Biochemical Characterization of the Cell-Biomaterial Interface by Quantitative Proteomics

Surface topography and texture of cell culture substrata can affect the differentiation and growth of adherent cells. The biochemical basis of the transduction of the physical and mechanical signals to cellular responses is not well understood. The lack of a systematic characterization of cell-biomaterial interaction is the major bottleneck. This study demonstrated the use of a novel subcellular fractionation method combined with quantitative MS-based proteomics to enable the robust and high-throughput analysis of proteins at the adherence interface of Madin-Darby canine kidney cells. This method revealed the enrichment of extracellular matrix proteins and membrane and stress fibers proteins at the adherence surface, whereas it shows depletion of extracellular matrix belonging to the cytoplasmic, nucleus, and lateral and apical membranes. The asymmetric distribution of proteins between apical and adherence sides was also profiled. Apart from classical proteins with clear involvement in cell-material interactions, proteins previously not known to be involved in cell attachment were also discovered.The growth and differentiation of cells in multicellular organisms are regulated by the complex interplay of biochemical and mechanical signals. In the past decades, a plethora of data on the roles of mechanical and structural cues in modulating cellular behaviors has emerged (1–5). It is increasingly evident that cell fates can be changed by engineering the physical properties of the microenvironment to which the cells are exposed (6–8). These observations have inspired the development of functionalized biomaterials that can directly and specifically interact with tissue components, and support or even direct the appropriate cellular activities (9, 10). Although promising progress has been observed in the past few years, several gaps in knowledge in this field have hindered the development of such ”intelligent” biomaterials. In particular, the understanding of the mechanism in which the cell orchestrates physiological and morphological changes by translating mechanical and structural information into biochemical signals is still very limited.As a standard experimental model, cell lines cultured in vitro as a monolayer over solid substrata are usually used to study the effects of biomaterial surfaces on cellular phenotypes. With this simple model system, ingenious experiments have shown that physical forces applied through the extracellular matrix (ECM)1 can induce changes in cell adhesion molecules and stress-induced ion channels, which then lead to changes in the cytoskeleton and gene expressions (11–13). We term the biochemical structure present at the interface between the substratum and the cellular interior the adherence surface (AS), which is composed of the basal plasma membrane with associated structures such as the ECM on one side and the focal adherence complexes on the other. In monolayer cell culture systems, the AS is the only part of the cells in direct contact with the substratum, and is therefore responsible for the first line of communication between the cells and the biomaterial. It is likely that the AS is the organelle that mediates the communication of mechanical and tectonic signals from the substratum to biochemical transducers in the cells. Given the complexity of this process, it is clear that the understanding of this phenomenon cannot be achieved merely by studying individual biological parts in isolation. It is necessary, therefore, to systematically characterize the biochemical factors that mediate the interactions between cells and materials to yield insights into intracellular signaling processes that are responsible for such cellular responses. Toward this goal, we seek to investigate the biochemical basis of how different biomaterials may impose changes in the composition of the AS of adherent cells.MS-based proteomics have recently emerged as a standard technique in modern cell biology. Various techniques based on the chemical conjugation of isotopically labeled reporters to proteins or peptides, such as the isobaric tag for relative and absolute quantitation (iTRAQ) and the isotope-coded affinity tags, enable MS-based proteomics to quantify and compare proteome changes between biological samples. As an attractive alternative, stable isotope labeling with amino acids in cell culture (SILAC) is a metabolic labeling technique that enables isotopically encoded cells to be mixed before lysis and fractionation, thus eliminating inherent quantification biases in these steps, and also enables a simpler procedure and more accurate quantitation (14). SILAC MS-based proteomics have recently contributed to organellar proteomes (15, 16), accurate measurement of protein-protein interactions (17), and the characterization of proteome dynamics during cell differentiation (18). The use of MS-based proteomics has enabled the systematic evaluation of proteome changes on the adhesion of cells to substrata of interest. Kantawong et al. (19) applied DIGE and LC-MS/MS to identify proteome changes in cells on surface with nanotopography. Xu et al. (20) investigated proteome differences of human osteoblasts on various nano-sized hydroxyapatite powders with different shapes and chemical compositions using iTRAQ-based two-dimensional LC-MS/MS.One advantage of proteomics is that it can effectively be combined with subcellular fractionation and allow the comprehensive characterization of the proteins enriched in targeted cellular structures. To yield new insight in molecular interactions in cell-biomaterial interfaces, we aimed to develop a robust protocol for the proteomic characterization of the AS of adherent cells on a biomaterial surface and use it for discovering new cell-biomaterial interface specific biomarkers. Our approach was to develop an isolation technique for AS with high yields and purity for proteomic analysis. The isolated AS on substratum was analyzed by confocal microscopy and Western blotting. SILAC was then used to characterize the fold-enrichment of proteins in the purified AS compared with whole cells and to discover new biomolecules in the cell-biomaterial interface. This study introduces a novel cell-biomaterial interface proteomic procedure, which can be used to identify the AS specific proteome in a high throughput manner and provide a simple and robust method to systematically analyze cell-biomaterial interactions at a molecular level.     

Morphological and physiological responses of canola (Brassica napus) siliquas and seeds to UVB and CO2 under controlled environment conditions

Combined effects of UVB radiation and CO₂ concentration on plant reproductive parts have received little attention. We studied morphological and physiological responses of siliquas and seeds of canola (Brassica napus L. cv. 46A65) to UVB and CO₂ under four controlled experimental conditions: UVB radiation (4.2 kJ m⁻² d⁻¹) with ambient level of CO₂ (370 μmol mol⁻¹) (control); UVB radiation (4.2 kJ m⁻² d⁻¹) with elevated level of CO₂ (740 μmol mol⁻¹); no UVB radiation (0 kJ m⁻² d⁻¹) with ambient level of CO₂ (370 μmol mol⁻¹); and no UVB radiation (0 kJ m⁻² d⁻¹) with elevated level of CO₂ (740 μmol mol⁻¹). UVB radiation affected the outer appearance of siliquas, such as colour, as well as their anatomical structures. At both CO₂ levels, the UVB radiation of 4.2 kJ m⁻² d⁻¹ reduced the size of seeds, which had different surface patterns than those from no UVB radiation. At both CO₂ levels, 4.2 kJ m⁻² d⁻¹ of UVB decreased net CO₂ assimilation (A_N) and water use efficiency (WUE), but had no effect on transpiration (E). Elevated CO₂ increased A_N and WUE, but decreased E, under both UVB conditions. At both CO₂ levels, the UVB radiation of 4.2 kJ m⁻² d⁻¹ decreased chlorophyll fluorescence, total chlorophyll (Chl), Chl a and Chl b, but had no effect on the ratio of Chl a/b and the concentration of UV-screening pigments. Elevated CO₂ increased total Chl and the concentration of UV-screening pigments under 4.2 kJ m⁻² d⁻¹ of UVB radiation. Neither UVB nor CO₂ affected wax content of siliqua surface. Many significant relationships were found between the above-mentioned parameters. This study revealed that UVB radiation exerts an adverse effect on canola siliquas and seeds, and some of the detrimental effects of UVB on these reproductive parts can partially be mitigated by CO₂.     

Mechanisms of the vasorelaxant effect of 1-hydroxy-2, 3, 5-trimethoxy-xanthone, isolated from a Tibetan herb, Halenia elliptica, on rat coronary artery

1-Hydroxy-2, 3, 5-trimethoxyxanthone (HM-1) is a xanthone isolated from Halenia elliptica, a Tibetan medicinal herb. HM-1 (0.33-42.1 microM) produced a concentration-dependent relaxation in rat coronary artery rings pre-contracted with 1 microM 5-hydroxytryptamine (5-HT), with an EC(50) of 1.67+/-0.27 microM. Removal of the endothelium significantly affected the vasodilator potency of HM-1, resulting in 46% decrease in E(max) value. The endothelium-dependent effects of HM-1 was confirmed when its vasorelaxant effect was inhibited after addition of nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (100 microM) or the soluble guanylate cyclase inhibitor 1H-[1, 2, 4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ, 10 microM). Atropine (100 nM), flurbiprofen (10 microM), propranolol (100 microM), pyrilamine (10 microM), cimetidine (10 microM) and SQ22536 (100 microM) had no effect on the vasorelaxant activity of HM-1 indicated the non-involvement of other receptor/enzyme systems. In endothelium-denuded coronary artery rings, the vasorelaxant effect of HM-1 was unaffected by potassium channel blockers such as tetraethylammonium (10 mM), iberiotoxin (100 nM), barium chloride (100 microM) and 4-aminopyridine (1 mM). The involvement of Ca(2+) channel in 5-HT-primed artery ring preparations incubated with Ca(2+)-free buffer was confirmed when HM-1 (9.93 microM) partially abolished the CaCl(2)-induced vasoconstriction (87% inhibition in intact-endothelium artery rings; 50% inhibition in endothelium-denuded rings). In the KCl-primed preparations incubated with Ca(2+)-free buffer, HM-1 (9.93 microM) produced a 27.3% inhibition in endothelium-denuded rings. HM-1 (3.31-33.1 microM) had minimal relaxant effects (14.4%-20.3%) on the contractile response generated by 10 microM phorbol 12,13-diacetate (PDA) in Ca(2+)-free solutions, suggesting minimal effects on intracellular Ca(2+) mechanisms. These findings suggest the vasodilator action of HM-1 involved both an endothelium-dependent mechanism involving NO and an endothelium-independent mechanism by inhibiting Ca(2+) influx through L-type voltage-operated Ca(2+) channels; a minor contribution to the effects of HM-1 may be related to inhibition of the protein kinase C-mediated release of intracellular Ca(2+) stores.     

Pharmacological and biophysical isolation of K+ currents encoded by ether-à-go-go-related genes in murine hepatic portal vein smooth muscle cells

The functional properties of the Saccharomyces cerevisiae bicarbonate transporter homolog Bor1p (YNL275wp) were characterized by measuring boron (H₃BO₃), Na⁺, and Cl^– fluxes. Neither Na⁺ nor Cl^– appears to be a transported substrate for Bor1p. Uphill efflux of boron mediated by Bor1p was demonstrated directly by loading cells with boron and resuspending in a low-boron medium. Cells with intact BOR1, but not the deletant strain, transport boron outward until the intracellular concentration is sevenfold lower than that in the medium. Boron efflux through Bor1p is a saturable function of intracellular boron (apparent K_m 1–2 mM). The extracellular pH dependences of boron distribution and efflux indicate that uphill efflux is driven by the inward H⁺ gradient. Addition of 30 mM HCO₃^– does not affect boron extrusion by Bor1p, indicating that HCO₃^– does not participate in Bor1p function. Functional Bor1p is present in cells grown in medium with no added boron, and overnight growth in 10 mM H₃BO₃ causes only a small increase in the levels of functional Bor1p and in BOR1 mRNA. The fact that Bor1p is expressed when there is no need for boron extrusion and is not strongly induced in the presence of growth-inhibitory boron concentrations is surprising if the main physiological function of yeast Bor1p is boron efflux. A possible role in vacuolar dynamics for Bor1p was recently reported by Decker and Wickner (10). Under the conditions used presently, there appears to be mildly abnormal vacuolar morphology in the deletant strain. boron; SLC4; YNL275w     

Quantitative Changes in Gimap3 and Gimap5 Expression Modify Mitochondrial DNA Segregation in Mice

Mammalian mitochondrial DNA (mtDNA) is a high-copy maternally inherited genome essential for aerobic energy metabolism. Mutations in mtDNA can lead to heteroplasmy, the co-occurence of two different mtDNA variants in the same cell, which can segregate in a tissue-specific manner affecting the onset and severity of mitochondrial dysfunction. To investigate mechanisms regulating mtDNA segregation we use a heteroplasmic mouse model with two polymorphic neutral mtDNA haplotypes (NZB and BALB) that displays tissue-specific and age-dependent selection for mtDNA haplotypes. In the hematopoietic compartment there is selection for the BALB mtDNA haplotype, a phenotype that can be modified by allelic variants of Gimap3. Gimap3 is a tail-anchored member of the GTPase of the immunity-associated protein (Gimap) family of protein scaffolds important for leukocyte development and survival. Here we show how the expression of two murine Gimap3 alleles from Mus musculus domesticus and M. m. castaneus differentially affect mtDNA segregation. The castaneus allele has incorporated a uORF (upstream open reading frame) in-frame with the Gimap3 mRNA that impairs translation and imparts a negative effect on the steady-state protein abundance. We found that quantitative changes in the expression of Gimap3 and the paralogue Gimap5, which encodes a lysosomal protein, affect mtDNA segregation in the mouse hematopoietic tissues. We also show that Gimap3 localizes to the endoplasmic reticulum and not mitochondria as previously reported. Collectively these data show that the abundance of protein scaffolds on the endoplasmic reticulum and lysosomes are important to the segregation of the mitochondrial genome in the mouse hematopoietic compartment.     

Srs2 enables checkpoint recovery by promoting disassembly of DNA damage foci from chromatin

Following DNA repair, checkpoint signalling must be abated to resume cell cycling in a phenomenon known as checkpoint recovery. Although a number of genes have been implicated in the recovery process, it is still unknown whether checkpoint recovery is caused by a signalling network activated by DNA repair or whether it is the result of the loss of DNA structures that elicit the checkpoint. Here we show that checkpoint recovery can be uncoupled from bulk chromosome DNA repair if single-stranded (ss) DNA persists. This situation occurs in cells that are deficient in the Srs2 helicase, a protein that antagonizes Rad51. We report that srs2Δ cells fail to eliminate Ddc2 and RPA subnuclear foci following bulk chromosome repair due to the persistence of ssDNA. In contrast to cells with DNA double-strand breaks that remain unrepaired, srs2Δ cells remove the 9-1-1 checkpoint clamp from chromatin after repair. However, despite the loss of the 9-1-1 clamp, Dpb11 remains associated with chromatin to promote checkpoint activity. Our work indicates that Srs2 promotes checkpoint recovery by removing Rad51 after DNA repair. A failure to remove Rad51 causes persistence of ssDNA and the checkpoint signal. Therefore, we conclude that cells initiate recovery when the DNA structures that elicit the checkpoint are eliminated.     

Identification of major clonal complexes and toxin producing strains among Staphylococcus aureus associated with atopic dermatitis

Staphylococcus aureus promotes the onset and severity of atopic dermatitis (AD), which is exacerbated by superantigen toxins SEB and SEC. The genetic identity of these isolates, and their relationship to common hospital- or community-associated methicillin resistant S. aureus (HA-MRSA and CA-MRSA) has not been defined. We conducted spa typing, partial multi-locus sequence typing (MLST), and toxin profiling (seb, sec, lukS-PV) of S. aureus from 119 pediatric and 40 adult AD patients. MLST clonal complexes CC45, CC5, CC15, CC1, CC8 and CC30 accounted for 79% of isolates, representing the same major groups reported for nosocomial S. aureus in hospital intensive care units. The highest disease severity was associated with CC1, which was significantly greater relative to CC15 (p?=?0.017) or CC30 (p?=?0.040), but with no significant difference relative to CC45, CC5 or CC8. Although there were two few lukS-PV, seb or sec isolates to infer a role in disease severity, CC45 was identified as a source of SEC producing strains, and lukS-PVL was associated with a small number of CC5 pediatric isolates. CC1 harbored the only CA-MRSA that was identified, and was a source of isolates that expressed both seb and sec, and closely resembled the USA400 strain of CA-MRSA.     

Cistrome: an integrative platform for transcriptional regulation studies

The increasing volume of ChIP-chip and ChIP-seq data being generated creates a challenge for standard, integrative and reproducible bioinformatics data analysis platforms. We developed a web-based application called Cistrome, based on the Galaxy open source framework. In addition to the standard Galaxy functions, Cistrome has 29 ChIP-chip- and ChIP-seq-specific tools in three major categories, from preliminary peak calling and correlation analyses to downstream genome feature association, gene expression analyses, and motif discovery. Cistrome is available at http://cistrome.org/ap/.