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Due to its preferred habitats in the environment, the free-living nematode

The global chemical inventory includes neurotoxins that are mostly interrogated concerning the biological response in developing organisms. Effects of pollutants on adults receive less attention, although vulnerabilities can be expected throughout the entire life span in young, middle-aged and old individuals. We use the animal model

Due to worldwide production, sales and application, neonicotinoids dominate the global use of insecticides. While, neonicotinoids are considered as pinpoint neurotoxicants that impair cholinergic neurotransmission in pest insects, the sublethal effects on nontarget organisms and other neurotransmitters remain poorly understood. Thus, we investigated long-term neurological outcomes in the decomposer nematode Caenorhabditis elegans. In the adult roundworm the neonicotinoid thiacloprid impaired serotonergic and dopaminergic neuromuscular behaviors, while respective exposures to thiamethoxam showed no effects. Thiacloprid caused a concentration-dependent delay of the transition between swimming and crawling locomotion that is controlled by dopaminergic and serotonergic neurotransmission. Age-resolved analyses revealed that impairment of locomotion occurred in young as well as middle-aged worms. Treatment with exogenous serotonin rescued thiacloprid-induced swimming deficits in young worms, whereas additional exposure with silica nanoparticles enhanced the reduction of swimming behavior. Delay of forward locomotion was partly caused by a new paralysis pattern that identified thiacloprid as an agent promoting a specific rigidity of posterior body wall muscle cells and peripheral neuropathy in the nematode (lowest-observed-effect-level 10 ng/ml). On the molecular level exposure with thiacloprid accelerated protein aggregation in body wall muscle cells of polyglutamine disease reporter worms indicating proteotoxic stress. The results from the soil nematode Caenorhabditis elegans show that assessment of neurotoxicity by neonicotinoids requires acknowledgment and deeper research into dopaminergic and serotonergic neurochemistry of nontarget organisms. Likewise, it has to be considered more that different neonicotinoids may promote diverse neural end points.

Delaying aging while prolonging health and lifespan is a major goal in aging research. While many resources have been allocated to find positive interventions with promising results, negative interventions such as pollution and their accelerating effect on age-related degeneration and disease have been mostly neglected. Here, we used the short-lived model organism C. elegans to analyze whether two candidate pollutants interfere with positive interventions by corrupting general aging pathways. We took advantage of the immense data sets describing the age-related remodeling of the proteome including increased protein insolubilities to complement our analysis. We show that the emergent pollutant silica nanoparticles (NP) and the classic xenobiotic inorganic mercury reduce lifespan and cause a premature protein aggregation phenotype. Silica NPs rescaled the longevity effect of genetic interventions targeting the IGF-1/insulin-like signaling pathway. Comparative mass spectrometry revealed that increased insolubility of proteins with important functions in proteostasis is a shared phenotype of intrinsic- and pollution-induced aging supporting the hypothesis that proteostasis is a central resilience pathway controlling lifespan and aging. The presented data demonstrate that pollutants corrupt intrinsic aging pathways, which results in premature aging phenotypes. Reducing pollution is therefore an important step to increase healthy aging and prolong life expectancies on a population level in humans and animals.

Prevalent environmental challenges are climate change, the biodiversity crisis, and the global scale of environmental pollution. We identified the cell nucleus as a sensitive sensor for bio-effects of pollutants such as mercury and nanoparticles. As a major route of pollutant uptake into organisms is ingestion, we have developed a test system that uses single intestinal cells of the nematode roundworm Caenorhabditis elegans. Microscopic observation of the cell nucleus in reporter worms for the methyltransferase fibrillarin (FIB-1::GFP) revealed nuclear staining patterns that are specific for pollutants such as silica nanoparticles, BULK silica particles, silver nanoparticles, ionic AgNO

How the environment contributes to neurodegenerative diseases such as Alzheimer’s is not well understood. In recent years, science has found augmenting evidence that nano-sized particles generated by transport (e.g., fuel combustion, tire wear and brake wear) may promote Alzheimer’s disease (AD). Individuals residing close to busy roads are at higher risk of developing AD, and nanomaterials that are specifically generated by traffic-related processes have been detected in human brains. Since AD represents a neurodegenerative disease characterized by amyloid protein aggregation, this review summarizes our current knowledge on the amyloid-generating propensity of traffic-related nanomaterials. Certain nanoparticles induce the amyloid aggregation of otherwise soluble proteins in in vitro laboratory settings, cultured neuronal cells and vertebrate or invertebrate animal models. We discuss the challenges for future studies, namely, strategies to connect the wet laboratory with the epidemiological data in order to elucidate the molecular bio-interactions of airborne nanomaterials and their effects on human health.

At present, the majority of investigations concerning nanotoxicology in the nematode C. elegans address short-term effects. While this approach allows for the identification of uptake pathways, exposition and acute toxicity, nanoparticle-organism interactions that manifest later in the adult life of C. elegans are missed. Here we show that a microhabitat composed of liquid S-medium and live bacteria in microtiter wells prolongs C. elegans longevity and is optimally suited to monitor chronic eNP-effects over the entire life span (about 34 days) of the nematode. Silver (Ag) nanoparticles reduced C. elegans life span in concentrations ≥10 μg/mL, whereas nano ZnO and CeO 2 (1–160 μg/mL) had no effect on longevity. Monitoring of locomotion behaviors throughout the entire life span of C. elegans showed that Ag NPs accelerate the age-associated decline of swimming and increase of uncoordinated movements at concentrations of ≥10 μg/mL, whereas neuromuscular defects did not occur in response to ZnO and CeO 2 NPs. By means of a fluorescing reporter worm expressing tryptophan hydroxylase-1 ::DsRed Ag NP-induced behavioral defects were correlated to axonal protein aggregation and neurodegeneration in single serotonergic HSN as well as sensory ADF neurons. Notably, serotonergic ADF neurons represented a sensitive target for Ag NPs in comparison to GABAergic neurons that showed no signs of degeneration under the same conditions. We conclude that due to its analogy to the jellylike boom culture of C. elegans on microbe-rich rotting plant material liquid S-medium culture in spatially confined microtiter wells represents a relevant as well as practical tool for comparative identification of age-resolved nanoparticle effects and vulnerabilities in a significant target organism. Consistent with this, specifically middle-aged nematodes showed premature neuromuscular defects after Ag NP-exposure.

This review addresses nanotoxicology in the nematode roundworm Caenorhabditis elegans. Special emphasis is on the natural habitats of wild C. elegans in correlation with modeled environmental sinks of engineered nanomaterials. Respective hypotheses about a first encounter are outlined and identify the worm as a relevant target organism of nanoparticles. In the laboratory C. elegans represents a genetically and experimentally amenable animal model that leads a boom life cycle consisting of several larval stages followed by an adult life span of only 2–3 weeks. The latter enables long-term observation of nanoparticle effects in young, middle-aged and old worms. A picture emerges that certain nanomaterials induce premature aging processes, thereby reducing the health span and longevity of C. elegans. The neural system of the worm turns out as a prominent target organ showing neuromuscular defects that among others manifest in neurodegeneration of single neurons in combination with altered locomotion, reduced fecundity and impaired olfaction. Notably, specific vulnerabilities occur at distinct ages of adult C. elegans. Conceivable consequences of neural defects for wild C. elegans are discussed, as are system biology-based strategies to identify common pathways of nanoparticle–bio-interactions from worm to men.

Prevalent environmental challenges are climate change, the biodiversity crisis, and the global scale of environmental pollution. We identified the cell nucleus as a sensitive sensor for bio-effects of pollutants such as mercury and nanoparticles. As a major route of pollutant uptake into organisms is ingestion, we have developed a test system that uses single intestinal cells of the nematode roundworm Caenorhabditis elegans. Microscopic observation of the cell nucleus in reporter worms for the methyltransferase fibrillarin (FIB-1::GFP) revealed nuclear staining patterns that are specific for pollutants such as silica nanoparticles, BULK silica particles, silver nanoparticles, ionic AgNO3, and inorganic mercury (HgCl2). While the underlying molecular mechanisms need further investigation, cultivation of the reporter worms in liquid culture on microtiter plates now enables cost-effective screening of more pollutants and samples from the environment, e.g., mesocosm analyses. As C. elegans leads a dual life in the lab and in ecosystems, alteration of nuclear structure and function may likewise explain how environmental pollutants reduce the fitness of wild worms and thus may negatively affect biodiversity.

Despite of the increasing application of silica nanoparticles and identification of oral exposure as a major entry portal, we lack understanding of nanosilica effects in the gut. Thus, we investigated biointeractions of nanosilica with single intestinal cells. The invertebrate nematode Caenorhabditis elegans was chosen as model organism with a tractable intestine and realistic target organism of nanomaterials in the environment. We found that nanosilica impairs the intestinal uptake of oligopeptides. Downstream to absorption by the apical OPT-2/PEP-2 transporter dipeptides were trapped in aberrant vesicles that grow over time and reach diameters of ≥6 μm. The peptide vesicles do not correspond to known organelles such as gut granules and form independently of related gene products GLO-1 or GLO-3. Formation of aberrant peptide vesicles also occurred independently of insulin/IGF-I receptor (DAF-2) signaling and daf-2 loss of function mutants showed specific vesicle patterns including distinct localization along the apical membrane of single intestinal cells. As malnutrition of exposed C. elegans manifested in reduced growth and a petite phenotype similar to OPT-2/PEP-2 transporter deficient mutants, we conclude that nanosilica-induced peptide vesicles represent a new compartment of di- and tripeptide trapping which disrupts hydrolysis of nutrient peptides and metabolism.

In aging societies increasing cases of neurodegenerative protein deposit diseases urge for the identification of the underlying mechanisms. Expectations are that in 2050 the percentage of population over age 60 is 42% in Japan, 34% in China, and 27% in the US. The cell nucleus is a major target of amyloid-like protein fibrillation in a variety of disorders that are characterized by widespread aggregation of proteins with instable homopolymeric amino acid repeats, ubiquitin, and other proteinaceous components. Additionally, accumulation of insoluble, SDS-resistant proteins has been identified as an intrinsic property of organismal aging. This review collects current knowledge about the composition and function of insoluble, nuclear protein inclusions from the protein homeostasis perspective. It discusses the occurrence and role of nuclear amyloid in the diseased as well as the healthy cell. Features of nuclear inclusions such as protein composition and locally active protein degradation may predict neural fitness and survival in a variety of health or disease settings.

Invertebrate animal models such as the nematode Caenorhabditis elegans (C. elegans) are increasingly used in nanotechnological applications. Research in this area covers a wide range from remote control of worm behavior by nanoparticles (NPs) to evaluation of organismal nanomaterial safety. Despite of the broad spectrum of investigated NP-bio interactions, little is known about the role of nanomaterials with respect to aging processes in C. elegans. We trace NPs in single cells of adult C. elegans and correlate particle distribution with the worm's metabolism and organ function. By confocal microscopy analysis of fluorescently labeled NPs in living worms, we identify two entry portals for the uptake of nanomaterials via the pharynx to the intestinal system and via the vulva to the reproductive system. NPs are localized throughout the cytoplasm and the cell nucleus in single intestinal, and vulval B and D cells. Silica NPs induce an untimely accumulation of insoluble ubiquitinated proteins, nuclear amyloid and reduction of pharyngeal pumping that taken together constitute a premature aging phenotype of C. elegans on the molecular and behavioral level, respectively. Screening of different nanomaterials for their effects on protein solubility shows that polystyrene or silver NPs do not induce accumulation of ubiquitinated proteins suggesting that alteration of protein homeostasis is a unique property of silica NPs. The nematode C. elegans represents an excellent model to investigate the effect of different types of nanomaterials on aging at the molecule, cell, and whole organism level.

Promyelocytic leukemia (PML) protein binds to and stabilizes CIITA at PML nuclear bodies, which promotes expression of the MHC class II gene locus in response to interferon-γ exposure., Promyelocytic leukemia (PML) nuclear bodies selectively associate with transcriptionally active genomic regions, including the gene-rich major histocompatibility (MHC) locus. In this paper, we have explored potential links between PML and interferon (IFN)-γ–induced MHC class II expression. IFN-γ induced a substantial increase in the spatial proximity between PML bodies and the MHC class II gene cluster in different human cell types. Knockdown experiments show that PML is required for efficient IFN-γ–induced MHC II gene transcription through regulation of the class II transactivator (CIITA). PML mediates this function through protection of CIITA from proteasomal degradation. We also show that PML isoform II specifically forms a stable complex with CIITA at PML bodies. These observations establish PML as a coregulator of IFN-γ–induced MHC class II expression.

While it is widely acknowledged that the ubiquitin– proteasome system plays an important role in transcription, little is known concerning the mechanistic basis, in particular the spatial organization of proteasome-dependent proteolysis at the transcription site. Here, we show that proteasomal activity and tetraubiquitinated proteins concentrate to nucleoplasmic microenvironments in the euchromatin. Such proteolytic domains are immobile and distinctly positioned in relation to transcriptional processes. Analysis of gene arrays and early genes in Caenorhabditis elegans embryos reveals that proteasomes and proteasomal activity are distantly located relative to transcriptionally active genes. In contrast, transcriptional inhibition generally induces local overlap of proteolytic microdomains with components of the transcription machinery and degradation of RNA polymerase II. The results establish that spatial organization of proteasomal activity differs with respect to distinct phases of the transcription cycle in at least some genes, and thus might contribute to the plasticity of gene expression in response to environmental stimuli.

Caseins are major constituents of mammalian milks that are thought to be exclusively expressed in mammary glands and to function primarily as a protein source, as well as to ameliorate intestinal calcium uptake. In addition, proinflammatory and immunomodulatory properties have been reported for bovine caseins. Our aim was to investigate whether human casein α s1 (CSN1S1) is expressed outside the mammary gland and possesses immunomodulatory functions in humans as well. For this purpose, CSN1S1 mRNA was detected in primary human monocytes and CD4+ and CD8+ T cells, but not in CD19+ B cells. CSN1S1 protein was traceable in supernatants of cultured primary human CD14+ monocytes by ELISA. Similarly, CSN1S1 mRNA and protein were detected in the human monocytic cell lines HL60, U937, and THP1 but not in Mono Mac 6 cells. Moreover, permeabilized human monocytes and HL60 cells could be stained by immunofluorescence, indicating intracellular expression. Recombinant human CSN1S1 was bound to the surface of Mono Mac 6 cells and upregulated the expression of GM-CSF mRNA in primary human monocytes and Mono Mac 6 cells in a time- and concentration-dependent manner. A similar increase in GM-CSF protein was found in the culture supernatants. CSN1S1-dependent upregulation of GM-CSF was specifically blocked by the addition of the p38 MAPK inhibitor ML3403. Our results indicated that human CSN1S1 may possess an immunomodulatory role beyond its nutritional function in milk. It is expressed in human monocytes and stimulates the expression of the proinflammatory cytokine GM-CSF.

The nucleus represents a cellular control unit that regulates all events concerning the storage and processing of DNA and RNA. It is organized by highly crowded, dynamic assemblies of proteins and nucleic acids in molecular machines, ribonucleoprotein complexes, clusters of ongoing nuclear processes, nuclear bodies, and chromatin. This review discusses the occurrence of nuclear protein aggregation with special emphasis on the functional architecture of the nucleus, and quality control by the ubiquitin-proteasome system.

Protein aggregates and nuclear inclusions (NIs) containing components of the ubiquitin–proteasome system (UPS), expanded polyglutamine (polyQ) proteins, and transcriptional coactivators characterize cellular responses to stress and are hallmarks of neurodegenerative diseases. The biological function of polyQ-containing aggregates is unknown. To analyze proteasomal activity within such aggregates, we present a nanoparticle (NP)-based method that enables controlled induction of sodium dodecyl sulfate–resistant inclusions of endogenous nuclear proteins while normal regulatory mechanisms remain in place. Consistent with the idea that the UPS maintains quality control, inhibition of proteasomal proteolysis promotes extra large protein aggregates (1.4–2 μm), whereas formation of NP-induced NIs is found to be inversely correlated to proteasome activation. We show that global proteasomal proteolysis increases in NP-treated nuclei and, on the local level, a subpopulation of NIs overlaps with focal domains of proteasome-dependent protein degradation. These results suggest that inclusions in the nucleus constitute active proteolysis modules that may serve to concentrate and decompose damaged, malfolded, or misplaced proteins.

Proteasomes are ATP-driven, multisubunit proteolytic machines that degrade endogenous proteins into peptides and play a crucial role in cellular events such as the cell cycle, signal transduction, maintenance of proper protein folding and gene expression. Recent evidence indicates that the ubiquitin-proteasome system is an active component of the cell nucleus. A characteristic feature of the nucleus is its organization into distinct domains that have a unique composition of macromolecules and dynamically form as a response to the requirements of nuclear function. Here, we show by systematic application of different immunocytochemical procedures and comparison with signature proteins of nuclear domains that during interphase endogenous proteasomes are localized diffusely throughout the nucleoplasm, in speckles, in nuclear bodies, and in nucleoplasmic foci. Proteasomes do not occur in the nuclear envelope region or the nucleolus, unless nucleoplasmic invaginations expand into this nuclear body. Confirmedly, proteasomal proteolysis is detected in nucleoplasmic foci, but is absent from the nuclear envelope or nucleolus. The results underpin the idea that the ubiquitin-proteasome system is not only located, but also proteolytically active in distinct nuclear domains and thus may be directly involved in gene expression, and nuclear quality control.

Nanotechnology makes use of the special surface properties of extremely small particles. In this rapidly growing field, many different materials are produced for a multitude of diverse applications. Possible adverse health effects of these materials however are so far scarcely investigated and are therefore a special task of toxicology. Although strategies for risk assessment have been suggested, the authors of the current review emphasize the fact that on the cellular, subcellular and molecular levels, interactions between nanoparticles (NP) and target cells relevant for the induction of possible adverse health effects are poorly understood. On the basis of existing literature, the potentially most relevant cellular target sites of NP as well as the so far known major molecular events specifically induced by these xenobiotics are reviewed. Starting with NP uptake across the cell membrane, mechanisms of generation of reactive oxygen species and the activation of redox-sensitive signalling cascades are des...

Mutations of mitochondrial (mt) DNA play a role in neurodegeneration, normal aging, premature aging of the skin (photoaging), and tumors. We and others could demonstrate that mtDNA mutations can be induced in skin cells in vitro and in normal human skin in vivo by repetitive, sublethal ultraviolet (UV)-A-irradiation. These mutations are mediated by singlet oxygen and persist in human skin as long-term biomarkers of UV exposure. Although mtDNA exclusively encodes for the respiratory chain, involvement of the energy metabolism in mtDNA mutagenesis and a protective role of the energy precursor creatine have thus far not been shown. We assessed the amount of a marker mutation of mtDNA, the so-called common deletion, by real-time PCR. Induction of the common deletion was paralleled by a measurable decrease of oxygen consumption, mitochondrial membrane potential, and ATP content, as well as an increase of matrix metalloproteinase-1. Mitochondrial mutagenesis as well as functional consequences could be normalized by increasing intracellular creatine levels. These data indicate that increase of the energy precursor creatine protects from functionally relevant, aging-associated mutations of mitochondrial DNA.

The cell nucleus is a prominent target of autoantibodies in systemic autoimmune disorders. Approximately 2% of the population in Europe and North America suffers from systemic rheumatic diseases, such as rheumatoid arthritis, systemic lupus erythematosus (SLE), and scleroderma. The molecular mechanisms of systemic autoimmunity are largely unknown despite its high prevalence. Contributing factors that have been considered include (1) genetic predisposition, (2) influence of hormones, and (3) environmental factors. The latter are mainly correlated with the generation of scleroderma, as xenobiotic-induced subsets of this disease have been observed in individuals exposed to silica (SiO(2)) dusts, organic solvents, heavy metals, and certain drugs. In addition to the epidemiological relevance, animal models of xenobiotic-induced autoimmunity serve as elegant tools for controlled induction of antigen-driven autoimmune responses. Because antigen processing and presentation constitute the basis for every antigen-driven autoimmune response, effects of xenobiotics on degradation of nuclear autoantigens have been characterized to elucidate molecular mechanisms of autoimmunity that target the cell nucleus. By means of a cell-based disease model, it has been shown that xenobiotics such as mercuric chloride, platinum salts, and silica (nano)particles specifically alter structure, function, and proteolysis in the cell nucleus. Signature proteins of the cell nucleus redistribute to aberrant, nucleoplasmic clusters, where they colocalize with proteasomes and are subjected to proteasomal proteolysis. Recruitment of nuclear autoantigens to proteasomal degradation is correlated with autoimmune responses that specifically target these antigens in both animal models of xenobiotic-induced autoimmunity and patients with idiopathic scleroderma.

Despite of their exponentially growing use, little is known about cell biological effects of nanoparticles. Here, we report uptake of silica (SiO2) nanoparticles to the cell nucleus where they induce aberrant clusters of topoisomerase I (topo I) in the nucleoplasm that additionally contain signature proteins of nuclear domains, and protein aggregation such as ubiquitin, proteasomes, cellular glutamine repeat (polyQ) proteins, and huntingtin. Formation of intranuclear protein aggregates (1) inhibits replication, transcription, and cell proliferation; (2) does not significantly alter proteasomal activity or cell viability; and (3) is reversible by Congo red and trehalose. Since SiO2 nanoparticles trigger a subnuclear pathology resembling the one occurring in expanded polyglutamine neurodegenerative disorders, we suggest that integrity of the functional architecture of the cell nucleus should be used as a read out for cytotoxicity and considered in the development of safe nanotechnology.

Autoimmune diseases are believed to rise from a breakdown of tolerance and a deregulation of the immune system to discriminate effectively between “self” and “non-self”. Although the origins of autoimmunity and the factors leading to overt autoimmune diseases are largely unclear, genetic background, hormones, and environmental agents are found to be involved. The production of autoantibodies against distinct “self”-proteins of the cell nucleus constitutes one of the autoimmune manifestations that characterize systemic autoimmunity. While molecular mechanisms of anti-nuclear autoimmune responses remain poorly understood, processing and presentation of intracellular autoantigens to T cells constitutes a central issue in the generation of antigen-driven autoimmunity. Intracellular proteins, including nuclear autoantigens, are degraded by the proteasomal pathway that clears away endogenous proteins, regulates numerous cellular processes, and delivers immunocompetent peptides to the antigen presenting machinery. Since antigen processing is a key step for the maintenance of peripheral tolerance, in this article, we discuss the role of proteasome-dependent antigen processing in systemic autoimmunity.

Mercury (Hg) is a bioaccumulating trace metal that globally circulates the atmosphere and waters in its elemental, inorganic and organic chemical forms. While Hg represents a notorious neurotoxicant, the underlying cellular pathways are insufficiently understood. We identify amyloid protein aggregation in the cell nucleus as a novel pathway of Hg-bio-interactions. By mass spectrometry of purified protein aggregates a subset of spliceosomal components and nucleoskeletal protein lamin B1 were detected as constituent parts of an Hg-induced nuclear aggregome network. The aggregome network was located by confocal imaging of amyloid-specific antibodies and dyes to amyloid cores within splicing-speckles that additionally recruit components of the ubiquitin-proteasome system. Hg significantly enhances global proteasomal activity in the nucleus suggesting that formation of amyloid speckles plays a role in maintenance of protein homeostasis. RNAi knock down showed that lamin B1 for its part regulates amyloid speckle formation and thus likewise participates in nuclear protein homeostasis. As the Hg-induced cascade of interactions between the nucleoskeleton and protein homeostasis reduces neuronal signalling, amyloid fibrillation in the cell nucleus is introduced as a feature of Hg-neurotoxicity that opens new avenues of future research. Similar to protein aggregation events in the cytoplasm that are controlled by the cytoskeleton, amyloid fibrillation of nuclear proteins may be driven by the nucleoskeleton.

Identifying nanomaterial-bio-interactions are imperative due to the broad introduction of nanoparticle (NP) applications and their distribution. Here, we demonstrate that silica NPs effect widespread protein aggregation in the soil nematode Caenorhabditis elegans ranging from induction of amyloid in nucleoli of intestinal cells to facilitation of protein aggregation in body wall muscles and axons of neural cells. Proteomic screening revealed that exposure of adult C. elegans with silica NPs promotes segregation of proteins belonging to the gene ontology (GO) group of “protein folding, proteolysis and stress response” to an SDS-resistant aggregome network. Candidate proteins in this group include chaperones, heat shock proteins and subunits of the 26S proteasome which are all decisively involved in protein homeostasis. The pathway of protein homeostasis was validated as a major target of silica NPs by behavioral phenotyping, as inhibitors of amyloid formation rescued NP-induced defects of locomotory patterns and egg laying. The analysis of a reporter worm for serotonergic neural cells revealed that silica NP-induced protein aggregation likewise occurs in axons of HSN neurons, where presynaptic accumulation of serotonin, e.g. disturbed axonal transport reduces the capacity for neurotransmission and egg laying. The results suggest that in C. elegans silica NPs promote a cascade of events including disturbance of protein homeostasis, widespread protein aggregation and inhibition of serotonergic neurotransmission which can be interrupted by compounds preventing amyloid fibrillation.

Xenobiotics, as well as intrinsic processes such as cellular aging, contribute to an environment that constantly challenges nuclear organization and function. While it becomes increasingly clear that proteasome-dependent proteolysis is a major player, the topology and molecular mechanisms of nuclear protein homeostasis remain largely unknown. We have shown previously that (1) proteasome-dependent protein degradation is organized in focal microenvironments throughout the nucleoplasm and (2) heavy metals as well as nanoparticles induce nuclear protein fibrillation with amyloid characteristics. Here, we describe methods to characterize the landscape of intranuclear amyloid on the global and local level in different systems such as cultures of mammalian cells and the soil nematode Caenorhabditis elegans. Application of discrete mathematics to imaging data is introduced as a tool to develop pattern recognition of intracellular protein fibrillation. Since stepwise fibrillation of otherwise soluble proteins to insoluble amyloid-like protein aggregates is a hallmark of neurodegenerative protein-misfolding disorders including Alzheimer's disease, CAG repeat diseases, and the prion encephalopathies, investigation of intracellular amyloid may likewise aid to a better understanding of the pathomechanisms involved. We consider aggregate profiling as an important experimental approach to determine if nuclear amyloid has toxic or protective roles in various disease processes.

Xenobiotics, as well as intrinsic processes such as cellular aging, contribute to an environment that constantly challenges nuclear organization and function. While it becomes increasingly clear that proteasome-dependent proteolysis is a major player, the topology and molecular mechanisms of nuclear protein homeostasis remain largely unknown. We have shown previously that (1) proteasome-dependent protein degradation is organized in focal microenvironments throughout the nucleoplasm and (2) heavy metals as well as nanoparticles induce nuclear protein fibrillation with amyloid characteristics. Here, we describe methods to characterize the landscape of intranuclear amyloid on the global and local level in different systems such as cultures of mammalian cells and the soil nematode Caenorhabditis elegans. Application of discrete mathematics to imaging data is introduced as a tool to develop pattern recognition of intracellular protein fibrillation. Since stepwise fibrillation of otherwise soluble proteins to insoluble amyloid-like protein aggregates is a hallmark of neurodegenerative protein-misfolding disorders including Alzheimer's disease, CAG repeat diseases, and the prion encephalopathies, investigation of intracellular amyloid may likewise aid to a better understanding of the pathomechanisms involved. We consider aggregate profiling as an important experimental approach to determine if nuclear amyloid has toxic or protective roles in various disease processes.

The spatial organization of transcription- associated proteins is an important control mechanism of eukaryotic gene expression. Here we analyzed the nuclear distribution of the transcriptional coactivators CREB-binding protein (CBP)/p300 in situ by confocal laser scanning microscopy, and in vivo complex formation by coimmunoprecipitation. A subpopulation of CBP and p300 is targeted to active sites of transcription and partially colocalizes with hyper- and hypophosphorylated RNA polymerase II (pol II) in discrete regions of variable size throughout the nucleus. However, the coactivators were found in tight association with hypophosphorylated, but not hyperphosphorylated pol II. Transcriptional inhibition induced a relocation of CBP/p300 and pol II into speckles. Moreover, double and triple immunofluorescence analyses revealed the presence of CBP, p300, and pol II in a subset of promyelocytic leukemia (PML) bodies. Our results provide evidence for a dynamic spacial link between coactivators of transcription and the basal transcription machinery in discrete nuclear domains dependent upon the transcriptional activity of the cell. The identification of pol II in CBP/PML-containing nuclear bodies supports the idea that transcription takes place at PML bodies.

Investigation of differential gene regulation by protein degradation requires analysis of the spatial and temporal association between proteolysis and transcription. Here, we describe the isochronal visualization of proteasomal proteolysis and transcription in cell culture or in vivo in the model organism Caenorhabditis elegans. This includes localization of proteasome-dependent proteolysis by fluorescent degradation products of model and endogenous substrates of the proteasome in combination with immunolabelling of RNA polymerase II and transcription in situ run-on assays.

Understanding of nanoparticle-bio-interactions within living cells requires knowledge about the dynamic behavior of nanomaterials during their cellular uptake, intracellular traffic and mutual reactions with cell organelles. Here, we introduce a protocol of combined kinetic imaging techniques that enables investigation of exemplary fluorochrome-labelled nanoparticles concerning their intracellular fate. By time-lapse confocal microscopy we observe fast, dynamin-dependent uptake of polystyrene and silica nanoparticles via the cell membrane within seconds. Fluorescence recovery after photobleaching (FRAP) experiments reveal fast and complete exchange of the investigated nanoparticles at mitochondria, cytoplasmic vesicles or the nuclear envelope. Nuclear translocation is observed within minutes by free diffusion and active transport. Fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS) indicate diffusion coefficients of polystyrene and silica nanoparticles in the nucleus and the cytoplasm that are consistent with particle motion in living cells based on diffusion. Determination of the apparent hydrodynamic radii by FCS and RICS shows that nanoparticles exert their cytoplasmic and nuclear effects mainly as mobile, monodisperse entities. Thus, a complete toolkit of fluorescence fluctuation microscopy is presented for the investigation of nanomaterial biophysics in subcellular microenvironments that contributes to develop a framework of intracellular nanoparticle delivery routes.

Investigation of differential gene regulation by protein degradation requires analysis of the spatial and temporal association between proteolysis and transcription. Here, we describe the isochronal visualization of proteasomal proteolysis and transcription in cell culture or in vivo in the model organism Caenorhabditis elegans. This includes localization of proteasome-dependent proteolysis by fluorescent degradation products of model and endogenous substrates of the proteasome in combination with immunolabelling of RNA polymerase II and transcription in situ run-on assays.

Autoantibodies targeted against cellular proteins and nucleic acids are a common feature of autoimmune diseases. In this study, we show that ribosomal protein L7 is a novel autoantigen in patients suffering from systemic lupus erythematosus (SLE) and other connective tissue diseases. From 24 patients diagnosed as having SLE, 18 produce antibodies which precipitate in vitro translated L7 protein. The anti-L7 titer appears to correlate with the active state of the disease. Anti-L7 autoantibodies were also detected in 7 of 13 patients with mixed connective tissue disease (MCTD), 2 of 7 patients with rheumatoid arthritis (RA), 1 of 4 patients with Sjogren's syndrome (SS) and in 1 patient with progressive systemic sclerosis (PSS). Anti-L7 autoantibodies belong to the IgG-class and detect specifically at least two epitopes on the L7 molecule, as shown by immunoprecipitation and immunoblotting. The epitope(s) of the highly conserved C-terminal region are preferentially recognized. Utilizing rabbit anti-L7 serum, autoimmune sera and affinity-purified anti-L7 autoantibodies in immunoblotting, and rabbit and chicken anti-L7 antibodies in indirect immunofluorescence, we detect L7 protein in the nuclei and in the cytoplasm of various cell-lines. Yet unlike most integral structural components of ribosomes, L7 is absent from nucleoli.

Nuclear inclusions that contain proteins with expanded polyglutamine (polyQ) repeats are observed in neurodegenerative aggregation diseases and are, therefore, viewed as a pathologic feature. However, a summary of research indicates that polyQ repeats are inherently both toxic and functional at the same time. PolyQ motifs occur in proteins involved in gene expression and promote nuclear assemblies such as the transcription initiation complex. Transition of these functional complexes to insoluble protein aggregates is constitutively prevented by proteasomal proteolysis. Thus, conditions that exhaust the ubiquitin–proteasome system, such as the extensive production of expanded polyQ proteins, aging and xenobiotic stress, induce a congested state in which nuclear proteins, including those with polyQ stretches, form amyloid-like aggregates. Because protein aggregation is preceded by a series of protein misfolding steps termed polyQ fibrillation, the characterization of distinct fibrillation steps correlating with nuclear function and identification of the respective genetic modifiers is essential for understanding both the biology and pathology of polyQ. Thus, the comprehension of the physiological role of polyQ repeats is a prerequisite for uncovering the underlying mechanisms of neurodegenerative aggregation diseases.

While expectations and applications of nanotechnologies grow exponentially, little is known about interactions of engineered nanoparticles with multicellular organisms. Here we propose the transparent roundworm Caenorhabditis elegans as a simple but anatomically and biologically well defined animal model that allows for whole organism analyses of nanoparticle-bio-interactions. Microscopic techniques showed that fluorescently labelled nanoparticles are efficiently taken up by the worms during feeding, and translocate to primary organs such as epithelial cells of the intestine, as well as secondary organs belonging to the reproductive tract. The life span of nanoparticle-fed Caenorhabditis elegans remained unchanged, whereas a reduction of progeny production was observed in silica-nanoparticle exposed worms versus untreated controls. This reduction was accompanied by a significant increase of the ‘bag of worms’ phenotype that is characterized by failed egg-laying and usually occurs in aged wild type worms. Experimental exclusion of developmental defects suggests that silica-nanoparticles induce an age-related degeneration of reproductive organs, and thus set a research platform for both, detailed elucidation of molecular mechanisms and high throughput screening of different nanomaterials by analyses of progeny production.

The 20S proteasome is part of a larger complex, the 26S proteasome, that is implicated in the ATP-dependent degradation of multiubiquitin-conjugated proteins (1). About 80% of intracellular protein breakdown occurs via the ubiquitin-proteasome system (UPS). Key proteins such as transcription factors, nuclear receptors, cyclins, cyclin-dependent kinase inhibitors, p53, and NF-kappaB are regulated by this pathway. Thus, the UPS has been implicated to play a role in multiple cellular events including the cell cycle, signal transduction, antigen presentation, and DNA repair and transcription (2, 3). In 1984 Varshavsky and co-workers discovered that ubiquitin-dependent pathways play a role in cell cycle control, and suggested that protein degradation is instrumental in regulation of gene expression (4). Consistent with this idea, Franke and colleagues had shown that proteasomes localize to the nuclei of Xenopus laevis oocytes and HeLa cells (5, 6). Subsequent work confirmed that (i) all components of the UPS that are required for protein degradation indeed reside in the cell nucleus (7); (ii) nuclear proteins are substrates for proteasomal degradation (8); and (iii) proteasome-dependent proteolysis occurs in distinct nucleoplasmic foci (9). The intricate balance between nuclear function and quality control through proteolysis is exemplified by reports that show a correlation of aberrant nuclear protein aggregates with inhibition of transcription in neurodegenerative diseases such as Huntington's chorea and animal and cell culture models of polyglutamine repeat disorders (10,11).Considering the central role of the UPS in nuclear processes, a detailed knowledge of the time and place at which a substrate is ubiquitinylated and degraded will be essential to our understanding of the cellular mechanisms that orchestrate the expression of thousands of genes or development of subnuclear pathologies. Here, we describe fluorescence-based localization methods for proteasomes, protein aggregates, and proteasomal proteolysis in the cell nucleus that may aid to analyse the UPS in housekeeping and disease conditions.

The 20S proteasome is part of a larger complex, the 26S proteasome, that is implicated in the ATP-dependent degradation of multiubiquitin-conjugated proteins (1). About 80% of intracellular protein breakdown occurs via the ubiquitin-proteasome system (UPS). Key proteins such as transcription factors, nuclear receptors, cyclins, cyclin-dependent kinase inhibitors, p53, and NF-kappaB are regulated by this pathway. Thus, the UPS has been implicated to play a role in multiple cellular events including the cell cycle, signal transduction, antigen presentation, and DNA repair and transcription (2, 3). In 1984 Varshavsky and co-workers discovered that ubiquitin-dependent pathways play a role in cell cycle control, and suggested that protein degradation is instrumental in regulation of gene expression (4). Consistent with this idea, Franke and colleagues had shown that proteasomes localize to the nuclei of Xenopus laevis oocytes and HeLa cells (5, 6). Subsequent work confirmed that (i) all components of the UPS that are required for protein degradation indeed reside in the cell nucleus (7); (ii) nuclear proteins are substrates for proteasomal degradation (8); and (iii) proteasome-dependent proteolysis occurs in distinct nucleoplasmic foci (9). The intricate balance between nuclear function and quality control through proteolysis is exemplified by reports that show a correlation of aberrant nuclear protein aggregates with inhibition of transcription in neurodegenerative diseases such as Huntington's chorea and animal and cell culture models of polyglutamine repeat disorders (10,11).Considering the central role of the UPS in nuclear processes, a detailed knowledge of the time and place at which a substrate is ubiquitinylated and degraded will be essential to our understanding of the cellular mechanisms that orchestrate the expression of thousands of genes or development of subnuclear pathologies. Here, we describe fluorescence-based localization methods for proteasomes, protein aggregates, and proteasomal proteolysis in the cell nucleus that may aid to analyse the UPS in housekeeping and disease conditions.

In eukaryotes, thousands of genes have to be organized and expressed in the cell nucleus. Conformational and kinetic instability of nuclear structure and components appear to enable cells to use the encoded information selectively. The ubiquitin-proteasome system is active in distinct nuclear domains and plays a major role controlling the initial steps of gene expression, DNA repair and nuclear quality-control mechanisms. Recent work indicates that a tuned balance of ubiquitylation and proteasome-dependent protein degradation of nuclear proteins is instrumental in nuclear function and, when deregulated, leads to the development of diseases such as polyQ disorders and other neurodegenerative conditions.