Vira V. Artym

Dynamic Interactions of Cortactin and Membrane Type 1 Matrix Metalloproteinase at Invadopodia: Defining the Stages of Invadopodia Formation and Function

Metastatic tumor cells that actively migrate and invade surrounding tissues rely on invadopodia to degrade extracellular matrix (ECM) barriers. Invadopodia are membrane protrusions that localize enzymes required for ECM degradation. Little is known about the formation, function, and regulation of invadopodia. Here, we show that invadopodia have two distinct aspects: (a) structural for organizing the cellular actin cytoskeleton to form membrane protrusions and (b) functional for using proteolytic enzyme(s) for ECM degradation. Small interfering RNA (siRNA) inhibition established that organization of invadopodia structure requires cortactin, whereas protease inhibitor studies identified membrane type 1 matrix metalloproteinase (MT1-MMP) as the key invadopodial enzyme responsible for gelatin matrix degradation in the breast carcinoma cell line MDA-MB-231. The inhibition of invadopodial structure assembly by cortactin depletion resulted in a block of matrix degradation due to failure of invadopodia formation. Either protease inhibition or MT1-MMP siRNA depletion moderately decreased the formation of invadopodial structures that were identified as actin-cortactin accumulations at the ventral cell membrane adherent to matrix. The invadopodia that were able to form upon MT1-MMP inhibition or depletion retained actin-cortactin accumulations but were unable to degrade matrix. Examination of cells at different time points as well as live-cell imaging revealed four distinct invadopodial stages: membrane cortactin aggregation at membranes adherent to matrix, MT1-MMP accumulation at the region of cortactin accumulation, matrix degradation at the invadopodia region, and subsequent cortactin dissociation from the area of continued MT1-MMP accumulation associated with foci of degraded matrix. Based on these results, we propose a stepwise model of invadopodia formation and function.

Src-Dependent Phosphorylation of ASAP1 Regulates Podosomes

ABSTRACT Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.

ECM Degradation Assays for Analyzing Local Cell Invasion

Proteolytic degradation of extracellular matrix (ECM) is a critical step during cell invasion and tissue transmigration that is required for many physiological and pathological processes. Cellular structures that mediate cell adhesion to, degradation of, and invasion into ECM are invadopodia of transformed and tumor cells and podosomes of normal monocytic, endothelial, and smooth muscle cells. Detecting the ability of the cell to form invadopodia and podosomes and to degrade ECM is required for studying the invasive capability of the cell. We have developed approximately 50 nm thick fluorescent gelatin matrices that provide a rapid, sensitive, and reliable in vitro system for detection of invadopodia and podosomes, and measurements of the extent of ECM degradation. In this chapter, we provide a detailed protocol for preparation of thin fluorescent gelatin matrices and for evaluation of the results from this degradation assay.

The Role of the Exocyst in Matrix Metalloproteinase Secretion and Actin Dynamics during Tumor Cell Invadopodia Formation

Invadopodia are actin-rich membrane protrusions formed by tumor cells that degrade the extracellular matrix for invasion. Invadopodia formation involves membrane protrusions driven by Arp2/3-mediated actin polymerization and secretion of matrix metalloproteinases (MMPs) at the focal degrading sites. The exocyst mediates the tethering of post-Golgi secretory vesicles at the plasma membrane for exocytosis and has recently been implicated in regulating actin dynamics during cell migration. Here, we report that the exocyst plays a pivotal role in invadopodial activity. With RNAi knockdown of the exocyst component Exo70 or Sec8, MDA-MB-231 cells expressing constitutively active c-Src failed to form invadopodia. On the other hand, overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst function by siEXO70 or siSEC8 treatment or by expression of a dominant negative fragment of Exo70 inhibited the secretion of MMPs. We have also found that the exocyst interacts with the Arp2/3 complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/3-mediated actin polymerization and invadopodia formation. Together, our results suggest that the exocyst plays important roles in cell invasion by mediating the secretion of MMPs at focal degrading sites and regulating Arp2/3-mediated actin dynamics.

Direct visualization of protease activity on cells migrating in three-dimensions

Determining the specific role(s) of proteases in cell migration and invasion will require high-resolution imaging of sites of protease activity during live-cell migration through extracellular matrices. We have designed a novel fluorescent biosensor to detect localized extracellular sites of protease activity and to test requirements for matrix metalloprotease (MMP) function as cells migrate and invade three-dimensional collagen matrices. This probe fluoresces after cleavage of a peptide site present in interstitial collagen by a variety of proteases including MMP-2, -9, and -14 (MT1-MMP) without requiring transfection or modification of the cells being characterized. Using matrices derivatized with this biosensor, we show that protease activity is localized at the polarized leading edge of migrating tumor cells rather than further back on the cell body. This protease activity is essential for cell migration in native cross-linked but not pepsin-treated collagen matrices. The new type of high-resolution probe described in this study provides site-specific reporting of protease activity and insights into mechanisms by which cells migrate through extracellular matrices; it also helps to clarify discrepancies between previous studies regarding the contributions of proteases to metastasis.

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

High-density fibrillar collagen matrix induces invadopodia formation in both fibroblasts and carcinoma cell lines through a kindlin2-dependent mechanism that drives local ECM remodeling.

Dynamic membrane remodeling at invadopodia differentiates invadopodia from podosomes

Invadopodia are specialized actin-rich protrusions of metastatic tumor and transformed cells with crucial functions in ECM degradation and invasion. Although early electron microscopy studies described invadopodia as long filament-like protrusions of the cell membrane adherent to the matrix, fluorescence microscopy studies have focused on invadopodia as actin-cortactin aggregates localized to areas of ECM degradation. The absence of a clear conceptual integration of these two descriptions of invadopodial structure has impeded understanding of the regulatory mechanisms that govern invadopodia. To determine the relationship between the membrane filaments identified by electron microscopy and the actin-cortactin aggregates of invadopodia, we applied rapid live-cell high-resolution TIRF microscopy to examine cell membrane dynamics at the cortactin core of the invadopodia of human carcinoma cells. We found that cortactin docking to the cell membrane adherent to 2D fibronectin matrix initiates invadopodium assembly associated with the formation of an invadopodial membrane process that extends from a ventral cell membrane lacuna toward the ECM. The tip of the invadopodial process flattens as it interacts with the 2D matrix, and it undergoes constant rapid ruffling and dynamic formation of filament-like protrusions as the invadopodium matures. To describe this newly discovered dynamic relationship between the actin-cortactin core and invadopodial membranes, we propose a model of the invadopodial complex. Using TIRF microscopy, we also established that - in striking contrast to the invadopodium - membrane at the podosome of a macrophage fails to form any process- or filament-like membrane protrusions. Thus, the undulation and ruffling of the invadopodial membrane together with the formation of dynamic filament-like extensions from the invadopodial cortactin core defines invadopodia as invasive superstructures that are distinct from the podosomes.

Imaging Cells in Three‐Dimensional Collagen Matrix

The use of in vitro three‐dimensional (3‐D) collagen matrices to mimic an in vivo cellular environment has become increasingly popular and is broadening our understanding of cellular processes and cell‐ECM interactions. To study cells in in vitro 3‐D collagen matrices, both cellular proteins and the collagen matrix must be visualized. In this unit, the authors describe the protocol and provide troubleshooting for immunolabeling of cells in 3‐D collagen gels to localize and visualize cellular proteins with high‐resolution fluorescence confocal microscopy. The authors then describe confocal reflection microscopy as a technique for direct imaging of 3‐D fibrillar collagen matrices by discussing the advantages and disadvantages of the technique. They also provide instrument settings required for simultaneous imaging of cellular proteins with fluorescence confocal imaging and 3‐D collagen fibrils with confocal reflection microscopy. Additionally, the authors provide protocols for a “cell sandwiching” technique to prepare cell cultures in 3‐D collagen matrices required for high‐resolution confocal imaging. Curr. Protoc. Cell Biol. 48:10.18.1‐10.18.20.

Tensin 2 modulates cell contractility in 3D collagen gels through the RhoGAP DLC1

Cytoskeletal proteins of the tensin family couple integrins to the actin cytoskeleton. They are found in both focal adhesions and the fibrillar adhesions formed between cells and the fibronectin matrix. There are four tensin genes which encode three large (∼200 kDa) tensin isoforms (tensin 1, 2, 3) and one short isoform (cten). However, the subcellular localization and function of the individual isoforms is poorly understood. Using human foreskin fibroblasts (HFFs), and imaging on both fixed and live cells, we show that GFP‐tensin 2 is enriched in dynamic focal adhesions at the leading edge of the cell, whereas GFP‐tensin 3 translocates rearward, and is enriched in fibrillar adhesions. To investigate the possible role of tensins in cell‐matrix remodeling, we used siRNAs to knockdown each tensin isoform. We discovered that tensin 2 knockdown significantly reduced the ability of HFFs to contract 3D collagen gels, whilst no effect on fibronectin fibrillogenesis was observed. This inhibition of collagen gel contraction was associated with a substantial reduction in Rho activity, and it was reversed by depletion of DLC1, a RhoGAP that binds to tensin in focal adhesions. These findings suggest that focal adhesion‐localized tensin 2 negatively regulates DLC1 to permit Rho‐mediated actomyosin contraction and remodeling of collagen fibers. J. Cell. Biochem. 109: 808–817, 2010.

Extracellular matrix protocols

Preface Contributors I. Molecular Biology Retroviral delivery of ECM genes Vitali Alexeev and Olga Igoucheva Tissue-Specific KO of ECM Proteins Mara Brancaccio, Emila Turco and Emilio Hirsch Recombinant Collagen Trimers from Insect Cells and Yeast- Johanna Myllyharju Eukaryotic Expression and Purification of Recombinant Extracellular Matrix Proteins Carrying the Strep II Tag Neil Smyth, Uwe Odenthal, Barbara Merkl, and Mats Paulsson Tissue Recombinants to Study Extracellular Matrix Targeting to Basement Membranes Patricia Simon-Assmann, Anne-Laure Bolcato-Bellemin, Annick Klein and Michele Kedinger Preparation of recombinant fibronectin fragments for functional and structural studies David Staunton, Christopher J Millard, Radu Aricescu and Iain D Campbell II. Biochemical and biophysical analysis Quantitative Determination of Collagen Cross-links Nicholas C Avery, Trevor J Sims and Allen J Bailey ECM Macromolecules: Height-mapping and Nano-mechanics Using Atomic Force Microscopy Nigel W. Hodson, Cay M. Kielty and Michael J. Sherratt Atomic Force Microscopy Measurements of Intermolecular Binding Forces- Gradimir N. Misevic, Yannis Karamanos and Nikola J. Misevic Mass-Mapping of ECM Macromolecules by Scanning Transmission Electron Microscopy Michael J. Sherratt, Helen K. Graham, Cay M. Kielty and David F. Holmes Chemical Microscopy of Biological Samples by Dynamic Mode Secondary Ion Mass Spectrometry (SIMS) Gradimir N. Misevic, Bernard Rasser, Vic Norris, Cedric Derue, David Gibouin, Fabrice Lefebvre, Marie-Claire Verdus, Anthony Delaune, Guillaume Legent and CamilleRipoll ECM Macromolecules: Rotary Shadowing and Transmission Electron Microscopy Michael J. Sherratt, Roger S. Meadows, Helen K. Graham, Cay M. Kielty and David F. Holmes Using Self-Assembled Monolayers to Pattern ECM Proteins and Cells on Substrates Emanuele Ostuni, George M. Whitesides, Donald E. Ingber and Christopher S. Chen Solid Phase Assays for Studying ECM Protein-Protein Interactions Paul A Mould III. Cell biology assays Cell Adhesion Assays Martin J. Humphries ECM Degradation Assays for Analyzing Local Cell Invasion Vira V. Artym, Kenneth M. Yamada, Susette C. Mueller Fluorescence-Based Assays for In Vitro Analysis of Cell Adhesion and Migration Paola Spessotto, Katia Lacrima, Pier Andrea Nicolosi, Eliana Pivetta, Martina Scapolan and Roberto Perris Fibrin Gel Model for Assessment of Cellular Contractility Sharona Even-Ram Fluorescent labeling techniques for investigation of fibronectin fibrillogenesis ( Labeling fibronectin fibrillogenesis) Roumen Pankov and Albena Momchilova Stromagenesis During Tumorigenesis: Characterization of Tumor-associated Fibroblasts and Stroma-derived 3D Matrices Remedios Castello-Cros and Edna Cukierman IV. Organ models ECM and FGF-dependent assay of embryonic SMG epithelial morphogenesis: investigating growth factor /matrix regulation of gene expression during submandibular gland development Ivan T. Rebustini and Matthew P. Hoffman Analyzing Cell-ECM Interactions in Adult Mammary Gland by Transplantation of Embryonic Mammary Tissue from Knockout Mice Teresa C. M. Klinowska and Charles H. Streuli V. Tissue