STRUCTURAL AND FUNCTIONAL HETEROGENEITY OF THE SURFACE OF RAT LEUKEMIA CELLS

Rat leukemia cells IRC 741 in suspension culture form single cytoplasmic protrusions by which the cells preferentially adhere to one another. The induction and/or maintenance of these protrusions is sensitive to changes in intercellular contact, pH, temperature, and nutritional conditions. The protrusions are stable, rigid structures which take part in intercellular adhesion but not in adhesion to substrata. Movement on substrata occurs by means of ruffling membranes formed on the main cell body. This asymmetry in cellular form and function is associated with specialized cell surface regions. Ultrastructurally, the cell surface over the protrusions lacks microvilli, and is covered with a 3,000–4,000-Å thick cell coat consisting of 200–500-Å electron-dense particles in an amorphous matrix. In contrast, the surface over the main cell body has microvilli and a 200-Å wide cell coat which lacks particles. The extracellular particles overlying the protrusions have electron-lucent cores, are protease- and pepsin-resistant, and do not stain with colloidal iron, while the matrix in which they are embedded is sensitive to proteolytic enzymes and contains acidic moieties. The negative surface charge density over the protrusions is higher than that over the main cell body, as shown by the orientation of the cells in an electric field. The unexpected observation that a region of higher charge density is one of increased intercellular adhesiveness might be explained, in part, by the rigidity of the protrusions and by the very small radius of curvature of the overlying extracellular particles. The protrusions permit the observation of discrete regions, differing in charge density, on the surface of living leukemia cells.     

Contractile forces regulate cell division in three-dimensional environments

Physical forces direct the orientation of the cell division axis for cells cultured on rigid, two-dimensional (2D) substrates. The extent to which physical forces regulate cell division in three-dimensional (3D) environments is not known. Here, we combine live-cell imaging with digital volume correlation to map 3D matrix displacements and identify sites at which cells apply contractile force to the matrix as they divide. Dividing cells embedded in fibrous matrices remained anchored to the matrix by long, thin protrusions. During cell rounding, the cells released adhesive contacts near the cell body while applying tensile forces at the tips of the protrusions to direct the orientation of the cell division axis. After cytokinesis, the daughter cells respread into matrix voids and invaded the matrix while maintaining traction forces at the tips of persistent and newly formed protrusions. Mechanical interactions between cells and the extracellular matrix constitute an important mechanism for regulation of cell division in 3D environments.     

Fluxes of Water through Aquaporin 9 Weaken Membrane-Cytoskeleton Anchorage and Promote Formation of Membrane Protrusions

All modes of cell migration require rapid rearrangements of cell shape, allowing the cell to navigate within narrow spaces in an extracellular matrix. Thus, a highly flexible membrane and a dynamic cytoskeleton are crucial for rapid cell migration. Cytoskeleton dynamics and tension also play instrumental roles in the formation of different specialized cell membrane protrusions, viz. lamellipodia, filopodia, and membrane blebs. The flux of water through membrane-anchored water channels, known as aquaporins (AQPs) has recently been implicated in the regulation of cell motility, and here we provide novel evidence for the role of AQP9 in the development of various forms of membrane protrusion. Using multiple imaging techniques and cellular models we show that: (i) AQP9 induced and accumulated in filopodia, (ii) AQP9-associated filopodial extensions preceded actin polymerization, which was in turn crucial for their stability and dynamics, and (iii) minute, local reductions in osmolarity immediately initiated small dynamic bleb-like protrusions, the size of which correlated with the reduction in osmotic pressure. Based on this, we present a model for AQP9-induced membrane protrusion, where the interplay of water fluxes through AQP9 and actin dynamics regulate the cellular protrusive and motile activity of cells.     

Functions and Regulation of Circular Dorsal Ruffles

Cells construct a number of plasma membrane structures to meet a range of physiological demands. Driven by juxtamembrane actin machinery, these actin-based membrane protrusions are essential for the operation and maintenance of cellular life. They are required for diverse cellular functions, such as directed cell motility, cell spreading, adhesion, and substrate/matrix degradation. Circular dorsal ruffles (CDRs) are one class of such structures characterized as F-actin-rich membrane projections on the apical cell surface. CDRs commence their formation minutes after stimulation as flat, open, and immature ruffles and progressively develop into fully enclosed circular ruffles. These “rings” then mature and contract centrifugally before subsiding. Serving a critical function in receptor internalization and cell migration, CDRs are thus highly dynamic but transient formations. Here, we review the current state of knowledge concerning the regulation of circular dorsal ruffles. We focus specifically on the biochemical pathways leading to CDR formation in order to better define the roles and functions of these enigmatic structures.     

Vitronectin Induces Phosphorylation of Ezrin/Radixin/Moesin Actin-binding Proteins through Binding to Its Novel Neuronal Receptor Telencephalin

Vitronectin (VN) is an extracellular matrix protein abundantly present in blood and a wide variety of tissues and plays important roles in a number of biological phenomena mainly through its binding to αV integrins. However, its definite function in the brain remains largely unknown. Here we report the identification of telencephalin (TLCN/ICAM-5) as a novel VN receptor on neuronal dendrites. VN strongly binds to TLCN, a unique neuronal member of the ICAM family, which is specifically expressed on dendrites of spiny neurons in the mammalian telencephalon. VN-coated microbeads induce the formation of phagocytic cup-like plasma membrane protrusions on dendrites of cultured hippocampal neurons and trigger the activation of TLCN-dependent intracellular signaling cascade including the phosphorylation of ezrin/radixin/moesin actin-binding proteins and recruitment of F-actin and phosphatidylinositol 4,5-bisphosphate for morphological transformation of the dendritic protrusions. These results suggest that the extracellular matrix molecule VN and its neuronal receptor TLCN play a pivotal role in the phosphorylation of ezrin/radixin/moesin proteins and the formation of phagocytic cup-like structures on neuronal dendrites.     

Focal adhesion and actin dynamics: a place where kinases and proteases meet to promote invasion

Integrin-linked focal adhesion complexes provide the main sites of cell adhesion to extracellular matrix and associate with the actin cytoskeleton to control cell movement. Dynamic regulation of focal adhesions and reorganization of the associated actin cytoskeleton are crucial determinants of cell migration. There are important roles for tyrosine kinases, extracellular signal-regulated protein kinase/mitogen-activated protein kinase signalling, and intracellular and extracellular proteases during actin and adhesion modulation. Dysregulation of these is associated with tumour cell invasion. In this article, we discuss established roles for these signalling pathways, as well as the functional interplay between them in controlling the migratory phenotype.     

Talin regulates moesin–NHE-1 recruitment to invadopodia and promotes mammary tumor metastasis

Invadopodia are actin-rich protrusions that degrade the extracellular matrix and are required for stromal invasion, intravasation, and metastasis. The role of the focal adhesion protein talin in regulating these structures is not known. Here, we demonstrate that talin is required for invadopodial matrix degradation and three-dimensional extracellular matrix invasion in metastatic breast cancer cells. The sodium/hydrogen exchanger 1 (NHE-1) is linked to the cytoskeleton by ezrin/radixin/moesin family proteins and is known to regulate invadopodium-mediated matrix degradation. We show that the talin C terminus binds directly to the moesin band 4.1 ERM (FERM) domain to recruit a moesin–NHE-1 complex to invadopodia. Silencing talin resulted in a decrease in cytosolic pH at invadopodia and blocked cofilin-dependent actin polymerization, leading to impaired invadopodium stability and matrix degradation. Furthermore, talin is required for mammary tumor cell motility, intravasation, and spontaneous lung metastasis in vivo. Thus, our findings provide a novel understanding of how intracellular pH is regulated and a molecular mechanism by which talin enhances tumor cell invasion and metastasis.     

SPARC (osteonectin/BM-40)

SPARC (Secreted ProteinAcidic and Rich in Cysteine) is a prototype of a family of biologically active glycoproteins that bind to cells and to extracellular matrix (ECM) components. It is expressed spatially and temporally during embryogenesis, tissue remodeling and repair. SPARC is a modular protein (34 kDa) comprised of three structural domains, one or more of which are implicated in the regulation of cell adhesion, proliferation, matrix synthesis/turnover. Rapid proteolysis of SPARC by extracellular proteases accounts for its transient detection in the extracellular environment. The proposed roles of SPARC in the development of cataracts and the regulation of angiogenesis during wound healing and tumor growth account for the recent attention it has received from the biomedical community.     

Fascins, and their roles in cell structure and function

The fascins are a structurally unique and evolutionarily conserved group of actin cross-linking proteins. Fascins function in the organisation of two major forms of actin-based structures: dynamic, cortical cell protrusions and cytoplasmic microfilament bundles. The cortical structures, which include filopodia, spikes, lamellipodial ribs, oocyte microvilli and the dendrites of dendritic cells, have roles in cell-matrix adhesion, cell interactions and cell migration, whereas the cytoplasmic actin bundles appear to participate in cell architecture. We discuss the current understanding of the cellular mechanisms that regulate the binding of fascin to actin and how these processes contribute to the organisation or disassembly of cell protrusions. Although the in vivo roles of fascin have been studied principally in Drosophila, several human diseases are associated with inherited or acquired alterations in the expression of fascins. Strategies to modulate fascin-containing protrusions and thereby cell adhesive and migratory behaviour could have potential for therapeutic intervention in these conditions. The supplementary material referred to in this section can be found at http://www.interscience.wiley.com/jpages/0265-9247/suppmat/2002/v24.350.html     

The location and cellular composition of the hemopoietic stem cell niche

While it is accepted that hemopoietic stem cells (HSC) are located in a three-dimensional microenvironment, termed a niche, the cellular and extracellular composition, as well as the multifaceted effects the components of the niche have on HSC regulation, remains undefined. Over the past four decades numerous advances in the field have led to the identification of roles for some cell types and propositions of potentially a number of HSC niches. We present evidence supporting the roles of multiple cell types and extracellular matrix molecules in the HSC niche, as well as discuss the potential significant overlap and intertwining of previously proposed distinct HSC niches.     

Covalent cross-linking of basement membrane-like matrices physically restricts invasive protrusions in breast cancer cells

The basement membrane (BM) provides a physical barrier to invasion in epithelial tumors, and alterations in the molecular makeup and structural integrity of the BM have been implicated in cancer progression. Invadopodia are the invasive protrusions that enable cancer cells to breach the nanoporous basement membrane, through matrix degradation and generation of force. However, the impact of covalent cross-linking on invadopodia extension into the BM remains unclear. Here, we examine the impact of covalent cross-linking of extracellular matrix on invasive protrusions using biomaterials that present ligands relevant to the basement membrane and provide a nanoporous, confining microenvironment. We find that increased covalent cross-linking of reconstituted basement membrane (rBM) matrix diminishes matrix mechanical plasticity, or the ability of the matrix to permanently retain deformation due to force. Covalently cross-linked rBM matrices, and rBM-alginate interpenetrating networks (IPNs) with covalent cross-links and low plasticity, restrict cell spreading and protrusivity. The reduced spreading and reduced protrusivity in response to low mechanical plasticity occurred independent of proteases. Mechanistically, our computational model reveals that the reduction in mechanical plasticity due to covalent cross-linking is sufficient to mechanically prevent cell protrusions from extending, independent of the impact of covalent cross-linking or matrix mechanical plasticity on cell signaling pathways. These findings highlight the biophysical role of covalent cross-linking in regulating basement membrane plasticity, as well as cancer cell invasion of this confining tissue layer.     

The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function

Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to - and degradation of - the extracellular matrix. The key proteins in these structures include the actin regulators cortactin and neural Wiskott-Aldrich syndrome protein (N-WASP), the adaptor proteins Tyr kinase substrate with four SH3 domains (TKS4) and Tyr kinase substrate with five SH3 domains (TKS5), and the metalloprotease membrane type 1 matrix metalloprotease (MT1MMP; also known as MMP14). Many cell types can produce these structures, including invasive cancer cells, vascular smooth muscle and endothelial cells, and immune cells such as macrophages and dendritic cells. Recently, progress has been made in our understanding of the regulatory and functional aspects of podosome and invadopodium biology and their role in human disease.     

Control of myofibroblast differentiation and function by cytoskeletal signaling

The cytoskeleton consists of three distinct types of protein polymer structures–microfilaments, intermediate filaments, and microtubules; each serves distinct roles in controlling cell shape, division, contraction, migration, and other processes. In addition to mechanical functions, the cytoskeleton accepts signals from outside the cell and triggers additional signals to extracellular matrix, thus playing a key role in signal transduction from extracellular stimuli through dynamic recruitment of diverse intermediates of the intracellular signaling machinery. This review summarizes current knowledge about the role of cytoskeleton in the signaling mechanism of fibroblast-to-myofibroblast differentiation–a process characterized by accumulation of contractile proteins and secretion of extracellular matrix proteins, and being critical for normal wound healing in response to tissue injury as well as for aberrant tissue remodeling in fibrotic disorders. Specifically, we discuss control of serum response factor and Hippo signaling pathways by actin and microtubule dynamics as well as regulation of collagen synthesis by intermediate filaments.     

Detyrosinated microtubule protrusions in suspended mammary epithelial cells promote reattachment

Breast tumor cells enter the bloodstream long before the development of clinically evident metastasis. However, the early presence of such bloodborne cells predicts poor patient outcome. Nearly 90% of human breast tumors arise as carcinomas from mammary epithelial cells, so it is important to study how these cells respond to the detached conditions that they would experience in the bloodstream. We report here that mammary epithelial cell lines produce long and dynamic protrusions of the plasma membrane when detached. Although human and mouse mammary epithelial cell lines die by apoptosis within 16 h of detachment, this protrusive response persists for days in cells overexpressing either Bcl-2 or Bcl-xL. Unlike actin-dependent invadopodia and podosomes, these protrusions are actually enhanced by actin depolymerization with Cytochalasin-D or Latrunculin-A. Immunofluorescence and Western blotting demonstrate that the protrusions are enriched in detyrosinated Glu-tubulin, a post-translationally modified form of alpha-tubulin that is found in stabilized microtubules. Video microscopy indicates that these protrusions promote cell-cell attachment, and inhibiting microtubule-based protrusions correlates with reduced extracellular matrix attachment. Since bloodborne metastasis depends on both cell-cell and cell-matrix attachment, microtubule-based protrusions in detached mammary epithelial cells provide a novel mechanism that could influence the metastatic spread of breast tumors.     

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.     

Actin cytoskeletal organisation in osteoclasts: a model to decipher transmigration and matrix degradation

Osteoclasts are large monocyte-derived multinucleated cells whose function is to resorb bone, i.e. a mineralised extracellular matrix. They exhibit two different actin cytoskeleton organisations according to their substratum. On non-mineralised substrates they form canonical podosomes, but on mineralised extracellular matrices they form a sealing zone. Podosomes consist of two functionally different actin subdomains: a podosome core, probably made of branched actin organised through a CD44 transmembrane receptor, and an actin cloud of actin cables organised around alphavbeta3 integrin. During osteoclast differentiation, podosome patterning is highly dynamic, and we propose that it ends up in a sealing zone in mature bone-resorbing osteoclasts after a complete reorganisation of the two subdomains. In addition to matrix degradation, osteoclasts share with tumour cells the ability to transmigrate through cell layers and-for that purpose-can arrange their cytoskeleton in long protrusions reminiscent of invadopodia. In this review, we discuss the relationships between podosomes and sealing zone, comparing their structures, their molecular composition and their abilities to degrade extracellular matrices. The dynamic actin remodelling in osteoclasts appears then as a major factor to understand their unusual abilities reminiscent of metastatic tumour cells.     

Membrane proteases: roles in tissue remodeling and tumour invasion.

The coordinated control of extracellular matrix degradation on the cell surface involves three crucial elements: secreted proteases and their inhibitors, surface protease receptors and integral membrane proteases. The roles that each of these elements play in cell surface proteolysis are described. The localization of proteases to the cell surface, protease activation, and regulation of cell surface proteolysis by protease inhibitors are key issues for elucidating the role of membrane proteases in tissue remodeling and tumour invasion.     

Receptor-type protein tyrosine phosphatase alpha (PTPα) mediates MMP14 localization and facilitates triple-negative breast cancer cell invasion

The ability of cancer cells to invade surrounding tissues requires degradation of the extracellular matrix (ECM). Invasive structures, such as invadopodia, form on the plasma membranes of cancer cells and secrete ECM-degrading proteases that play crucial roles in cancer cell invasion. We have previously shown that the protein tyrosine phosphatase alpha (PTPα) regulates focal adhesion formation and migration of normal cells. Here we report a novel role for PTPα in promoting triple-negative breast cancer cell invasion in vitro and in vivo. We show that PTPα knockdown reduces ECM degradation and cellular invasion of MDA-MB-231 cells through Matrigel. PTPα is not a component of TKS5-positive structures resembling invadopodia; rather, PTPα localizes with endosomal structures positive for MMP14, caveolin-1, and early endosome antigen 1. Furthermore, PTPα regulates MMP14 localization to plasma membrane protrusions, suggesting a role for PTPα in intracellular trafficking of MMP14. Importantly, we show that orthotopic MDA-MB-231 tumors depleted in PTPα exhibit reduced invasion into the surrounding mammary fat pad. These findings suggest a novel role for PTPα in regulating the invasion of triple-negative breast cancer cells.