Ariane De Ganck

A Llama-Derived Gelsolin Single-Domain Antibody Blocks Gelsolin-G-Actin Interaction.

RNA interference has tremendously advanced our understanding of gene function but recent reports have exposed undesirable side-effects. Recombinant Camelid single-domain antibodies (VHHs) provide an attractive means for studying protein function without affecting gene expression. We raised VHHs against gelsolin (GsnVHHs), a multifunctional actin-binding protein that controls cellular actin organization and migration. GsnVHH-induced delocalization of gelsolin to mitochondria or the nucleus in mammalian cells reveals distinct subpopulations including free gelsolin and actin-bound gelsolin complexes. GsnVHH 13 specifically recognizes Ca2+-activated gelsolin (Kd ~10xa0nM) while GsnVHH 11 binds gelsolin irrespective of Ca2+ (Kd ~5xa0nM) but completely blocks its interaction with G-actin. Both GsnVHHs trace gelsolin in membrane ruffles of EGF-stimulated MCF-7 cells and delay cell migration without affecting F-actin severing/capping or actin nucleation activities by gelsolin. We conclude that VHHs represent a potent way of blocking structural proteins and that actin nucleation by gelsolin is more complex than previously anticipated.

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

An ER-directed gelsolin nanobody targets the first step in amyloid formation in a gelsolin amyloidosis mouse model

Hereditary gelsolin amyloidosis is an autosomal dominantly inherited amyloid disorder. A point mutation in the GSN gene (G654A being the most common one) results in disturbed calcium binding by the second gelsolin domain (G2). As a result, the folding of G2 is hampered, rendering the mutant plasma gelsolin susceptible to a proteolytic cascade. Consecutive cleavage by furin and MT1-MMP-like proteases generates 8 and 5 kDa amyloidogenic peptides that cause neurological, ophthalmological and dermatological findings. To this day, no specific treatment is available to counter the pathogenesis. Using GSN nanobody 11 as a molecular chaperone, we aimed to protect mutant plasma gelsolin from furin proteolysis in the trans-Golgi network. We report a transgenic, GSN nanobody 11 secreting mouse that was used for crossbreeding with gelsolin amyloidosis mice. Insertion of the therapeutic nanobody gene into the gelsolin amyloidosis mouse genome resulted in improved muscle contractility. X-ray crystal structure determination of the gelsolin G2:Nb11 complex revealed that Nb11 does not directly block the furin cleavage site. We conclude that nanobodies can be used to shield substrates from aberrant proteolysis and this approach might establish a novel therapeutic strategy in amyloid diseases.

A monopartite nuclear localization sequence regulates nuclear targeting of the actin binding protein myopodin

Myopodin is an actin bundling protein that shuttles between nucleus and cytoplasm in response to cell stress or during differentiation. Here, we show that the myopodin sequence 58KKRRRRARK66, when tagged to either enhanced green fluorescent protein (EGFP) or to enhanced cyan fluorescent protein‐CapG (ECFPCapG), is able to target these proteins to the nucleolus in HeLa or HEK293T cells. By contrast, 58KKRR61‐ECFP‐CapG accumulates in the nucleus. Mutation of 58KKRRRRARK66 into alanine residues blocks myopodin nuclear import and promotes formation of cytoplasmic actin filaments. A second putative nuclear localization sequence, 612KTSKKKGKK620, displays much weaker activity in a heterologous context, and appears not to be functional in the full length protein. Thus myopodin nuclear translocation is dependent on a monopartite nuclear localization sequence.

The Tandem PDZ Protein Syntenin Interacts with the Aminoacyl tRNA Synthetase Complex in a Lysyl-tRNA Synthetase-Dependent Manner

Syntenin-1 is a tandem PDZ protein that binds a diverse array of signaling molecules that are often associated with cell adhesion and intracellular trafficking. With the use of a MS-based functional proteomics approach, we identified several members of the aminoacyl-tRNA synthetase macromolecular (ARS) complex in a syntenin-1 pull down assay. Interaction of these proteins with syntenin-1 was confirmed by co-immunoprecipitation from cultured cells. We demonstrate a direct interaction of syntenin-1 with lysyl-tRNA synthetase (KRS), which contains a PDZ binding motif at its C-terminus. This motif is important for the interaction of the entire complex with syntenin-1. A point mutation in the PDZ2 domain of syntenin-1 abrogates interaction with KRS. As a result, other components of the ARS complex no longer co-immunoprecipitate with syntenin-1. We further show that syntenin-1 regulates KRS activity. These findings suggest that syntenin-1 is an adaptor modulating the activity of KRS.

Multiple isoforms of the tumor suppressor myopodin are simultaneously transcribed in cancer cells.

Expression of myopodin, an actin associated protein, is frequently lost in invasive prostate cancers due to partial or complete deletion of the gene. Screening of public databases reveals that two human myopodin isoforms have been proposed. Remarkably both isoforms deviate profoundly from the human or mouse isoforms examined to date. Here, we investigated expression of human myopodin. Rapid amplification of cDNA ends revealed a new myopodin transcript, hitherto unpredicted by public databases. RT-PCR analysis indicates that the new isoform (Myo2), in addition to the two predicted isoforms (Myo1 and Myo3), are transcribed in various mammalian cell lines. The three isoforms (Myo1-3) are translated into full length proteins of 1093, 1109, and 1261 amino acids, respectively, when expressed in cells. Thus, mammalian cells simultaneously express at least three myopodin isoforms with a common N-terminal PDZ domain, but a dissimilar carboxy-terminal amino acid tract. These findings shed new light on the expression of this tumor suppressor gene and necessitate closer examination of both mouse and human myopodin polypeptides currently under study.

A new survivin tracer tracks, delocalizes and captures endogenous survivin at different subcellular locations and in distinct organelles

Survivin, the smallest member of the inhibitor of apoptosis protein family, plays a central role during mitosis and exerts a cytoprotective function. Survivin is highly expressed in most cancer types and contributes to multiple facets of carcinogenesis. The molecular mechanisms underlying its highly diverse functions need to be extensively explored, which is crucial for rational design of future personalized therapeutics. In this study, we have generated an alpaca survivin nanobody (SVVNb8) that binds with low nanomolar affinity to its target. When expressed as an intrabody in HeLa cells, SVVNb8 faithfully tracks survivin during different phases of mitosis without interfering with survivin function. Furthermore, coupling SVVNb8 with a subcellular delocalization tag efficiently redirects endogenous survivin towards the nucleus, the cytoplasm, peroxisomes and even to the intermembrane space of mitochondria where it presumably interacts with resident mitochondrial survivin. Based on our findings, we believe that SVVNb8 is an excellent instrument to further elucidate survivin biology and topography, and can serve as a model system to investigate mitochondrial and peroxisomal (survivin) protein import.

Model-based classification for digital PCR : your Umbrella for rain

Standard data analysis pipelines for digital PCR estimate the concentration of a target nucleic acid by digitizing the end-point fluorescence of the parallel micro-PCR reactions, using an automated hard threshold. While it is known that misclassification has a major impact on the concentration estimate and substantially reduces accuracy, the uncertainty of this classification is typically ignored. We introduce a model-based clustering method to estimate the probability that the target is present (absent) in a partition conditional on its observed fluorescence and the distributional shape in no-template control samples. This methodology acknowledges the inherent uncertainty of the classification and provides a natural measure of precision, both at individual partition level and at the level of the global concentration. We illustrate our method on genetically modified organism, inhibition, dynamic range, and mutation detection experiments. We show that our method provides concentration estimates of similar accuracy or better than the current standard, along with a more realistic measure of precision. The individual partition probabilities and diagnostic density plots further allow for some quality control. An R implementation of our method, called Umbrella, is available, providing a more objective and automated data analysis procedure for absolute dPCR quantification.

Flexible analysis of digital PCR experiments using generalized linear mixed models.

The use of digital PCR for quantification of nucleic acids is rapidly growing. A major drawback remains the lack of flexible data analysis tools. Published analysis approaches are either tailored to specific problem settings or fail to take into account sources of variability. We propose the generalized linear mixed models framework as a flexible tool for analyzing a wide range of experiments. We also introduce a method for estimating reference gene stability to improve accuracy and precision of copy number and relative expression estimates. We demonstrate the usefulness of the methodology on a complex experimental setup.