Gabor Csucs

Focal adhesion size controls tension-dependent recruitment of α-smooth muscle actin to stress fibers

Expression of α-smooth muscle actin (α-SMA) renders fibroblasts highly contractile and hallmarks myofibroblast differentiation. We identify α-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-μm-long “supermature” focal adhesions (suFAs), which exert a stress approximately fourfold higher (∼12 nN/μm2) on micropatterned deformable substrates than 2–6-μm-long classical FAs. Inhibition of suFA formation by growing myofibroblasts on substrates with a compliance of ≤11 kPa and on rigid micropatterns of 6-μm-long classical FA islets confines α-SMA to the cytosol. Reincorporation of α-SMA into stress fibers is established by stretching 6-μm-long classical FAs to 8.1-μm-long suFA islets on extendable membranes; the same stretch producing 5.4-μm-long classical FAs from initially 4-μm-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.

Optical grating coupler biosensors.

By incorporating a grating in a planar optical waveguide one creates a device with which the spectrum of guided lightmodes can he measured. When the surface of the waveguide is exposed to different solutions, the peaks in the spectrum shift due to molecular interactions with the surface. Optical waveguide lightmode spectroscopy (OWLS) is a highly sensitive technique that is capable of real-time monitoring of these interactions. Since this integrated optical method is based on the measurement of the polarizability density (i.e., refractive index) in the vicinity of the waveguide surface, radioactive, fluorescent or other kinds of labeling are not required. In addition, measurement of at least two guided modes enables the absolute mass of adsorbed molecules to be determined. In this article, the technique will be described in some detail, and applications from different areas will be discussed. Selected examples will be presented to demonstrate how monitoring the modification of different metal oxides with polymers and the response of the coated oxides to biofluids help in the design of novel biomaterials; how OWLS is useful for accurate bioaffinity sensing, which is a key issue in the development of new drugs; and how the quantitative study of protein-DNA/RNA and cell surface interactions can enhance the understanding of processes in molecular and cellular biology.

Tocopherol cyclase (VTE1) localization and vitamin E accumulation in chloroplast plastoglobule lipoprotein particles.

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.

Microcontact printing of novel co-polymers in combination with proteins for cell-biological applications.

Microcontact printing (microcP) is a cost effective and simple method to create chemically micropatterned surfaces for cell biological applications. We have combined the technique with the spontaneous molecular assembly of a polycationic PEG-grafted copolymer, poly-L-lysine-g-poly(ethylene glycol) (PLL-g-PEG). PLL-g-PEG with omega-functionalized PEG chains was print-transferred onto tissue culture polystyrene (TCPS) or glass substrates, resulting in patterns with a lateral resolution down to 1 microm. Subsequently, dipping in an aqueous solution of non-functionalized PLL-g-PEG was used to backfill the non-printed regions of the surface, rendering them highly protein and thus cell resistant. In a second approach, proteins were stamped and a PLL-g-PEG backfill was applied for passivation of the bare surface regions. Printing of peptide(RGD)-functionalized PLL-g-PEG or proteins combined with a subsequent PLL-g-PEG backfill can be applied to a wide variety of substrate materials with negatively charged surfaces such as TCPS, glass and many metal oxides. We have tested the printed surfaces with human foreskin fibroblasts for cell adhesion and long-term performance and with fish epidermal keratocytes for cell motility and short-time behaviour. Both cell types reacted selectively to the surface micropatterns. Fibroblasts adhered to the printed (adhesive) regions only, where they remained attached up to at least 1 week and were even able to proliferate. Keratocyte spreading and motility were also directed by the geometry of the underlying patterns. The results prove that microcP in conjunction with the use of PLL-g-PEG and its derivatives provides a simple and robust alternative to previously reported micropatterning methods for future cell biological and biotechnological applications.

A Protein Inventory of Human Ribosome Biogenesis Reveals an Essential Function of Exportin 5 in 60S Subunit Export

A systematic search for human ribosome biogenesis factors shows conservation of many aspects of eukaryotic ribosome synthesis with the well-studied process in yeast and identifies an export route of 60S subunits that is specific for higher eukaryotes.

Ligand-Specific Targeting of Microspheres to Phagocytes by Surface Modification with Poly(L-Lysine)-Grafted Poly(Ethylene Glycol) Conjugate

AbstractPurpose. The purpose of this study was to demonstrate specific receptor-mediated targeting of phagocytes by functional surface coatings of microparticles, shielding from nonspecific phagocytosis and allowing ligand-specific interactions via molecular recognition. Methods. Coatings of the comb polymer poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) were investigated for potential to inhibit 1) nonspecific spreading of human blood-derived macrophages (MOs) and dendritic cells (DCs) on glass and 2) nonspecific phagocytosis of PLL-g-PEG-coated, carboxylated polystyrene (PS) or biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres. Coating was performed by adsorption of positively charged PLL-g-PEG on negatively charged microparticles or plasma-cleaned glass through electrostatic interaction. The feasibility of ligand-specific interactions was tested with a model ligand, RGD, conjugated to PEG chains of PLL-g-PEG to form PLL-g-PEG-RGD and compared with inactive ligand conjugate, PLL-g-PEG-RDG. Results. Coatings with PLL-g-PEG largely impaired the adherence and spreading of MOs and DCs on glass. The repellent character of PLL-g-PEG coatings drastically reduced phagocytosis of coated PS and PLGA microparticles to 10% in presence of serum. With both MOs and DCs, we observed ligand-specific interactions with PLL-g-PEG-RGD coatings on glass and PS and PLGA microspheres. Ligand specificity was abolished when using inactive ligand conjugate PLL-g-PEG-RDG, whereas repellency of coating was maintained. Conclusions. Coatings of PLL-g-PEG-ligand conjugates provide a novel technology for ligand specific targeting of microspheres to MOs and DCs while reducing nonspecific phagocytosis.

A classical NLS and the SUN domain contribute to the targeting of SUN2 to the inner nuclear membrane

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.

Machine Learning Improves the Precision and Robustness of High-Content Screens: Using Nonlinear Multiparametric Methods to Analyze Screening Results

Imaging-based high-content screens often rely on single cell-based evaluation of phenotypes in large data sets of microscopic images. Traditionally, these screens are analyzed by extracting a few image-related parameters and use their ratios (linear single or multiparametric separation) to classify the cells into various phenotypic classes. In this study, the authors show how machine learning–based classification of individual cells outperforms those classical ratio-based techniques. Using fluorescent intensity and morphological and texture features, they evaluated how the performance of data analysis increases with increasing feature numbers. Their findings are based on a case study involving an siRNA screen monitoring nucleoplasmic and nucleolar accumulation of a fluorescently tagged reporter protein. For the analysis, they developed a complete analysis workflow incorporating image segmentation, feature extraction, cell classification, hit detection, and visualization of the results. For the classification task, the authors have established a new graphical framework, the Advanced Cell Classifier, which provides a very accurate high-content screen analysis with minimal user interaction, offering access to a variety of advanced machine learning methods.

CIDRE: an illumination-correction method for optical microscopy

Uneven illumination affects every image acquired by a microscope. It is often overlooked, but it can introduce considerable bias to image measurements. The most reliable correction methods require special reference images, and retrospective alternatives do not fully model the correction process. Our approach overcomes these issues for most optical microscopy applications without the need for reference images.

Genome-wide RNAi Screening Identifies Protein Modules Required for 40S Subunit Synthesis in Human Cells

Ribosome biogenesis is a highly complex process requiring many assisting factors. Studies in yeast have yielded comprehensive knowledge of the cellular machinery involved in this process. However, many aspects of ribosome synthesis are different in higher eukaryotes, and the global set of mammalian ribosome biogenesis factors remains unexplored. We used an imaging-based, genome-wide RNAi screen to find human proteins involved in 40S ribosomal subunit biogenesis. Our analysis identified ∼ 300 factors, many part of essential protein modules such as the small subunit (SSU) processome, the eIF3 and chaperonin complexes, and the ubiquitin-proteasome system. We demonstrate a role for the vertebrate-specific factor RBIS in ribosome synthesis, uncover a requirement for the CRL4 E3 ubiquitin ligase in nucleolar ribosome biogenesis, and reveal that intracellular glutamine synthesis supports 40S subunit production.