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.
WASH and exocyst promote pericellular matrix degradation and tumor cell invasion by enabling localized exocytosis of MT1-MMP from late endosomes.
Remodeling of the extracellular matrix by carcinoma cells during metastatic dissemination requires formation of actin-based protrusions of the plasma membrane called invadopodia, where the trans-membrane type 1 matrix metalloproteinase (MT1-MMP) accumulates. Here, we describe an interaction between the exocyst complex and the endosomal Arp2/3 activator Wiskott-Aldrich syndrome protein and Scar homolog (WASH) on MT1-MMP–containing late endosomes in invasive breast carcinoma cells. We found that WASH and exocyst are required for matrix degradation by an exocytic mechanism that involves tubular connections between MT1-MMP–positive late endosomes and the plasma membrane in contact with the matrix. This ensures focal delivery of MT1-MMP and supports pericellular matrix degradation and tumor cell invasion into different pathologically relevant matrix environments. Our data suggest a general mechanism used by tumor cells to breach the basement membrane and for invasive migration through fibrous collagen-enriched tissues surrounding the tumor.
Invasive carcinoma cells form actin-rich matrix-degrading protrusions called invadopodia. These structures resemble podosomes produced by some normal cells and play a crucial role in extracellular matrix remodeling. In cancer, formation of invadopodia is strongly associated with invasive potential. Although deregulated signals from the receptor tyrosine kinase Met (also known as hepatocyte growth factor are linked to cancer metastasis and poor prognosis, its role in invadopodia formation is not known. Here we show that stimulation of breast cancer cells with the ligand for Met, hepatocyte growth factor, promotes invadopodia formation, and in aggressive gastric tumor cells where Met is amplified, invadopodia formation is dependent on Met activity. Using both GRB2-associated-binding protein 1 (Gab1)-null fibroblasts and specific knockdown of Gab1 in tumor cells we show that Met-mediated invadopodia formation and cell invasion requires the scaffold protein Gab1. By a structure–function approach, we demonstrate that two proline-rich motifs (P4/5) within Gab1 are essential for invadopodia formation. We identify the actin regulatory protein, cortactin, as a direct interaction partner for Gab1 and show that a Gab1–cortactin interaction is dependent on the SH3 domain of cortactin and the integrity of the P4/5 region of Gab1. Both cortactin and Gab1 localize to invadopodia rosettes in Met-transformed cells and the specific uncoupling of cortactin from Gab1 abrogates invadopodia biogenesis and cell invasion downstream from the Met receptor tyrosine kinase. Met localizes to invadopodia along with cortactin and promotes phosphorylation of cortactin. These findings provide insights into the molecular mechanisms of invadopodia formation and identify Gab1 as a scaffold protein involved in this process.
Invadopodia; Met RTK; Gab1; Cortactin; Matrix remodeling; Cell invasion
Polarized exocytosis is important for morphogenesis and cell growth. The exocyst is a multiprotein complex implicated in tethering secretory vesicles at specific sites of the plasma membrane for exocytosis. In the budding yeast, the exocyst is localized to sites of bud emergence or the tips of small daughter cells, where it mediates secretion and cell surface expansion. To understand how exocytosis is spatially controlled, we systematically analyzed the localization of Sec15p, a member of the exocyst complex and downstream effector of the rab protein Sec4p, in various mutants. We found that the polarized localization of Sec15p relies on functional upstream membrane traffic, activated rab protein Sec4p, and its guanine exchange factor Sec2p. The initial targeting of both Sec4p and Sec15p to the bud tip depends on polarized actin cable. However, different recycling mechanisms for rab and Sec15p may account for the different kinetics of polarization for these two proteins. We also found that Sec3p and Sec15p, though both members of the exocyst complex, rely on distinctive targeting mechanisms for their localization. The assembly of the exocyst may integrate various cellular signals to ensure that exocytosis is tightly controlled. Key regulators of cell polarity such as Cdc42p are important for the recruitment of the exocyst to the budding site. Conversely, we found that the proper localization of these cell polarity regulators themselves also requires a functional exocytosis pathway. We further report that Bem1p, a protein essential for the recruitment of signaling molecules for the establishment of cell polarity, interacts with the exocyst complex. We propose that a cyclical regulatory network contributes to the establishment and maintenance of polarized cell growth in yeast.
RhoGTPases have been implicated in the regulation of cancer metastasis. Invasive carcinoma cells form invadopodia, F-actin-rich matrix degrading protrusions that are thought to be important for tumor cell invasion and intravasation. Regulation of actin dynamics at invadopodial protrusions is crucial to drive invasion. This process requires the severing activity of cofilin to generate actin-free barbed ends. Previous work demonstrates that cofilin’s severing activity is tightly regulated through multiple mechanisms including regulation of cofilin serine phosphorylation by Rho GTPases. However, it is not known which Rho GTPase is involved in regulating cofilin’s phosphorylation status at invadopodia.
We show here, for the first time, how RhoC activation is controlled at invadopodia and how this activation regulates cofilin phosphorylation to control cofilin’s generation of actin-free barbed ends. Live-cell imaging of fluorescent RhoC biosensor reveals that RhoC activity is spatially confined to areas surrounding invadopodia. This spatiotemporal restriction of RhoC activity is controlled by “spatially distinct regulatory elements” that confines RhoC activation within this compartment. p190RhoGEF localizes around invadopodia to activate RhoC, while p190RhoGAP localizes inside invadopodia to deactivate the GTPase within the structure. RhoC activation enhances cofilin phosphorylation outside invadopodia.
These results show how RhoC activity is spatially regulated at invadopodia by p190RhoGEF and p190RhoGAP. RhoC activation in areas surrounding invadopodia restricts cofilin activity to within the invadopodium core resulting in a focused invadopodial protrusion. This mechanism likely enhances tumor cell invasion during metastasis.
Metastasis; Invadopodia; RhoC; p190RhoGEF; p190RhoGAP; Cofilin; Cofilin phosphorylation; tumor invasion
Invasion and metastasis are aggressive cancer phenotypes that are highly related to the ability of cancer cells to degrade extracellular matrix (ECM). At the cellular level, specialized actin-rich structures called invadopodia mediate focal matrix degradation by serving as exocytic sites for ECM-degrading proteinases. Adhesion signaling is likely to be a critical regulatory input to invadopodia, but the mechanism and location of such adhesion signaling events are poorly understood. Here, we report that adhesion rings surround invadopodia shortly after formation and correlate strongly with invadopodium activity on a cell-by-cell basis. By contrast, there was little correlation of focal adhesion number or size with cellular invadopodium activity. Prevention of adhesion ring formation by inhibition of RGD-binding integrins or knockdown (KD) of integrin-linked kinase (ILK) reduced the number of ECM-degrading invadopodia and reduced recruitment of IQGAP to invadopodium actin puncta. Furthermore, live cell imaging revealed that the rate of extracellular MT1-MMP accumulation at invadopodia was greatly reduced in both integrin-inhibited and ILK-KD cells. Conversely, KD of MT1-MMP reduced invadopodium activity and dynamics but not the number of adhesion-ringed invadopodia. These results suggest a model in which adhesion rings are recruited to invadopodia shortly after formation and promote invadopodium maturation by enhancing proteinase secretion. Since adhesion rings are a defining characteristic of podosomes, similar structures formed by normal cells, our data also suggest further similarities between invadopodia and podosomes.
Invadopodia; Adhesion rings; MT1-MMP; ILK; Integrin; Invasion
β1 integrin is a major regulator of invadopodium maturation. Studies reveal that β1 integrin–mediated adhesion is a key upstream switch that induces Arg-dependent cortactin phosphorylation, actin polymerization, and MMP recruitment to invadopodia for extracellular matrix degradation.
β1 integrin has been shown to promote metastasis in a number of tumor models, including breast, ovarian, pancreatic, and skin cancer; however, the mechanism by which it does so is poorly understood. Invasive membrane protrusions called invadopodia are believed to facilitate extracellular matrix degradation and intravasation during metastasis. Previous work showed that β1 integrin localizes to invadopodia, but its role in regulating invadopodial function has not been well characterized. We find that β1 integrin is required for the formation of mature, degradation-competent invadopodia in both two- and three-dimensional matrices but is dispensable for invadopodium precursor formation in metastatic human breast cancer cells. β1 integrin is activated during invadopodium precursor maturation, and forced β1 integrin activation enhances the rate of invadopodial matrix proteolysis. Furthermore, β1 integrin interacts with the tyrosine kinase Arg and stimulates Arg-dependent phosphorylation of cortactin on tyrosine 421. Silencing β1 integrin with small interfering RNA completely abrogates Arg-dependent cortactin phosphorylation and cofilin-dependent barbed-end formation at invadopodia, leading to a significant decrease in the number and stability of mature invadopodia. These results describe a fundamental role for β1 integrin in controlling actin polymerization–dependent invadopodial maturation and matrix degradation in metastatic tumor cells.
Invadopodia are actin-rich subcellular protrusions with associated proteases used by cancer cells to degrade extracellular matrix (ECM) . Molecular components of invadopodia include branched actin assembly proteins, membrane trafficking proteins, signaling proteins and transmembrane proteinases. Similar structures exist in nontransformed cells, such as osteoclasts and dendritic cells, but are generally called podosomes and are thought to be more involved in cell-matrix adhesion than invadopodia [2–4]. Despite intimate contact with their ECM substrates, it is unknown whether physical or chemical ECM signals regulate invadopodia function. Here, we report that ECM rigidity directly increases both the number and activity of invadopodia. Transduction of ECM rigidity signals depends on the cellular contractile apparatus [5–7], as inhibition of nonmuscle myosin II, myosin light chain kinase, and Rho kinase all abrogate invadopodia-associated ECM degradation. Whereas myosin IIA, IIB, and phosphorylated myosin light chain do not localize to invadopodia puncta, active phosphorylated forms of the mechanosensing proteins p130Cas (Cas) and focal adhesion kinase (FAK) are present in actively degrading invadopodia and the levels of phospho-Cas and phospho-FAK in invadopodia are sensitive to myosin inhibitors. Overexpression of Cas or FAK further enhances invadopodia activity in cells plated on rigid polyacrylamide substrates. Thus, in invasive cells, ECM rigidity signals lead to increased matrix-degrading activity at invadopodia, via a myosin II-FAK/Cas pathway. These data suggest a potential mechanism, via invadopodia, for the reported correlation of tissue density with cancer aggressiveness.
Invadopodia; Extracellular matrix; rigidity; mechanotransduction; cell contractility; tumor invasion
The exocyst complex localizes to distinct foci at the plasma membrane of Arabidopsis thaliana cells. Their localization at the plasma membrane is insensitive to BFA treatment but is decreased in an exocyst-subunit mutant. In turn, exocyst-subunit mutants show decreased exocytosis.
The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6–green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.
Head and neck squamous cell carcinoma (HNSCC) has a proclivity for locoregional invasion. HNSCC mediates invasion in part through invadopodia-based proteolysis of the extracellular matrix (ECM). Activation of Src, Erk1/2, Abl and Arg downstream of epidermal growth factor receptor (EGFR) modulates invadopodia activity through phosphorylation of the actin regulatory protein cortactin. In MDA-MB-231 breast cancer cells, Abl and Arg function downstream of Src to phosphorylate cortactin, promoting invadopodia ECM degradation activity and thus assigning a pro-invasive role for Ableson kinases. We report that Abl kinases have an opposite, negative regulatory role in HNSCC where they suppress invadopodia and tumor invasion. Impairment of Abl expression or Abl kinase activity with imatinib mesylate enhanced HNSCC matrix degradation and 3D collagen invasion, functions that were impaired in MDA-MB-231. HNSCC lines with elevated EGFR and Src activation did not contain increased Abl or Arg kinase activity, suggesting Src could bypass Abl/Arg to phosphorylate cortactin and promote invadopodia ECM degradation. Src transformed Abl−/−/Arg−/− fibroblasts produced ECM degrading invadopodia containing pY421 cortactin, indicating that Abl/Arg are dispensable for invadopodia function in this system. Imatinib treated HNSCC cells had increased EGFR, Erk1/2 and Src activation, enhancing cortactin pY421 and pS405/418 required for invadopodia function. Imatinib stimulated shedding of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) from HNSCC cells, where soluble HB-EGF enhanced invadopodia ECM degradation in HNSCC but not in MDA-MB-231. HNSCC cells treated with inhibitors of the EGFR invadopodia pathway indicated that EGFR and Src are required for invadopodia function. Collectively our results indicate that Abl kinases negatively regulate HNSCC invasive processes through suppression of an HB-EGF autocrine loop responsible for activating a EGFR-Src-cortactin cascade, in contrast to the invasion promoting functions of Abl kinases in breast and other cancer types. Our results provide mechanistic support for recent failed HNSCC clinical trials utilizing imatinib.
Abl; imatinib mesylate; invadopodia; invasion; head and neck cancer; cortactin
Invadopodia or invasive feet, which are actin-rich membrane protrusions with matrix degradation activity formed by invasive cancer cells, are a key determinant in the malignant invasive progression of tumors and represent an important target for cancer therapies. In this work, we presented a microfluidic 3D culture device with continuous supplement of fresh media via a syringe pump. The device mimicked tumor microenvironment in vivo and could be used to assay invadopodia formation and to study the mechanism of human lung cancer invasion. With this device, we investigated the effects of epidermal growth factor (EGF) and matrix metalloproteinase (MMP) inhibitor, GM6001 on invadopodia formation by human non-small cell lung cancer cell line A549 in 3D matrix model. This device was composed of three units that were capable of achieving the assays on one control group and two experimental groups' cells, which were simultaneously pretreated with EGF or GM6001 in parallel. Immunofluorescence analysis of invadopodia formation and extracellular matrix degradation was conducted using confocal imaging system. We observed that EGF promoted invadopodia formation by A549 cells in 3D matrix and that GM6001 inhibited the process. These results demonstrated that epidermal growth factor receptor (EGFR) signaling played a significant role in invadopodia formation and related ECM degradation activity. Meanwhile, it was suggested that MMP inhibitor (GM6001) might be a powerful therapeutic agent targeting invadopodia formation in tumor invasion. This work clearly demonstrated that the microfluidic-based 3D culture device provided an applicable platform for elucidating the mechanism of cancer invasion and could be used in testing other anti-invasion agents.
MT1-MMP is a potent invasion-promoting membrane protease employed by aggressive cancer cells. MT1-MMP localizes preferentially at membrane protrusions called invadopodia where it plays a central role in degradation of the surrounding extracellular matrix (ECM). Previous reports suggested a role for a continuous supply of MT1-MMP in ECM degradation. However, the turnover rate of MT1-MMP and the extent to which the turnover contributes to the ECM degradation at invadopodia have not been clarified. To approach this problem, we first performed FRAP (Fluorescence Recovery after Photobleaching) experiments with fluorescence-tagged MT1-MMP focusing on a single invadopodium and found very rapid recovery in FRAP signals, approximated by double-exponential plots with time constants of 26 s and 259 s. The recovery depended primarily on vesicle transport, but negligibly on lateral diffusion. Next we constructed a computational model employing the observed kinetics of the FRAP experiments. The simulations successfully reproduced our FRAP experiments. Next we inhibited the vesicle transport both experimentally, and in simulation. Addition of drugs inhibiting vesicle transport blocked ECM degradation experimentally, and the simulation showed no appreciable ECM degradation under conditions inhibiting vesicle transport. In addition, the degree of the reduction in ECM degradation depended on the degree of the reduction in the MT1-MMP turnover. Thus, our experiments and simulations have established the role of the rapid turnover of MT1-MMP in ECM degradation at invadopodia. Furthermore, our simulations suggested synergetic contributions of proteolytic activity and the MT1-MMP turnover to ECM degradation because there was a nonlinear and marked reduction in ECM degradation if both factors were reduced simultaneously. Thus our computational model provides a new in silico tool to design and evaluate intervention strategies in cancer cell invasion.
Prevention of invasion is important in cancer therapy. MT1-MMP is a membrane protein involved in degradation of ECM (extracellular matrix) that is highly expressed at invadopodia, which are small protrusions of cancer cells. ECM degradation by MT1-MMP at invadopodia is hypothesized as the initial step of cancer cell invasion. However, MT1-MMP is inhibited by the endogenous inhibitor TIMP-2, so continuous turnover of MT1-MMP at the surface of invadopodia would be required. In agreement, it has been reported that the blockade of vesicle transport, which is one mechanism involved in the turnover, blocked the ECM degradation. However, the turnover rate of MT1-MMP at invadopodia and the extent to which the turnover is critical for the degradation of ECM have not been clarified. In this report we measured the turnover rate of MT1-MMP at a single invadopodium and found rapid turnover rates with time constants of 26 s and 259 s, which primarily depended on the vesicle transport. A computational model was constructed based on the observed kinetics. If we blocked the rapid turnover, the ECM degradation was blocked both experimentally and in simulations. These results established the role of the rapid turnover of MT1-MMP in the ECM degradation at invadopodia.
Changes in cellular behavior that cause epithelial cells to lose adhesiveness, acquire a motile, invasive phenotype and metastasize to secondary sites are complex and poorly understood. Molecules that normally function to integrate adhesive spatial information with cytoskeleton dynamics and membrane trafficking likely serve important functions in cellular transformation. One such complex is the Exocyst, which is essential for targeted delivery of membrane and secretory proteins to specific plasma membrane sites to maintain epithelial cell polarity. Upon loss of cadherin-mediated adhesion in Dunning R3327-5′A prostate tumor cells, Exocyst localization shifts from lateral membranes to tips of protrusive membrane extensions. Here, it co-localizes and co-purifies with focal complex proteins that regulate membrane trafficking and cytoskeleton dynamics. These sites are the preferred destination of post-Golgi transport vesicles ferrying biosynthetic cargo, such as α5-integrin, which mediates adhesion of cells to the substratum, a process essential to cell motility. Interference with Exocyst activity impairs integrin delivery to plasma membrane and inhibits tumor cell motility and matrix invasiveness. Localization of Exocyst, and by extension targeting of Exocyst-dependent cargo, is dependent on Ral GTPases, which control association between Sec5 and paxillin. Overexpression of Ral-uncoupled Sec5 mutants inhibited Exocyst interaction with paxillin in 5′A cells, as did RNAi-mediated reduction of either RalA or RalB. Reduction of neither GTPase significantly altered steady state levels of assembled Exocyst in these cells, but did change the observed localization of Exocyst proteins.
In fission yeast, long-range transport and vesicle tethering by the exocyst are individually dispensable but together essential for cell morphogenesis. Both pathways function downstream of Cdc42. The exocyst localizes to growing cell tips independently of the cytoskeleton and instead depends on PIP2.
Cell morphogenesis depends on polarized exocytosis. One widely held model posits that long-range transport and exocyst-dependent tethering of exocytic vesicles at the plasma membrane sequentially drive this process. Here, we describe that disruption of either actin-based long-range transport and microtubules or the exocyst did not abolish polarized growth in rod-shaped fission yeast cells. However, disruption of both actin cables and exocyst led to isotropic growth. Exocytic vesicles localized to cell tips in single mutants but were dispersed in double mutants. In contrast, a marker for active Cdc42, a major polarity landmark, localized to discreet cortical sites even in double mutants. Localization and photobleaching studies show that the exocyst subunits Sec6 and Sec8 localize to cell tips largely independently of the actin cytoskeleton, but in a cdc42 and phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2)–dependent manner. Thus in fission yeast long-range cytoskeletal transport and PIP2-dependent exocyst represent parallel morphogenetic modules downstream of Cdc42, raising the possibility of similar mechanisms in other cell types.
Our recent studies implicated key and distinct roles for the highly related RalA and RalB small GTPases (82% sequence identity) in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis and invasive and metastatic growth, respectively. How RalB may promote PDAC invasion and metastasis has not been determined. In light of known Ral effector functions in regulation of actin organization and secretion, we addressed a possible role for RalB in formation of invadopodia, actin-rich membrane protrusions that contribute to tissue invasion and matrix remodeling. We determined that a majority of KRAS mutant PDAC cell lines exhibited invadopodia and that expression of activated K-Ras is both necessary and sufficient for invadopodium formation. Invadopodium formation was not dependent on the canonical Raf-MEK-ERK effector pathway and was instead dependent on the Ral effector pathway. However, this process was more dependent on RalB than on RalA. Surprisingly, RalB-mediated invadopodium formation was dependent on RalBP1/RLIP76 but not Sec5 and Exo84 exocyst effector function. Unexpectedly, the requirement for RalBP1 was independent of its best known function as a GTPase-activating protein for Rho small GTPases. Instead, disruption of the ATPase function of RalBP1 impaired invadopodium formation. Our results identify a novel RalB-mediated biochemical and signaling mechanism for invadopodium formation.
Invadopodia are protrusive structures used by tumor cells for degradation of the extracellular matrix to promote invasion . Invadopodia formation and function are regulated by cytoskeletal remodeling pathways and the oncogenic kinase Src. The guanine nucleotide exchange factor Vav1, which is an activator of Rho family GTPases, is ectopically expressed in many pancreatic cancers, where it promotes tumor cell survival and migration [2, 3]. We have now determined that Vav1 is also a potent regulator of matrix degradation by pancreatic tumor cells, as depletion of Vav1 by siRNA-mediated knockdown inhibits the formation of invadopodia. This requires the exchange function of Vav1 toward the GTPase Cdc42, which is required for invadopodia assembly [4, 5]. In addition, we have determined that Src-mediated phosphorylation and activation of Vav1 is both required for, and, unexpectedly, sufficient for, invadopodia formation. Expression of Vav1 Y174F, which mimics its activated state, is a potent inducer of invadopodia formation through Cdc42, even in the absence of Src activation and phosphorylation of other Src substrates, such as cortactin. Thus, these data identify a novel mechanism by which Vav1 can enhance the tumorigenicity and invasive potential of cancer cells. These data suggest that Vav1 promotes the matrix-degrading processes underlying tumor cell migration, and further, under conditions of ectopic Vav1 expression, that Vav1 is a central regulator and major driver of invasive matrix remodeling by pancreatic tumor cells.
The exocyst complex plays a critical role in targeting and tethering vesicles to specific sites of the plasma membrane. These events are crucial for polarized delivery of membrane components to the cell surface, which is critical for cell motility and division. Though Rho GTPases are involved in regulating actin dynamics and membrane trafficking, their role in exocyst-mediated vesicle targeting is not very clear. Herein, we present evidence that depletion of GEF-H1, a guanine nucleotide exchange factor for Rho proteins, affects vesicle trafficking. Interestingly, we found that GEF-H1 directly binds to exocyst component Sec5 in a Ral GTPase-dependent manner. This interaction promotes RhoA activation, which then regulates exocyst assembly/localization and exocytosis. Taken together, our work defines a mechanism for RhoA activation in response to RalA-Sec5 signaling and involvement of GEF-H1/RhoA pathway in the regulation of vesicle trafficking.
Genetic analyses in zebrafish identify a novel physical signaling mechanism that drives formation of invadopodia-like structures and promotes cell invasion in vivo.
The signals that initiate cell invasion are not well understood, but there is increasing evidence that extracellular physical signals play an important role. Here we show that epithelial cell invasion in the intestine of zebrafish meltdown (mlt) mutants arises in response to unregulated contractile tone in the surrounding smooth muscle cell layer. Physical signaling in mlt drives formation of membrane protrusions within the epithelium that resemble invadopodia, matrix-degrading protrusions present in invasive cancer cells. Knockdown of Tks5, a Src substrate that is required for invadopodia formation in mammalian cells blocked formation of the protrusions and rescued invasion in mlt. Activation of Src-signaling induced invadopodia-like protrusions in wild type epithelial cells, however the cells did not migrate into the tissue stroma, thus indicating that the protrusions were required but not sufficient for invasion in this in vivo model. Transcriptional profiling experiments showed that genes responsive to reactive oxygen species (ROS) were upregulated in mlt larvae. ROS generators induced invadopodia-like protrusions and invasion in heterozygous mlt larvae but had no effect in wild type larvae. Co-activation of oncogenic Ras and Wnt signaling enhanced the responsiveness of mlt heterozygotes to the ROS generators. These findings present the first direct evidence that invadopodia play a role in tissue cell invasion in vivo. In addition, they identify an inducible physical signaling pathway sensitive to redox and oncogenic signaling that can drive this process.
The epithelial cells lining the digestive tract are separated from the connective tissue stroma by a thin layer of extracellular matrix called the basement membrane. During cell invasion, as occurs during cancer metastasis, epithelial cells breach the basement membrane and invade the tissue stroma. The proteases used by invasive cells to degrade basement membrane in vitro are localized in specialized plasma membrane protrusions known as invadopodia. It is not known, however, whether invadopodia are required for cell invasion in vivo or what triggers their formation. Here, we show that epithelial cells in the intestine of the zebrafish mutant meltdown form invadopodia-like protrusions and invade the tissue stroma in response to unregulated contractile tone in the surrounding smooth muscle layer. The invadopodia-like protrusions that form in response to this physical signal are required for epithelial cell invasion in this in vivo model, and they can be induced when unregulated smooth muscle contraction is induced by oxidative stress. These findings provide the first direct evidence that invadopodia play a role in tissue cell invasion in vivo and identify a novel inducible physical signaling mechanism that can drive this process.
Podoplanin overexpression has been reported in various cancers, however, the precise mechanism for podoplanin to promote tumor progression remains elusive. In the present study, podoplanin overexpression was found associated with invasiveness both in OSCC tissues and cell lines. Moreover, the cell invasiveness increased with forced podoplanin expression and decreased when podoplanin was knockdown, indicating podoplanin-mediated cell invasion during OSCC progression. To further identify the role of podoplanin in tumor invasion, cell spreading and immunofluorescence assay were performed firstly. It was found that podoplanin knockdown caused an impaired cell spreading with reduced filopodia and the premature assembly of stress fibers while podoplanin overexpression induced an increase in cellular protrusions and stress fibers with extensive parallel bundles. Then, pull-down assays revealed forced podoplanin expression increased Cdc42 activity and reduced RhoA activity while podoplanin knockdown decreased Cdc42 and increased RhoA markedly. Moreover, a hierarchy of crosstalk between RhoA and Cdc42 was confirmed in podoplanin-mediated cell motility. On the other hand, a significant correlation between podoplanin and MT1-MMP expression in OSCCs was found both in vivo and in vitro, co-located in invasive cells and cellular protrusions. Furthermore, our data showed MT1-MMP knockdown significantly blocked the upregulation of cell motility by forced podoplanin expression, indicating that MT1-MMP played a role in podoplanin-mediated tumor invasion. To further confirm the interaction between RhoA/Cdc42 complex, MT1-MMP and podoplanin, co-precipitation experiments were performed. Both the co-precipitation of podoplanin with MT1-MMP and the podoplanin-induced specific binding of MT1-MMP to Cdc42 were found, and immunofluorescence revealed the co-location of podoplanin, MT1-MMP and Cdc42 at the plasma membrane and filopodia induced an increase in cellular protrusion and stress fibers formation. Moreover, MT1-MMP inhibition could partly rescue the increase of Cdc42 activity caused by forced podoplanin expression. Taken together, our data demonstrated a hierarchy of crosstalk between RhoA and Cdc42 was involved in podoplanin-mediated cytoskeleton remodeling and invasion; the co-location and co-ordination of podoplanin, Cdc42 and MT1-MMP in the invadopodia might induce cytoskeleton remodeling, ECM degradation and tumor invasion, while podoplanin-induced EMT may not be indispensible during OSCC progression.
Podoplanin; invasion; Cdc42/RhoA; MT1-MMP; oral squamous cell carcinoma
The invasiveness of cells is correlated with the presence of dynamic actin-rich membrane structures called invadopodia, which are membrane protrusions that are associated with localized polymerization of sub-membrane actin filaments. Similar to focal adhesions and podosomes, invadopodia are cell matrix adhesion sites. Indeed, invadopodia share several features with podosomes, but whether they are distinct structures is still a matter of debate. Invadopodia are built upon an N-WASP-dependent branched actin network, and the Rho GTPase Cdc42 is involved in inducing invadopodial-membrane protrusion, which is mediated by actin filaments that are organized in bundles to form an actin core. Actin-core formation is thought to be an early step in invadopodium assembly, and the actin core is perpendicular to the extracellular matrix and the plasma membrane; this contrasts with the tangential orientation of actin stress fibers anchored to focal adhesions. In this Commentary, we attempt to summarize recent insights into the actin dynamics of invadopodia and podosomes, and the forces that are transmitted through these invasive structures. Although the mechanisms underlying force-dependent regulation of invadopodia and podosomes are largely unknown compared with those of focal adhesions, these structures do exhibit mechanosensitivity. Actin dynamics and associated forces might be key elements in discriminating between invadopodia, podosomes and focal adhesions. Targeting actin regulatory molecules that specifically promote invadopodium formation is an attractive strategy against cancer-cell invasion.
Actins; Animals; Cell Adhesion; Cell Movement; Cell-Matrix Junctions; Extracellular Matrix; Focal Adhesions; Humans; Integrins; Models, Biological; Podosomes; invadopodia; invasion; cancer; osteoporosis
The SNAREs SNAP23, Syntaxin4, and VAMP7 associate to target the delivery of MT1-MMP to sites of invadopodium formation in breast tumor cells. The interaction of these SNAREs correlates with decreased phosphorylation of Syntaxin4. The targeted delivery of MT1-MMP is required for efficient ECM degradation and cell invasion.
Movement through the extracellular matrix (ECM) requires cells to degrade ECM components, primarily through the action of matrix metalloproteinases (MMPs). Membrane type 1–matrix metalloproteinase (MT1-MMP) has an essential role in matrix degradation and cell invasion and localizes to subcellular degradative structures termed invadopodia. Trafficking of MT1-MMP to invadopodia is required for the function of these structures, and here we examine the role of N-ethylmaleimide–sensitive factor–activating protein receptor (SNARE)–mediated membrane traffic in the transport of MT1-MMP to invadopodia. During invadopodium formation in MDA-MB-231 human breast cancer cells, increased association of SNAP23, Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) is detected by coimmunoprecipitation. Blocking the function of these SNAREs perturbs invadopodium-based ECM degradation and cell invasion. Increased level of SNAP23-Syntaxin4-VAMP7 interaction correlates with decreased Syntaxin4 phosphorylation. These results reveal an important role for SNARE-regulated trafficking of MT1-MMP to invadopodia during cellular invasion of ECM.
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.
Cell interactions with the extracellular matrix (ECM) can regulate multiple cellular activities and the matrix itself in dynamic, bidirectional processes. One such process is local proteolytic modification of the ECM. Invadopodia of tumor cells are actin-rich proteolytic protrusions that locally degrade matrix molecules and mediate invasion. We report that a novel high-density fibrillar collagen (HDFC) matrix is a potent inducer of invadopodia, both in carcinoma cell lines and in primary human fibroblasts. In carcinoma cells, HDFC matrix induced formation of invadopodia via a specific integrin signaling pathway that did not require growth factors or even altered gene and protein expression. In contrast, phosphoproteomics identified major changes in a complex phosphosignaling network with kindlin2 serine phosphorylation as a key regulatory element. This kindlin2-dependent signal transduction network was required for efficient induction of invadopodia on dense fibrillar collagen and for local degradation of collagen. This novel phosphosignaling mechanism regulates cell surface invadopodia via kindlin2 for local proteolytic remodeling of the ECM.
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.
invadopodia; podosomes; cortactin; focal adhesions; invasion
Rho GTPases are important regulators of polarity in eukaryotic cells. In yeast they are involved in regulating the docking and fusion of secretory vesicles with the cell surface. Our analysis of a Rho3 mutant that is unable to interact with the Exo70 subunit of the exocyst reveals a normal polarization of the exocyst complex as well as other polarity markers. We also find that there is no redundancy between the Rho3–Exo70 and Rho1–Sec3 pathways in the localization of the exocyst. This suggests that Rho3 and Cdc42 act to polarize exocytosis by activating the exocytic machinery at the membrane without the need to first recruit it to sites of polarized growth. Consistent with this model, we find that the ability of Rho3 and Cdc42 to hydrolyze GTP is not required for their role in secretion. Moreover, our analysis of the Sec3 subunit of the exocyst suggests that polarization of the exocyst may be a consequence rather than a cause of polarized exocytosis.
Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires matrix degrading protrusions called invadopodia. The Na+/H+ exchanger (NHE1) has recently been shown to be fundamental in the regulation of invadopodia actin cytoskeleton dynamics and activity. However, the structural link between the invadopodia cytoskeleton and NHE1 is still unknown. A candidate could be ezrin, a linker between the NHE1 and the actin cytoskeleton known to play a pivotal role in invasion and metastasis. However, the mechanistic basis for its role remains unknown. Here, we demonstrate that ezrin phosphorylated at T567 is highly overexpressed in the membrane of human breast tumors and positively associated with invasive growth and HER2 overexpression. Further, in the metastatic cell line, MDA-MB-231, p-ezrin was almost exclusively expressed in invadopodia lipid rafts where it co-localized in a functional complex with NHE1, EGFR, ß1-integrin and phosphorylated-NHERF1. Manipulation by mutation of ezrins T567 phosphorylation state and/or PIP2 binding capacity or of NHE1s binding to ezrin or PIP2 demonstrated that p-ezrin expression and binding to PIP2 are required for invadopodia-mediated ECM degradation and invasion and identified NHE1 as the membrane protein that p-ezrin regulates to induce invadopodia formation and activity.