Inhibitory cortactin nanobodies delineate the role of NTA- and SH3-domain-specific functions during invadopodium formation and cancer cell invasion.

Cancer cells exploit different strategies to escape from the primary tumor, gain access to the circulation, disseminate throughout the body, and form metastases, the leading cause of death by cancer. Invadopodia, proteolytically active plasma membrane extensions, are essential in this escape mechanism. Cortactin is involved in every phase of invadopodia formation, and its overexpression is associated with increased invadopodia formation, extracellular matrix degradation, and cancer cell invasion. To analyze endogenous cortactin domain function in these processes, we characterized the effects of nanobodies that are specific for the N-terminal acidic domain of cortactin and expected to target small epitopes within this domain. These nanobodies inhibit cortactin-mediated actin-related protein (Arp)2/3 activation, and, after their intracellular expression in cancer cells, decrease invadopodia formation, extracellular matrix degradation, and cancer cell invasion. In addition, one of the nanobodies affects Arp2/3 interaction and invadopodium stability, and a nanobody targeting the Src homology 3 domain of cortactin enabled comparison of 2 functional regions in invadopodium formation or stability. Given their common and distinct effects, we validate cortactin nanobodies as an instrument to selectively block and study distinct domains within a protein with unprecedented precision, aiding rational future generation of protein domain-selective therapeutic compounds.-Bertier, L., Boucherie, C., Zwaenepoel, O., Vanloo, B., Van Troys, M., Van Audenhove, I., Gettemans, J. Inhibitory cortactin nanobodies delineate the role of NTA- and SH3-domain-specific functions during invadopodium formation and cancer cell invasion.

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 (K(d)~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 (K(d)~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.

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

INTRODUCTION: Aberrant 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. METHODS: We 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. RESULTS: With 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). CONCLUSIONS: CapG 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.

Mapping cytoskeletal protein function in cells by means of nanobodies.

Nanobodies or VHHs are single domain antigen binding fragments derived from heavy-chain antibodies naturally occurring in species of the Camelidae. Due to their ease of cloning, high solubility and intrinsic stability, they can be produced at low cost. Their small size, combined with high affinity and antigen specificity, enables recognition of a broad range of structural (undruggable) proteins and enzymes alike. Focusing on two actin binding proteins, gelsolin and CapG, we summarize a general protocol for the generation, cloning and production of nanobodies. Furthermore, we describe multiple ways to characterize antigen-nanobody binding in more detail and we shed light on some applications with recombinant nanobodies. The use of nanobodies as intrabodies is clarified through several case studies revealing new cytoskeletal protein properties and testifying to the utility of nanobodies as intracellular bona fide protein inhibitors. Moreover, as nanobodies can traverse the plasma membrane of eukaryotic cells by means of the enteropathogenic E. coli type III protein secretion system, we show that in this promising way of nanobody delivery, actin pedestal formation can be affected following nanobody injection.

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.

An alpaca single-domain antibody blocks filopodia formation by obstructing L-plastin-mediated F-actin bundling.

L-plastin, a conserved modular F-actin bundling protein, is ectopically expressed in tumor cells and contributes to cell malignancy and invasion. The underlying molecular mechanisms involved remain unclear, in part, because specific inhibitors of L-plastin are lacking. We used recombinant alpaca-derived L-plastin single-domain antibodies (nanobodies) as effector of L-plastin function in cells.

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)P (2)) 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.

The mouse thymosin beta15 gene family displays unique complexity and encodes a functional thymosin repeat.

We showed earlier that human beta-thymosin 15 (Tb15) is up-regulated in prostate cancer, confirming studies from others that propagated Tb15 as a prostate cancer biomarker. In this first report on mouse Tb15, we show that, unlike in humans, four Tb15-like isoforms are present in mouse. We used phylogenetic analysis of deuterostome beta-thymosins to show that these four new isoforms cluster within the vertebrate Tb15-clade. Intriguingly, one of these mouse beta-thymosins, Tb15r, consists of two beta-thymosin domains. The existence of such a repeat beta-thymosin is so far unique in vertebrates, though common in lower eukaryotes. Biochemical data indicate that Tb15r potently sequesters actin. In a cellular context, Tb15r behaves as a bona fide beta-thymosin, lowering central stress fibre content. We reveal that a complex genomic organization underlies Tb15r expression: Tb15r results from read-through transcription and alternative splicing of two tandem duplicated mouse Tb15 genes. Transcript profiling of all mouse beta-thymosin isoforms (Tb15s, Tb4 and Tb10) reveals that two isoform switches occur between embryonic and adult tissues, and indicates Tb15r as the major mouse Tb15 isoform in adult cells. Tb15r is present also in mouse prostate cancer cell lines. This insight into the mouse Tb15 family is fundamental for future studies on Tb15 in mouse (prostate) cancer models.

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 GTPgammaS 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.

The tandem PDZ domains of syntenin promote cell invasion.

Syntenin is a tandem PDZ protein that has recently been shown to be overexpressed in several cancer cells and tissues, and that might play an active role in tumor cell invasion and metastasis. Here we show that overexpression of the tandem PDZ domains of syntenin in non-invasive cells is necessary and sufficient to stimulate these cells to invade a collagen I matrix, and this effect can be regulated by ligand binding to the PDZ domains. Furthermore, we show that syntenin-induced invasion requires signaling through ras, rho and PI3K/MAPK signaling pathways and involves changes in cell-cell adhesion. Inversely, when we used RNA interference to inhibit syntenin expression in different invasive cancer cell lines, we observed a drastically decreased ability of these cells to migrate and invade into collagen type I or Matrigel. RNAi-treated cells also show increased cell aggregation, indicating that syntenin is important for cell-cell adhesion in epithelial cells. Together, these results suggest that downregulation of syntenin by RNA interference could provide a means of inhibiting tumor invasion and possibly metastasis in different cancers, and point to syntenin as a potential cancer biomarker and drug target.

Lysophosphatidic acid affinity chromatography reveals pyruvate kinase as a specific LPA-binding protein.

Lysophosphatidic acid is a pleiotropic lipid signaling molecule that evokes a broad array of cellular responses including proliferation, tumor cell invasion, neurite retraction, cytoskeletal rearrangements and smooth muscle contraction. Generally, lysophosphatidic acid triggers physiological responses through interaction with specific plasma membrane receptors called LPA 1-4. There is, however, increasing evidence in support of intracellular proteins that interact with LPA. We employed Affigel-immobilized LPA to isolate cytoplasmic proteins that interact with this lysophospholipid. Among the proteins retained by this affinity matrix, pyruvate kinase, clathrin heavy chain and heat shock protein 70 (Hsp70) were identified by mass spectrometry. Isothermal titration calorimetry showed that pyruvate kinase contains one binding site for LPA (Ka approx. 10(6) M(-1)). Furthermore, LPA dissociates enzymatically active pyruvate-kinase tetramers into less active dimers, and is maximally active at concentrations close to its critical micelle concentration. These effects were not mimicked by other lysophospholipids. Co-immunoprecipitation experiments showed that pyruvate kinase interacts with clathrin, and confocal imaging revealed co-localization between clathrin and pyruvate kinase in the perinuclear region of cells. Our data suggest that pyruvate kinase partly exists in complex with clathrin in subcellular membranous areas, and that locally increased LPA levels can trigger inactivation of the metabolic enzyme.

Poly-l-glutamic acid derivatives as multifunctional vectors for gene delivery. Part B. Biological evaluation.

Cationic polymers, such as poly-l-lysine (pLL) and polyethyleneimine (pEI), are receiving growing attention as vectors for gene therapy. They form polyelectrolyte complexes with DNA, resulting in a reduced size of the DNA and an enhanced stability toward nucleases. The major disadvantages of using both polymers for in vivo purposes are their cytotoxicity and, in the case of pEI, the fact that it's not biodegradable. In this work, we investigated the interaction between a series of cationic, glutamic acid based polymers and red blood cells. The MTT test was used to investigate the cytotoxicity of the complexes. The ability of the polymers to stabilize DNA toward nucleases was investigated. Transfection studies were carried out on Cos-1 cells. The results from the haemolysis studies, the haemagglutination studies, and the MTT assay show that the polymers are substantially less toxic than pLL and pEI. The polymers are able to protect the DNA from digestion by DNase I. The transfection studies show that the polymer-DNA complexes are capable of transfecting cells, most of them with poor efficiency compared to pEI-DNA complexes.

Physicochemical and biological evaluation of cationic polymethacrylates as vectors for gene delivery.

We report here the physicochemical and biological evaluation of a series of polymethacrylates with side groups of different pK(a) values, such as tertiary amines, pyridine groups, acid functions and imidazole groups as synthetic vectors for gene delivery. The ability of the different polymers to condense DNA was studied by ethidium bromide exclusion tests and agarose gel electrophoresis. The results show that all polymers are able to condense DNA. Both the molecular weight and the chemical composition of the polymers have an influence on the DNA condensation process. Furthermore, the biological properties of the polymer-DNA complexes were investigated, including their haemolytic activity, cytotoxicity and in vitro transfection efficiency. Complexes based on polymers containing only tertiary amines, have a transfection efficiency similar to that of poly(ethyleneimine) (PEI). Polymers containing pyridine groups have a reduced transfection efficiency compared to polymers containing tertiary amines. Introduction of imidazole groups or acid functions results in a loss of the transfection efficiency of the corresponding complexes with DNA. In general, the viability of cells incubated with complexes based on the polymethacrylates is higher than with PEI. Polymers with high transfection efficiency induce erythrocyte lysis.

Characterization of the ABCA transporter subfamily: identification of prokaryotic and eukaryotic members, phylogeny and topology.

An alignment of the mammalian ABCA transporters enabled the identification of sequence segments, specific to the ABCA subfamily, which were used as queries to search for eukaryotic and prokaryotic homologues. Thirty-seven eukaryotic half and full-length transporters were found, and a close relationship with prokaryotic subfamily 7 transporters was detected. Each half of the ABCA full-transporters is predicted to comprise a membrane-spanning domain (MSD) composed of six helices and a large extracellular loop, followed by a nucleotide-binding domain (NBD) and a conserved cytoplasmic 80-residue sequence, which might have a regulatory function. The topology predicted for the ABCA transporters was compared to the crystal structures of the MsbA and BtuCD bacterial transporters. The alignment of the MSD and NBD domains provided an estimate of the degree of residue conservation in the cytoplasmic, extracellular and transmembrane domains of the ABCA transporter subfamily. The phylogenic tree of eukaryotic ABCA transporters based upon the NBD sequences, consists of three major clades, corresponding to the half-transporter single NBDs and to the full-transporter NBDls and NBD2s. A phylogenic tree of prokaryotic transporters and the eukaryotic ABCA transporters confirmed the evolutionary relationship between prokaryotic subfamily 7 transporters and eukaryotic ABCA half and full-transporters.

Fragmin60 encodes an actin-binding protein with a C2 domain and controls actin Thr-203 phosphorylation in Physarum plasmodia and sclerotia.

We report the isolation of a cDNA clone encoding a 60-kDa protein termed fragmin60 that cross-reacts with fragmin antibodies. Unlike other gelsolin-related proteins, fragmin60 contains a unique N-terminal domain that shows similarity with C2 domains of aczonin, protein kinase C, and synaptotagmins. The fragmin60 C2 domain binds three calcium ions, one with nanomolar affinity and two with micromolar affinity. Actin binding by fragmin60 requires higher calcium concentrations than does binding of actin by a fragmin60 mutant lacking the C2 domain, suggesting that the C2 domain secures the actin binding moiety in a conformation preventing actin binding at low calcium concentrations. The fragmin60 C2 domain does not bind phospholipids but interacts with the endogenous homologue of Saccharomyces cerevisiae S-phase kinase-associated protein (Skp1), as shown by pull-down assays and co-expression in mammalian cells. Recombinant fragmin60 promotes in vitro phosphorylation of actin Thr-203 by the actin-fragmin kinase. We further show that in vivo phosphorylation of actin in the fragmin60-actin complex occurs in sclerotia, a dormant stage of Physarum development, as well as in plasmodia. Our findings indicate that we have cloned a novel type of gelsolin-related actin-binding protein that is involved in controlling regulation of actin phosphorylation in vivo.

Tryptophan fluorescence study of the interaction of penetratin peptides with model membranes.

Penetratin is a 16-amino-acid peptide, derived from the homeodomain of antennapedia, a Drosophila transcription factor, which can be used as a vector for the intracellular delivery of peptides or oligonucleotides. To study the relative importance of the Trp residues in the wild-type penetratin peptide (RQIKIWFQNRRMKWKK) two analogues, the W48F (RQIKIFFQNRRMKWKK) and the W56F (RQI KIWFQNRRMKFKK) variant peptides were synthesized. Binding of the three peptide variants to different lipid vesicles was investigated by fluorescence. Intrinsic Trp fluorescence emission showed a decrease in quantum yield and a blue shift of the maximal emission wavelength upon interaction of the peptides with negatively charged phosphatidylserine, while no changes were recorded with neutral phosphatidylcholine vesicles. Upon binding to phosphatidylcholine vesicles containing 20% (w/w) phosphatidylserine the fluorescence blue shift induced by the W56F-penetratin variant was larger than for the W48F-penetratin. Incorporation of cholesterol into the negatively charged lipid bilayer significantly decreased the binding affinity of the peptides. The Trp mean lifetime of the three peptides decreased upon binding to negatively charged phospholipids, and the Trp residues were shielded from acrylamide and iodide quenching. CD measurements indicated that the peptides are random in buffer, and become alpha helical upon association with negatively charged mixed phosphatidylcholine/phosphatidylserine vesicles, but not with phosphatidylcholine vesicles. These data show that wild-type penetratin and the two analogues interact with negatively charged phospholipids, and that this is accompanied by a conformational change from random to alpha helical structure, and a deeper insertion of W48 compared to W56, into the lipid bilayer.