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High-content image-based cell phenotyping provides fundamental insights in a broad variety of life science areas. Striving for accurate conclusions and meaningful impact demands high reproducibility standards, even more importantly with the advent of data sharing initiatives. However, the sources and degree of biological and technical variability, and thus the reproducibility and usefulness of meta-analysis of results from live-cell microscopy have not been systematically investigated. Here, using high content data describing features of cell migration and morphology, we determine the sources of variability across different scales, including between laboratories, persons, experiments, technical repeats, cells and time points. Significant technical variability occurred between laboratories, providing low value to direct meta-analysis on the data from different laboratories. However, batch effect removal markedly improved the possibility to combine image-based datasets of perturbation experiments. Thus, reproducible quantitative high-content cell image data and meta-analysis depend on standardized procedures and batch correction applied to studies of perturbation effects.

We describe furan as a triggerable 'warhead' for site-specific cross-linking using the actin and thymosin β4 (Tβ4)-complex as model of a weak and dynamic protein-protein interaction (PPI) with known 3D structure and with application potential in disease contexts. The identified cross-linked residues demonstrate that lysine is a target for the furan warhead. The presented in vitro validation of covalently acting 'furan-armed' Tβ4-variants provides initial proof to further exploit furan-technology for covalent drug design targeting lysines.

Actins form a strongly conserved family of proteins that are central to the functioning of the actin cytoskeleton partaking in natural processes such as cell division, adhesion, contraction and migration. These processes, however, also occur during the various phases of cancer progression. Yet, surprisingly, alterations in the six human actin genes in cancer studies have received little attention and the focus was mostly on deregulated expression levels of actins and even more so of actin-binding or regulatory proteins. Starting from the early mutation work in the 1980s, we propose based on reviewing literature and data from patient cancer genomes that alterations in actin genes are different in distinct cancer subtypes, suggesting some specificity. These actin gene alterations include (missense) mutations, gene fusions and copy number alterations (deletions and amplifications) and we illustrate their occurrence for a limited number of examples including actin mutations in lymphoid cancers and nonmelanoma skin cancer and actin gene copy number alterations for breast, prostate and liver cancers. A challenge in the future will be to further sort out the specificity per actin gene, alteration type and cancer subtype. Even more challenging is (experimentally) distinguishing between cause and consequence: which alterations are passengers and which are involved in tumor progression of particular cancer subtypes?

Autonomous migration is a central characteristic of immune cells, and changes in this function have been correlated to the progression and severity of diseases. Hence, the identification of pathologically altered leukocyte migration patterns might be a promising approach for disease surveillance and prognostic scoring. However, because of the lack of standardized and robust assays, migration patterns have not been clinically exploited so far. In this study, we introduce an easy-to-use and cross-laboratory, standardized two-dimensional migration assay for neutrophil granulocytes from peripheral blood. By combining time-lapse video microscopy and automated cell tracking, we calculated the average migration of neutrophils from 111 individual participants of the German Heinz Nixdorf Recall MultiGeneration study under steady-state, formyl-methionyl-leucyl-phenylalanine–, CXCL1-, and CXCL8-stimulated conditions. Comparable values were obtained in an independent laboratory from a cohort in Belgium, demonstrating the robustness and transferability of the assay. In a double-blinded retrospective clinical analysis, we found that neutrophil migration strongly correlated with the Revised International Prognostic Scoring System scoring and risk category of myelodysplastic syndrome (MDS) patients. In fact, patients suffering from high-risk subtypes MDS with excess blasts I or II displayed highly significantly reduced neutrophil migration. Hence, the determination of neutrophil migration patterns might represent a useful tool in the surveillance of MDS. Taken together, we suggest that standardized migration assays of neutrophils and other leukocyte subtypes might be broadly applicable as prognostic and surveillance tools for MDS and potentially for other diseases.

Contains fulltext : 220755.pdf (Publisher’s version ) (Open Access) Cell migration research has become a high-content field. However, the quantitative information encapsulated in these complex and high-dimensional datasets is not fully exploited owing to the diversity of experimental protocols and non-standardized output formats. In addition, typically the datasets are not open for reuse. Making the data open and Findable, Accessible, Interoperable, and Reusable (FAIR) will enable meta-analysis, data integration, and data mining. Standardized data formats and controlled vocabularies are essential for building a suitable infrastructure for that purpose but are not available in the cell migration domain. We here present standardization efforts by the Cell Migration Standardisation Organisation (CMSO), an open community-driven organization to facilitate the development of standards for cell migration data. This work will foster the development of improved algorithms and tools and enable secondary analysis of public datasets, ultimately unlocking new knowledge of the complex biological process of cell migration.

The Maillard conjugation of whey protein isolate (WPI) by dry heat treatment (74% relative humidity at 60 °C) to either the water-soluble fraction of almond gum (SFAG) or flaxseed mucilage (SFM) was compared. Depending on the protein to polysaccharide ratio, carbohydrate type, and incubation time, different degrees of substitutions of the amino groups were obtained. The characterization of the conjugates by TNBS, SDS-PAGE, size exclusion chromatography, and circular dichroism analysis confirmed the formation of conjugates. SFAG was found to have less tendency for the formation of grafted WPI than SFM, which could be attributed to both the polysaccharide composition and/or a higher molecular weight. Ultimately, the emulsions stabilized by conjugates (pH 5.0 and 6.5) remained homogenous with no droplet size variation after heating, indicating that the conjugation of WPI to SFAG and SFM substantially improved its heat stability.

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.

Lichenized Nostoc cells isolated from the lichen Peltigera canina develop chemotaxis towards a lectin purified from the lichen thallus. Similar movements in unicellular eukaryotes require actin and myosin to generate contraction and relaxation along the chemotactic axis. We provide evidences for prokaryotic actin-like and myosin-like proteins in the cyanobiont Nostoc sp using cross-reacting antibodies against α- and β-actin and non-muscle myosin II light and heavy chains, and two-dimensional gel electrophoresis to determine the isoelectric point (IP) of the actin-like protein. Actin antibodies bound to a single reactive Nostoc polypeptide of an approximate molecular mass of 50 kD with IP values between 4 and 7 pH, similar to eukaryotic actin. The myosin light chain antibody reacted with a Nostoc protein with an apparent molecular weight of 20 kDa and another of 48 kDa. Immunoprecipitation of cell free extracts using anti-heavy chain myosin separately yielded only one signal corresponding to a protein of a molecular weight around 200 kDa. A bioinformatics analysis indicated that (cyano)bacterial actins are rare but do exist. Our results are consistent with the possible existence of protein homologues of actins and myosins in cyanobionts of P. canina, suggesting the existence of an actin-like apparatus that supports chemotactic swimming.

The industrial application of whey proteins as a food emulsifier is still limited due to its susceptibility towards heating. In this study, dry heat conjugation of whey protein and lactose was investigated to improve the heat stability of WPC-stabilised oil in water (O/W) emulsions. A commercially available whey protein concentrate (WPC), containing 80 % protein and 12 % lactose, was dry heat incubated. Hereby, the effect of incubation temperature (60, 70, and 80 °C), relative humidity (64, 74, and 79 %), and preconditioning pH (4, 6, 8, 10) were determined. Temperature variation showed a pronounced effect on the required duration to obtain conjugates with excellent emulsion heat stabilising capacity. Four hours of incubation at 80 °C was sufficient to retain the original particle size and consistency after heating the emulsions at 80 °C for 20 min. Whereas circular dichroism spectroscopy revealed that this dry heat incubation condition (80 °C, 4 h) maintained the secondary structure of WPC, pulsed-field gradient NMR revealed that about 40 % of the lactose present should become protein-bound to generate conjugates with the desired functionality. The results indicated that a heat stable WPC with about 40 % of bound lactose could be obtained by some hours of dry heat treatment at 80 °C.

Whey protein–lactose conjugates were prepared by dry heat incubation (60 °C, 74% relative humidity) at 1:1 M ratio of protein amino groups to reducing sugar. Pulsed-field gradient-nuclear magnetic resonance (PFG-NMR) was introduced as a new and absolute method to determine the degree of conjugation without any sample pretreatment, based on the different diffusion behaviour of free and protein-bound lactose. Whereas the TNBS method revealed 73% conjugation, PFG-NMR results indicated that only 37% of lactose was bound to the protein after 8 h incubation. Circular dichroism spectra showed no change of WPI secondary structure upon dry heat incubation, whereas the electrophoretic mobility shifted towards more negative values. The emulsifying and, more importantly, heat stabilising properties of the conjugates were subsequently evaluated in oil/water emulsions. Eight hours of incubation was sufficient to produce conjugates that successfully stabilised the emulsion against heating (80 °C, 20 min), regarding the particle size and viscosity.

Interactions between G protein-coupled receptors and their ligands hold extensive potential for drug discovery. Studying these interactions poses technical problems due to their transient nature and the inherent difficulties when working with G protein-coupled receptors (GPCR) that are only functional in a membrane setting. Here, we describe the use of a furan-based chemical cross-linking methodology to achieve selective covalent coupling between a furan-modified peptide ligand and its native GPCR present on the surface of living cells under normal cell culture conditions. This methodology relies on the oxidation of the furan moiety, which can be achieved by either addition of an external oxidation signal or by the reactive oxygen species produced by the cell. The cross-linked ligand-GPCR complex is subsequently detected by Western blotting based on the biotin label that is incorporated in the peptide ligand.

Mutations in actins have been linked to several developmental diseases. Their occurrence across different cancers has, however, not been investigated. Using the cBioPortal database we show that human actins are infrequently mutated in patient samples of various cancers types. Nevertheless, ranking these studies by mutational frequency suggest that some have a higher percentage of patients with ACTB and ACTG1 mutations. Within studies on hematological cancers, mutations in ACTB and ACTG1 are associated with lymphoid cancers since none have currently been reported in myeloid cancers. Within the different types of lymphoid cancers ACTB mutations are most frequent in diffuse large B-cell lymphoma (DLBCL) and ACTG1 mutations in multiple myeloma. We mapped the ACTB and ACTG1 mutations found in these two cancer types on the 3D-structure of actin showing they are in regions important for actin polymer formation or binding to myosin. The potential effects of the mutations on actin properties imply that mutations in cytoplasmic actins deserve dedicated research in DLBCL and multiple myeloma.

Triple-negative breast cancer (TNBC) is the most challenging subtype to treat due to the lack of estrogen receptor, progesterone receptor, and HER2 expression, which excludes the usage of directed targeted therapy against them. Promising therapeutic targets are the hepatocyte growth factor receptor (MET) and epidermal growth factor receptor (EGFR), which expression is frequently elevated in TNBC. Inhibitors of these receptors used as monotherapy are often ineffective. Due to that, we studied the efficacy of combined therapy targeting MET and EGFR simultaneously. Two TNBC cell lines were treated with lapatinib (a dual EGFR and HER2 inhibitor), foretinib (a MET inhibitor), or a combination of the two. After the inhibitors treatment, we verified the cell viability (XTT assay), distribution of the cell cycle phases, the activation of signaling pathways (Western blotting), distribution of invadopodia, fluorescent gelatin digestion (immunofluorescence), and the invasion capacity of cells. A combination of foretinib and lapatinib effectively reduced the viability of examined cells, led to G2/M arrest and reduction of pAKT. There was also a decreasein number of invadopodia formed by cells, their ability to digest gelatin and reduction of cells migration/invasion capacity. Therapy targeting of both EGFR and MET receptors was much more effective against tested cells than monotherapy. We selected a combination of drugs that could be successfully used against this breast cancer subtype.

In vitro tests of cancer cell invasion are the "first line" tools of preclinical researchers for screening the multitude of chemical compounds or cell perturbations that may aid in halting or treating cancer malignancy. In order to have predictive value or to contribute to designing personalized treatment regimes, these tests need to take into account the cancer cell environment and measure effects on invasion in sufficient detail. The in vitro invasion assays presented here are a trade-off between feasibility in a multisample format and mimicking the complexity of the tumor microenvironment. They allow testing multiple samples and conditions in parallel using 3D-matrix-embedded cells and deal with the heterogeneous behavior of an invading cell population in time. We describe the steps to take, the technical problems to tackle and useful software tools for the entire workflow: from the experimental setup to the quantification of the invasive capacity of the cells. The protocol is intended to guide researchers to standardize experimental set-ups and to annotate their invasion experiments in sufficient detail. In addition, it provides options for image processing and a solution for storage, visualization, quantitative analysis, and multisample comparison of acquired cell invasion data.

Cell migration is central to the development and maintenance of multicellular organisms. Fundamental understanding of cell migration can, for example, direct novel therapeutic strategies to control invasive tumor cells. However, the study of cell migration yields an overabundance of experimental data that require demanding processing and analysis for results extraction. Computational methods and tools have therefore become essential in the quantification and modeling of cell migration data. We review computational approaches for the key tasks in the quantification of in vitro cell migration: image pre-processing, motion estimation and feature extraction. Moreover, we summarize the current state-of-the-art for in silico modeling of cell migration. Finally, we provide a list of available software tools for cell migration to assist researchers in choosing the most appropriate solution for their needs.

Invadopodia are actin-rich protrusions formed by mesenchymally migrating cancer cells. They are mainly composed of actin, actin-associated proteins, integrins and proteins of signaling machineries. These protrusions display focalized proteolytic activity towards the extracellular matrix. It is well known that polymerized (F-)actin is present in these structures, but the nature of the actin isoform has not been studied before. We here show that both cytoplasmic actin isoforms, β- and γ-actin, are present in the invadopodia of MDA-MB-231 breast cancer cells cultured on a 2D-surface, where they colocalize with the invadopodial marker cortactin. Invadopodial structures formed by the cells in a 3D-collagen matrix also contain β- and γ-actin. We demonstrate this using isoform-specific antibodies and expression of fluorescently-tagged actin isoforms. Additionally, using simultaneous expression of differentially tagged β- and γ-actin in cells, we show that the actin isoforms are present together in a single invadopodium. Cells with an increased level of β- or γ-actin, display a similar increase in the number and size of invadopodia in comparison to control cells. Moreover, increasing the level of either actin isoforms also increases invasion velocity.

In vitro tests of cancer cell invasion are the "first line" tools of preclinical researchers for screening the multitude of chemical compounds or cell perturbations that may aid in halting or treating cancer malignancy. In order to have predictive value or to contribute to designing personalized treatment regimes, these tests need to take into account the cancer cell environment and measure effects on invasion in sufficient detail. The in vitro invasion assays presented here are a trade-off between feasibility in a multisample format and mimicking the complexity of the tumor microenvironment. They allow testing multiple samples and conditions in parallel using 3D-matrix-embedded cells and deal with the heterogeneous behavior of an invading cell population in time. We describe the steps to take, the technical problems to tackle and useful software tools for the entire workflow: from the experimental setup to the quantification of the invasive capacity of the cells. The protocol is intended to guide researchers to standardize experimental set-ups and to annotate their invasion experiments in sufficient detail. In addition, it provides options for image processing and a solution for storage, visualization, quantitative analysis, and multisample comparison of acquired cell invasion data.

Chemical cross-linking is well-established for investigating protein–protein interactions. Traditionally, photo cross-linking is used but is associated with problems of selectivity and UV toxicity in a biological context. We here describe, with live cells and under normal growth conditions, selective cross-linking of a furan-modified peptide ligand to its membrane-presented receptor with zero toxicity, high efficiency, and spatio-specificity. Furan-modified kisspeptin-10 is covalently coupled to its glycosylated membrane receptor, GPR54(KISS1R). This newly expands the applicability of furan-mediated cross-linking not only to protein–protein cross-linking but also to cross-linking in situ. Moreover, in our earlier reports on nucleic acid interstrand cross-linking, furan activation required external triggers of oxidation (via addition of N-bromo succinimide or singlet oxygen). In contrast, we here show, for multiple cell lines, the spontaneous endogenous oxidation of the furan moiety with concurrent selective cross-link formation. We propose that reactive oxygen species produced by NADPH oxidase (NOX) enzymes form the cellular source establishing furan oxidation.

The multimodular nature of many eukaryotic proteins underlies their temporal or spatial engagement in a range of protein cocomplexes. Using the multimodule protein testin (TES), we here report a proteomics approach to increase insight in cocomplex diversity. The LIM-domain containing and tumor suppressor protein TES is present at different actin cytoskeleton adhesion structures in cells and influences cell migration, adhesion and spreading. TES module accessibility has been proposed to vary due to conformational switching and variants of TES lacking specific domains target to different subcellular locations. By applying iMixPro AP-MS ("intelligent Mixing of Proteomes"-affinity purification-mass spectrometry) to a set of tagged-TES modular variants, we identified proteins residing in module-specific cocomplexes. The obtained distinct module-specific interactomes combine to a global TES interactome that becomes more extensive and richer in information. Applying pathway analysis to the module interactomes revealed expected actin-related canonical pathways and also less expected pathways. We validated two new TES cocomplex partners: TGFB1I1 and a short form of the glucocorticoid receptor. TES and TGFB1I1 are shown to oppositely affect cell spreading providing biological validity for their copresence in complexes since they act in similar processes.

Database surveys in the vertebrate model organisms: chicken (Gallus gallus), western clawed frog (Xenopus tropicalis), anole lizard (Anolis carolinensis) and zebrafish (Danio rerio) indicate that in some of these species the number of actin paralogues differs from the well-established six paralogues in mouse (Mus musculus). To investigate differential functions of actins and for establishing disease models it is important to know how actins in the different model organisms relate to each other and whether the vertebrate actin family is truly limited to six groups. Primarily through synteny analyses we discovered that the vertebrate actin family consists of eight instead of six orthologous actin groups for which we propose improved gene nomenclature. We also established that α-skeletal muscle, γ-enteric smooth muscle and γ-cytoplasmic actin genes originated prior to tetrapods contradicting an earlier and widely accepted model of actin evolution. Our findings allow a more reliable predictive classification of actin paralogues in (non-mammalian) vertebrates and contribute to a better understanding of actin evolution as basis for biomedical research on actin-related diseases.

β-actin depletion from mouse embryonic fibroblasts results in an altered transcriptional response rendering these cells a myofibroblast like phenotype. The proteins and upstream regulatory factors responsible for this acquired phenotype, with prominent focal adhesions and stress fibres, are unknown. Data-mining of the changed proteome revealed that actin binding proteins associated with stress fiber or focal adhesion formation are overexpressed in the β-actin knock-out cells and that many of these contain CH-, LIM- or EFh- domains. Furthermore in silico analysis predicts potential common upstream regulators that may, at least partly, coordinate the altered transcriptional response.

The conserved alphaherpesviral serine/threonine kinase US3 causes dramatic actin rearrangements, associated with increased viral spread. Here, we show that US3 of pseudorabies virus (PRV) leads to activation (dephosphorylation) of the central actin regulator cofilin. A mutation that impairs US3 kinase activity and the group I p21-activated kinase inhibitor IPA-3 inhibited US3-mediated cofilin activation. Additionally, expression of phosphomimetic S3D cofilin significantly suppressed the ability of US3 to cause cell projections and cell rounding. In conclusion, the US3 kinase of PRV leads to activation (dephosphorylation) of cofilin, and cofilin contributes to US3-mediated actin rearrangements.

The systematic study of single-cell migration requires the availability of software for assisting data inspection, quality control and analysis. This is especially important for high-throughput experiments, where multiple biological conditions are tested in parallel. Although the field of cell migration can count on different computational tools for cell segmentation and tracking, downstream data visualization, parameter extraction and statistical analysis are still left to the user and are currently not possible within a single tool. This article presents a completely new module for the open-source, cross-platform CellMissy software for cell migration data management. This module is the first tool to focus specifically on single-cell migration data downstream of image processing. It allows fast comparison across all tested conditions, providing automated data visualization, assisted data filtering and quality control, extraction of various commonly used cell migration parameters, and non-parametric statistical analysis. Importantly, the module enables parameters computation both at the trajectory- and at the step-level. Moreover, this single-cell analysis module is complemented by a new data import module that accommodates multiwell plate data obtained from high-throughput experiments, and is easily extensible through a plugin architecture. In conclusion, the end-to-end software solution presented here tackles a key bioinformatics challenge in the cell migration field, assisting researchers in their high-throughput data processing.

Actin is the central building block of the actin cytoskeleton, a highly regulated filamentous network enabling dynamic processes of cells and simultaneously providing structure. Mammals have six actin isoforms that are very conserved and thus share common functions. Tissue-specific expression in part underlies their differential roles, but actin isoforms also coexist in various cell types and tissues, suggesting specific functions and preferential interaction partners. Gene deletion models, antibody-based staining patterns, gene silencing effects, and the occurrence of isoform-specific mutations in certain diseases have provided clues for specificity on the subcellular level and its consequences on the organism level. Yet, the differential actin isoform functions are still far from understood in detail. Biochemical studies on the different isoforms in pure form are just emerging, and investigations in cells have to deal with a complex and regulated system, including compensatory actin isoform expression.

Cell proliferation, a main target in cancer therapy, is influenced by the surrounding three-dimensional (3D) extracellular matrix (ECM). In vitro drug screening is, thus, optimally performed under conditions in which cells are grown (embedded or trapped) in dense 3D matrices, as these most closely mimic the adhesive and mechanical properties of natural ECM. Measuring cell proliferation under these conditions is, however, technically more challenging compared with two-dimensional (2D) culture and other "3D culture conditions," such as growth on top of a matrix (pseudo-3D) or in spongy scaffolds with large pore sizes. Consequently, such measurements are only slowly applied on a wider scale. To advance this, we report on the equal quality (dynamic range, background, linearity) of measuring the proliferation of cell layers embedded in dense 3D matrices (collagen, Matrigel) compared with cells in 2D culture using the easy (one-step) and in 2D well-validated, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)-assay. The comparison stresses the differences in proliferation kinetics and drug sensitivity of matrix-embedded cells versus 2D culture. Using the specific cell-layer-embedded 3D matrix setup, quantitative measurements of cell proliferation and cell invasion are shown to be possible in similar assay conditions, and cytostatic, cytotoxic, and anti-invasive drug effects can thus be reliably determined and compared in physiologically relevant settings. This approach in the 3D matrix holds promise for improving early-stage, high-throughput drug screening, targeting either highly invasive or highly proliferative subpopulations of cancers or both.

The actin-binding proteins of the actin-depolymerisation factor (ADF)/cofilin family were first described more than three decades ago, but research on these proteins still occupies a front role in the actin and cell migration field. Moreover, cofilin activity is implicated in the malignant, invasive properties of cancer cells. The effects of ADF/cofilins on actin dynamics are diverse and their regulation is complex. In stimulated cells, multiple signalling pathways can be initiated resulting in different activation/deactivation switches that control ADF/cofilin activity. The output of this entire regulatory system, in combination with spatial and temporal segregation of the activation mechanisms, underlies the contribution of ADF/cofilins to various cell migration/invasion phenotypes. In this framework, we describe current views on how ADF/cofilins function in migrating and invading cells.

Actin is the central building block of the actin cytoskeleton, a highly regulated filamentous network enabling dynamic processes of cells and simultaneously providing structure. Mammals have six actin isoforms that are very conserved and thus share common functions. Tissue-specific expression in part underlies their differential roles, but actin isoforms also coexist in various cell types and tissues, suggesting specific functions and preferential interaction partners. Gene deletion models, antibody-based staining patterns, gene silencing effects, and the occurrence of isoform-specific mutations in certain diseases have provided clues for specificity on the subcellular level and its consequences on the organism level. Yet, the differential actin isoform functions are still far from understood in detail. Biochemical studies on the different isoforms in pure form are just emerging, and investigations in cells have to deal with a complex and regulated system, including compensatory actin isoform expression.

Actin filament bundling, i.e. the formation of actin cables, is an important process that relies on proteins able to directly bind and cross-link subunits of adjacent actin filaments. Animal cysteine-rich proteins and their plant counterparts are two LIM domain-containing proteins that were recently suggested to define a new family of actin cytoskeleton regulators involved in actin filament bundling. We here identified the LIM domains as responsible for F-actin binding and bundling activities of the tobacco WLIM1. The deletion of one of the two LIM domains reduced significantly, but did not entirely abolish, the ability of WLIM1 to bind actin filaments. Individual LIM domains were found to interact directly with actin filaments, although with a reduced affinity compared with the native protein. Variants lacking the C-terminal or the inter-LIM domain were only weakly affected in their F-actin stabilizing and bundling activities and trigger the formation of thick cables containing tightly packed actin filaments as does the native protein. In contrast, the deletion of one of the two LIM domains negatively impacted both activities and resulted in the formation of thinner and wavier cables. In conclusion, we demonstrate that the LIM domains of WLIM1 are new autonomous actin binding and bundling modules that cooperate to confer WLIM1 high actin binding and bundling activities.

We used confocal microscopy and in vitro analyses to show that Nicotiana tabacum WLIM1, a LIM domain protein related to animal Cys-rich proteins, is a novel actin binding protein in plants. Green fluorescent protein (GFP)–tagged WLIM1 protein accumulated in the nucleus and cytoplasm of tobacco BY2 cells. It associated predominantly with actin cytoskeleton, as demonstrated by colabeling and treatment with actin-depolymerizing latrunculin B. High-speed cosedimentation assays revealed the ability of WLIM1 to bind directly to actin filaments with high affinity. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed a highly dynamic in vivo interaction of WLIM1-GFP with actin filaments. Expression of WLIM1-GFP in BY2 cells significantly delayed depolymerization of the actin cytoskeleton induced by latrunculin B treatment. WLIM1 also stabilized actin filaments in vitro. Importantly, expression of WLIM1-GFP in Nicotiana benthamiana leaves induces significant changes in actin cytoskeleton organization, specifically, fewer and thicker actin bundles than in control cells, suggesting that WLIM1 functions as an actin bundling protein. This hypothesis was confirmed by low-speed cosedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of WLIM1. Taken together, these data identify WLIM1 as a novel actin binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.

The HeadPiece (HP) domain, present in several F-actin-binding multi-domain proteins, features a well-conserved, solvent-exposed PXWK motif in its C-terminal subdomain. The latter is an autonomously folding subunit comprised of three alpha-helices organised around a hydrophobic core, with the sequence motif preceding the last helix. We report the contributions of each conserved residue in the PXWK motif to human villin HP function and structure, as well as the structural implications of the naturally occurring Pro to Ala mutation in dematin HP. NMR shift perturbation mapping reveals that substitution of each residue by Ala induces only minor, local perturbations in the full villin HP structure. CD spectroscopic thermal analysis, however, shows that the Pro and Trp residues in the PXWK motif afford stabilising interactions. This indicates that, in addition to the residues in the hydrophobic core, the Trp-Pro stacking within the motif contributes to HP stability. This is reinforced by our data on isolated C-terminal HP subdomains where the Pro is also essential for structure formation, since the villin, but not the dematin, C-terminal subdomain is structured. Proper folding can be induced in the dematin C-terminal subdomain by exchanging the Ala for Pro. Conversely, the reverse substitution in the villin C-terminal subdomain leads to loss of structure. Thus, we demonstrate a crucial role for this proline residue in structural stability and folding potential of HP (sub)domains consistent with Pro-Trp stacking as a more general determinant of protein stability.

Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegans protein tetraThymosinbeta expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosinbeta has four repeats, each related to the actin monomer-sequestering protein thymosinbeta 4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosinbeta binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G- and F-actin interaction sites, tetraThymosinbeta may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosinbeta is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosinbeta is of crucial importance at specific developmental stages requiring actin polymerization.

Headpiece (HP) is a 76-residue F-actin-binding module at the C terminus of many cytoskeletal proteins. Its 35-residue C-terminal subdomain is one of the smallest known motifs capable of autonomously adopting a stable, folded structure in the absence of any disulfide bridges, metal ligands, or unnatural amino acids. We report the three-dimensional solution structures of the C-terminal headpiece subdomains of human villin (HVcHP) and human advillin (HAcHP), determined by two-dimensional 1H-NMR. They represent the second and third structures of such C-terminal headpiece subdomains to be elucidated so far. A comparison with the structure of the chicken villin C-terminal subdomain reveals a high structural conservation. Both C-terminal subdomains bind specifically to F-actin. Mutagenesis is used to demonstrate the involvement of Trp 64 in the F-actin-binding surface. The latter residue is part of a conserved structural feature, in which the surface-exposed indole ring is stacked on the proline and lysine side chain embedded in a PXWK sequence motif. On the basis of the structural and mutational data concerning Trp 64 reported here, the results of a cysteine-scanning mutagenesis study of full headpiece, and a phage display mutational study of the 69–74 fragment, we propose a modification of the model, elaborated by Vardar and coworkers, for the binding of headpiece to F-actin.

We propose phage display combined with enzyme-linked immunosorbent assay as a tool for the systematic analysis of protein-protein interactions by investigating the binding behavior of variants to a partner protein. Via enzyme-linked immunosorbent assay we determine both the amount of fusion protein presented at the phage surface and the amount of complex formed, the ratio of which is proportional to the affinity. Hence this method enables us to calculate the relative affinities of a large number of mutants. As model systems, we investigated actin-binding motifs conserved in a number of proteins binding monomeric or filamentous actin. The hexapeptide motifs LKKTET, present in thymosin औ4, and LKKEKG, present in the villin headpiece, were mutated, and the variants were analyzed. Study of the positional tolerance allows postulating that the motifs, although similar in primary structures adopt different conformations when bound to actin. In addition, our data show that the second and the fourth amino acid of the thymosin औ4 motif and the first three residues of the villin headpiece motif are most important for actin binding. The latter result challenges the charged crown hypothesis for the villin headpiece filamentous actin interaction.

Hepatocellular carcinoma (HCC) is characterized by the accumulation of unfolded proteins in the endoplasmic reticulum (ER), which activates the unfolded protein response (UPR). However, the role of ER stress in tumor initiation and progression is controversial. To determine the impact of ER stress, we applied tauroursodeoxycholic acid (TUDCA), a bile acid with chaperone properties. The effects of TUDCA were assessed using a diethylnitrosamine-induced mouse HCC model in preventive and therapeutic settings. Cell metabolic activity, proliferation and invasion were investigated in vitro. Tumor progression was assessed in the HepG2 xenograft model. Administration of TUDCA in the preventive setting reduced carcinogen-induced elevation of alanine and aspartate aminotransferase levels, apoptosis of hepatocytes and tumor burden. TUDCA also reduced eukaryotic initiation factor 2a (eIf2a) phosphorylation, C/EBP homologous protein expression and caspase-12 processing. Thus, TUDCA suppresses carcinogen-induced pro-apoptotic UPR. TUDCA alleviated hepatic inflammation by increasing NF-kappa B inhibitor I kappa Ba. Furthermore, TUDCA altered the invasive phenotype and enhanced metabolic activity but not proliferation in HCC cells. TUDCA administration after tumor development did not alter orthotopic tumor or xenograft growth. Taken together, TUDCA attenuates hepatocarcinogenesis by suppressing carcinogen-induced ER stress-mediated cell death and inflammation without stimulating tumor progression. Therefore, this chemical chaperone could represent a novel chemopreventive agent.

The number of actin binding proteins for which (part of) the three-dimensional structure is known, is steadily increasing. This has led to a picture in which defined structural modules with actin binding capacity are shared between different actin binding proteins. A classification of these based on their common three-dimensional modules appears a logical future step and in this review we provide an initial list starting from the currently known structures. The discussed cases illustrate that a comparison of the similarities and variations within the common structural actin binding unit of different members of a particular class may ultimately provide shortcuts for defining their actin target site and for understanding their effect on actin dynamics. Within this concept, the multitude of possible interactions by an extensive, and still increasing, list of actin binding proteins becomes manageable because they can be presented as variations upon a limited number of structural themes. We discuss the possible evolutionary routes that may have produced the present array of actin binding modules.

Cofilin is representative for a family of low molecular weight actin filament binding and depolymerizing proteins. Recently the three-dimensional structure of yeast cofilin and of the cofilin homologs destrin and actophorin were resolved, and a striking similarity to segments of gelsolin and related proteins was observed (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagaka, F. (1996) Cell 85, 1047-1055; Fedorov, A. A., Lappalainen, P., Fedorov, E. V., Drubin, D. G., and Almo, S. C. (1997) Nat. Struct. Biol. 4, 366-369; Leonard, S. A., Gittis, A. G., Petrella, E. C., Pollard, T. D., and Lattman, E. E. (1997) Nat. Struct. Biol. 4, 369-373). Using peptide mimetics, we show that the actin binding site stretches over the entire cofilin alpha-helix 112-128. In addition, we demonstrate that cofilin and its actin binding peptide compete with gelsolin segments 2-3 for binding to actin filaments. Based on these competition data, we propose that cofilin and segment 2 of gelsolin use a common structural topology to bind to actin and probably share a similar target site on the filament. This adds a functional dimension to their reported structural homology, and this F-actin binding mode provides a basis to further enlighten the effect of members of the cofilin family on actin filament dynamics.

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., This work was supported by grants from the League against Cancer (Stichting tegen Kanker, Belgium), Ghent University (BOF-GOA), the Research Foundation-Flanders (FWO-Vlaanderen), the Interuniversity Attraction Poles program (IUAP), the Spanish Ministry of Science and Innovation, (BIO2011-26689), the Autonomous Community of Madrid (S2010-BMD-2312), “la Caixa” Foundation, the Fund for Scientific Spearheads of Ghent University Hospital and NKP-29, and the Flanders Institute for Biotechnology (VIB). This work further received funding from the European Community’s Health 7thFramework Programme (PRIME-XS project, grant agreement number 262067). JB is supported by the Agency for Innovation by Science and Technology in Flanders (IWT-Vlaanderen). FI is a Postdoctoral Fellow of the Research Foundation-Flanders (FWO-Vlaanderen). DRG is supported by a contract of “Personal Investigador de Apoyo” from the Autonomous Community of Madrid. SC is supported by SonoDrugs (FP7 programme) and Ghent University.

β-actin depletion from mouse embryonic fibroblasts results in an altered transcriptional response rendering these cells a myofibroblast like phenotype. The proteins and upstream regulatory factors responsible for this acquired phenotype, with prominent focal adhesions and stress fibres, are unknown. Data-mining of the changed proteome revealed that actin binding proteins associated with stress fiber or focal adhesion formation are overexpressed in the β-actin knock-out cells and that many of these contain CH-, LIM- or EFh- domains. Furthermore in silico analysis predicts potential common upstream regulators that may, at least partly, coordinate the altered transcriptional response.

Thymosin beta 4 is acknowledged as a major G-actin binding protein maintaining a pool of unassembled actin in motile vertebrate cells. We have examined the function of Tbeta 4 in actin assembly in the high range of concentrations (up to 300 micron) at which Tbeta 4 is found in highly motile blood cells. Tbeta 4 behaves as a simple G-actin sequestering protein only in a range of low concentrations (20 micron). As the concentration of Tbeta 4 increases, its ability to depolymerize F-actin decreases, due to its interaction with F-actin. The Tbeta 4-actin can be incorporated, in low molar ratios, into F-actin, and can be cross-linked in F-actin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. As a result of the copolymerization of actin and Tbeta 4-actin complex, the critical concentration is the sum of free G-actin and Tbeta 4-G-actin concentrations at steady state, and the partial critical concentration of G-actin is decreased by Tbeta 4-G-actin complex. The incorporation of Tbeta 4-actin in F-actin is associated to a structural change of the filaments and eventually leads to their twisting around each other. In conclusion, Tbeta 4 is not a simple passive actin-sequestering agent, and at high concentrations the ability of Tbeta 4-actin to copolymerize with actin reduces the sequestering activity of G-actin-binding proteins. These results question the evaluation of the unassembled actin in motile cells. They account for observations made on living fibroblasts overexpressing beta-thymosins.

International audience; We previously demonstrated that the Bcr-Abl oncogene, p210(bcr-abl), through its unique GEF domain, specifically activates RhoA and induces spontaneous amoeboid motility. We intend to study the pathways downstream RhoA controlling amoeboid motility. Mouse prolymphoblastic cells (Ba/F3 cell line) expressing different forms of Bcr-Abl were embedded in 3-dimensional (3D) Matrigel to study motility and explore the effects of inhibiting Rho pathway (inhibitors and siRNAs). The phosphorylation levels of cofilin-1 and destrin were analyzed by 2-dimensional electrophoresis. Composition of Bcr-Abl signalplex in different conditions was determined by coimmunoprecipitation. Ba/F3p190 and Ba/F3 expressing a mutant form of p210(bcr-abl) (unable to activate RhoA) cells presented a spontaneous motility, but not an amoeboid type. p210(bcr-abl)-induced amoeboid motility in a 3D matrix requires isoform-specific RhoA/ROCK-1/destrin signaling. Next to the conventional Rho/ROCK/MLC/myosin pathway, this pathway is a crucial determinant for amoeboid motility, specific for the destrin isoform (and not its coexpressed homologue cofilin-1). Also, the presence of destrin (and not cofilin-1) in the p210(bcr-abl) complex is dependent on ROCK1, and this signalplex is required for amoeboid motility. This underscores isoform-specific function within the ADF/cofilin family and provides new insight into Bcr-Abl signaling to amoeboid motility and possible impact on understanding chronic myeloid leukemia progression.

Evolutionary conservation for structure function relations is commonly accepted. Here we hypothesize that closely related single domain paralogous proteins, having similar expression profiles and redundant biochemical core functions, additionally evolved to allow and maintain isoform specific differential regulation by single conserved amino acid substitutions. To substantiate this, we considered two families of closely related actin binding proteins combined with data mining of phosphorylated residues in human and mouse proteins. We show that such residues are identical in other orthologs whereas paralogs have a different, but also conserved, non-phosphorylatable residue at the equivalent positions.

Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinson's disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase- and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function.