Individual plasmonic nanogaps : controlled assembly and detailed investigation

Diese Arbeit befasst sich mit den optischen Resonanzen metallischer Nanopartikel im Abstand weniger Nanometer von einer metallischen Grenzfläche. Die elektromagnetische Wechselwirkung dieser „Kugel-vor-Fläche“ Geometrie ruft interessante optische Phänomene hervor. Sie erzeugt eine spezielle elektromagnetische Eigenmode, auch Spaltmode genannt, die im Wesentlichen auf den Nanospalt zwi-schen Kugel und Oberfläche lokalisiert ist. In der quasistatischen Näherung hängt die Resonanzposition nur vom Material, der Umgebung, dem Film-Kugel Abstand und dem Kugelradius selbst ab. Theoretische Berechnungen sagen für diese Region unter Resonanzbedingungen eine große Verstärkung des elektro-magnetischen Feldes voraus. rnUm die optischen Eigenschaften dieser Systeme zu untersuchen, wurde ein effizienter plasmonenver-mittelnder Dunkelfeldmodus für die konfokale Rastermikroskopie durch dünne Metallfilme entwickelt, der die Verstärkung durch Oberflächenplasmonen sowohl im Anregungs- als auch Emissionsprozess ausnutzt. Dadurch sind hochwertige Dunkelfeldaufnahmen durch die Metallfilme der Kugel-vor-Fläche Systeme garantiert, und die Spektroskopie einzelner Resonatoren wird erleichtert. Die optischen Untersuchungen werden durch eine Kombination von Rasterkraft- und Rasterelektronenmikroskopie vervollständigt, so dass die Form und Größe der untersuchten Resonatoren in allen drei Dimensionen bestimmt und mit den optischen Resonanzen korreliert werden können. Die Leistungsfähigkeit des neu entwickelten Modus wird für ein Referenzsystem aus Polystyrol-Kugeln auf einem Goldfilm demonstriert. Hierbei zeigen Partikel gleicher Größe auch die erwartete identische Resonanz.rnFür ein aus Gold bestehendes Kugel-vor-Fläche System, bei dem der Spalt durch eine selbstorganisierte Monolage von 2-Aminoethanthiol erzeugt wird, werden die Resonanzen von Goldpartikeln, die durch Reduktion mit Chlorgoldsäure erzeugt wurden, mit denen von idealen Goldkugeln verglichen. Diese ent-stehen aus den herkömmlichen Goldpartikeln durch zusätzliche Bestrahlung mit einem Pikosekunden Nd:Yag Laser. Bei den unbestrahlten Partikeln mit ihrer Unzahl an verschiedenen Formen zeigen nur ein Drittel der untersuchten Resonatoren ein Verhalten, das von der Theorie vorhergesagt wird, ohne das dies mit ihrer Form oder Größe korrelieren würde. Im Fall der bestrahlten Goldkugeln tritt eine spürbare Verbesserung ein, bei dem alle Resonatoren mit den theoretischen Rechnungen übereinstimmen. Eine Änderung der Oberflächenrauheit des Films zeigt hingegen keinen Einfluß auf die Resonanzen. Obwohl durch die Kombination von Goldkugeln und sehr glatten Metallfilmen eine sehr definierte Probengeometrie geschaffen wurde, sind die experimentell bestimmten Linienbreiten der Resonanzen immer noch wesentlich größer als die berechneten. Die Streuung der Daten, selbst für diese Proben, deutet auf weitere Faktoren hin, die die Spaltmoden beeinflußen, wie z.B. die genaue Form des Spalts. rnDie mit den Nanospalten verbundenen hohen Feldverstärkungen werden untersucht, indem ein mit Farbstoff beladenes Polyphenylen-Dendrimer in den Spalt eines aus Silber bestehenden Kugel-vor-Fläche Systems gebracht wird. Das Dendrimer in der Schale besteht lediglich aus Phenyl-Phenyl Bindungen und garantiert durch die damit einhergende Starrheit des Moleküls eine überragende Formstabiliät, ohne gleichzeitig optisch aktiv zu sein. Die 16 Dithiolan Endgruppen sorgen gleichzeitig für die notwendige Affinität zum Silber. Dadurch kann der im Inneren befindliche Farbstoff mit einer Präzision von wenigen Nanometern im Spalt zwischen den Metallstrukturen platziert werden. Der gewählte Perylen Farbstoff zeichnet sich wiederum durch hohe Photostabilität und Fluoreszenz-Quantenausbeute aus. Für alle untersuchten Partikel wird ein starkes Fluoreszenzsignal gefunden, das mindestens 1000-mal stärker ist, als das des mit Farbstoff überzogenen Metallfilms. Das Profil des Fluoreszenz-Anregungsspektrums variiert zwischen den Partikeln und zeigt im Vergleich zum freien Farbstoff eine zusätzliche Emission bei höheren Frequenzen, was in der Literatur als „hot luminescence“ bezeichnet wird. Bei der Untersuchung des Streuverhaltens der Resonatoren können wieder zwei unterschiedliche Arten von Resonatoren un-terschieden werden. Es gibt zunächst die Fälle, die bis auf die beschriebene Linienverbreiterung mit einer idealen Kugel-vor-Fläche Geometrie übereinstimmen und dann andere, die davon stark abweichen. Die Veränderungen der Fluoreszenz-Anregungsspektren für den gebundenen Farbstoffs weisen auf physikalische Mechanismen hin, die bei diesen kleinen Metall/Farbstoff Abständen eine Rolle spielen und die über eine einfache wellenlängenabhängige Verstärkung hinausgehen.

Molecular basis for class V beta-tubulin effects on microtubule assembly and paclitaxel resistance.

Vertebrates produce at least seven distinct beta-tubulin isotypes that coassemble into all cellular microtubules. The functional differences among these tubulin isoforms are largely unknown, but recent studies indicate that tubulin composition can affect microtubule properties and cellular microtubule-dependent behavior. One of the isotypes whose incorporation causes the largest change in microtubule assembly is beta5-tubulin. Overexpression of this isotype can almost completely destroy the microtubule network, yet it appears to be required in smaller amounts for normal mitotic progression. Moderate levels of overexpression can also confer paclitaxel resistance. Experiments using chimeric constructs and site-directed mutagenesis now indicate that the hypervariable C-terminal region of beta5 plays no role in these phenotypes. Instead, we demonstrate that two residues found in beta5 (Ser-239 and Ser-365) are each sufficient to inhibit microtubule assembly and confer paclitaxel resistance when introduced into beta1-tubulin; yet the single mutation of residue Ser-239 in beta5 eliminates its ability to confer these phenotypes. Despite the high degree of conservation among beta-tubulin isotypes, mutations affecting residue 365 demonstrate that amino acid substitutions can be context sensitive; i.e. an amino acid change in one isotype will not necessarily produce the same phenotype when introduced into a different isotype. Modeling studies indicate that residue Cys-239 of beta1-tubulin is close to a highly conserved Cys-354 residue suggesting the possibility that disulfide formation could play a significant role in the stability of microtubules formed with beta1- but not with beta5-tubulin.

Identification of Trypanosoma brucei components involved in trypanolysis by normal human serum

Normal human serum (NHS) confers human resistance to infection by the parasite Trypanosoma brucei owing to the trypanolytic activity of apolipoprotein L1 (APOL1), present in two serum complexes termed Trypanolytic Factors (TLF-1 and -2). In order to identify parasite components involved in the intracellular trafficking and activity of TLFs, an inducible RNA interference (RNAi) genomic DNA library constructed in bloodstream form T. brucei was subjected to RNAi induction and selection for resistant parasites under NHS conditions favouring either TLF-1 or TLF-2 uptake. While TLF-1 conditions readily selected the haptoglobin-haemoglobin (HP-HB) surface receptor TbHpHbR as expected, given its known ability to bind TLF-1, under TLF-2 conditions no specific receptor for TLF-2 was identified. Instead, the screen allowed the identification of five distinct factors expected to be involved in the assembly of the vacuolar proton pump V-ATPase and consecutive endosomal acidification. These data confirm that lowering the pH during endocytosis is required for APOL1 toxic activity.

C9ORF72 Regulates Stress Granule Formation and Its Deficiency Impairs Stress Granule Assembly, Hypersensitizing Cells to Stress

Hexanucleotide repeat expansions in the C9ORF72 gene are causally associated with frontotemporal lobar dementia (FTLD) and/or amyotrophic lateral sclerosis (ALS). The physiological function of the normal C9ORF72 protein remains unclear. In this study, we characterized the subcellular localization of C9ORF72 to processing bodies (P-bodies) and its recruitment to stress granules (SGs) upon stress-related stimuli. Gain of function and loss of function experiments revealed that the long isoform of C9ORF72 protein regulates SG assembly. CRISPR/Cas9-mediated knockdown of C9ORF72 completely abolished SG formation, negatively impacted the expression of SG-associated proteins such as TIA-1 and HuR, and accelerated cell death. Loss of C9ORF72 expression further compromised cellular recovery responses after the removal of stress. Additionally, mimicking the pathogenic condition via the expression of hexanucleotide expansion upstream of C9ORF72 impaired the expression of the C9ORF72 protein, caused an abnormal accumulation of RNA foci, and led to the spontaneous formation of SGs. Our study identifies a novel function for normal C9ORF72 in SG assembly and sheds light into how the mutant expansions might impair SG formation and cellular-stress-related adaptive responses.

A pyrimidine-rich exonic splicing suppressor binds multiple RNA splicing factors and inhibits spliceosome assembly

The bovine papillomavirus type 1 (BPV-1) exonic splicing suppressor (ESS) is juxtaposed immediately downstream of BPV-1 splicing enhancer 1 and negatively modulates selection of a suboptimal 3′ splice site at nucleotide 3225. The present study demonstrates that this pyrimidine-rich ESS inhibits utilization of upstream 3′ splice sites by blocking early steps in spliceosome assembly. Analysis of the proteins that bind to the ESS showed that the U-rich 5′ region binds U2AF65 and polypyrimidine tract binding protein, the C-rich central part binds 35- and 54–55-kDa serine/arginine-rich (SR) proteins, and the AG-rich 3′ end binds alternative splicing factor/splicing factor 2. Mutational and functional studies indicated that the most critical region of the ESS maps to the central C-rich core (GGCUCCCCC). This core sequence, along with additional nonspecific downstream nucleotides, is sufficient for partial suppression of spliceosome assembly and splicing of BPV-1 pre-mRNAs. The inhibition of splicing by the ESS can be partially relieved by excess purified HeLa SR proteins, suggesting that the ESS suppresses pre-mRNA splicing by interfering with normal bridging and recruitment activities of SR proteins.

Differential Epitope Tagging of Actin in Transformed Drosophila Produces Distinct Effects on Myofibril Assembly and Function of the Indirect Flight Muscle

We have tested the impact of tags on the structure and function of indirect flight muscle (IFM)-specific Act88F actin by transforming mutant Drosophila melanogaster, which do not express endogenous actin in their IFMs, with tagged Act88F constructs. Epitope tagging is often the method of choice to monitor the fate of a protein when a specific antibody is not available. Studies addressing the functional significance of the closely related actin isoforms rely almost exclusively on tagged exogenous actin, because only few antibodies exist that can discriminate between isoforms. Thereby it is widely presumed that the tag does not significantly interfere with protein function. However, in most studies the tagged actin is expressed in a background of endogenous actin and, as a rule, represents only a minor fraction of the total actin. The Act88F gene encodes the only Drosophila actin isoform exclusively expressed in the highly ordered IFM. Null mutations in this gene do not affect viability, but phenotypic effects in transformants can be directly attributed to the transgene. Transgenic flies that express Act88F with either a 6x histidine tag or an 11-residue peptide derived from vesicular stomatitis virus G protein at the C terminus were flightless. Overall, the ultrastructure of the IFM resembled that of the Act88F null mutant, and only low amounts of C-terminally tagged actins were found. In contrast, expression of N-terminally tagged Act88F at amounts comparable with that of wild-type flies yielded fairly normal-looking myofibrils and partially reconstituted flight ability in the transformants. Our findings suggest that the N terminus of actin is less sensitive to modifications than the C terminus, because it can be tagged and still polymerize into functional thin filaments.

Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian CellsV⃞

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

Kinesin-II Is Preferentially Targeted to Assembling Cilia and Is Required for Ciliogenesis and Normal Cytokinesis in TetrahymenaV⃞

We cloned two genes, KIN1 and KIN2, encoding kinesin-II homologues from the ciliate Tetrahymena thermophila and constructed strains lacking either KIN1 or KIN2 or both genes. Cells with a single disruption of either gene showed partly overlapping sets of defects in cell growth, motility, ciliary assembly, and thermoresistance. Deletion of both genes resulted in loss of cilia and arrests in cytokinesis. Mutant cells were unable to assemble new cilia or to maintain preexisting cilia. Double knockout cells were not viable on a standard medium but could be grown on a modified medium on which growth does not depend on phagocytosis. Double knockout cells could be rescued by transformation with a gene encoding an epitope-tagged Kin1p. In growing cells, epitope-tagged Kin1p preferentially accumulated in cilia undergoing active assembly. Kin1p was also detected in the cell body but did not show any association with the cleavage furrow. The cell division arrests observed in kinesin-II knockout cells appear to be induced by the loss of cilia and resulting cell paralysis.

Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly

At least 11 complementation groups (CGs) have been identified for the peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome, for which seven pathogenic genes have been elucidated. We have isolated a human PEX19 cDNA (HsPEX19) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary cell line, ZP119, defective in import of both matrix and membrane proteins. This cDNA encodes a hydrophilic protein (Pex19p) comprising 299 amino acids, with a prenylation motif, CAAX box, at the C terminus. Farnesylated Pex19p is partly, if not all, anchored in the peroxisomal membrane, exposing its N-terminal part to the cytosol. A stable transformant of ZP119 with HsPEX19 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX19 expression also restored peroxisomal protein import in fibroblasts from a patient (PBDJ-01) with Zellweger syndrome of CG-J. This patient (PBDJ-01) possessed a homozygous, inactivating mutation: a 1-base insertion, A764, in a codon for Met255, resulted in a frameshift, inducing a 24-aa sequence entirely distinct from normal Pex19p. These results demonstrate that PEX19 is the causative gene for CG-J PBD and suggest that the C-terminal part, including the CAAX homology box, is required for the biological function of Pex19p. Moreover, Pex19p is apparently involved at the initial stage in peroxisome membrane assembly, before the import of matrix protein.

Posttranslational modification of TEL and TEL/AML1 by SUMO-1 and cell-cycle-dependent assembly into nuclear bodies

The E-26 transforming specific (ETS)-related gene, TEL, also known as ETV6, encodes a strong transcription repressor that is rearranged in several recurring chromosomal rearrangements associated with leukemia and congenital fibrosarcoma. TEL is a nuclear phosphoprotein that is widely expressed in all normal tissues. TEL contains a DNA-binding domain at the C terminus and a helix–loop–helix domain (also called a pointed domain) at the N terminus. The pointed domain is necessary for homotypic dimerization and for interaction with the ubiquitin-conjugating enzyme UBC9. Here we show that the interaction with UBC9 leads to modification of TEL by conjugating it to SUMO-1. The SUMO-1-modified TEL localizes to cell-cycle-specific nuclear speckles that we named TEL bodies. We also show that the leukemia-associated fusion protein TEL/AML1 is modified by SUMO-1 and found in the TEL bodies, in a pattern quite different from what we observe and report for AML1. Therefore, SUMO-1 modification of TEL could be a critical signal necessary for normal functioning of the protein. In addition, the modification by SUMO-1 of TEL/AML1 could lead to abnormal localization of the fusion protein, which could have consequences that include contribution to neoplastic transformation.

Fourier transform infrared spectroscopy reveals a rigid α-helical assembly for the tetrameric Streptomyces lividans K+ channel

The structure of the tetrameric K+ channel from Streptomyces lividans in a lipid bilayer environment was studied by polarized attenuated total reflection Fourier transform infrared spectroscopy. The channel displays approximately 43% α-helical and 25% β-sheet content. In addition, H/D exchange experiments show that only 43% of the backbone amide protons are exchangeable with solvent. On average, the α-helices are tilted 33° normal to the membrane surface. The results are discussed in relationship to the lactose permease of Escherichia coli, a membrane transport protein.

Assembly of the neutrophil respiratory burst oxidase: A direct interaction between p67PHOX and cytochrome b558

Activation of the phagocyte NADPH oxidase complex requires the assembly of the cytosolic factors p47PHOX, p67PHOX, p40PHOX, and Rac1 or Rac2, with the membrane-bound cytochrome b558. Whereas the interaction of p47PHOX with cytochrome b558 is well established, an interaction between p67PHOX and cytochrome b558 has never been investigated. We report here a direct interaction between p67PHOX and cytochrome b558. First, labeled p67PHOX recognizes a 91-kDa band in specific granules from a normal patient but not from a cytochrome b558-deficient patient. Second, p67PHOX binds to cytochrome b558 that has been bound to nitrocellulose. Third, GTP-p67PHOX bound to glutathione agarose is able to pull down cytochrome b558. Rac1-GTP or Rac1-GDP increased the binding of p67PHOX to cytochrome b558, suggesting that at least one of the oxidase-related functions of Rac1 is to promote the interaction between p67PHOX and cytochrome b558.

Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis

Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55/57 are required, whereas either Rad52 or Rad55/57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30°C but not 20°C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G1, consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.

Mass assembly and morphological transformations since z ∼ 3 from CANDELS

We quantify the evolution of the stellar mass functions (SMFs) of star-forming and quiescent galaxies as a function of morphology from z ∼ 3 to the present. Our sample consists of ∼50 000 galaxies in the CANDELS fields (∼880 arcmin^2), which we divide into four main morphological types, i.e. pure bulge-dominated systems, pure spiral disc-dominated, intermediate two-component bulge+disc systems and irregular disturbed galaxies. At z ∼ 2, 80 per cent of the stellar mass density of star-forming galaxies is in irregular systems. However, by z ∼ 0.5, irregular objects only dominate at stellar masses below 10^9 M_⊙. A majority of the star-forming irregulars present at z ∼ 2 undergo a gradual transformation from disturbed to normal spiral disc morphologies by z ∼ 1 without significant interruption to their star formation. Rejuvenation after a quenching event does not seem to be common except perhaps for the most massive objects, because the fraction of bulge-dominated star-forming galaxies with M^*/M_⊙ > 10^10.7 reaches 40 per cent at z < 1. Quenching implies the presence of a bulge: the abundance of massive red discs is negligible at all redshifts over 2 dex in stellar mass. However, the dominant quenching mechanism evolves. At z > 2, the SMF of quiescent galaxies above M^* is dominated by compact spheroids. Quenching at this early epoch destroys the disc and produces a compact remnant unless the star-forming progenitors at even higher redshifts are significantly more dense. At 1 < z < 2, the majority of newly quenched galaxies are discs with a significant central bulge. This suggests that mass quenching at this epoch starts from the inner parts and preserves the disc. At z < 1, the high-mass end of the passive SMF is globally in place and the evolution mostly happens at stellar masses below 10^10 M_⊙. These low-mass galaxies are compact, bulge-dominated systems, which were environmentally quenched: destruction of the disc through ram-pressure stripping is the likely process.