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1.  Dynamic Membrane Remodeling at Invadopodia Differentiates Invadopodia from Podosomes 
European journal of cell biology  2010;90(2-3):172-180.
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 a 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.
doi:10.1016/j.ejcb.2010.06.006
PMCID: PMC3153956  PMID: 20656375
invadopodia; podosomes; cortactin; focal adhesions; invasion
2.  Imaging Cells in Three-Dimensional Collagen Matrix 
The use of in vitro three-dimensional (3D) 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 3D collagen matrices, both cellular proteins and the collagen matrix must be visualized. In this unit, the authors describe the protocol and provide troubleshooting for immuno-labeling of cells in 3D 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 3D fibrilar 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 3D collagen fibrils with confocal reflection microscopy. Additionally, the authors provide protocols for a “cell sandwiching” technique to prepare cell cultures in 3D collagen matrices required for high resolution confocal imaging.
doi:10.1002/0471143030.cb1018s48
PMCID: PMC2988473  PMID: 20853341
three-dimensional collagen matrix; collagen type I; Nutragen; confocal reflection microscopy; immunostainig of 3D cell samples; invasion
3.  Tensin 2 modulates cell contractility in 3D collagen gels through the RhoGAP DLC1 
Journal of cellular biochemistry  2010;109(4):808-817.
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.
doi:10.1002/jcb.22460
PMCID: PMC3164319  PMID: 20069572
Tensin; Fibronectin matrix assembly; Fibrillar adhesion; Collagen contraction; DLC1; RhoGAP
4.  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.
doi:10.1016/j.matbio.2008.10.001
PMCID: PMC2661756  PMID: 19010413
Cell motility; Collagenase; Invasion; Metastasis; MMP; TIMP; MT1-MMP
5.  The Role of the Exocyst in Matrix Metalloproteinase Secretion and Actin Dynamics during Tumor Cell Invadopodia Formation 
Molecular Biology of the Cell  2009;20(16):3763-3771.
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.
doi:10.1091/mbc.E08-09-0967
PMCID: PMC2777935  PMID: 19535457
6.  Molecular Proximity of Kv1.3 Voltage-gated Potassium Channels and β1-Integrins on the Plasma Membrane of Melanoma Cells 
Tumor cell membranes have multiple components that participate in the process of metastasis. The present study investigates the physical association of β1-integrins and Kv1.3 voltage-gated potassium channels in melanoma cell membranes using resonance energy transfer (RET) techniques. RET between donor-labeled anti–β1-integrin and acceptor-labeled anti-Kv1.3 channels was detected on LOX cells adherent to glass and fibronectin-coated coverslips. However, RET was not observed on LOX cells in suspension, indicating that molecular proximity of these membrane molecules is adherence-related. Several K+ channel blockers, including tetraethylammonium, 4-aminopyridine, and verapamil, inhibited RET between β1-integrins and Kv1.3 channels. However, the irrelevant K+ channel blocker apamin had no effect on RET between β1-integrins and Kv1.3 channels. Based on these findings, we speculate that the lateral association of Kv1.3 channels with β1-integrins contributes to the regulation of integrin function and that channel blockers might affect tumor cell behavior by influencing the assembly of supramolecular structures containing integrins.
doi:10.1085/jgp.20028607
PMCID: PMC2311400  PMID: 12084773
Kv1.3 channel; integrin; resonance energy transfer; spectroscopy; receptors, signaling

Results 1-6 (6)