Sven Tågerud

Actomyosin motility on nanostructured surfaces

We have here, for the first time, used nanofabrication techniques to reproduce aspects of the ordered actomyosin arrangement in a muscle cell. The adsorption of functional heavy meromyosin (HMM) to five different resist polymers was first assessed. One group of resists (MRL-6000.1XP and ZEP-520) consistently exhibited high quality motility of actin filaments after incubation with HMM. A second group (PMMA-200, PMMA-950, and MRI-9030) generally gave low quality of motility with only few smoothly moving filaments. Based on these findings electron beam lithography was applied to a bi-layer resist system with PMMA-950 on top of MRL-6000.1XP. Grooves (100-200nm wide) in the PMMA layer were created to expose the MRL-6000.1XP surface for adsorption of HMM and guidance of actin filament motility. This guidance was quite efficient allowing no U-turns of the filaments and approximately 20 times higher density of moving filaments in the grooves than on the surrounding PMMA.

Dopamine release from the rat substantia nigra in vitro. Effect of raphe lesions and veratridine stimulation.

Abstract In order to eliminate the 5-hydroxytryptaminergic input to the substantia nigra lesions were placed in the dorsal and medial raphe nuclei in a number of rats. The release of exogenously applied [ 3 H]dopamine from the partially denervated substantia nigra was determined in vitro and found to be very similar to the release observed from slices of control substantia nigra. These results lend further support to the theory that the release of exogenously applied [ 3 H]dopamine at the level of the substantia nigra occurs mainly from dopaminergic dendrites, rather than from terminals of 5-hydroxytryptamine-containing neurons. A veratridine-induced release of [ 3 H]dopamine from the pars reticulata of the substantia nigra is also described. An almost complete blockade of veratridine (3.0 μM) stimulation was observed with 100 nM tetrodotoxin. Similar effects of veratridine and tetrodotoxin were also observed on [ 3 H]dopamine release from slices of corpus striatum. These results suggest that dendrites of the dopaminergic neurones in the substantia nigra contain fast, tetrodotoxin-sensitive sodium channels.

Denervation-induced alterations in gene expression in mouse skeletal muscle

Motoneurons are important for regulating the function and properties of skeletal muscle. In the present study high‐density oligonucleotide arrays have been used to compare gene expression in innervated and six‐days denervated NMRI mouse skeletal muscle. To avoid looking at genes mainly participating in the process of atrophy, both hind‐limb muscles (atrophic after denervation) and hemidiaphragm muscle (transiently hypertrophic after denervation) were used. Only genes previously not known to respond to denervation and with potential roles in DNA/RNA interactions/transcription and/or cellular communication/signalling are presented. Data for additional genes are provided as supplementary material. Thirty‐two genes, up‐regulated by a factor of two or down‐regulated to the same extent after denervation, are presented. These include genes that may act through chromatin remodelling and/or as transcription factors/regulators (Cdkn1a, Cdr2, Hrmt1l2, Idb2, Myc/c‐myc, L‐myc1, Rb1, Sap30 and Tgif), genes possibly involved in the regulation of muscle membrane properties and/or excitation‐contraction coupling (Cacng1, Camk2d, Hrmt1l2, Kcnj12, Kcna7 and Rrad) and genes potentially involved in neuromuscular interactions and/or receptor signalling (Acvr2b, Adam19, D0H4S114, Kai1, Maged1, Mt2, Prkcabp, Ptp4a3, Ramp1, Rras, Timp1, Vegfa and Zfp145). A set of five genes with altered expression after denervation (Fzd9, Nr4a1, Frat2, Ctgf and Cyr61) indicate that Wnt signalling may be reduced in denervated skeletal muscle.

Guiding motor-propelled molecules with nanoscale precision through silanized bi-channel structures

Guiding motor-propelled molecules with nanoscale precision through silanized bi-channel structures

Rhodamine B, a fluorescent probe for acidic organelles in denervated skeletal muscle.:

We describe a very efficient method for fluorescent labeling of acidic structures in denervated skeletal muscle with rhodamine B. Rhodamine B at 50 ng/ml gave selective and distinct segmental labeling of denervated muscle fibers after 5-min incubation at room temperature. Labeling was also achieved at 4 degrees C. The labeling was disrupted by the ionophores monensin and nigericin, suggesting a labeling confined to acidic structures. Rhodamine B co-localized with the lysosomotropic dye Lyso Tracker Green and a marker for endocytosis (fluorescein isothiocyanate-labeled dextran). Rhodamine B, which is highly lipophilic, showed pH-dependent fluorescence emission in saturated aqueous N-octanol. Tetramethylrhodamine showed similar characteristics for labeling of denervated muscle fibers and pH-dependent fluorescence in N-octanol. The carboxyl group present in these two compounds appears important, because structurally related compounds that either lack this group or have it esterified failed to label denervated muscle fibers and showed no pH-dependent fluorescence in N-octanol. The results suggest that rhodamine B labels acidic organelles belonging to the endosomal/lyosomal system of denervated skeletal muscle fibers. Nevertheless, it failed to label such organelles in a number of mammalian cell types other than denervated skeletal muscle fibers.

Nogo (Reticulon 4) expression in innervated and denervated mouse skeletal muscle

The nogo gene encodes at least three different proteins, which share a high C-terminal homology with other members of the Reticulon family. Nogo (Reticulon 4) expression has been studied in innervated and denervated mouse hind-limb and hemidiaphragm muscles. A common Nogo A, B, and C probe hybridized to three transcripts, in accordance with human and rat data. Denervation caused decreased Nogo C and increased Nogo A mRNA expression, while Nogo B was not substantially altered. Western blots and immunohistochemistry confirmed the presence of Nogo A-like and Nogo B-like immunoreactivity in muscle. Nogo A-like immunoreactivity increased after denervation and was also present in intramuscular nerves in both innervated and denervated muscle. Nogo B-like immunoreactivity was observed in connective tissue surrounding muscle fibres and nerves. The different Nogo transcripts are produced by both alternative splicing (A and B) and alternative promoter usage (C); both mechanisms seem to be under neural control in skeletal muscle.

Role of the proteasome in rat indomethacin-induced gastropathy☆☆☆

BACKGROUND & AIMS Intercellular adhesion molecule (ICAM)-dependent adhesion of circulating neutrophils to microvascular endothelial cells is thought to be critical in causing indomethacin (nonsteroidal anti-inflammatory drug [NSAID])-induced gastropathy. Indomethacin stimulates tumor necrosis factor (TNF)-alpha expression, which may enhance adhesiveness of gastric capillaries for neutrophils by activating ICAM expression on endothelial cells. Stimulation of ICAM expression is mediated by activation of the transcription factor NF-kappaB. Because activation of NF-kappaB requires proteolytic degradation of IkappaB by the ubiquitin-proteasome pathway of intracellular proteolysis, treatment with proteasome inhibitors was evaluated for efficacy in preventing NSAID gastropathy. METHODS The effect of proteasome inhibitors on gastric injury caused by oral indomethacin was measured, along with their effects on gastric mucosal permeability measured by the blood to lumen EDTA clearance. Gastric ICAM expression was measured in vivo using infusion of a labeled rat ICAM antibody. RESULTS Proteasome inhibitors prevented NSAID gastropathy if administered from 0 to 12 hours before indomethacin. Equivalent efficacy was observed with intravenous and oral administration of proteasome inhibitors. There was a strong correlation between the potency of proteasome inhibitors in preventing NSAID gastropathy and their potency in inhibiting intracellular proteolysis or their anti-inflammatory potency. All three classes of proteasome inhibitors, peptide boronates, aldehydes, and the mechanistically different lactacystin, prevented NSAID gastropathy. Proteasome inhibitor treatment also abolished the increase in gastric mucosal permeability and the increase in gastric endothelial ICAM expression induced by indomethacin. CONCLUSIONS Indomethacin-induced gastric injury and increased ICAM expression are inhibited by inhibition of the proteasome.

Tetrahydroaminoacridine (tacrine) stimulates neurosecretion at mammalian motor endplates

1 Tacrine (20 μm) induced, like 4‐aminoquinoline (4‐AQ, 200 μm), the appearance of a population of miniature endplate potentials (m.e.p.ps) with more than twice the normal amplitude or time‐to‐peak. The times‐to‐peak of nerve impulse‐evoked endplate potentials were not similarly affected. 2 Cholinesterase inhibition by edrophonium (25 μm) did not prevent tacrine or 4‐AQ from inducing this population of m.e.p.ps. 3 Nerve‐muscle preparations in which the normal calcium‐sensitive quantal release of acetylcholine had been blocked by botulinum neurotoxin type A also responded to tacrine by an increase in the frequency of giant or slow m.e.p.ps. 4 Reduction of the temperature from 30° to 14°C reduced the frequency of giant or slow m.e.p.ps induced either by tacrine or by 4‐AQ. A similar effect was obtained by colchicine (5 mm). This supports the idea that proximo‐distal axonal transport is required for the secretory activity. 5 The neurosecretion evoked by tacrine could explain the therapeutic effects of the drug claimed in the treatment of Alzheimers type of dementia.

Multivariate statistics in analysis of data from the in vitro motility assay.

A novel approach is described for classification of filaments as stationary or moving and for extraction of velocity data for smooth actin filament sliding in vitro. Moving and stationary filaments were effectively classified using four discriminating variables in a multivariate statistical analysis. The variables were (1) two different measures of the average filament distance from its starting point, (2) a measure of the variability in sliding direction, and (3) the coefficient of variation (CV) of the frame-to-frame sliding velocity (v(mean)). On the basis of this multivariate analysis we obtained correct classification of 98% of the stationary filaments and 94% of the moving filaments in a cross-validation data set. The same classification functions were useful throughout despite a 10-fold variation in the average sliding velocity in the cross-validation data. Further analysis of motile filaments suggested that the velocity of smooth sliding should, ideally, be obtained from the intercept on the velocity axis of a plot of v(mean) against CV. The velocity, so obtained, was between 10 and 30% (mean 20+/-3%; n=7; p<0.001) higher than if average sliding velocity was obtained for all moving filaments with CV<0.5.

Diffusion Dynamics of Motor-Driven Transport: Gradient Production and Self-Organization of Surfaces.

The interaction between cytoskeletal filaments (e.g., actin filaments) and molecular motors (e.g., myosin) is the basis for many aspects of cell motility and organization of the cell interior. In the in vitro motility assay (IVMA), cytoskeletal filaments are observed while being propelled by molecular motors adsorbed to artificial surfaces (e.g., in studies of motor function). Here we integrate ideas that cytoskeletal filaments may be used as nanoscale templates in nanopatterning with a novel approach for the production of surface gradients of biomolecules and nanoscale topographical features. The production of such gradients is challenging but of increasing interest (e.g., in cell biology). First, we show that myosin-induced actin filament sliding in the IVMA can be approximately described as persistent random motion with a diffusion coefficient (D) given by a relationship analogous to the Einstein equation (D = kT/gamma). In this relationship, the thermal energy (kT) and the drag coefficient (gamma) are substituted by a parameter related to the free-energy transduction by actomyosin and the actomyosin dissociation rate constant, respectively. We then demonstrate how the persistent random motion of actin filaments can be exploited in conceptually novel methods for the production of actin filament density gradients of predictable shapes. Because of regularly spaced binding sites (e.g., lysines and cysteines) the actin filaments act as suitable nanoscale scaffolds for other biomolecules (tested for fibronectin) or nanoparticles. This forms the basis for secondary chemical and topographical gradients with implications for cell biological studies and biosensing.