The WD40 protein Caf4p is a component of the mitochondrial fission machinery and recruits Dnm1p to mitochondria

The mitochondrial division machinery regulates mitochondrial dynamics and consists of Fis1p, Mdv1p, and Dnm1p. Mitochondrial division relies on the recruitment of the dynamin-related protein Dnm1p to mitochondria. Dnm1p recruitment depends on the mitochondrial outer membrane protein Fis1p. Mdv1p interacts with Fis1p and Dnm1p, but is thought to act at a late step during fission because Mdv1p is dispensable for Dnm1p localization. We identify the WD40 repeat protein Caf4p as a Fis1p-associated protein that localizes to mitochondria in a Fis1p-dependent manner. Caf4p interacts with each component of the fission apparatus: with Fis1p and Mdv1p through its NH2-terminal half and with Dnm1p through its COOH-terminal WD40 domain. We demonstrate that mdv1{Delta} yeast contain residual mitochondrial fission due to the redundant activity of Caf4p. Moreover, recruitment of Dnm1p to mitochondria is disrupted in mdv1{Delta} caf4{Delta} yeast, demonstrating that Mdv1p and Caf4p are molecular adaptors that recruit Dnm1p to mitochondrial fission sites. Our studies support a revised model for assembly of the mitochondrial fission apparatus

A SILAC-based Approach Identifies Substrates of Caspase-dependent Cleavage upon TRAIL-induced Apoptosis

The extracellular ligand-induced extrinsic pathway of apoptosis is executed via caspase protease cascades that activate downstream effectors by means of site-directed proteolysis. Here we identify proteome changes upon the induction of apoptosis by the cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a Jurkat T cell line. We detected caspase-dependent cleavage substrates by quantifying protein intensities before and after TRAIL induction in SDS gel slices. Apoptotic protein cleavage events are identified by a characteristic stable isotope labeling with amino acids in cell culture (SILAC) ratio pattern across gel slices that results from differential migration of the cleaved and uncleaved proteins. We applied a statistical test to define apoptotic substrates in the proteome. Our approach identified more than 650 of these cleaved proteins in response to TRAIL-induced apoptosis, including many previously unknown substrates and cleavage sites. Inhibitor treatment combined with triple SILAC demonstrated that the detected cleavage events were caspase dependent. Proteins located in the lumina of organelles such as mitochondria and endoplasmic reticulum were significantly underrepresented in the substrate population. Interestingly, caspase cleavage is generally observed in not only one but several members of stable complexes, but often with lower stoichiometry. For instance, all five proteins of the condensin I complex were cleaved upon TRAIL treatment. The apoptotic substrate proteome data can be accessed and visualized in the MaxQB database and might prove useful for basic and clinical research into TRAIL-induced apoptosis. The technology described here is extensible to a wide range of other proteolytic cleavage events

The Performance of Alfalfa Synthetics in the First and Advanced Generations

During alfalfa breeding investigations conducted at the Nebraska Agricultural Experiment Station, numerous superior clones were selected and tested as clones, and in polycross progeny tests. Information was needed on the performance of synthetic varieties in the first and advanced generations, on the optimum number of clones to include in a synthetic variety, and on parent-progeny relationships. Clones with high general combining ability for forage yield as measured by polycross progeny tests, and in certain instances specific combining ability based on single-cross tests, were intercrossed in various ways to produce synthetic varieties. A group of synthetics varying in number of parents from 2 to 6 clones, having in some instances certain clones as common parents, was tested initially in the first generation of synthesis (referred to as Syn-1 from here on), later in the Syn-1 versus the Syn-2, and in some instances in the Syn-1, Syn-2, and Syn-3, and ultimately in the Syn-1,-2,-3, and -4 generations. The purposes of this bulletin are to report (1) comparative results obtained in yield trials involving the Syn-1,-2,-3, and -4 generations of 5 two-clone and 14 multiple-clone synthetics at Lincoln, Nebraska, and Ithaca, New York, and (2) parent-progeny relationships

Intelligent optical methods in image analysis for human detection

This thesis introduces the concept of a person recognition system for use on an integrated autonomous surveillance camera. Developed to enable generic surveillance tasks without the need for complex setup procedures nor operator assistance, this is achieved through the novel use of a simple dynamic noise reduction and object detection algorithm requiring no previous knowledge of the installation environment and without any need to train the system to its installation. The combination of this initial processing stage with a novel hybrid neural network structure composed of a SOM mapper and an MLP classifier using a combination of common and individual input data lines has enabled the development of a reliable detection process, capable of dealing with both noisy environments and partial occlusion of valid targets. With a final correct classification rate of 94% on a single image analysis, this provides a huge step forwards as compared to the reported 97% failure rate of standard camera surveillance systems.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair

BACKGROUND: Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells. RESULTS: A deletion of the sbcC gene rendered Bacillus subtilis cells sensitive to DNA damage caused by Mitomycin C (MMC) or by gamma irradiation. The deletion of the sbcC gene in a recN mutant background increased the sensitivity of the single recN mutant strain. SbcC was also non-epistatic with AddAB (analog of Escherichia coli RecBCD), but epistatic with RecA. A deletion of the ykoV gene encoding the B. subtilis Ku protein in a sbcC mutant strain did not resulted in an increase in sensitivity towards MMC and gamma irradiation, but exacerbated the phenotype of a recN or a recA mutant strain. In exponentially growing cells, SbcC-GFP was present throughout the cells, or as a central focus in rare cases. Upon induction of DNA damage, SbcC formed 1, rarely 2, foci on the nucleoids. Different to RecN protein, which forms repair centers at any location on the nucleoids, SbcC foci mostly co-localized with the DNA polymerase complex. In contrast to this, AddA-GFP or AddB-GFP did not form detectable foci upon addition of MMC. CONCLUSION: Our experiments show that SbcC plays an important role in the repair of DNA inter-strand cross-links (induced by MMC), most likely through HR, and suggest that NHEJ via Ku serves as a backup DNA repair system. The cell biological experiments show that SbcC functions in close proximity to the replication machinery, suggesting that SbcC may act on stalled or collapsed replication forks. Our results show that different patterns of localization exist for DNA repair proteins, and that the B. subtilis SMC proteins RecN and SbcC play distinct roles in the repair of DNA damage

Marker gene tethering by nucleoporins affects gene expression in plants

In non-plant systems, chromatin association with the nuclear periphery affects gene expression, where interactions with nuclear envelope proteins can repress and interactions with nucleoporins can enhance transcription. In plants, both hetero- and euchromatin can localise at the nuclear periphery, but the effect of proximity to the nuclear periphery on gene expression remains largely unknown. This study explores the putative function of Seh1 and Nup50a nucleoporins on gene expression by using the Lac Operator / Lac Repressor (LacI-LacO) system adapted to Arabidopsis thaliana. We used LacO fused to the luciferase reporter gene (LacO:Luc) to investigate whether binding of the LacO:Luc transgene to nucleoporin:LacI protein fusions alters luciferase expression. Two separate nucleoporin-LacI-YFP fusions were introduced into single insert, homozygous LacO:Luc Arabidopsis plants. Homozygous plants carrying LacO:Luc and a single insert of either Seh1-LacI-YFP or Nup50a-LacI-YFP were tested for luciferase activity and compared to plants containing LacO:Luc only. Seh1-LacI-YFP increased, while Nup50a-LacI-YFP decreased luciferase activity. Seh1-LacI-YFP accumulated at the nuclear periphery as expected, while Nup50a-LacI-YFP was nucleoplasmic and was not selected for further study. Protein and RNA levels of luciferase were quantified by western blotting and RT-qPCR, respectively. Increased luciferase activity in LacO:Luc+Seh1-LacI-YFP plants was correlated with increased luciferase protein and RNA levels. This change of luciferase expression was abolished by disruption of LacI-LacO binding by treating with IPTG in young seedlings, rosette leaves and inflorescences. This study suggests that association with the nuclear periphery is involved in the regulation of gene expression in plants

A novel family of plant nuclear envelope associated proteins

This paper describes the characterisation of a new family of higher plant nuclear envelope associated proteins (NEAPs) that interact with proteins of the nuclear envelope. In the model plant Arabidopsis thaliana, the family consists of three genes expressed ubiquitously (AtNEAP1-3) and a pseudogene (AtNEAP4). NEAPs consist of extensive coiled-coil domains, followed by a nuclear localisation signal and a C-terminal predicted transmembrane domain. Domain deletion mutants confirm the presence of a functional nuclear localisation signal and transmembrane domain. AtNEAP proteins localise to the nuclear periphery as part of stable protein complexes, are able to form homo- and heteromers and interact with the SUN domain proteins AtSUN1 and AtSUN2, involved in the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. An A. thaliana cDNA library screen identified a putative transcription factor called AtbZIP18 as a novel interactor of AtNEAP1, which suggest a connection between NEAP and chromatin. An Atneap1 Atneap3 double knock out mutant showed reduced root growth and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as INM anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure

Silicate bonding of sapphire to SESAMs: adjustable thermal lensing for high-power lasers

Silicate bonding is a flexible bonding method that enables room-temperature bonding of many types of materials with only moderate flatness constraints. It is a promising approach for bonding components in high power laser systems, since it results in a thin and low-absorption interface layer between the bonded materials. Here we demonstrate for the first time silicate bonding of a sapphire window to a SEmiconductor Saturable Absorber Mirror (SESAM) and use the composite structure to mode-lock a high-power thin-disk laser. We characterize the fabricated devices both theoretically and experimentally and show how the thermally induced lens of the composite structure can be tuned both in magnitude and sign via the thickness of the sapphire window. We demonstrate mode-locking of a high-power thin-disk laser oscillator with these devices. The altered thermal lens allows us to increase the output power to 233 W, a 70-W-improvement compared to the results achieved with a state-of-the-art SESAM in the same cavity