Automated Analysis of Single-Molecule Photobleaching Data by Statistical Modeling of Spot Populations

The number of fluorophores within a molecule complex can be revealed by single-molecule photobleaching imaging. A widely applied strategy to analyze intensity traces over time is the quantification of photobleaching step counts. However, several factors can limit and bias the detection of photobleaching steps, including noise, high numbers of fluorophores, and the possibility that several photobleaching events occur almost simultaneously. In this study, we propose a new approach, to our knowledge, to determine the fluorophore number that correlates the intensity decay of a population of molecule complexes with the decay of the number of visible complexes. We validated our approach using single and fourfold Atto-labeled DNA strands. As an example we estimated the subunit stoichiometry of soluble CD95L using GFP fusion proteins. To assess the precision of our method we performed in silico experiments showing that the estimates are not biased for experimentally observed intensity fluctuations and that the relative precision remains constant with increasing number of fluorophores. In case of fractional fluorescent labeling, our simulations predicted that the fluorophore number estimate corresponds to the product of the true fluorophore number with the labeling fraction. Our method, denoted by spot number and intensity correlation (SONIC), is fully automated, robust to noise, and does not require the counting of photobleaching events.     

Complete genome sequence of Mahella australiensis type strain (50-1 BON)

Mahella australiensis Bonilla Salinas et al. 2004 is the type species of the genus Mahella, which belongs to the family Thermoanaerobacteraceae. The species is of interest because it differs from other known anaerobic spore-forming bacteria in its G+C content, and in certain phenotypic traits, such as carbon source utilization and relationship to temperature. Moreover, it has been discussed that this species might be an indigenous member of petroleum and oil reservoirs. This is the first completed genome sequence of a member of the genus Mahella and the ninth completed type strain genome sequence from the family Thermoanaerobacteraceae. The 3,135,972 bp long genome with its 2,974 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Substrate and inhibitor specificities differ between human cytosolic and mitochondrial thioredoxin reductases: Implications for development of specific inhibitors

The cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and thioredoxins (Trx1 and Trx2) are key components of the mammalian thioredoxin system, which is important for antioxidant defense and redox regulation of cell function. TrxR1 and TrxR2 are selenoproteins generally considered to have comparable properties, but to be functionally separated by their different compartments. To compare their properties we expressed recombinant human TrxR1 and TrxR2 and determined their substrate specificities and inhibition by metal compounds. TrxR2 preferred its endogenous substrate Trx2 over Trx1, whereas TrxR1 efficiently reduced both Trx1 and Trx2. TrxR2 displayed strikingly lower activity with dithionitrobenzoic acid (DTNB), lipoamide, and the quinone substrate juglone compared to TrxR1, and TrxR2 could not reduce lipoic acid. However, Sec-deficient two-amino-acid-truncated TrxR2 was almost as efficient as full-length TrxR2 in the reduction of DTNB. We found that the gold(I) compound auranofin efficiently inhibited both full-length TrxR1 and TrxR2 and truncated TrxR2. In contrast, some newly synthesized gold(I) compounds and cisplatin inhibited only full-length TrxR1 or TrxR2 and not truncated TrxR2. Surprisingly, one gold(I) compound, [Au(d2pype)(2)]Cl, was a better inhibitor of TrxR1, whereas another, [(iPr(2)Im)(2)Au]Cl, mainly inhibited TrxR2. These compounds also inhibited TrxR activity in the cytoplasm and mitochondria of cells, but their cytotoxicity was not always dependent on the proapoptotic proteins Bax and Bak. In conclusion, this study reveals significant differences between human TrxR1 and TrxR2 in substrate specificity and metal compound inhibition in vitro and in cells, which may be exploited for development of specific TrxR1- or TrxR2-targeting drugs.     

Biofunctionalization of a generic collagenous triple helix with the α2β1 integrin binding site allows molecular force measurements

The integrin α2β1 plays an important role in force-transmitting cell-matrix interactions. It recognizes the peptide sequence GFOGER (O=4-hydroxy-proline) presented as trimer within a collagenous triple-helical framework. We produced the recombinant non-hydroxylated mini-collagen, termed FC3, which harbors the α2β1 integrin recognition site. FC3 consists of a foldon-stabilized host triple helix of three chains with 10 GPP-repeats, into which the integrin binding motif was inserted. The triple-helical structure could further be stabilized by covalently cross-linking the three chains. Unlike collagen-I, FC3 lacks binding sites for matrix proteins and cellular receptors other than the collagen-binding integrins. It showed a preference for α2β1 over α1β1 integrin, especially when the chains were neither cross-linked nor prolyl-hydroxylated. Using FC3 as substratum for primary skin fibroblasts, we showed that the loss of α2β1 integrin could not be compensated by other collagen-binding integrins, suggesting a major role of α2β1 integrin in exerting sufficient mechanical force to induce or sustain cell spreading. Atomic force microscopy revealed that a single α2β1 integrin can withstand tensile forces of up to approximately 160pN before it releases FC3. Moreover, FC3 is fully competent to agonistically elicit α2β1 integrin-induced cell reactions, such as recruitment of α2β1 integrin into focal adhesions and lamellipodia formation. The biofunctionalized mini-collagen sheds light on the molecular forces of the α2β1 integrin-collagen interaction, which affects tissue homeostasis by contracting the connective tissue and by contributing to interstitial tissue pressure regulation. Additionally, biofunctionalized mini-collagens can be useful in force-resistant cell attachment to biomedical materials.     

Protein phosphatase 2A (PP2A)-specific ubiquitin ligase MID1 is a sequence-dependent regulator of translation efficiency controlling 3-phosphoinositide-dependent protein kinase-1 (PDPK-1)

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells.     

The effect of charcoal production on carbon cycling in African biomes

Using biomass for charcoal production in sub-Saharan Africa (SSA) may change carbon stock dynamics and lead to irreversible changes in the carbon balance, yet we have little understanding of whether these dynamics vary by biome in this region. Currently, charcoal production contributes up to 7% of yearly deforestation in tropical regions, with carbon emissions corresponding to 71.2 million tonnes of CO₂ and 1.3 million tonnes of CH₄. With a projected increased demand for charcoal in the coming decades, even low harvest rates may throw the carbon budget off-balance due to legacy effects. Here, we parameterized the dynamic global vegetation model LPJ-GUESS for six SSA biomes and examined the effect of charcoal production on net ecosystem exchange (NEE), carbon stock sizes and recovery time for tropical rain forest, montane forest, moist savanna, dry savanna, temperate grassland and semi-desert. Under historical charcoal regimes, tropical rain forests and montane forests transitioned from net carbon sinks to net sources, that is, mean cumulative NEE from −3.56 ± 2.59 kg C/m² to 2.46 ± 3.43 kg C/m² and −2.73 ± 2.80 kg C/m² to 1.87 ± 4.94 kg C/m² respectively. Varying charcoal production intensities resulted in tropical rain forests showing at least two times higher carbon losses than the other biomes. Biome recovery time varied by carbon stock, with tropical and montane forests taking about 10 times longer than the fast recovery observed for semi-desert and temperate grasslands. Our findings show that high biomass biomes are disproportionately affected by biomass harvesting for charcoal, and even low harvesting rates strongly affect vegetation and litter carbon and their contribution to the carbon budget. Therefore, the prolonged biome recoveries imply that current charcoal production practices in SSA are not sustainable, especially in tropical rain forests and montane forests, where we observe longer recovery for vegetation and litter carbon stocks.     

Identification of a bone sialoprotein receptor in osteosarcoma cells

Bone sialoprotein (BSP) is an extracellular matrix glycoprotein associated with the mineral bone matrix. The amino acid sequence of BSP contains an Arg-Gly-Asp (RGD) sequence which confers to the protein cell binding properties (Oldberg, A., Franzén, A., and Heineg?rd, D. (1988) J. Biol. Chem. 263, 19430-19432). When BSP was used as an affinity matrix to isolate a cell surface receptor from rat osteosarcoma cells, a protein composed of polypeptides similar in size to those of a previously characterized vitronectin receptor was obtained. This putative BSP receptor, like the vitronectin receptor, bound also to an affinity matrix made of an RGD-containing heptapeptide. Moreover, similar patterns of inhibition of cell attachment to BSP and vitronectin was obtained with variant RGD-containing peptides, with BSP and with vitronectin. Finally, an anti-vitronectin receptor antiserum immunoprecipitated a receptor identical in size to the receptor bound to a BSP affinity matrix. These results show that BSP is recognized by an RGD-directed receptor and that both vitronectin and BSP can bind to this receptor.     

Targeting the retinoblastoma protein by MC007L, gene product of the molluscum contagiosum virus: detection of a novel virus-cell interaction by a member of the poxviruses

The human pathogenic poxvirus molluscum contagiosum virus (MCV) is the causative agent of benign neoplasm, with worldwide incidence, characterized by intraepidermal hyperplasia and hypertrophy of cells. Here, we present evidence that the MC007L protein of MCV targets retinoblastoma protein (pRb) via a conserved LxCxE motif, which is present in many viral oncoproteins. The deregulation of the pRb pathway plays a central role in tumor pathogenesis. The oncoproteins of small DNA viruses contain amino acid sequences that bind to and inactivate pRb. Isolated expression of these oncoproteins induces apoptosis, cell proliferation, and cellular transformation. The MC007L gene displays no homology to other genes within the poxvirus family. The protein anchors into the outer mitochondrial membrane via an N-terminal mitochondrial targeting sequence. Through the LxCxE motifs, MC007L induces a cytosolic sequestration of pRb at mitochondrial membranes, leading to the inactivation of the protein by mislocalization. MC007L precipitates the endogenous pRb/E2F-1 complex. Moreover, MC007L is able to cooperate to transform primary rat kidney cells. The interaction between MC007L and pRb provides a novel mechanism by which a virus can perturb the cell cycle.