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2.  ADAM8 as a drug target in Pancreatic Cancer 
Nature communications  2015;6:6175.
Pancreatic ductal adenocarcinoma (PDAC) has a grim prognosis with less than 5% survivors after 5 years. High expression levels of ADAM8, a metalloprotease-disintegrin, are correlated with poor clinical outcome. We show that ADAM8 expression is associated with increased migration and invasiveness of PDAC cells caused by activation of ERK 1/2 and higher MMP activities. For biological function, ADAM8 requires multimerisation and associates with β1-integrin on the cell surface. A peptidomimetic ADAM8 inhibitor, BK-1361, designed by structural modelling of the disintegrin domain, prevents ADAM8 multimerisation. In PDAC cells, BK-1361 affects ADAM8 function leading to reduced invasiveness, and less ERK 1/2 and MMP activation. BK-1361 application in mice decreased tumour burden and metastasis of implanted pancreatic tumour cells and provides improved metrics of clinical symptoms and survival in a KrasG12D-driven mouse model of PDAC. Thus, our data integrate ADAM8 in pancreatic cancer signalling and validate ADAM8 as a target for PDAC therapy.
doi:10.1038/ncomms7175
PMCID: PMC5014123  PMID: 25629724
3.  Fascin Regulates Nuclear Movement and Deformation in Migrating Cells 
Developmental Cell  2016;38(4):371-383.
Summary
Fascin is an F-actin-bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells, and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers. Here, we identified the nuclear envelope protein nesprin-2 as a binding partner for fascin in a range of cell types in vitro and in vivo. Nesprin-2 interacts with fascin through a direct, F-actin-independent interaction, and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery. Moreover, disrupting the interaction between fascin and nesprin-2 C-terminal domain leads to specific defects in F-actin coupling to the nuclear envelope, nuclear movement, and the ability of cells to deform their nucleus to invade through confined spaces. Together, our results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion.
Graphical Abstract
Highlights
•Fascin binds directly to nesprin-2 at the nuclear envelope•Fascin-nesprin-2 binding occurs independently of fascin-actin bundling•The fascin-nesprin-2 complex regulates nuclear movement in migration•Uncoupling the fascin-nesprin complex reduces nuclear deformation and cell invasion
Fascin is an F-actin-bundling protein that is significantly upregulated in most cancers and correlates with poor prognosis. Jayo et al. show that fascin can associate directly with the nuclear envelope protein nesprin-2. This complex is required for nuclear movement and deformation during cell invasion through 3D matrices.
doi:10.1016/j.devcel.2016.07.021
PMCID: PMC4997957  PMID: 27554857
4.  TNFα promotes CAR-dependent migration of leukocytes across epithelial monolayers 
Scientific Reports  2016;6:26321.
Trans-epithelial migration (TEpM) of leukocytes during inflammation requires engagement with receptors expressed on the basolateral surface of the epithelium. One such receptor is Coxsackie and Adenovirus Receptor (CAR) that binds to Junctional Adhesion Molecule-like (JAM-L) expressed on leukocytes. Here we provide the first evidence that efficient TEpM of monocyte-derived THP-1 cells requires and is controlled by phosphorylation of CAR. We show that TNFα acts in a paracrine manner on epithelial cells via a TNFR1-PI3K-PKCδ pathway leading to CAR phosphorylation and subsequent transmigration across cell junctions. Moreover, we show that CAR is hyper-phosphorylated in vivo in acute and chronic lung inflammation models and this response is required to facilitate immune cell recruitment. This represents a novel mechanism of feedback between leukocytes and epithelial cells during TEpM and may be important in controlling responses to pro-inflammatory cytokines in pathological settings.
doi:10.1038/srep26321
PMCID: PMC4872059  PMID: 27193388
5.  Podoplanin mediates ECM degradation by squamous carcinoma cells through control of invadopodia stability 
Oncogene  2014;34(34):4531-4544.
Invadopodia are actin-rich cell membrane projections used by invasive cells to penetrate the basement membrane. Control of invadopodia stability is critical for efficient degradation of the extracellular matrix (ECM); however, the underlying molecular mechanisms remain poorly understood. Here, we uncover a new role for podoplanin, a transmembrane glycoprotein closely associated with malignant progression of squamous cell carcinomas (SCCs), in the regulation of invadopodia-mediated matrix degradation. Podoplanin downregulation in SCC cells impairs invadopodia stability, thereby reducing the efficiency of ECM degradation. We report podoplanin as a novel component of invadopodia-associated adhesion rings, where it clusters prior to matrix degradation. Early podoplanin recruitment to invadopodia is dependent on lipid rafts, whereas ezrin/moesin proteins mediate podoplanin ring assembly. Finally, we demonstrate that podoplanin regulates invadopodia maturation by acting upstream of the ROCK-LIMK-Cofilin pathway through the control of RhoC GTPase activity. Thus, podoplanin plays a key role in the regulation of invadopodia function in SCC cells, controlling the initial steps of cancer cell invasion.
doi:10.1038/onc.2014.388
PMCID: PMC4430312  PMID: 25486435
matrix degradation; cancer cell invasion; podoplanin; SCCs; invadopodia
6.  A direct interaction between fascin and microtubules contributes to adhesion dynamics and cell migration 
Journal of Cell Science  2015;128(24):4601-4614.
ABSTRACT
Fascin is an actin-binding and bundling protein that is highly upregulated in most epithelial cancers. Fascin promotes cell migration and adhesion dynamics in vitro and tumour cell metastasis in vivo. However, potential non-actin bundling roles for fascin remain unknown. Here, we show for the first time that fascin can directly interact with the microtubule cytoskeleton and that this does not depend upon fascin-actin bundling. Microtubule binding contributes to fascin-dependent control of focal adhesion dynamics and cell migration speed. We also show that fascin forms a complex with focal adhesion kinase (FAK, also known as PTK2) and Src, and that this signalling pathway lies downstream of fascin–microtubule association in the control of adhesion stability. These findings shed light on new non actin-dependent roles for fascin and might have implications for the design of therapies to target fascin in metastatic disease.
Summary: Fascin associates directly with microtubules independently of F-actin binding, and this contributes to microtubule dynamics, adhesion assembly and cell migration.
doi:10.1242/jcs.175760
PMCID: PMC4696496  PMID: 26542021
Fascin; Cytoskeleton; Actin; Microtubule; Focal adhesion; Migration; Focal adhesion kinase
7.  Mutually Exclusive Roles of SHARPIN in Integrin Inactivation and NF-κB Signaling 
PLoS ONE  2015;10(11):e0143423.
SHANK-associated RH domain interactor (SHARPIN) inhibits integrins through interaction with the integrin α-subunit. In addition, SHARPIN enhances nuclear factor-kappaB (NF-κB) activity as a component of the linear ubiquitin chain assembly complex (LUBAC). However, it is currently unclear how regulation of these seemingly different roles is coordinated. Here, we show that SHARPIN binds integrin and LUBAC in a mutually exclusive manner. We map the integrin binding site on SHARPIN to the ubiquitin-like (UBL) domain, the same domain implicated in SHARPIN interaction with LUBAC component RNF31 (ring finger protein 31), and identify two SHARPIN residues (V267, L276) required for both integrin and RNF31 regulation. Accordingly, the integrin α-tail is capable of competing with RNF31 for SHARPIN binding in vitro. Importantly, the full SHARPIN RNF31-binding site contains residues (F263A/I272A) that are dispensable for SHARPIN-integrin interaction. Importantly, disrupting SHARPIN interaction with integrin or RNF31 abolishes SHARPIN-mediated regulation of integrin or NF-κB activity, respectively. Altogether these data suggest that the roles of SHARPIN in inhibiting integrin activity and supporting linear ubiquitination are (molecularly) distinct.
doi:10.1371/journal.pone.0143423
PMCID: PMC4658161  PMID: 26600301
8.  Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells 
Biomedical Optics Express  2015;6(10):3842-3854.
We present a novel integrated multimodal fluorescence microscopy technique for simultaneous fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging (FAIM). This approach captures a series of polarization-resolved fluorescence lifetime images during a FRAP recovery, maximizing the information available from a limited photon budget. We have applied this method to analyse the behaviour of GFP-labelled coxsackievirus and adenovirus receptor (CAR) in living human epithelial cells. Our data reveal that CAR exists in oligomeric states throughout the cell, and that these complexes occur in conjunction with high immobile fractions of the receptor at cell-cell junctions. These findings shed light on previously unknown molecular associations between CAR receptors in intact cells and demonstrate the power of combined FRAP, FLIM and FAIM microscopy as a robust method to analyse complex multi-component dynamics in living cells.
doi:10.1364/BOE.6.003842
PMCID: PMC4605044  PMID: 26504635
(180.0180) Microscopy; (300.0300) Spectroscopy; (170.2520) Fluorescence microscopy; (170.6920) Time-resolved imaging; (260.2160) Energy transfer; (260.5430) Polarization
9.  Cadherin Switch during EMT in Neural Crest Cells Leads to Contact Inhibition of Locomotion via Repolarization of Forces 
Developmental Cell  2015;34(4):421-434.
Summary
Contact inhibition of locomotion (CIL) is the process through which cells move away from each other after cell-cell contact, and it contributes to malignant invasion and developmental migration. Various cell types exhibit CIL, whereas others remain in contact after collision and may form stable junctions. To investigate what determines this differential behavior, we study neural crest cells, a migratory stem cell population whose invasiveness has been likened to cancer metastasis. By comparing pre-migratory and migratory neural crest cells, we show that the switch from E- to N-cadherin during EMT is essential for acquisition of CIL behavior. Loss of E-cadherin leads to repolarization of protrusions, via p120 and Rac1, resulting in a redistribution of forces from intercellular tension to cell-matrix adhesions, which break down the cadherin junction. These data provide insight into the balance of physical forces that contributes to CIL in cells in vivo.
Graphical Abstract
Highlights
•Neural crest cells acquire contact inhibition of locomotion (CIL) during EMT•An E- to N-cadherin switch controls CIL•E-cadherin represses CIL by controlling Rac1-dependent protrusions via p120•During CIL, forces are redistributed from intercellular junctions to cell matrix
Cell-cell adhesions are transiently formed during contact inhibition of locomotion. Scarpa et al. show that an E- to N-cadherin switch controls CIL acquisition upon EMT. E-cadherin loss leads to repolarization of protrusions via p120 and Rac1, resulting in redistribution of forces from intercellular tension to cell-matrix adhesions, triggering junction disassembly.
doi:10.1016/j.devcel.2015.06.012
PMCID: PMC4552721  PMID: 26235046
10.  An open access microfluidic device for the study of the physical limits of cancer cell deformation during migration in confined environments 
Microelectronic Engineering  2015;144:42-45.
Graphical abstract
Highlights
•A microfluidic device to study cell and nuclear deformation during translocation.•Adherent cells protrude their cytoplasm regardless of the channel cross-section.•The nucleus acts as a limiting factor when the channel area is below 7 × 5 μm2.
During metastasis, cancerous cells leave the primary tumour, pass into the circulatory system, and invade into new tissues. To migrate through the wide variety of environments they encounter, the cells must be able to remodel their cell shape efficiently to squeeze through small gaps in the extracellular matrix or extravasate into the blood stream or lymphatic system. Several studies have shown that the nucleus is the main limiting factor to migration through small gaps (Wolf et al., 2013; Harada et al., 2014; Mak et al., 2013). To understand the physical limits of cancer cell translocation in confined environments, we have fabricated a microfluidic device to study their ability to adapt their nuclear and cellular shape when passing through small gaps. The device is open access for ease of use and enables examination of the effect of different levels of spatial confinement on cell behaviour and morphology simultaneously. The results show that increasing cell confinement decreases the ability of cells to translocate into small gaps and that cells cannot penetrate into the microchannels below a threshold cross-section.
doi:10.1016/j.mee.2015.02.022
PMCID: PMC4567073  PMID: 26412914
Microfluidics; Cell deformation; Breast cancer cells; Multilayer photolithography
11.  Prostaglandins regulate nuclear localization of Fascin and its function in nucleolar architecture 
Molecular Biology of the Cell  2015;26(10):1901-1917.
Fascin, a conserved actin-bundling protein, is not only cytoplasmic but also localizes to the nucleus and nuclear periphery in both Drosophila and mammalian cell contexts. In Drosophila, prostaglandin signaling regulates this localization. In addition, Fascin plays a critical role in nucleolar architecture in both Drosophila and mammalian cells.
Fascin, a highly conserved actin-bundling protein, localizes and functions at new cellular sites in both Drosophila and multiple mammalian cell types. During Drosophila follicle development, in addition to being cytoplasmic, Fascin is in the nuclei of the germline-derived nurse cells during stages 10B–12 (S10B–12) and at the nuclear periphery during stage 13 (S13). This localization is specific to Fascin, as other actin-binding proteins, Villin and Profilin, do not exhibit the same subcellular distribution. In addition, localization of fascin1 to the nucleus and nuclear periphery is observed in multiple mammalian cell types. Thus the regulation and function of Fascin at these new cellular locations is likely to be highly conserved. In Drosophila, loss of prostaglandin signaling causes a global reduction in nuclear Fascin and a failure to relocalize to the nuclear periphery. Alterations in nuclear Fascin levels result in defects in nucleolar morphology in both Drosophila follicles and cultured mammalian cells, suggesting that nuclear Fascin plays an important role in nucleolar architecture. Given the numerous roles of Fascin in development and disease, including cancer, our novel finding that Fascin has functions within the nucleus sheds new light on the potential roles of Fascin in these contexts.
doi:10.1091/mbc.E14-09-1384
PMCID: PMC4436834  PMID: 25808493
12.  Epithelial inflammation resulting from an inherited loss-of-function mutation in EGFR 
Epidermal growth factor receptor (EGFR) signaling is fundamentally important for tissue homeostasis through EGFR/ligand interactions that stimulate numerous signal transduction pathways. Aberrant EGFR signaling has been reported in inflammatory and malignant diseases but thus far no primary inherited defects in EGFR have been recorded. Using whole-exome sequencing, we identified a homozygous loss-of-function missense mutation in EGFR (c.1283G>A; p.Gly428Asp) in a male infant with life-long inflammation affecting the skin, bowel and lungs. During the first year of life, his skin showed erosions, dry scale, and alopecia. Subsequently, there were numerous papules and pustules – similar to the rash seen in patients receiving EGFR inhibitor drugs. Skin biopsy demonstrated an altered cellular distribution of EGFR in the epidermis with reduced cell membrane labeling, and in vitro analysis of the mutant receptor revealed abrogated EGFR phosphorylation and EGF-stimulated downstream signaling. Microarray analysis on the patient’s skin highlighted disturbed differentiation/premature terminal differentiation of keratinocytes and upregulation of several inflammatory/innate immune response networks. The boy died aged 2.5 years from extensive skin and chest infections as well as electrolyte imbalance. This case highlights the major mechanism of epithelial dysfunction following EGFR signaling ablation and illustrates the broader impact of EGFR inhibition on other tissues.
doi:10.1038/jid.2014.164
PMCID: PMC4090136  PMID: 24691054
13.  Alternative Mechanisms for Talin to Mediate Integrin Function 
Current Biology  2015;25(7):847-857.
Summary
Cell-matrix adhesion is essential for building animals, promoting tissue cohesion, and enabling cells to migrate and resist mechanical force. Talin is an intracellular protein that is critical for linking integrin extracellular-matrix receptors to the actin cytoskeleton. A key question raised by structure-function studies is whether talin, which is critical for all integrin-mediated adhesion, acts in the same way in every context. We show that distinct combinations of talin domains are required for each of three different integrin functions during Drosophila development. The partial function of some mutant talins requires vinculin, indicating that recruitment of vinculin allows talin to duplicate its own activities. The different requirements are best explained by alternative mechanisms of talin function, with talin using one or both of its integrin-binding sites. We confirmed these alternatives by showing that the proximity between the second integrin-binding site and integrins differs, suggesting that talin adopts different orientations relative to integrins. Finally, we show that vinculin and actomyosin activity help change talin’s orientation. These findings demonstrate that the mechanism of talin function differs in each developmental context examined. The different arrangements of the talin molecule relative to integrins suggest that talin is able to sense different force vectors, either parallel or perpendicular to the membrane. This provides a paradigm for proteins whose apparent uniform function is in fact achieved by a variety of distinct mechanisms involving different molecular architectures.
Graphical Abstract
Highlights
•Integrin function requires distinct sets of talin domains in three different tissues•Vinculin helps talin retain function when domains are removed•Talin IBS2 is separated from integrins in muscle but not wing adhesion sites•Vinculin and actomyosin contribute to separating IBS2 from integrins
Klapholz et al. show that talin domains have variable importance in different developmental events in Drosophila, suggesting distinct mechanisms of action, with altered configurations of talin, vinculin, and integrins. These were validated by advanced imaging in vivo. Such diverse configurations may allow talin to sense a variety of forces.
doi:10.1016/j.cub.2015.01.043
PMCID: PMC4386027  PMID: 25754646
14.  Golgi Anti-apoptotic Proteins Are Highly Conserved Ion Channels That Affect Apoptosis and Cell Migration* 
The Journal of Biological Chemistry  2015;290(18):11785-11801.
Background: GAAPs regulate intracellular Ca2+ fluxes, cell migration, and apoptosis.
Results: GAAP forms a cation-selective channel, and residues involved in its ion-conducting properties were identified.
Conclusion: Mutations within the pore demonstrate that GAAP effects on apoptosis and migration are separable.
Significance: Characterization of the pore region of GAAP provides insight into the mechanism of action of this novel and highly conserved ion channel.
Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca2+ content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently.
doi:10.1074/jbc.M115.637306
PMCID: PMC4416878  PMID: 25713081
Electrophysiology; Ion Channel; Lipid Bilayer; Membrane Protein; Viral Protein
15.  In vivo collective cell migration requires an LPAR2-dependent increase in tissue fluidity 
The Journal of Cell Biology  2014;206(1):113-127.
Neural crest epithelial–mesenchymal transition (EMT) and collective cell migration rely on a solid-to-liquid-like transition triggered by internalization of N-cadherin downstream of lysophosphatidic acid receptor 2.
Collective cell migration (CCM) and epithelial–mesenchymal transition (EMT) are common to cancer and morphogenesis, and are often considered to be mutually exclusive in spite of the fact that many cancer and embryonic cells that have gone through EMT still cooperate to migrate collectively. Here we use neural crest (NC) cells to address the question of how cells that have down-regulated cell–cell adhesions can migrate collectively. NC cell dissociation relies on a qualitative and quantitative change of the cadherin repertoire. We found that the level of cell–cell adhesion is precisely regulated by internalization of N-cadherin downstream of lysophosphatidic acid (LPA) receptor 2. Rather than promoting the generation of single, fully mesenchymal cells, this reduction of membrane N-cadherin only triggers a partial mesenchymal phenotype. This intermediate phenotype is characterized by an increase in tissue fluidity akin to a solid-like–to–fluid-like transition. This change of plasticity allows cells to migrate under physical constraints without abolishing cell cooperation required for collectiveness.
doi:10.1083/jcb.201402093
PMCID: PMC4085712  PMID: 25002680
16.  An antagonistic interaction between PlexinB2 and Rnd3 controls RhoA activity and cortical neuron migration 
Nature communications  2014;5:3405.
A transcriptional programme initiated by the proneural factors Neurog2 and Ascl1 controls successive steps of neurogenesis in the embryonic cerebral cortex. Previous work has shown that proneural factors also confer a migratory behaviour to cortical neurons by inducing the expression of the small GTP-binding proteins Rnd2 and Rnd3. However, the directionality of radial migration suggests that migrating neurons also respond to extracellular signal-regulated pathways. Here we show that the Plexin B2 receptor interacts physically and functionally with Rnd3 and stimulates RhoA activity in migrating cortical neurons. Plexin B2 competes with p190RhoGAP for binding to Rnd3, thus blocking the Rnd3-mediated inhibition of RhoA and also recruits RhoGEFs to directly stimulate RhoA activity. Thus, an interaction between a cell-extrinsic Plexin signaling pathway and the cell-intrinsic Ascl1-Rnd3 pathway determines the level of RhoA activity appropriate for cortical neuron migration.
doi:10.1038/ncomms4405
PMCID: PMC3939360  PMID: 24572910
17.  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
18.  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
19.  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
20.  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
21.  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
23.  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
24.  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
25.  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

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