Ciska Boucherie

Functional complexes between YAP2 and ZO-2 are PDZ domain-dependent, and regulate YAP2 nuclear localization and signalling.

The Hippo pathway regulates the size of organs by controlling two opposing processes: proliferation and apoptosis. YAP2 (Yes kinase-associated protein 2), one of the three isoforms of YAP, is a WW domain-containing transcriptional co-activator that acts as the effector of the Hippo pathway in mammalian cells. In addition to WW domains, YAP2 has a PDZ-binding motif at its C-terminus. We reported previously that this motif was necessary for YAP2 localization in the nucleus and for promoting cell detachment and apoptosis. In the present study, we show that the tight junction protein ZO (zonula occludens)-2 uses its first PDZ domain to form a complex with YAP2. The endogenous ZO-2 and YAP2 proteins co-localize in the nucleus. We also found that ZO-2 facilitates the nuclear localization and pro-apoptotic function of YAP2, and that this activity of ZO-2 is PDZ-domain-dependent. The present paper is the first report on a PDZ-based nuclear translocation mechanism. Moreover, since the Hippo pathway acts as a tumour suppressor pathway, the YAP2-ZO-2 complex could represent a target for cancer therapy.

Gelsolin-induced epithelial cell invasion is dependent on Ras-Rac signaling.

Gelsolin is a widely distributed actin binding protein involved in controlling cell morphology, motility, signaling and apoptosis. The role of gelsolin in tumor progression, however, remains poorly understood. Here we show that expression of green fluorescent pro tein (GFP)‐tagged gelsolin in MDCK‐AZ, MDCKtsSrc or HEK293T cells promotes invasion into collagen type I. In organ culture assays, MDCK cells expressing gelsolin–GFP invaded pre‐cultured chick heart fragments. Gelsolin expression inhibited E‐cadherin‐mediated cell aggregation but did not disrupt the E‐cadherin–catenin complex. Co‐expression of dominant‐negative Rac1N17, but not RhoAN19 or Cdc42N17, counteracted gelsolin‐induced invasion, suggesting a requirement for Rac1 activity. Increased ARF6, PLD or PIP5K 1α activity canceled out gelsolin‐induced invasion. Furthermore, we found that invasion induced by gelsolin is dependent on Ras activity, acting through the PI3K–Rac pathway via the Ras guanine nucleotide exchange factor Sos‐1. These findings establish a connection between gelsolin and the Ras oncogenic signaling pathway.

Increased importin-beta-dependent nuclear import of the actin modulating protein CapG promotes cell invasion

CapG (gCap39) is a ubiquitous gelsolin-family actin modulating protein involved in cell signalling, receptor-mediated membrane ruffling, phagocytosis and motility. CapG is the only gelsolin-related actin binding protein that localizes constitutively to both nucleus and cytoplasm. Structurally related proteins like severin and fragmin are cytoplasmic because they contain a nuclear export sequence that is absent in CapG. Increased CapG expression has been reported in some cancers but a causal role for CapG in tumour development, including invasion and metastasis, has not been explored. We show that moderate expression of green fluorescent protein-tagged CapG (CapG-EGFP) in epithelial cells induces invasion into collagen type I and precultured chick heart fragments. Nuclear export sequence-tagged CapG-EGFP fails to induce invasion, whereas point mutations in the nuclear export sequence permitting nuclear re-entry restore cellular invasion. Nuclear import of CapG is energy-dependent and requires the cytosolic receptor importin β but not importin α. Nuclear CapG does not possess intrinsic transactivation activity but suppresses VP16 transactivation of a luciferase reporter gene in a dose-dependent manner. Furthermore, invasion requires signalling through the Ras-phosphoinositide 3-kinase pathway and Cdc42 or RhoA, but not Rac1. We show for the first time active nuclear import of an actin binding protein, and our findings point to a role for nuclear CapG in eliciting invasion, possibly through interfering with the cellular transcription machinery.

TAZ interacts with zonula occludens-1 and -2 proteins in a PDZ-1 dependent manner.

MINT‐7994937: ZO‐2 (uniprotkb:Q95168) binds (MI:0407) to TAZ (uniprotkb:Q9EPK5) by pull down (MI:0096) MINT‐7994900, MINT‐7994835, MINT‐7994885: ZO‐1 (uniprotkb:Q07157) physically interacts (MI:0915) with TAZ (uniprotkb:Q9EPK5) by pull down (MI:0096) MINT‐7995020: ZO‐2 (uniprotkb:Q9UDY2) and TAZ (uniprotkb:Q9GZV5) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT‐7994953: ZO‐1 (uniprotkb:Q07157) binds (MI:0407) to TAZ (uniprotkb:Q9EPK5) by pull down (MI:0096) MINT‐7994970: TAZ (uniprotkb:73990382) and ZO‐2 (uniprotkb:Q95168) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT‐7994867: TAZ (uniprotkb:Q9EPK5) physically interacts (MI:0915) with ZO‐2 (uniprotkb:Q9UDY2) by pull down (MI:0096) MINT‐7994988: TAZ (uniprotkb:Q9GZV5) and ZO‐1 (uniprotkb:Q07157) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT‐7994999: TAZ (uniprotkb:Q9EPK5) and ZO‐2 (uniprotkb:Q95168) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT‐7994922, MINT‐7994853: ZO‐2 (uniprotkb:Q95168) physically interacts (MI:0915) with TAZ (uniprotkb:Q9EPK5) by pull down (MI:0096)

A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis

IntroductionAberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer. Indirect evidence suggests that CapG is involved in tumor cell dissemination and metastasis. In this study, we used llama-derived CapG single-domain antibodies or nanobodies in a breast cancer metastasis model to address whether inhibition of CapG activity holds therapeutic merit.MethodsWe raised single-domain antibodies (nanobodies) against human CapG and used these as intrabodies (immunomodulation) after lentiviral transduction of breast cancer cells. Functional characterization of nanobodies was performed to identify which biochemical properties of CapG are perturbed. Orthotopic and tail vein in vivo models of metastasis in nude mice were used to assess cancer cell spreading.ResultsWith G-actin and F-actin binding assays, we identified a CapG nanobody that binds with nanomolar affinity to the first CapG domain. Consequently, CapG interaction with actin monomers or actin filaments is blocked. Intracellular delocalization experiments demonstrated that the nanobody interacts with CapG in the cytoplasmic environment. Expression of the nanobody in breast cancer cells restrained cell migration and Matrigel invasion. Notably, the nanobody prevented formation of lung metastatic lesions in orthotopic xenograft and tail-vein models of metastasis in immunodeficient mice. We showed that CapG nanobodies can be delivered into cancer cells by using bacteria harboring a type III protein secretion system (T3SS).ConclusionsCapG inhibition strongly reduces breast cancer metastasis. A nanobody-based approach offers a fast track for gauging the therapeutic merit of drug targets. Mapping of the nanobody-CapG interface may provide a platform for rational design of pharmacologic compounds.

The PDZ2 domain of zonula occludens-1 and -2 is a phosphoinositide binding domain

Zonula occludens proteins (ZO) are postsynaptic density protein-95 discs large-zonula occludens (PDZ) domain-containing proteins that play a fundamental role in the assembly of tight junctions and establishment of cell polarity. Here, we show that the second PDZ domain of ZO-1 and ZO-2 binds phosphoinositides (PtdInsP) and we identified critical residues involved in the interaction. Furthermore, peptide and PtdInsP binding of ZO PDZ2 domains are mutually exclusive. Although lipid binding does not seem to be required for plasma membrane localisation of ZO-1, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) binding to the PDZ2 domain of ZO-2 regulates ZO-2 recruitment to nuclear speckles. Knockdown of ZO-2 expression disrupts speckle morphology, indicating that ZO-2 might play an active role in formation and stabilisation of these subnuclear structures. This study shows for the first time that ZO isoforms bind PtdInsPs and offers an alternative regulatory mechanism for the formation and stabilisation of protein complexes in the nucleus.

Stratifying fascin and cortactin function in invadopodium formation using inhibitory nanobodies and targeted subcellular delocalization

Invadopodia are actin‐rich protrusions arising through the orchestrated regulation of precursor assembly, stabilization, and maturation, endowing cancer cells with invasive properties. Using nanobodies (antigen‐binding domains of Camelid heavy‐chain antibodies) as perturbators of intracellular functions and/or protein domains at the level of the endogenous protein, we examined the specific contribution of fascin and cortactin during invadopodium formation in MDA‐MB‐231 breast and PC‐3 prostate cancer cells. A nanobody (Kd~35 nM, 1:1 stoichiometry) that disrupts fascin F‐actin bundling emphasizes the importance of stable actin bundles in invadopodium array organization and turnover, matrix degradation, and cancer cell invasion. Cortactin‐SH3 dependent WIP recruitment toward the plasma membrane was specifically inhibited by a cortactin nanobody (Kd~75 nM, 1:1 stoichiometry). This functional domain is shown to be important for formation of properly organized invadopodia, MMP‐9 secretion, matrix degradation, and cancer cell invasion. Notably, using a subcellular delocalization strategy to trigger protein loss of function, we uncovered a fascin‐bundling‐independent role in MMP‐9 secretion. Hence, we demonstrate that nanobodies enable high resolution protein function mapping in cells.—Van Audenhove, I., Boucherie, C., Pieters, L., Zwaenepoel, O., Vanloo, B., Martens, E., Verbrugge, C., Hassanzadeh‐Ghassabeh, G., Vandekerckhove, J., Cornelissen, M., De Ganck, A., Gettemans, J. Stratifying fascin and cortactin function in invadopodium formation using inhibitory nanobodies and targeted subcellular delocalization. FASEB J. 28, 1805–1818 (2014). www.fasebj.org

A New Role for Nuclear Transport Factor 2 and Ran: Nuclear Import of CapG

The small GTPase Ran plays a central role in nucleocytoplasmic transport. Nuclear transport of Ran itself depends on nuclear transport factor 2 (NTF2). Here, we report that NTF2 and Ran control nuclear import of the filamentous actin capping protein CapG. In digitonin‐permeabilized cells, neither GTPγS nor the GTP hydrolysis‐deficient Ran mutant RanQ69L affect transit of CapG to the nucleus in the presence of cytosol. Obstruction of nucleoporins prevents nuclear transport of CapG, and we show that CapG binds to nucleoporin62. In addition, CapG interacts with NTF2, associates with Ran and is furthermore able to bind the NTF2–Ran complex. NTF2–Ran interaction is required for CapG nuclear import. This is corroborated by a NTF2 mutant with reduced affinity for Ran and a Ran mutant that does not bind NTF2, both of which prevent CapG import. Thus, a ubiquitously expressed protein shuttles to the nucleus through direct association with NTF2 and Ran. The role of NTF2 may therefore not be solely confined to sustaining the Ran gradient in cells.

L-plastin nanobodies perturb matrix degradation, podosome formation, stability and lifetime in THP-1 macrophages.

Podosomes are cellular structures acting as degradation ‘hot-spots’ in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein. The presence of the capping protein CapG in podosomes has not yet been investigated. We used an innovative approach to investigate the role of these proteins in macrophage podosomes by means of nanobodies or Camelid single domain antibodies. Nanobodies directed against distinct domains of gelsolin, L-plastin or CapG were stably expressed in macrophage-like THP-1 cells. CapG was not enriched in podosomes. Gelsolin nanobodies had no effect on podosome formation or function but proved very effective in tracing distinct gelsolin populations. One gelsolin nanobody specifically targets actin-bound gelsolin and was effectively enriched in podosomes. A gelsolin nanobody that blocks gelsolin-G-actin interaction was not enriched in podosomes demonstrating that the calcium-activated and actin-bound conformation of gelsolin is a constituent of podosomes. THP-1 cells expressing inhibitory L-plastin nanobodies were hampered in their ability to form stable podosomes. Nanobodies did not perturb Ser5 phosphorylation of L-plastin although phosphorylated L-plastin was highly enriched in podosomes. Furthermore, nanobody-induced inhibition of L-plastin function gave rise to an irregular and unstable actin turnover of podosomes, resulting in diminished degradation of the underlying matrix. Altogether these results indicate that L-plastin is indispensable for podosome formation and function in macrophages.

Molecular Basis for Dissimilar Nuclear Trafficking of the Actin‐Bundling Protein Isoforms T‐ and L‐Plastin

T‐ and L‐plastin are highly similar actin‐bundling proteins implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. We show that T‐plastin localizes predominantly to the cytoplasm, whereas L‐plastin distributes between nucleus and cytoplasm in HeLa or Cos cells. T‐plastin shows nuclear accumulation upon incubation of cells with the CRM1 antagonist leptomycin B (LMB). We identified a Rev‐like nuclear export sequence (NES) in T‐plastin that is able to export an otherwise nuclear protein in an LMB‐dependent manner. Deletion of the NES promotes nuclear accumulation of T‐plastin. Mutation of residues L17, F21 or L26 in the T‐plastin NES inhibits nuclear efflux. L‐plastin harbors a less conserved NES and lacks the F21 T‐plastin residue. Insertion of a Phe residue in the L‐plastin NES specifically enhances its export activity. These findings explain why both isoforms exhibit specific distribution patterns in eukaryotic cells.