@article{MTMT:1498564, title = {Automated suppression of sample-related artifacts in Fluorescence Correlation Spectroscopy}, url = {https://m2.mtmt.hu/api/publication/1498564}, author = {Ries, J and Bayer, M and Csúcs, Gábor and Dirkx, R and Solimena, M and Ewers, H and Schwille, P}, journal-iso = {OPT EXPRESS}, journal = {OPTICS EXPRESS}, volume = {18}, unique-id = {1498564}, issn = {1094-4087}, abstract = {Fluorescence Correlation Spectroscopy (FCS) in cells often suffers from artifacts caused by bright aggregates or vesicles, depletion of fluorophores or bleaching of a fluorescent background. The common practice of manually discarding distorted curves is time consuming and subjective. Here we demonstrate the feasibility of automated FCS data analysis with efficient rejection of corrupted parts of the signal. As test systems we use a solution of fluorescent molecules, contaminated with bright fluorescent beads, as well as cells expressing a fluorescent protein (ICA512-EGFP), which partitions into bright secretory granules. This approach improves the accuracy of FCS measurements in biological samples, extends its applicability to especially challenging systems and greatly simplifies and accelerates the data analysis. (C) 2010 Optical Society of America}, year = {2010}, pages = {11073-11082} } @article{MTMT:1498565, title = {A classical NLS and the SUN domain contribute to the targeting of SUN2 to the inner nuclear membrane}, url = {https://m2.mtmt.hu/api/publication/1498565}, author = {Turgay, Y and Ungricht, R and Rothballer, A and Kiss, A and Csúcs, Gábor and Horváth, Péter and Kutay, U}, doi = {10.1038/emboj.2010.119}, journal-iso = {EMBO J}, journal = {EMBO JOURNAL}, volume = {29}, unique-id = {1498565}, issn = {0261-4189}, abstract = {Integral membrane proteins of the inner nuclear membrane (INM) are inserted into the endoplasmic reticulum membrane during their biogenesis and are then targeted to their final destination. We have used human SUN2 to delineate features that are required for INM targeting and have identified multiple elements that collectively contribute to the efficient localization of SUN2 to the nuclear envelope (NE). One such targeting element is a classical nuclear localization signal (cNLS) present in the N-terminal, nucleoplasmic domain of SUN2. A second motif proximal to the cNLS is a cluster of arginines that serves coatomer-mediated retrieval of SUN2 from the Golgi. Unexpectedly, also the C-terminal, lumenal SUN domain of SUN2 supports NE localization, showing that targeting elements are not limited to cytoplasmic or transmembrane domains of INM proteins. Together, SUN2 represents the first mammalian INM protein relying on a functional cNLS, a Golgi retrieval signal and a perinuclear domain to mediate targeting to the INM. The EMBO Journal (2010) 29, 2262-2275. doi:10.1038/emboj.2010.119; Published online 15 June 2010}, year = {2010}, eissn = {1460-2075}, pages = {2262-2275} } @article{MTMT:1498566, title = {Workflow-Based Software Environment for Large-Scale Biological Experiments}, url = {https://m2.mtmt.hu/api/publication/1498566}, author = {Kozak, K and Bakos, G and Hoff, A and Bennett, E and Dunican, D and Davies, A and Kelleher, D and Long, A and Csúcs, Gábor}, doi = {10.1177/1087057110377354}, journal-iso = {J BIOMOL SCREEN}, journal = {JOURNAL OF BIOMOLECULAR SCREENING}, volume = {15}, unique-id = {1498566}, issn = {1087-0571}, abstract = {High-content screening (HCS) technologies are becoming increasingly used in both large-scale drug discovery and basic research programs. These automated imaging and analysis technologies enable the researcher to elucidate the complex biology that underlies the functions of genes, proteins, and other biomolecules at the cellular level. HCS combines the power of automated digital microscopy and advanced software-based image analysis algorithms to detect and quantify biological changes in cells and tissues. This technology is a particularly powerful tool when used to interrogate the cellular effects of exogenously applied agents such as RNAi and/or small molecules. HCS allows for the evaluation of cellular perturbations that occur both at the level of the single cell and within cellular populations. In a multivariate approach, multiple cellular parameters are collected, allowing for more complex analysis. However, in these scenarios, data flow and management still represent substantial bottlenecks in HCS projects. HCS data include a diversity of information from multiple sources such as details pertaining to screening libraries (e.g., siRNA and small molecules), image stacks acquired from automated microscopes (of which there may be up to several million), and the image analysis data. From this, postprocessing algorithms are required to generate statistical, quality control bioinformatic information and ultimately a final hit list. To accomplish these individual tasks, numerous tools can be used to perform each analytical step; however, management of the entire information flow currently requires the use of commercially available proprietary software, the scope of which is often limited, or bespoke customized scripts. In this article, the authors introduce an open-source research tool that allows for the management of the entire data flow of the HCS data chain, by handling and linking information and providing many powerful postprocessing and visualization tools. (Journal of Biomolecular Screening 2010:892-899)}, year = {2010}, eissn = {1552-454X}, pages = {892-899} } @article{MTMT:1498567, title = {A Protein Inventory of Human Ribosome Biogenesis Reveals an Essential Function of Exportin 5 in 60S Subunit Export}, url = {https://m2.mtmt.hu/api/publication/1498567}, author = {Wild, T and Horváth, Péter and Wyler, E and Widmann, B and Badertscher, L and Zemp, I and Kozak, K and Csúcs, Gábor and Lund, E and Kutay, U}, doi = {10.1371/journal.pbio.1000522}, journal-iso = {PLOS BIOL}, journal = {PLOS BIOLOGY}, volume = {8}, unique-id = {1498567}, issn = {1544-9173}, abstract = {The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.}, year = {2010}, eissn = {1545-7885} } @article{MTMT:1498568, title = {Kernelized Z' factor in multiparametric screening technology}, url = {https://m2.mtmt.hu/api/publication/1498568}, author = {Kozak, K and Csúcs, Gábor}, journal-iso = {RNA BIOL}, journal = {RNA BIOLOGY}, volume = {7}, unique-id = {1498568}, issn = {1547-6286}, abstract = {RNA interference (RNAi) high-content screening (HCS) enables massive parallel gene silencing and is increasingly being used to reveal novel connections between genes and disease-relevant phenotypes. The application of genome-scale RNAi relies on the development of high quality HCS assays. The Z' factor statistic provides a way to evaluate whether or not screening run conditions (reagents, protocols, instrumentation, kinetics and other conditions not directly related to the test compounds) are optimized. Z' factor, introduced by Zhang et al. 1 is a dimensionless value that represents both the variability and the dynamic range between two sets of sample control data. This paper describes a new extension of the Z' factor, which integrates multiple readouts for screening quality assessment. Currently presented multivariate Z' factor is based on linear projection, which may not be suitable for data with nonlinear structure. This paper proposes an algorithm which extends existing algorithm to deal with nonlinear data by using the kernel function. Using kernel methods for projections, multiple readouts are condensed to a single parameter, based on which the screening run quality is monitored.}, year = {2010}, eissn = {1555-8584}, pages = {615-620} } @article{MTMT:1498569, title = {Locomotion of fish epidermal keratocytes on spatially selective adhesion patterns}, url = {https://m2.mtmt.hu/api/publication/1498569}, author = {Csúcs, Gábor and Quirin, K and Danuser, G}, doi = {10.1002/cm.20230}, journal-iso = {CELL MOTIL CYTOSKEL}, journal = {CELL MOTILITY AND THE CYTOSKELETON}, volume = {64}, unique-id = {1498569}, issn = {0886-1544}, abstract = {Cell migration results from forces generated by assembly, contraction, and adhesion of the cytoskeleton. To address how these forces integrate in space and time, novel assays are required that allow spatial separation of the different force categories. We used micro-contact printing of fibronectin on glass substrates to study the effect of adhesion patterns on fish epidermal keratocytes locomotion. Cells migrated at similar speeds on homogeneously adhesive substrates and on patterns with 5 pm-wide adhesive stripes interleaved by non-adhesive stripes with a width varied between 5 and 13 pm. The leading edge protruded on adhesive stripes and lagged behind on non-adhesive stripes. On patterns with non-adhesive stripes wider than 13 pm cells halted, although the lamellipodium did not collapse. High correlation was found between the widths of protruding and lagging edge segments and the widths of the underlying stripes. We explain our data by the force balances between actin polymerization, contraction and adhesion on fibronectin stripes; and between actin polymerization, contraction and lamellipodium-internal elastic tension on non-adhesive stripes. We tested our model further by blocking lamellipodium actin network contraction and polymerization. In both experiments we observed that cells eventually lost their ability to move. However, the two perturbations induced distinct morphological responses. The data suggested that forces powering forward motion of keratocytes are largely associated with network assembly whereas contraction maintains cell polarity. This study establishes spatially selective adhesion substrates and cell morphological readouts as a means to elucidate the mechanical balance between substrate adhesion and cytoskeleton-internal tension in cell migration.}, year = {2007}, eissn = {1097-0169}, pages = {856-867} } @article{MTMT:1498571, title = {Surface engineering approaches to micropattern surfaces for cell-based assays}, url = {https://m2.mtmt.hu/api/publication/1498571}, author = {Falconnet, D and Csúcs, Gábor and Grandin, HM and Textor, M}, doi = {10.1016/j.biomaterials.2005.12.024}, journal-iso = {BIOMATERIALS}, journal = {BIOMATERIALS}, volume = {27}, unique-id = {1498571}, issn = {0142-9612}, abstract = {The ability to produce patterns of single or multiple cells through precise surface engineering of cell culture substrates has promoted the development of cellular bioassays that provide entirely new insights into the factors that control cell adhesion to material surfaces, cell proliferation, differentiation and molecular signaling pathways. The ability to control shape and spreading of attached cells and cell-cell contacts through the form and dimension of the cell-adhesive patches with high precision is important. Commitment of stem cells to different specific lineages depends strongly on cell shape, implying that controlled microenvironments through engineered surfaces may not only be a valuable approach towards fundamental cell-biological studies, but also of great importance for the design of cell culture substrates for tissue engineering. Furthermore, cell patterning is an important tool for organizing cells on transducers for cell-based sensing and cell-based drug discovery concepts. From a material engineering standpoint, patterning approaches have greatly profited by combining microfabrication technologies, such as photolithography, with biochemical functionalization to present to the cells biological cues in spatially controlled regions where the background is rendered non-adhesive ("non-fouling") by suitable chemical modification. The focus of this review is on the surface engineering aspects of biologically motivated micropatterning of two-dimensional (flat) surfaces with the aim to provide an introductory overview and critical assessment of the many techniques described in the literature. In particular, the importance of non-fouling surface chemistries, the combination of hard and soft lithography with molecular assembly techniques as well as a number of less well known, but useful patterning approaches, including direct cell writing, are discussed. (c) 2006 Elsevier Ltd. All rights reserved.}, year = {2006}, eissn = {1878-5905}, pages = {3044-3063} } @article{MTMT:1498572, title = {Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers}, url = {https://m2.mtmt.hu/api/publication/1498572}, author = {Goffin, JM and Pittet, P and Csúcs, Gábor and Lussi, JW and Meister, JJ and Hinz, B}, doi = {10.1083/jcb.200506179}, journal-iso = {J CELL BIOL}, journal = {JOURNAL OF CELL BIOLOGY}, volume = {172}, unique-id = {1498572}, issn = {0021-9525}, abstract = {Expression of alpha-smooth muscle actin (alpha-SMA) renders fibroblasts highly contractile and hallmarks myofibroblast differentiation. We identify a-SMA as a mechanosensitive protein that is recruited to stress fibers under high tension. Generation of this threshold tension requires the anchoring of stress fibers at sites of 8-30-mu m-long "supermature" focal adhesions (suFAs), which exert a stress approximately fourfold higher (similar to 12nN/mu m(2)) on micropatterned deformable substrates than 2-6-mu m-long classical FAs. Inhibition of suFA formation by growing myobroblasts on substrates with a compliance of <= 11 kPa and on rigid micropatterns of 6-mu m-long classical FA islets confines alpha-SMA to the cytosol. Reincorporation of alpha-SMA into stress fibers is established by stretching 6-mu m-long classical FAs to 8.1-mu m-long suFA islets on extendable membranes; the same stretch producing 5.4-mu m-long classical FAs from initially 4-mu m-long islets is without effect. We propose that the different molecular composition and higher phosphorylation of FAs on supermature islets, compared with FAs on classical islets, accounts for higher stress resistance.}, year = {2006}, eissn = {1540-8140}, pages = {259-268} } @article{MTMT:1498573, title = {Pattern stability under cell culture conditions - A comparative study of patterning methods based on PLL-g-PEG background passivation}, url = {https://m2.mtmt.hu/api/publication/1498573}, author = {Lussi, JW and Falconnet, D and Hubbell, JA and Textor, M and Csúcs, Gábor}, doi = {10.1016/j.biomaterials.2005.11.027}, journal-iso = {BIOMATERIALS}, journal = {BIOMATERIALS}, volume = {27}, unique-id = {1498573}, issn = {0142-9612}, abstract = {Despite the rapidly increasing number of publications on the fabrication and use of micro-patterns for cell studies, comparatively little is know about the long-term stability of such patterns under cell culture conditions. Here, we report on the long-term stability of cellular patterns created by three different patterning techniques: selective molecular assembly patterning, micro-contact printing and molecular assembly patterning by lift-off. We demonstrate that although all three techniques were combined with the same background passivation chemistry based on assembly of a PEG-graft copolymer, there are considerable differences in the long-term stability between the three different pattern types under cell culture conditions. Our results suggest that these differences are not cell-dependent but are due to different (substrate-dependent) interactions between the patterned substrate, the passivating molecule and the serum containing cellular medium. (c) 2005 Elsevier Ltd. All rights reserved.}, year = {2006}, eissn = {1878-5905}, pages = {2534-2541} } @article{MTMT:1498575, title = {Tocopherol cyclase (VTE1) localization and vitamin E accumulation in chloroplast plastoglobule lipoprotein particles}, url = {https://m2.mtmt.hu/api/publication/1498575}, author = {Vidi, PA and Kanwischer, M and Baginsky, S and Austin, JR and Csúcs, Gábor and Dormann, P and Kessler, F and Brehelin, C}, doi = {10.1074/jbc.M511939200}, journal-iso = {J BIOL CHEM}, journal = {JOURNAL OF BIOLOGICAL CHEMISTRY}, volume = {281}, unique-id = {1498575}, issn = {0021-9258}, abstract = {Chloroplasts contain lipoprotein particles termed plastoglobules. Plastoglobules are generally believed to have little function beyond lipid storage. Here we report on the identification of plastoglobule proteins using mass spectrometry methods in Arabidopsis thaliana. We demonstrate specific plastoglobule association of members of the plastid lipid-associated proteins/fibrillin family as well as known metabolic enzymes, including the tocopherol cyclase (VTE1), a key enzyme of tocopherol ( vitamin E) synthesis. Moreover, comparative analysis of chloroplast membrane fractions shows that plastoglobules are a site of vitamin E accumulation in chloroplasts. Thus, in addition to their lipid storage function, we propose that plastoglobules are metabolically active, taking part in tocopherol synthesis and likely other pathways.}, year = {2006}, eissn = {1083-351X}, pages = {11225-11234} }