Dynamics of active lamellae in cultured epithelial cells: effects of expression of exogenous N-ras oncogene.

We examined the functional consequences of cellular transformation of rat IAR-2 epithelial cells, by a mutant N-ras oncogene, on the dynamics of active lamellae, structures that play an important role in cell motility, adhesion, and surface-receptor capping. Lamellar activity was assessed by measuring the rate of outer-edge pseudopodial activity and by analyzing the motility of Con A-coated beads placed on lamellar surfaces with optical tweezers. Although transformation dramatically affected the shape and size of active cellular lamellae, there was little detectable effect on either pseudopodial activity or bead movement. To investigate the potential relationship between functional lamellar activity and the microtubule cytoskeleton, lamellar activity was examined in nontransformed and transformed cells treated with the microtubule-disrupting drug nocodazole. In the absence of microtubules, transformed cells were less polarized and possessed decreased rates of pseudopodial and bead motility. On the basis of these observations, it is suggested that ras-induced transformation of epithelial cells consists of two cytoskeletal modifications: overall diminished actin cytoskeletal dynamics in lamellae and reorganization of the microtubule cytoskeleton that directs pseudopodial activity to smaller polarized lamellae.

Apolipoprotein E and Alzheimer disease.

Inheritance of specific apolipoprotein E (apoE) alleles determines, in large part, the risk and mean age of onset of late-onset familial and sporadic Alzheimer disease. The mechanism by which the apoE isoforms differentially contribute to disease expression is, however, unknown. Isoform-specific differences have been identified in the binding of apoE to the microtubule-associated protein tau, which forms the paired helical filament and neurofibrillary tangles, and to amyloid beta peptide, a major component of the neuritic plaque. These and other isoform-specific interactions of apoE give rise to testable hypotheses for the mechanism(s) of pathogenesis of Alzheimer disease. An unresolved issue of increasing importance is the relationship between the structural pathological lesions and the cellular pathogenesis responsible for the clinical disease phenotype, progressive dementia. The identification of apoE in the cytoplasm of human neurons and the characterization of isoform-specific binding of apoE to the microtubule-associated proteins tau and MAP-2 present the possibility that apoE may affect microtubule function in the Alzheimer brain.

Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.

Eg5, a member of the bimC subfamily of kinesin-like microtubule motor proteins, localizes to spindle microtubules in mitosis but not to interphase microtubules. We investigated the molecular basis for spindle localization by transient transfection of Xenopus A6 cells with myc-tagged derivatives of Eg5. Expressed at constitutively high levels from a cytomegalovirus promoter, mycEg5 protein is cytoplasmic throughout interphase, begins to bind microtubules in early prophase, and remains localized to spindle and/or midbody microtubules through mitosis to the end of telophase. Both N- and C-terminal regions of Eg5 are required for this cell-cycle-regulated targeting. Eg5 also contains within its C-terminal domain a sequence conserved among bimC subfamily proteins that includes a potential p34cdc2 phosphorylation site. We show that mutation of a single threonine (T937) within this site to nonphosphorylatable alanine abolishes localization of the mutant protein to the spindle, whereas mutation of T937 to serine preserves spindle localization. We hypothesize that phosphorylation of Eg5 may regulate its localization to the spindle in the cell cycle.

Applications of EPR with an Emphasis on Tau Fibril Structure

Substances containing unpaired electrons have been studied by electron paramagnetic resonance (EPR) for nearly 70 years. With continual development and enhancement of EPR techniques, questions have arisen regarding optimum method selection for a given sample based on its properties. In this work, radiation defects, natural lattice defects, solid organic radicals, radicals in solution, and spin-labeled proteins were analyzed using CW, pulse, and rapid scan EPR to compare methods. Studies of solid BDPA, EOe in quartz, Ns0 in diamond, and a-Si:H, showed that rapid scan could overcome many obstacles presented by other techniques, cementing rapid scan as an effective alternative to CW and pulse methods. Relaxation times of six nitroxide radicals were characterized from 0.25-34 GHz, guiding synthesis of improved nitroxides for in vivo imaging experiments. Processes contributing to T1 of DPPH in polystyrene were found through variable temperature measurements at X- and Q-band, resolving previously-reported discrepancies in relaxation properties and providing new insight into this commonly-used standard. In the history of EPR, the study of proteins is relatively new. Double electron-electron resonance (DEER) has emerged as a powerful technique for the study of amyloid fibrils, a class of protein aggregates implicated in a number of neurodegenerative disorders. Microtubule-associated protein tau forms fibrils linked to Alzheimerfs disease through seeded conversion of monomer. Self-assembly is mediated by the microtubule binding repeats in tau, and there are either three or four repeats present depending on the isoform. DEER was used to show that filaments of 3R and 4R tau are conformationally distinct and that 4R fibrils adopt a heterogeneous mixture of conformations. Populations of 4R fibril conformations, which were independently validated using a model system, can be modulated by introduction of mutations to the primary sequence or by varying fibril growth conditions. These findings provided unprecedented insights into the seed selection of tau monomers and established conformational compatibility as an important driving force in tau fibril propagation. Lastly, DEER acquisition was improved through addition of paramagnetic metal to spin-labeled protein, decreasing collection time, and through use of a novel spin label with increased T2, thereby lengthening the available acquisition window.

The Effects of Molecular Chaperones on Tau Fibril Assembly

The accumulation of microtubule-associated protein tau into fibrillar aggregates is the hallmark of Alzheimer’s disease and other neurodegenerative disorders, collectively referred to as tauopathies. Fibrils can propagate from one cell to the next and spread throughout the brain. However, a study shows that only small aggregates can be taken up by cultured neuronal cells. The mechanisms that lead to the breakage of fibrils into smaller fragments remain unknown. In yeast, the AAA+ chaperone HSP104 processes the reactivation of protein aggregates and is responsible for fragmentation of fibrils. This study focused on investigating the effects of molecular chaperones on tau fibrils and using HSP104 as a model system to test whether we can monitor fibril fracturing. The assays used to detect the chaperone’s actions on tau utilized acrylodan fluorescence, thioflavin T fluorescence, and sedimentation. Tau fibrils were either formed with a cofactor, heparin, to accelerate assembly or without a cofactor. In the process of investigating the effects of HSP104 on tau fibrils, this study established an assay to determine the effects of breakage on the seeding properties of tau fibrils. Our findings demonstrated that the sonication of tau fibrils produces smaller fragments (seeds) that accelerate the conversion of monomeric tau into fibrils. The use of this assay with HSP104 provided evidence that HSP104 inhibits the elongation of tau fibrils. Indeed, HSP104 inhibits the aggregation of soluble tau into aggregates. However, tau fibril breakage and dissociation were not observed with HSP104, either alone or in combination with co-chaperones (HSP70 and HSP40). Our findings provide insights into the seeding properties of tau fibrils, and suggest that fragmentation is a critical part of tau assembly. This knowledge should be valuable for understanding tau fibril aggregation and propagation in the brain, which is necessary to identify new treatments for neurodegenerative diseases.

Sowing the Seeds of Centromeres

The centromere is a chromatin-based platform that accumulates microtubule-binding proteins that drive chromosome segregation during cell division. Despite their size (on the order of megabases of DNA in mammals) and conserved role, centromeres have the remarkable capacity to leave their usual comfort zone and to reform at a new chromosomal site (1). Although found rarely, these so-called neocentromeres are by most measures bona fide and segregate chromosomes with high fidelity. What accounts for this nomadic behavior?

Drosophila melanogaster as a model for basal body research

The fruit fly, Drosophila melanogaster, is one of the most extensively studied organisms in biological research and has centrioles/basal bodies and cilia that can be modelled to investigate their functions in animals generally. Centrioles are nine-fold symmetrical microtubule-based cylindrical structures required to form centrosomes and also to nucleate the formation of cilia and flagella. When they function to template cilia, centrioles transition into basal bodies. The fruit fly has various types of basal bodies and cilia, which are needed for sensory neuron and sperm function. Genetics, cell biology and behaviour studies in the fruit fly have unveiled new basal body components and revealed different modes of assembly and functions of basal bodies that are conserved in many other organisms, including human, green algae and plasmodium. Here we describe the various basal bodies of Drosophila, what is known about their composition, structure and function.

Activities of fenbendazole in comparison with albendazole against Echinococcus multilocularis metacestodes in vitro and in a murine infection model.

The current chemotherapeutic treatment of alveolar echinococcosis (AE) in humans is based on albendazole and/or mebendazole. However, the costs of treatment, life-long consumption of drugs, parasitostatic rather than parasiticidal activity of chemotherapy, and high recurrence rates after treatment interruption warrant more efficient treatment options. Experimental treatment of mice infected with Echinococcus multilocularis metacestodes with fenbendazole revealed similar efficacy to albendazole. Inspection of parasite tissue from infected and benzimidazole-treated mice by transmission electron microscopy (TEM) demonstrated drug-induced alterations within the germinal layer of the parasites, and most notably an almost complete absence of microtriches. On the other hand, upon in vitro exposure of metacestodes to benzimidazoles, no phosphoglucose isomerase activity could be detected in medium supernatants during treatment with any of these drugs, indicating that in vitro treatment did not severely affect the viability of metacestode tissue. Corresponding TEM analysis also revealed a dramatic shortening/retraction of microtriches as a hallmark of benzimidazole action, and as a consequence separation of the acellular laminated layer from the cellular germinal layer. Since TEM did not reveal any microtubule-based structures within Echinococcus microtriches, this effect cannot be explained by the previously described mechanism of action of benzimidazoles targeting β-tubulin, thus benzimidazoles must interact with additional targets that have not been yet identified. In addition, these results indicate the potential usefulness of fenbendazole for the chemotherapy of AE.

Tau haplotypes regulate transcription and are associated with Parkinson's disease

A primary haplotype (H1) of the microtubule-associated protein Tau (MAPT) gene is associated with Parkinson's disease (PD). However, the mechanism for disease susceptibility remains unknown. We examined the promoter region of MAPT and identified single nucleotide polymorphisms and insertions of 1 to 11 nucleotides. These polymorphisms corresponded to the previously characterized haplotypes, H1 and H2, as well as a novel variant of the H1 haplotype, H1'. As observed in other studies, we demonstrated a significant association with the H1/H1 promoter genotype and PD in a cohort of 206 idiopathic late-onset cases. This is in contrast with a panel of 13 early-onset PD patients, for whom we did not detect any mutations in MAPT. By examining single nucleotide polymorphisms in adjacent genes, we showed that linkage disequilibrium does not extend beyond the MAPT haplotype to neighboring genes. To define the mechanism of disease susceptibility, we examined the transcriptional activity of the promoter haplotypes using a luciferase reporter assay. We demonstrated in two human cell lines, SK-N-MC and 293, that the H1 haplotype was more efficient at driving gene expression than the H2 haplotype. Our data suggest that an increase in expression of the MAPT gene is a susceptibility factor in idiopathic PD.

Dynamic and regulated association of caveolin with lipid bodies: Modulation of lipid body motility and function by a dominant negative mutant

Caveolins are a crucial component of caveolae but have also been localized to the Golgi complex, and, under some experimental conditions, to lipid bodies (LBs). The physiological relevance and dynamics of LB association remain unclear. We now show that endogenous caveolin-1 and caveolin-2 redistribute to LBs in lipid loaded A431 and FRT cells. Association with LBs is regulated and reversible; removal of fatty acids causes caveolin to rapidly leave the lipid body. We also show by subcellular fractionation, light and electron microscopy that during the first hours of liver regeneration, caveolins show a dramatic redistribution from the cell surface to the newly formed LBs. At later stages of the regeneration process (when LBs are still abundant), the levels of caveolins in LBs decrease dramatically. As a model system to study association of caveolins with LBs we have used brefeldin A (BFA). BFA causes rapid redistribution of endogenous caveolins to LBs and this association was reversed upon BFA washout. Finally, we have used a dominant negative LB-associated caveolin mutant (cav(DGV)) to study LB formation and to examine its effect on LB function. We now show that the cav(DGV) mutant inhibits microtubule-dependent LB motility and blocks the reversal of lipid accumulation in LBs.

Association of stomatin with lipid bodies

The oligomeric lipid raft-associated integral protein stomatin normally localizes to the plasma membrane and the late endosomal compartment. Similar to the caveolins, it is targeted to lipid bodies (LBs) on overexpression. Endogenous stomatin also associates with LBs to a small extent. Green fluorescent protein-tagged stomatin (StomGFP) and the dominant-negative caveolin-3 mutant DGV(cav3)(HA) occupy distinct domains on LB surfaces but eventually intermix. Studies of StomGFP deletion mutants reveal that the region for membrane association but not oligomerization and raft association is essential for LB targeting. Blocking protein synthesis leads to the redistribution of StomGFP from LBs to LysoTracker-positive vesicles indicating a connection with the late endosomal/ lysosomal pathway. Live microscopy of StomGFP reveals multiple interactions between LBs and microtubule-associated vesicles possibly representing signaling events and/or the exchange of cargo. Proteomic analysis of isolated LBs identifies adipophilin and TIP47, various lipid-specific enzymes, cytoskeletal components, chaperones, Ras-related proteins, protein kinase D2, and other regulatory proteins. The association of the Rab proteins 1, 6, 7, 10, and 18 with LBs indicates various connections to other compartments. Our data suggest that LBs are not only involved in the storage of lipids but also participate actively in the cellular signaling network and the homeostasis of lipids.

Chromosomal genotoxicity of nitrobenzene and benzonitrile

In order to investigate the chromosomal genotoxicity of nitrobenzene and benzonitrile, we studied the induction of micronuclei (MN) by these test compounds in V79 cells, as well as effects on the formation and stability of microtubules and on motor protein functions. No cytotoxicity was seen in V79 cell cultures in terms of Neutral red uptake after 18 h treatment with up to 1 mM nitrobenzene or 1 mM benzonitrile. Subsequently, a concentration range up to 100 muM was used in the experiments on induction of MN. Both test compounds exhibit a weak, but definitely positive test result compared to the solvent (DMSO) control. Minimal effect concentrations of nitrobenzene and benzonitrile appeared as low as 0.01 muM, and no-effect-concentrations were between 0.001 and 0.005 muM. Clearly enhanced MN rates were found at 0.1 muM and higher. Both, nitrobenzene and benzonitrile, induced mostly kinetochor (CREST)-positive micronuclei, thus characterising the chromosomal effects as aneugenic. In cell-free assays, a slight effect on tubulin assembly was observed at 1 mM nitrobenzene without addition of DMSO. Higher concentrations (5 mM) led to secondary effects. In presence of 1% DMSO, nitrobenzene exerted no detectable effect on tubulin assembly up to the solubility limit in water of about 15 mM. For benzonitrile in presence of DMSO, a clear dose-response of inhibition of tubulin assembly at 37degreesC was seen above the no-effect-concentration of 2 mM, with an IC50 of 13 mM and protein denaturation starting above a level of about 20 mM. The nature of the effects of nitrobenzene and benzonitrile on the association of tubulin to form microtubules was confirmed by electron microscopy. Treatment by either 5 mM nitrobenzene or 13 mM benzonitrile plus 1% DMSO left the microtubular structure intact whereas 5 mM nitrobenzene, in absence of DMSO, led to irregular cluster formations. The experiments demonstrate that both nitrobenzene and benzonitrile, in millimolar concentration ranges, may lead to interference with tubulin assembly in a cell-free system. The functionality of the tubulin-kinesin motor protein system was assessed using the microtubule gliding assay. Nitrobenzene affected the gliding velocity in a concentration-dependent manner, starting at about 7.5 muM and reaching complete inhibition of motility at 30 muM, whereas benzonitrile up to 200 muM did not affect the kinesin-driven gliding velocity. The micronucleus assay data demonstrate a chromosomal endpoint of genotoxicity of nitrobenzene and benzonitrile. Aneugenic effects of both compounds occur at remarkably low concentrations, with lowest-effect-concentrations being 0.1 muM. This points to the relevance of interactions with the cellular spindle apparatus.

Tyrosine 394 is phosphorylated in Alzheimer's paired helical filament tau and in fetal tau with c-Abl as the candidate tyrosine kinase

Tau is a major microtubule-associated protein of axons and is also the principal component of the paired helical filaments (PHFs) that comprise the neurofibrillary tangles found in Alzheimer's disease and other tauopathies. Besides phosphorylation of tau on serine and threonine residues in both normal tau and tau from neurofibrillary tangles, Tyr-18 was reported to be a site of phosphorylation by the Src-family kinase Fyn. We examined whether tyrosine residues other than Tyr-18 are phosphorylated in tau and whether other tyrosine kinases might phosphorylate tau. Using mass spectrometry, we positively identified phosphorylated Tyr-394 in PHF-tau from an Alzheimer brain and in human fetal brain tau. When wild-type human tau was transfected into fibroblasts or neuroblastoma cells, treatment with pervanadate caused tau to become phosphorylated on tyrosine by endogenous kinases. By replacing each of the five tyrosines in tau with phenylalanine, we identified Tyr-394 as the major site of tyrosine phosphorylation in tau. Tyrosine phosphorylation of tau was inhibited by PP2 (4-amino-5-(4-chlorophenyl-7-(t-butyl) pyrazolo[3,4-d] pyrimidine), which is known to inhibit Src-family kinases and c-Abl. Cotransfection of tau and kinases showed that Tyr-18 was the major site for Fyn phosphorylation, but Tyr-394 was the main residue for Abl. In vitro, Abl phosphorylated tau directly. Abl could be coprecipitated with tau and was present in pretangle neurons in brain sections from Alzheimer cases. These results show that phosphorylation of tau on Tyr-394 is a physiological event that is potentially part of a signal relay and suggest that Abl could have a pathogenic role in Alzheimer's disease.

Changes in neuronal protein 22 expression and cytoskeletal association in the alcohol-dependent and withdrawn rat brain

The action of alcohol on neuronal pathways has been an issue of increasing research focus, with numerous findings contradicting the previously accepted idea that its effect is nonspecific. The human NP22 (hNP22) gene was revealed by its elevated expression in the frontal cortex of the human alcoholic. The sequences of hNP22 and the rat orthologue rNP22 contain a number of domains consistent with those of cytoskeletal-interacting proteins. Localization of rNP22 is restricted to the cytoplasm and processes of neurons and it colocalizes with elements of the microfilament and microtubule matrices including filamentous actin (F-actin), alpha-tubulin, tau, and microtubule-associated protein 2 (MAP2). Withdrawal of Wistar rats after alcohol dependence induced by alcohol vapor produced elevated levels of rNP22 mRNA and protein in the cortex, CA2, and dentate gyrus regions of the hippocampus. In contrast, there was decreased rNP22 expression in the striatum after chronic ethanol exposure. Chronic ethanol exposure did not markedly alter rNP22 colocalization with F-actin, alpha-tubulin, or MAP2, although colocalization at the periphery of the neuronal soma with F-actin was observed only after chronic ethanol exposure and withdrawal. Rat NP22 colocalization with MAP2 was reduced during withdrawal, whereas association with alpha-tubulin and actin was maintained. These findings suggest that the effect of chronic ethanol exposure and withdrawal on rNP22 expression is region selective. Rat NP22 may affect microtubule or microfilament function, thereby regulating the neuroplastic changes associated with the development of alcohol dependence and physical withdrawal.