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1.  hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2 
The Journal of Cell Biology  2013;202(4):699-713.
hGAAP promotes cell adhesion and migration by increasing localized Ca2+-dependent activation of calpain, leading to increased focal adhesion dynamics.
Golgi antiapoptotic proteins (GAAPs) are highly conserved Golgi membrane proteins that inhibit apoptosis and promote Ca2+ release from intracellular stores. Given the role of Ca2+ in controlling cell adhesion and motility, we hypothesized that human GAAP (hGAAP) might influence these events. In this paper, we present evidence that hGAAP increased cell adhesion, spreading, and migration in a manner that depended on the C-terminal domain of hGAAP. We show that hGAAP increased store-operated Ca2+ entry and thereby the activity of calpain at newly forming protrusions. These hGAAP-dependent effects regulated focal adhesion dynamics and cell migration. Indeed, inhibition or knockdown of calpain 2 abrogated the effects of hGAAP on cell spreading and migration. Our data reveal that hGAAP is a novel regulator of focal adhesion dynamics, cell adhesion, and migration by controlling localized Ca2+-dependent activation of calpain.
doi:10.1083/jcb.201301016
PMCID: PMC3747308  PMID: 23940116
2.  ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis 
EMBO Molecular Medicine  2013;6(2):278-294.
The transmembrane metalloprotease-disintegrin ADAM8 mediates cell adhesion and shedding of ligands, receptors and extracellular matrix components. Here, we report that ADAM8 is abundantly expressed in breast tumors and derived metastases compared to normal tissue, especially in triple-negative breast cancers (TNBCs). Furthermore, high ADAM8 levels predicted poor patient outcome. Consistently, ADAM8 promoted an aggressive phenotype of TNBC cells in culture. In a mouse orthotopic model, tumors derived from TNBC cells with ADAM8 knockdown failed to grow beyond a palpable size and displayed poor vascularization. Circulating tumor cells and brain metastases were also significantly reduced. Mechanistically, ADAM8 stimulated both angiogenesis through release of VEGF-A and transendothelial cell migration via β1-integrin activation. In vivo, treatment with an anti-ADAM8 antibody from the time of cell inoculation reduced primary tumor burden and metastases. Furthermore, antibody treatment of established tumors profoundly decreased metastases in a resection model. As a non-essential protein under physiological conditions, ADAM8 represents a promising novel target for treatment of TNBCs, which currently lack targeted therapies and frequently progress with fatal dissemination.
Subject Category Cancer
doi:10.1002/emmm.201303373
PMCID: PMC3927960  PMID: 24375628
ADAM8; cancer progression; metastasis; therapeutic target; triple-negative; breast cancer
3.  Fascin Regulates the Migration of Subventricular Zone-Derived Neuroblasts in the Postnatal Brain 
The Journal of Neuroscience  2013;33(30):12171-12185.
After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). This migration is a fundamental event controlling the proper integration of new neurons in a pre-existing synaptic network. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we show that the actin-bundling protein fascin is highly upregulated in mouse SVZ-derived migratory neuroblasts. Fascin-1ko mice display an abnormal RMS and a smaller OB. Bromodeoxyuridine labeling experiments show that lack of fascin significantly impairs neuroblast migration, but does not appear to affect cell proliferation. Moreover, fascin depletion substantially alters the polarized morphology of rat neuroblasts. Protein kinase C (PKC)-dependent phosphorylation of fascin on Ser39 regulates its actin-bundling activity. In vivo postnatal electroporation of phosphomimetic (S39D) or nonphosphorylatable (S39A) fascin variants followed by time-lapse imaging of brain slices demonstrates that the phospho-dependent modulation of fascin activity ensures efficient neuroblast migration. Finally, fluorescence lifetime imaging microscopy studies in rat neuroblasts reveal that the interaction between fascin and PKC can be modulated by cannabinoid signaling, which controls neuroblast migration in vivo. We conclude that fascin, whose upregulation appears to mark the transition to the migratory neuroblast stage, is a crucial regulator of neuroblast motility. We propose that a tightly regulated phospho/dephospho-fascin cycle modulated by extracellular signals is required for the polarized morphology and migration in neuroblasts, thus contributing to efficient neurogenesis.
doi:10.1523/JNEUROSCI.0653-13.2013
PMCID: PMC3721833  PMID: 23884926
4.  FAK-heterozygous mice display enhanced tumour angiogenesis 
Nature communications  2013;4:2020.
Genetic ablation of endothelial Focal Adhesion Kinase (FAK) can inhibit pathological angiogenesis, suggesting that loss of endothelial FAK is sufficient to reduce neovascularisation. Here we show that reduced stromal-FAK expression in FAK-heterozygous mice unexpectedly enhances both B16F0 and CMT19T tumour growth and angiogenesis. We further demonstrate that cell proliferation and microvessel sprouting, but not migration, are increased in serum-stimulated FAK-heterozygous endothelial cells. FAK-heterozygous endothelial cells display an imbalance in FAK phosphorylation at pY397 and pY861 without changes in Pyk2 or Erk1/2 activity. By contrast, serum-stimulated phosphorylation of Akt is enhanced in FAK-heterozygous endothelial cells and these cells are more sensitive to Akt inhibition. Additionally, low doses of a pharmacological FAK inhibitor, although too low to affect FAK autophosphorylation in vitro, can enhance angiogenesis ex vivo and tumor growth in vivo. Our results highlight a potential novel role for FAK as a non-linear, dose-dependent regulator of angiogenesis where heterozygous levels of FAK enhance angiogenesis.
doi:10.1038/ncomms3020
PMCID: PMC3712492  PMID: 23799510
5.  CAR regulates epithelial cell junction stability through control of E-cadherin trafficking 
Scientific Reports  2013;3:2889.
CAR (Coxsackie and Adenovirus Receptor) is the primary docking receptor for typeB coxsackie viruses and subgroup C adenoviruses. CAR is a member of the JAM family of adhesion receptors and is located to both tight and adherens junctions between epithelial cells where it can assemble adhesive contacts through homodimerisation in trans. However, the role of CAR in controlling epithelial junction dynamics remains poorly understood. Here we demonstrate that levels of CAR in human epithelial cells play a key role in determining epithelial cell adhesion through control of E-cadherin stability at cell-cell junctions. Mechanistically, we show that CAR is phosphorylated within the C-terminus by PKCδ and that this in turn controls Src-dependent endocytosis of E-cadherin at cell junctions. This data demonstrates a novel role for CAR in regulating epithelial homeostasis.
doi:10.1038/srep02889
PMCID: PMC3791454  PMID: 24096322
7.  Integrin-Specific Control of Focal Adhesion Kinase and RhoA Regulates Membrane Protrusion and Invasion 
PLoS ONE  2013;8(9):e74659.
Cell invasion through extracellular matrix (ECM) is a hallmark of the metastatic cascade. Cancer cells require adhesion to surrounding tissues for efficient migration to occur, which is mediated through the integrin family of receptors. Alterations in expression levels of β1 and β3 integrins have previously been reported in a number of human cancers. However, whether there are specific roles for these ubiquitous receptors in mediating cell invasion remains unclear. Here we demonstrate that loss of β1 but not β3 integrins leads to increased spread cell area and focal adhesion number in cells on 2D immobilized fibronectin. Increased adhesion numbers in β1 knockdown cells correlated with decreased cell migration on 2D surfaces. Conversely, cells depleted of β1 integrins showed increased migration speed on 3D cell-derived matrix as well as in 3D organotypic cultures and inverted invasion assays. This increased invasive potential was also seen in cells lacking β3 integrin but only in 3D cultures containing fibroblasts. Mechanistically, in situ analysis using FRET biosensors revealed that enhanced invasion in cells lacking β1 integrins was directly coupled with reduced activation of focal adhesion kinase (FAK) and the small GTPase RhoA resulting in formation of enhanced dynamic protrusions and increased invasion. These reductions in FAK-RhoA signal activationwere not detected in β3 knockdown cells under the same conditions. This data demonstrates a specific role for β1 integrins in the modulation of a FAK-RhoA-actomyosin signaling axis to regulate cell invasion through complex ECM environments.
doi:10.1371/journal.pone.0074659
PMCID: PMC3767638  PMID: 24040310
8.  Phosphorylation of the Actin Binding Protein Drebrin at S647 Is Regulated by Neuronal Activity and PTEN 
PLoS ONE  2013;8(8):e71957.
Defects in actin dynamics affect activity-dependent modulation of synaptic transmission and neuronal plasticity, and can cause cognitive impairment. A salient candidate actin-binding protein linking synaptic dysfunction to cognitive deficits is Drebrin (DBN). However, the specific mode of how DBN is regulated at the central synapse is largely unknown. In this study we identify and characterize the interaction of the PTEN tumor suppressor with DBN. Our results demonstrate that PTEN binds DBN and that this interaction results in the dephosphorylation of a site present in the DBN C-terminus - serine 647. PTEN and pS647-DBN segregate into distinct and complimentary compartments in neurons, supporting the idea that PTEN negatively regulates DBN phosphorylation at this site. We further demonstrate that neuronal activity increases phosphorylation of DBN at S647 in hippocampal neurons in vitro and in ex vivo hippocampus slices exhibiting seizure activity, potentially by inducing rapid dissociation of the PTEN:DBN complex. Our results identify a novel mechanism by which PTEN is required to maintain DBN phosphorylation at dynamic range and signifies an unusual regulation of an actin-binding protein linked to cognitive decline and degenerative conditions at the CNS synapse.
doi:10.1371/journal.pone.0071957
PMCID: PMC3733845  PMID: 23940795
9.  Stromal Claudin14-Heterozygosity, but Not Deletion, Increases Tumour Blood Leakage without Affecting Tumour Growth 
PLoS ONE  2013;8(5):e62516.
The maintenance of endothelial cell-cell junctions is vital for the control of blood vessel leakage and is known to be important in the growth and maturation of new blood vessels during angiogenesis. Here we have investigated the role of a tight junction molecule, Claudin14, in tumour blood vessel leakage, angiogenesis and tumour growth. Using syngeneic tumour models our results showed that genetic ablation of Claudin14 was not sufficient to affect tumour blood vessel morphology or function. However, and surprisingly, Claudin14-heterozygous mice displayed several blood vessel-related phenotypes including: disruption of ZO-1-positive cell-cell junctions in tumour blood vessels; abnormal distribution of basement membrane laminin around tumour blood vessels; increased intratumoural leakage and decreased intratumoural hypoxia. Additionally, although total numbers of tumour blood vessels were increased in Claudin14-heterozygous mice, and in VEGF-stimulated angiogenesis ex vivo, the number of lumenated vessels was not changed between genotypes and this correlated with no difference in syngeneic tumour growth between wild-type, Claudin14-heterozygous and Claudin14-null mice. Lastly, Claudin14-heterozygosity, but not complete deficiency, also enhanced endothelial cell proliferation significantly. These data establish a new role for Claudin14 in the regulation of tumour blood vessel integrity and angiogenesis that is evident only after the partial loss of this molecule in Claudin14-heterozyous mice but not in Claudin14-null mice.
doi:10.1371/journal.pone.0062516
PMCID: PMC3652830  PMID: 23675413
10.  Fascin promotes filopodia formation independent of its role in actin bundling 
The Journal of Cell Biology  2012;197(4):477-486.
Mutation of a critical residue of fascin eliminates the protein’s actin-bundling activity but maintains its positive role in filopodia formation
Fascin is an evolutionarily conserved actin-binding protein that plays a key role in forming filopodia. It is widely thought that this function involves fascin directly bundling actin filaments, which is controlled by an N-terminal regulatory serine residue. In this paper, by studying cellular processes in Drosophila melanogaster that require fascin activity, we identify a regulatory residue within the C-terminal region of the protein (S289). Unexpectedly, although mutation (S289A) of this residue disrupted the actin-bundling capacity of fascin, fascin S289A fully rescued filopodia formation in fascin mutant flies. Live imaging of migrating macrophages in vivo revealed that this mutation restricted the localization of fascin to the distal ends of filopodia. The corresponding mutation of human fascin (S274) similarly affected its interaction with actin and altered filopodia dynamics within carcinoma cells. These data reveal an evolutionarily conserved role for this regulatory region and unveil a function for fascin, uncoupled from actin bundling, at the distal end of filopodia.
doi:10.1083/jcb.201110135
PMCID: PMC3352952  PMID: 22564415
11.  A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability 
BMC Biology  2012;10:72.
Background
Fascin-1 is an actin crosslinking protein that is important for the assembly of cell protrusions in neurons, skeletal and smooth muscle, fibroblasts, and dendritic cells. Although absent from most normal adult epithelia, fascin-1 is upregulated in many human carcinomas, and is associated with poor prognosis because of its promotion of carcinoma cell migration, invasion, and metastasis. Rac and Cdc42 small guanine triphosphatases have been identified as upstream regulators of the association of fascin-1 with actin, but the possible role of Rho has remained obscure. Additionally, experiments have been hampered by the inability to measure the fascin-1/actin interaction directly in intact cells. We investigated the hypothesis that fascin-1 is a functional target of Rho in normal and carcinoma cells, using experimental approaches that included a novel fluorescence resonance energy transfer (FRET)/fluorescence lifetime imaging (FLIM) method to measure the interaction of fascin-1 with actin.
Results
Rho activity modulates the interaction of fascin-1 with actin, as detected by a novel FRET method, in skeletal myoblasts and human colon carcinoma cells. Mechanistically, Rho regulation depends on Rho kinase activity, is independent of the status of myosin II activity, and is not mediated by promotion of the fascin/PKC complex. The p-Lin-11/Isl-1/Mec-3 kinases (LIMK), LIMK1 and LIMK2, act downstream of Rho kinases as novel binding partners of fascin-1, and this complex regulates the stability of filopodia.
Conclusions
We have identified a novel activity of Rho in promoting a complex between fascin-1 and LIMK1/2 that modulates the interaction of fascin-1 with actin. These data provide new mechanistic insight into the intracellular coordination of contractile and protrusive actin-based structures. During the course of the study, we developed a novel FRET method for analysis of the fascin-1/actin interaction, with potential general applicability for analyzing the activities of actin-binding proteins in intact cells.
doi:10.1186/1741-7007-10-72
PMCID: PMC3488970  PMID: 22883572
12.  Dissecting cell adhesion architecture using advanced imaging techniques 
Cell Adhesion & Migration  2011;5(4):351-359.
Cell adhesion to extracellular matrix proteins or to other cells is essential for the control of embryonic development, tissue integrity, immune function and wound healing. Adhesions are tightly spatially regulated structures containing over one hundred different proteins that coordinate both dynamics and signaling events at these sites. Extensive biochemical and morphological analysis of adhesion types over the past three decades has greatly improved understanding of individual protein contributions to adhesion signaling and, in some cases, dynamics. However, it is becoming increasingly clear that these diverse macromolecular complexes contain a variety of protein sub-networks, as well as distinct sub-domains that likely play important roles in regulating adhesion behavior. Until recently, resolving these structures, which are often less than a micron in size, was hampered by the limitations of conventional light microscopy. However, recent advances in optical techniques and imaging methods have revealed exciting insight into the intricate control of adhesion structure and assembly. Here we provide an overview of the recent data arising from such studies of cell:matrix and cell:cell contact and an overview of the imaging strategies that have been applied to study the intricacies and hierarchy of proteins within adhesions.
doi:10.4161/cam.5.4.16915
PMCID: PMC3210303  PMID: 21785274
adhesion; migration; microscopy; dynamics; cytoskeleton; photobleaching; super-resolution imaging; fluorescence
13.  Quantitation of integrin receptor agonism by fluorescence lifetime imaging 
Journal of cell science  2008;121(Pt 3):265-271.
Both spatiotemporal analyses of adhesion signalling and the development of pharmacological inhibitors of integrin adhesion receptors currently suffer from the lack of an assay to measure integrin-effector binding and the response of these interactions to agonists. Here, we have expressed integrin-GFP and effector-mRFP pairs in living cells and quantified their association using FLIM to measure FRET. Talin-β1 and paxillin-α4 association was both ligand- and receptor activation state-dependent, and sensitive to inhibition with small molecule RGD and LDV mimetics, respectively. An adaptation of the assay revealed the agonistic activity of these small molecules and provides a new, quantitative assay for the screening of activity of small molecule integrin inhibitors.
doi:10.1242/jcs.018440
PMCID: PMC3328206  PMID: 18216331
14.  Differential Regulation of Adhesion Complex Turnover by ROCK1 and ROCK2 
PLoS ONE  2012;7(2):e31423.
Background
ROCK1 and ROCK2 are serine/threonine kinases that function downstream of the small GTP-binding protein RhoA. Rho signalling via ROCK regulates a number of cellular functions including organisation of the actin cytoskeleton, cell adhesion and cell migration.
Methodology/Principal Findings
In this study we use RNAi to specifically knockdown ROCK1 and ROCK2 and analyse their role in assembly of adhesion complexes in human epidermal keratinocytes. We observe that loss of ROCK1 inhibits signalling via focal adhesion kinase resulting in a failure of immature adhesion complexes to form mature stable focal adhesions. In contrast, loss of ROCK2 expression results in a significant reduction in adhesion complex turnover leading to formation of large, stable focal adhesions. Interestingly, loss of either ROCK1 or ROCK2 expression significantly impairs cell migration indicating both ROCK isoforms are required for normal keratinocyte migration.
Conclusions
ROCK1 and ROCK2 have distinct and separate roles in adhesion complex assembly and turnover in human epidermal keratinocytes.
doi:10.1371/journal.pone.0031423
PMCID: PMC3278444  PMID: 22348083
15.  SHARPIN is an endogenous inhibitor of beta1-integrin activation 
Nature Cell Biology  2011;13(11):1315-1324.
Regulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and Kindlins activate β1-integrins, but the counteracting inhibiting mechanisms are poorly defined. Here we identified SHARPIN as an important inactivator of β1-integrins in an RNAi-screen. SHARPIN inhibited β1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased β1-integrin activity which was fully rescued by re-expression of SHARPIN. SHARPIN directly bound to a conserved cytoplasmic region of integrin α-subunits and inhibited recruitment of Talin and Kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of β1-integrins from inactive to active conformations.
doi:10.1038/ncb2340
PMCID: PMC3257806  PMID: 21947080
16.  Complement Fragment C3a Controls Mutual Cell Attraction during Collective Cell Migration 
Developmental Cell  2011;21(6):1026-1037.
Summary
Collective cell migration is a mode of movement crucial for morphogenesis and cancer metastasis. However, little is known about how migratory cells coordinate collectively. Here we show that mutual cell-cell attraction (named here coattraction) is required to maintain cohesive clusters of migrating mesenchymal cells. Coattraction can counterbalance the natural tendency of cells to disperse via mechanisms such as contact inhibition and epithelial-to-mesenchymal transition. Neural crest cells are coattracted via the complement fragment C3a and its receptor C3aR, revealing an unexpected role of complement proteins in early vertebrate development. Loss of coattraction disrupts collective and coordinated movements of these cells. We propose that coattraction and contact inhibition act in concert to allow cell collectives to self-organize and respond efficiently to external signals, such as chemoattractants and repellents.
Graphical Abstract
Highlights
► Neural crest (NC) cells mutually attract each other (coattraction) ► NC coattraction is mediated by the complement fragment C3a and its receptor C3aR ► Coattraction is required for the swarm-like behavior of migratory NC cells ► Coattraction and contact inhibition suffice to generate collective migration
doi:10.1016/j.devcel.2011.10.012
PMCID: PMC3272547  PMID: 22118769
17.  CAR Modulates E-Cadherin Dynamics in the Presence of Adenovirus Type 5 
PLoS ONE  2011;6(8):e23056.
Adenovirus (Ad) serotype 5 (Ad5) fiber competitively binds to the coxsackievirus and Ad receptor (CAR) to attach Ad5 to target cells and also disrupts cell junctions and facilitates virus escape at a late stage in Ad5 infection. Here we demonstrate that paracellular permeability in MCF7 and CAR overexpressing MCF7 (FLCARMCF7) cells is increased within minutes following the addition of Ad5 to cells. This is brought about, at least in part, by altering the molecular dynamics of E-cadherin, a key component of the cell-cell adhesion complex. We also demonstrate that the increase in E-cadherin mobility is constitutively altered by the presence of CAR at FLCARMCF7 cell junctions. As increased paracellular permeability was observed early after the addition of Ad5 to cells, we postulate that this may represent a mechanism by which Ad5 could disrupt cell junctions to facilitate further access to its cell receptors.
doi:10.1371/journal.pone.0023056
PMCID: PMC3151283  PMID: 21850251
18.  Proneural Transcription Factors Regulate Different Steps of Cortical Neuron Migration through Rnd-Mediated Inhibition of RhoA Signaling 
Neuron  2011;69-540(6-7):1069-1084.
Summary
Little is known of the intracellular machinery that controls the motility of newborn neurons. We have previously shown that the proneural protein Neurog2 promotes the migration of nascent cortical neurons by inducing the expression of the atypical Rho GTPase Rnd2. Here, we show that another proneural factor, Ascl1, promotes neuronal migration in the cortex through direct regulation of a second Rnd family member, Rnd3. Both Rnd2 and Rnd3 promote neuronal migration by inhibiting RhoA signaling, but they control distinct steps of the migratory process, multipolar to bipolar transition in the intermediate zone and locomotion in the cortical plate, respectively. Interestingly, these divergent functions directly result from the distinct subcellular distributions of the two Rnd proteins. Because Rnd proteins also regulate progenitor divisions and neurite outgrowth, we propose that proneural factors, through spatiotemporal regulation of Rnd proteins, integrate the process of neuronal migration with other events in the neurogenic program.
Highlights
► The small GTPase Rnd3 is a direct target of the proneural transcription factor Ascl1 ► Rnd3 promotes cortical neuron migration by inhibiting RhoA signaling ► Rnd3 and the related protein Rnd2 have distinct roles in neuronal migration ► Rnd3 and Rnd2 have distinct subcellular distributions in cortical neurons
doi:10.1016/j.neuron.2011.02.018
PMCID: PMC3383999  PMID: 21435554
19.  Podoplanin Associates with CD44 to Promote Directional Cell Migration 
Molecular Biology of the Cell  2010;21(24):4387-4399.
Podoplanin, a cancer-associated glycoprotein, interacts with CD44. Both glycoproteins are coordinately upregulated during tumor progression. Podoplanin–CD44 interaction in the cell membrane occurs mainly in migrating cells, and it seems to be required for podoplanin-mediated cell migration and directionality.
Podoplanin is a transmembrane glycoprotein up-regulated in different human tumors, especially those derived from squamous stratified epithelia (SCCs). Its expression in tumor cells is linked to increased cell migration and invasiveness; however, the mechanisms underlying this process remain poorly understood. Here we report that CD44, the major hyaluronan (HA) receptor, is a novel partner for podoplanin. Expression of the CD44 standard isoform (CD44s) is coordinately up-regulated together with that of podoplanin during progression to highly aggressive SCCs in a mouse skin model of carcinogenesis, and during epithelial-mesenchymal transition (EMT). In carcinoma cells, CD44 and podoplanin colocalize at cell surface protrusions. Moreover, CD44 recruitment promoted by HA-coated beads or cross-linking with a specific CD44 antibody induced corecruitment of podoplanin. Podoplanin–CD44s interaction was demonstrated both by coimmunoprecipitation experiments and, in vivo, by fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM), the later confirming its association on the plasma membrane of cells with a migratory phenotype. Importantly, we also show that podoplanin promotes directional persistence of motility in epithelial cells, a feature that requires CD44, and that both molecules cooperate to promote directional migration in SCC cells. Our results support a role for CD44-podoplanin interaction in driving tumor cell migration during malignancy.
doi:10.1091/mbc.E10-06-0489
PMCID: PMC3002391  PMID: 20962267
20.  Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics 
Nature  2010;466(7303):263-266.
Mechanical forces are central to developmental, physiological and pathological processes1. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with pico-Newton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy2. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent3. We show that tension across vinculin in stable FAs is ~2.5 pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently.
doi:10.1038/nature09198
PMCID: PMC2901888  PMID: 20613844
21.  Endothelial FAK is required for tumour angiogenesis 
EMBO Molecular Medicine  2010;2(12):516-528.
Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a fundamental role in integrin and growth factor mediated signalling and is an important player in cell migration and proliferation, processes vital for angiogenesis. However, the role of FAK in adult pathological angiogenesis is unknown. We have generated endothelial-specific tamoxifen-inducible FAK knockout mice by crossing FAK-floxed (FAKfl/fl) mice with the platelet derived growth factor b (Pdgfb)-iCreER mice. Tamoxifen-treatment of Pdgfb-iCreER;FAKfl/fl mice results in FAK deletion in adult endothelial cells (ECs) without any adverse effects. Importantly however, endothelial FAK-deletion in adult mice inhibited tumour growth and reduced tumour angiogenesis. Furthermore, in in vivo angiogenic assays FAK deletion impairs vascular endothelial growth factor (VEGF)-induced neovascularization. In addition, in vitro deletion of FAK in ECs resulted in reduced VEGF-stimulated Akt phosphorylation and correlating reduced cellular proliferation as well as increased cell death. Our data suggest that FAK is required for adult pathological angiogenesis and validates FAK as a possible target for anti-angiogenic therapies.
doi:10.1002/emmm.201000106
PMCID: PMC3377344  PMID: 21154724
angiogenesis; cancer; endothelial; FAK; tumour
22.  αvβ3 integrin spatially regulates VASP and RIAM to control adhesion dynamics and migration 
The Journal of Cell Biology  2010;189(2):369-383.
Loss of β3 integrin enhances turnover of focal adhesions and cell migration speed due to increased β1 integrin–talin interactions.
Integrins are fundamental to the control of protrusion and motility in adherent cells. However, the mechanisms by which specific members of this receptor family cooperate in signaling to cytoskeletal and adhesion dynamics are poorly understood. Here, we show that the loss of β3 integrin in fibroblasts results in enhanced focal adhesion turnover and migration speed but impaired directional motility on both 2D and 3D matrices. These motility defects are coupled with an increased rate of actin-based protrusion. Analysis of downstream signaling events reveals that loss of β3 integrin results in a loss of protein kinase A–dependent phosphorylation of the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP). Dephosphorylated VASP in β3-null cells is preferentially associated with Rap1-GTP–interacting adaptor molecule (RIAM) both in vitro and in vivo, which leads to enhanced formation of a VASP–RIAM complex at focal adhesions and subsequent increased binding of talin to β1 integrin. These data demonstrate a novel mechanism by which αvβ3 integrin acts to locally suppress β1 integrin activation and regulate protrusion, adhesion dynamics, and persistent migration.
doi:10.1083/jcb.200912014
PMCID: PMC2856911  PMID: 20404115
23.  Collective Chemotaxis Requires Contact-Dependent Cell Polarity 
Developmental Cell  2010;19(1):39-53.
Summary
Directional collective migration is now a widely recognized mode of migration during embryogenesis and cancer. However, how a cluster of cells responds to chemoattractants is not fully understood. Neural crest cells are among the most motile cells in the embryo, and their behavior has been likened to malignant invasion. Here, we show that neural crest cells are collectively attracted toward the chemokine Sdf1. While not involved in initially polarizing cells, Sdf1 directionally stabilizes cell protrusions promoted by cell contact. At this cell contact, N-cadherin inhibits protrusion and Rac1 activity and in turn promotes protrusions and activation of Rac1 at the free edge. These results show a role for N-cadherin during contact inhibition of locomotion, and they reveal a mechanism of chemoattraction likely to function during both embryogenesis and cancer metastasis, whereby attractants such as Sdf1 amplify and stabilize contact-dependent cell polarity, resulting in directional collective migration.
Graphical Abstract
Highlights
► Neural crest (NC) cells undergo collective chemotaxis toward Sdf1 ► N-cadherin-dependent contacts are required for NC cell chemotaxis toward Sdf1 ► N-cadherin regulates contact inhibition of locomotion by controlling small GTPases ► Sdf1 reinforces cell polarity induced by N-cadherin/contact inhibition
doi:10.1016/j.devcel.2010.06.012
PMCID: PMC2913244  PMID: 20643349
SIGNALING; CELLBIO; PROTEINS
24.  MyosinV controls PTEN function and neuronal cell size 
Nature cell biology  2009;11(10):1191-1196.
The tumour suppressor PTEN can inhibit proliferation and migration as well as control cell growth in different cell types1. PTEN functions predominately as a lipid phsophatase, converting PI(3,4,5)P3 to PI(4,5)P2, thereby antagonizing PI3K (Phosphoinositide 3-kinase) and its established downstream effector pathways2. However, much is unclear concerning the mechanisms that regulate PTEN movement to the cell membrane necessary for PTEN’s activity towards PI(3,4,5)P33-5.
Here we show a requirement for functional motor proteins in the control of PI3K signalling, involving a previously unknown association between PTEN and MyosinV. FRET measurements revealed that PTEN interacts directly with MyosinV, dependent on PTEN phosphorylation mediated by CK2 and/or GSK3. Inactivation of MyosinV-transport function in neurons increased cell size, which – in line with known attributes of PTEN-loss6, 7 - required PI3K and mTor. Our data demonstrate a myosin-based transport mechanism regulating PTEN function, providing new insights into the signalling networks regulating cell growth.
doi:10.1038/ncb1961
PMCID: PMC2756284  PMID: 19767745
25.  R-Ras regulates β1-integrin trafficking via effects on membrane ruffling and endocytosis 
BMC Cell Biology  2010;11:14.
Background
Integrin-mediated cell adhesion and spreading is dramatically enhanced by activation of the small GTPase, R-Ras. Moreover, R-Ras localizes to the leading edge of migrating cells, and regulates membrane protrusion. The exact mechanisms by which R-Ras regulates integrin function are not fully known. Nor is much known about the spatiotemporal relationship between these two molecules, an understanding of which may provide insight into R-Ras regulation of integrins.
Results
GFP-R-Ras localized to the plasma membrane, most specifically in membrane ruffles, in Cos-7 cells. GFP-R-Ras was endocytosed from these ruffles, and trafficked via multiple pathways, one of which involved large, acidic vesicles that were positive for Rab11. Cells transfected with a dominant negative form of GFP-R-Ras did not form ruffles, had decreased cell spreading, and contained numerous, non-trafficking small vesicles. Conversely, cells transfected with the constitutively active form of GFP-R-Ras contained a greater number of ruffles and large vesicles compared to wild-type transfected cells. Ruffle formation was inhibited by knock-down of endogenous R-Ras with siRNA, suggesting that activated R-Ras is not just a component of, but also an architect of ruffle formation. Importantly, β1-integrin co-localized with endogenous R-Ras in ruffles and endocytosed vesicles. Expression of dominant negative R-Ras or knock down of R-Ras by siRNA prevented integrin accumulation into ruffles, impaired endocytosis of β1-integrin, and decreased β1-integrin-mediated adhesion. Knock-down of R-Ras also perturbed the dynamics of another membrane-localized protein, GFP-VSVG, suggesting a more global role for R-Ras on membrane dynamics. However, while R-Ras co-internalized with integrins, it did not traffic with VSVG, which instead moved laterally out of ruffles within the plane of the membrane, suggesting multiple levels of regulation of and by R-Ras.
Conclusions
Our results suggest that integrin function involves integrin trafficking via a cycle of membrane protrusion, ruffling, and endocytosis regulated by R-Ras, providing a novel mechanism by which integrins are linked to R-Ras through control of membrane dynamics.
doi:10.1186/1471-2121-11-14
PMCID: PMC2830936  PMID: 20167113

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