B-cells stimulated on nanopatterned surfaces exhibit intracellular calcium oscillations with frequencies that depend on topography. Our results point to the importance of physical aspects of ligand presentation, in particular, nanotopography for B-cell activation and antigen gathering.

Plasma Membrane Diffusion Modes of FcεRI Receptor for Immunoglobin E Measured with Imaging Fluorescence Correlation Spectroscopy

Spatio-temporal organization of the plasma membrane plays a significant role in cell signaling. A prevailing view is that the plasma membrane spatially segregates into ordered lipid (Lo-like) nanodomains co-existing with disordered lipid (Ld-like) regions. Our laboratory, using super-resolution fluorescence imaging, previously showed that immunoglobin E bound to its receptor, FceRI, (IgE-FceRI complex) undergoes time-dependent redistribution on the membrane surface after multivalent ligand stimulation, upon which the receptor is phosphorylated by membrane-anchored Lyn kinase. A large body of evidence shows that this interaction of IgE-FceRI and Lyn occurs in the ordered regions of the plasma membrane enriched in cholesterol. We are examining the nature and dynamics of plasma membrane organization as experienced by IgE-FceRI in resting state and after antigen stimulation. We employ imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS), which maps lateral diffusion with pixel resolution, to investigate the diffusion distribution of IgE-FceRI complexes on the ventral surface of live RBL cells in native and experimentally modulated (e.g., cholesterol depleted) conditions. We also conduct spot variation FCS (svFCS), which analyzes space-dependence of diffusion coefficients from the same set of ITIR-FCS data, to reveal the existence of nanoscopic obstacles (such as nanodomains) that affect the diffusion of IgE-FceRI complexes. These observations are compared with the diffusion behavior of protein markers that are known to prefer ordered or disordered regions of plasma membrane. These results, in conjunction with super-resolution imaging, which determines the size and density of these complexes, will provide a new level of insight into plasma membrane organization and its dynamic remodeling upon ligand-receptor stimulation.

ARF6 promotes the formation of Rac1 and WAVE-dependent ventral F-actin rosettes in breast cancer cells in response to epidermal growth factor.

Coordination between actin cytoskeleton assembly and localized polarization of intracellular trafficking routes is crucial for cancer cell migration. ARF6 has been implicated in the endocytic recycling of surface receptors and membrane components and in actin cytoskeleton remodeling. Here we show that overexpression of an ARF6 fast-cycling mutant in MDA-MB-231 breast cancer-derived cells to mimick ARF6 hyperactivation observed in invasive breast tumors induced a striking rearrangement of the actin cytoskeleton at the ventral cell surface. This phenotype consisted in the formation of dynamic actin-based podosome rosette-like structures expanding outward as wave positive for F-actin and actin cytoskeleton regulatory components including cortactin, Arp2/3 and SCAR/WAVE complexes and upstream Rac1 regulator. Ventral rosette-like structures were similarly induced in MDA-MB-231 cells in response to epidermal growth factor (EGF) stimulation and to Rac1 hyperactivation. In addition, interference with ARF6 expression attenuated activation and plasma membrane targeting of Rac1 in response to EGF treatment. Our data suggest a role for ARF6 in linking EGF-receptor signaling to Rac1 recruitment and activation at the plasma membrane to promote breast cancer cell directed migration.

Flat clathrin lattices: stable features of the plasma membrane.

Clathrin-mediated endocytosis (CME) is a fundamental property of eukaryotic cells. Classical CME proceeds via the formation of clathrin-coated pits (CCPs) at the plasma membrane, which invaginate to form clathrin-coated vesicles, a process that is well understood. However, clathrin also assembles into flat clathrin lattices (FCLs); these structures remain poorly described, and their contribution to cell biology is unclear. We used quantitative imaging to provide the first comprehensive description of FCLs and explore their influence on plasma membrane organization. Ultrastructural analysis by electron and superresolution microscopy revealed two discrete populations of clathrin structures. CCPs were typified by their sphericity, small size, and homogeneity. FCLs were planar, large, and heterogeneous and present on both the dorsal and ventral surfaces of cells. Live microscopy demonstrated that CCPs are short lived and culminate in a peak of dynamin recruitment, consistent with classical CME. In contrast, FCLs were long lived, with sustained association with dynamin. We investigated the biological relevance of FCLs using the chemokine receptor CCR5 as a model system. Agonist activation leads to sustained recruitment of CCR5 to FCLs. Quantitative molecular imaging indicated that FCLs partitioned receptors at the cell surface. Our observations suggest that FCLs provide stable platforms for the recruitment of endocytic cargo.

Arf1 and Arf6 promote ventral actin structures formed by acute activation of protein kinase C and Src.

Arf proteins regulate membrane traffic and organelle structure. Although Arf6 is known to initiate actin-based changes in cell surface architecture, Arf1 may also function at the plasma membrane. Here we show that acute activation of protein kinase C (PKC) induced by the phorbol ester PMA led to the formation of motile actin structures on the ventral surface of Beas-2b cells, a lung bronchial epithelial cell line. Ventral actin structures also formed in PMA-treated HeLa cells that had elevated levels of Arf activation. For both cell types, formation of the ventral actin structures was enhanced by expression of active forms of either Arf1 or Arf6 and by the expression of guanine nucleotide exchange factors that activate these Arfs. By contrast, formation of these structures was blocked by inhibitors of PKC and Src and required phosphatidylinositol 4, 5-bisphosphate, Rac, Arf6, and Arf1. Furthermore, expression of ASAP1, an Arf1 GTPase activating protein (GAP) was more effective at inhibiting the ventral actin structures than was ACAP1, an Arf6 GAP. This study adds to the expanding role for Arf1 in the periphery and identifies a requirement for Arf1, a "Golgi Arf," in the reorganization of the cortical actin cytoskeleton on ventral surfaces, against the substratum.

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.

Distinct Stages of Stimulated FcεRI Receptor Clustering and Immobilization Are Identified through Superresolution Imaging

Recent advances in fluorescence localization microscopy have made it possible to image chemically fixed and living cells at 20 nm lateral resolution. We apply this methodology to simultaneously record receptor organization and dynamics on the ventral surface of live RBL-2H3 mast cells undergoing antigen-mediated signaling. Cross-linking of IgE bound to FcεRI by multivalent antigen initiates mast cell activation, which leads to inflammatory responses physiologically. We quantify receptor organization and dynamics as cells are stimulated at room temperature (22°C). Within 2 min of antigen addition, receptor diffusion coefficients decrease by an order of magnitude, and single-particle trajectories are confined. Within 5 min of antigen addition, receptors organize into clusters containing ∼100 receptors with average radii of ∼70 nm. By comparing simultaneous measurements of clustering and mobility, we determine that there are two distinct stages of receptor clustering. In the first stage, which precedes stimulated Ca2+ mobilization, receptors slow dramatically but are not tightly clustered. In the second stage, receptors are tightly packed and confined. We find that stimulation-dependent changes in both receptor clustering and mobility can be reversed by displacing multivalent antigen with monovalent ligands, and that these changes can be modulated through enrichment or reduction in cellular cholesterol levels.

Detection of CD133 (prominin‐1) in a human hepatoblastoma cell line (HuH‐6 clone 5)

We examined CD133 distribution in a human hepatoblastoma cell line (HuH-6 clone 5). We directly observed the cultured cells on a pressure-resistant thin film (silicon nitride thin film) in a buffer solution by using the newly developed atmospheric scanning electron microscope (ASEM), which features an open sample dish with a silicon nitride thin film window at its base, through which the scanning electron microscope beam scans samples in solution, from below. The ASEM enabled observation of the ventral cell surface, which could not be observed using standard SEM. However, observation of the dorsal cell surface was difficult with the ASEM. Therefore, we developed a new method to observe the dorsal side of cells by using Aclar® plastic film. In this method, cells are cultured on Aclar plastic film and the dorsal side of cells is in contact with the thin silicon nitride film of the ASEM dish. A preliminary study using the ASEM showed that CD133 was mainly localized in membrane ruffles in the peripheral regions of the cell. Standard transmission electron microscopy and scanning electron microscopy revealed that CD133 was preferentially concentrated in a complex structure comprising filopodia and the leading edge of lamellipodia. We also observed co-localization of CD133 with F-actin. An antibody against CD133 decreased cell migration. Methyl-β-cyclodextrin treatment decreased cell adhesion as well as lamellipodium and filopodium formation. A decrease in the cholesterol level may perturb CD133 membrane localization. The results suggest that CD133 membrane localization plays a role in tumor cell adhesion and migration.

Three‐dimensional invasion of macrophages is mediated by cysteine cathepsins in protrusive podosomes

Podosomes, specialized actin-rich structures in macrophages (Mfs), degrade the extra-cellular matrix (ECM) and are involved in cell migration. On two-dimensional (2D) surfaces Mfs form spot-like podosomes at the ventral cell surface that develop into protrusive structures in a three-dimensional (3D) environment resembling the ECM. We have shown that the tips of these protrusive podosomes are characterized by increased accumulation of cysteine cathepsins (Cts) B, X, S, H, and L, both in human blood Mfs and in human monocytic cell line U-937. Monocyte-to-Mf differentiation induces an increase in cysteine cathepsin expression and activity, promoting their translocation to the cell surface, where they interact with ECM. This group of proteases is crucial for the extracellular as well as intracellular degradation of ECM, as demonstrated by quantitative monitoring of collagen IV degradation. Furthermore, inhibiting CtsB, X, and S significantly impairs Mf invasion through the 3D matrix. Time-lapse live-cell imaging of CtsB activity revealed that the extracellular and the intracellular ECM degradation are associated with extensive endocytosis at the tip of protrusive podosomes. The targeting of cysteine cathepsins, as the major mediators of human Mf 3D invasion, could be an approach to the treatment of inflammatory and cancerous diseases.

MEK/ERK pathway mediates PKC activation‐induced recruitment of PKCζ and MMP‐9 to podosomes

Podosomes are adhesive structures on the ventral surface of cells that invade and degrade the extracellular matrix. Recently, we reported that phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator, induced podosome formation in normal human bronchial epithelial (NHBE) cells, and atypical PKCζ regulated MMP-9 recruitment to podosomes for its release and activation. The objective of this study was to explore signaling pathways that are involved in PKC activation-induced podosome formation and matrix degradation. Herein, we found that PDBu increased phosphorylation of PI3K p85, Akt, Src, ERK1/2, and JNK. Inhibitors for PI3K, Akt, and Src suppressed PDBu-induced podosome formation and matrix degradation. In contrast, blockers for MEK/ERK or JNK did not inhibit podosome formation but reduced proteolytic activity of podosomes. Inhibition of PKCζ activity with its pseudosubstrate peptide (PS)-inhibited PDBu-induced phosphorylation of MEK/ERK and JNK. On the other hand, inhibition of MEK/ERK or JNK pathway did not affect PKCζ phosphorylation, but reduced the recruitment of PKCζ and MMP-9 to podosomes. We conclude that PKCζ may regulate MEK/ERK and JNK phosphorylation and in turn activated MEK/ERK and JNK may regulate the proteolytic activity of PDBu-induced podosomes by influencing the recruitment of PKCζ and MMP-9 to podosomes.

3D Coupling of Fibronectin Fibril Arrangement with Topology of Ventral Plasma Membrane

Interaction of integrins with extracellular matrices is essential for cell adhesion to substrata. Ventral surfaces of fibroblasts adhering to flat substrata are not flat but have uneven 3D topology. However, spatial relationship between the topology of the ventral cell surface and arrangement of extracellular matrix fibrils remains unclear. Here, we report a novel and simple method based on total internal reflection fluorescence microscopy to quantify the distance between the ventral plasma membrane and the glass substratum. We observe that the distance varies from < 25 nm at focal adhesions to 40-50 nm at close contacts and > 80 nm in other regions. Furthermore, by applying this novel method, we show that fibronectin fibrils are also separated from the substratum in regions where the ventral cell surface-substratum distance is > 80 nm. Our results reveal that fibronectin fibrils are not simply adsorbed to the glass substratum but follow the ventral cell surface topology.

Cell migration and invasion in human disease: the Tks adaptor proteins.

Cell invasion plays a central role in a wide variety of biological phenomena and is the cause of tumour growth and metastasis. Understanding the biochemical mechanisms that control cell invasion is one of the major goals of our laboratory. Podosomes and invadopodia are specialized cellular structures present in cells with physiological or pathological invasive behaviours. These transient structures are localized at the ventral cell surface, contain an array of different proteins and facilitate cell-substrate adhesion, as well as the local proteolytic activity necessary for extracellular matrix remodelling and subsequent cellular invasion. We have shown previously that the adaptor proteins and Src substrates Tks4 and Tks5 are required for podosome and invadopodia formation, for cancer cell invasion in vitro, and for tumour growth in vivo. We have also defined a role for the Tks-mediated generation of ROS (reactive oxygen species) in both podosome and invadopodia formation, and invasive behaviour. Tks4 and Tks5 are also required for proper embryonic development, probably because of their roles in cell migration. Finally, we recently implicated podosome formation as part of the synthetic phenotype of vascular smooth muscle cells. Inhibitors of podosome and invadopodia formation might have utility in the treatment of vascular diseases and cancer. We have therefore developed a high-content cell-based high-throughput screening assay that allows us to identify inhibitors and activators of podosome/invadopodia formation. We have used this assay to screen for small-molecule inhibitors and defined novel regulators of invadopodia formation. In the present paper, I review these recent findings.

Focal adhesion disassembly is regulated by a RIAM to MEK-1 pathway

Cell migration and invasion require regulated turnover of integrin-dependent adhesion complexes. Rap1-GTP-interacting adaptor molecule (RIAM) is an adaptor protein that mediates talin recruitment to the cell membrane, and whose depletion leads to defective melanoma cell migration and invasion. In this study, we investigated the potential involvement of RIAM in focal adhesion (FA) dynamics. RIAM-depleted melanoma and breast carcinoma cells displayed an increased number, size and stability of FAs, which accumulated centrally at the ventral cell surface, a phenotype caused by defective FA disassembly. Impairment in FA disassembly resulting from RIAM knockdown correlated with deficient integrin-dependent mitogen-activated protein kinase kinase (MEK)-Erk1/2 activation and, importantly, overexpression of constitutively active MEK resulted in rescue of FA disassembly and recovery of cell invasion. Furthermore, RIAM-promoted Ras homologue gene family, member A (RhoA) activation following integrin engagement was needed for subsequent Erk1/2 activation. In addition, RhoA overexpression partially rescued the FA phenotype in RIAM-depleted cells, also suggesting a functional role for RhoA downstream of RIAM, but upstream of Erk1/2. RIAM knockdown also led to enhanced phosphorylation of paxillin Tyr118 and Tyr31. However, expression of phosphomimetic and nonphosphorylatable mutants at these paxillin residues indicated that paxillin hyperphosphorylation is a subsequent consequence of the blockade of FA disassembly, but does not cause the FA phenotype. RIAM depletion also weakened the association between FA proteins, suggesting that it has important adaptor roles in the correct assembly of adhesion complexes. Our data suggest that integrin-triggered, RIAM-dependent MEK activation represents a key feedback event required for efficient FA disassembly, which could help explain the role of RIAM in cell migration and invasion.

Actin dynamics counteract membrane tension during clathrin-mediated endocytosis

Clathrin-mediated endocytosis is independent of actin dynamics in many circumstances but requires actin polymerization in others. We show that membrane tension determines the actin dependence of clathrin-coat assembly. As found previously, clathrin assembly supports formation of mature coated pits in the absence of actin polymerization on both dorsal and ventral surfaces of non-polarized mammalian cells, and also on basolateral surfaces of polarized cells. Actin engagement is necessary, however, to complete membrane deformation into a coated pit on apical surfaces of polarized cells and, more generally, on the surface of any cell in which the plasma membrane is under tension from osmotic swelling or mechanical stretching. We use these observations to alter actin dependence experimentally and show that resistance of the membrane to propagation of the clathrin lattice determines the distinction between "actin-dependent" and "actin-independent". We also find that light-chain bound Hip1R mediates actin engagement. These data thus provide a unifying explanation for the role of actin dynamics in coated-pit budding.

Real-Time Imaging of Clathrin Dynamics in Three Dimensions

Clathrin mediated endocytosis is a crucial factor in maintaining cellular dynamics. So far, real-time observation of this phenomenon has been achieved using methods that are limited to two dimensional spatial analysis even though cells have three dimensional geometries. Here we have used a fast, confocal based, three dimensional automated tracking scheme to analyze clathrin coat dynamics at the ventral and dorsal surfaces of mammalian cells. This technique presents a direct observation of a clathrin mediated virus entry at the apical surface of polarized cells for the first time.

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.