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Find video protocols related to scientific articles indexed in Pubmed.
Thioredoxin and its reductase are present on synaptic vesicles, and their inhibition prevents the paralysis induced by botulinum neurotoxins.
Cell Rep
PUBLISHED: 06-03-2014
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Botulinum neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E. Specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner and are also very effective inhibitors of the paralysis of the neuromuscular junction. We found that this group of inhibitors of botulinum neurotoxins is very effective in vivo. Most of them are nontoxic and are good candidates as preventive and therapeutic drugs for human botulism.
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Exercise training induces mitochondrial biogenesis and glucose uptake in subcutaneous adipose tissue through eNOS-dependent mechanisms.
Diabetes
PUBLISHED: 03-12-2014
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Insulin resistance and obesity are associated with a reduction of mitochondrial content in various tissues of mammals. Moreover, a reduced nitric oxide (NO) bioavailability impairs several cellular functions, including mitochondrial biogenesis and insulin-stimulated glucose uptake, two important mechanisms of body adaptation in response to physical exercise. Although these mechanisms have been thoroughly investigated in skeletal muscle and heart, few studies have focused on the effects of exercise on mitochondria and glucose metabolism in adipose tissue. In this study, we compared the in vivo effects of chronic exercise in subcutaneous adipose tissue of wild-type (WT) and endothelial NO synthase (eNOS) knockout (eNOS(-/-)) mice after a swim training period. We then investigated the in vitro effects of NO on mouse 3T3-L1 and human subcutaneous adipose tissue-derived adipocytes after a chronic treatment with an NO donor: diethylenetriamine-NO (DETA-NO). We observed that swim training increases mitochondrial biogenesis, mitochondrial DNA content, and glucose uptake in subcutaneous adipose tissue of WT but not eNOS(-/-) mice. Furthermore, we observed that DETA-NO promotes mitochondrial biogenesis and elongation, glucose uptake, and GLUT4 translocation in cultured murine and human adipocytes. These results point to the crucial role of the eNOS-derived NO in the metabolic adaptation of subcutaneous adipose tissue to exercise training.
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Inflammation and pancreatic cancer: molecular and functional interactions between S100A8, S100A9, NT-S100A8 and TGF?1.
Cell Commun. Signal
PUBLISHED: 03-08-2014
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In order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGF?1 and the inflammatory proteins S100A8, S100A9 and NT-S100A8, a PDAC-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT). NF-?B, Akt and mTOR pathways, Cai2+ and EMT were studied in well (Capan1 and BxPC3) and poorly differentiated (Panc1 and MiaPaCa2) cell lines.
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Evidence for a radial SNARE super-complex mediating neurotransmitter release at the Drosophila neuromuscular junction.
J. Cell. Sci.
PUBLISHED: 05-17-2013
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The SNARE proteins VAMP/synaptobrevin, SNAP-25 and syntaxin are core components of the apparatus that mediates neurotransmitter release. They form a heterotrimeric complex, and an undetermined number of SNARE complexes assemble to form a super-complex. Here, we present a radial model of this nanomachine. Experiments performed with botulinum neurotoxins led to the identification of one arginine residue in SNAP-25 and one aspartate residue in syntaxin (R206 and D253 in Drosophila melanogaster). These residues are highly conserved and predicted to play a major role in the protein-protein interactions between SNARE complexes by forming an ionic couple. Accordingly, we generated transgenic Drosophila lines expressing SNAREs mutated in these residues and performed an electrophysiological analysis of their neuromuscular junctions. Our results indicate that SNAP-25-R206 and syntaxin-D253 play a major role in neuroexocytosis and support a radial assembly of several SNARE complexes interacting via the ionic couple formed by these two residues.
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Sodium channel ?2 subunit promotes filopodia-like processes and expansion of the dendritic tree in developing rat hippocampal neurons.
Front Cell Neurosci
PUBLISHED: 01-07-2013
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The ?2 auxiliary subunit of voltage-gated sodium channels (VGSCs) appears at early stages of brain development. It is abundantly expressed in the mammalian central nervous system where it forms complexes with different channel isoforms, including Na(v)1.2. From the structural point of view, ?2 is a transmembrane protein: at its extracellular N-terminus an Ig-like type C2 domain mediates the binding to the pore-forming alpha subunit with disulfide bonds and the interactions with the extracellular matrix. Given this structural versatility, ?2 has been suggested to play multiple functions ranging from channel targeting to the plasma membrane and gating modulation to control of cell adhesion. We report that, when expressed in Chinese Hamster Ovary cells CHO-K1, the subunit accumulates at the perimetral region of adhesion and particularly in large lamellipodia-like membrane processes where it induces formation of filopodia-like structures. When overexpressed in developing embryonic rat hippocampal neurons in vitro, ?2 specifically promotes formation of filopodia-like processes in dendrites leading to expansion of the arborization tree, while axonal branching remains unaltered. In contrast to this striking and highly specific effect on dendritic morphology, the targeting of functional sodium channels to the plasma membrane, including the preferential localization of Na(v)1.2 at the axon, and their gating properties are only minimally affected. From these and previously reported observations it is suggested that ?2, among its multiple functions, may contribute to promote dendritic outgrowth and to regulate neuronal wiring at specific stages of neuronal development.
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Mitochondrial Ca2+-handling in fast skeletal muscle fibers from wild type and calsequestrin-null mice.
PLoS ONE
PUBLISHED: 01-01-2013
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Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca(2+)-binding protein inside SR, calsequestrin. Mitochondrial free Ca(2+)-concentration ([Ca(2+)]mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca(2+) release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca(2+)]mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca(2+) concentration. The rise in [Ca(2+)]mito during electrical stimulation occurred in 20-30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s(-1). Accordingly, frequency-dependent increase in [Ca(2+)]mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca(2+)]mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca(2+)]mito transients were increased by inhibition of mitochondrial Na(+)/Ca(2+) exchanger (mNCX). These results provide direct evidence for fast Ca(2+) accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca(2+)-handling and a dependence of mitochondrial Ca(2+)-handling on intracellular (SR) and external Ca(2+) stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca(2+)-leakage.
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Defhc1.1, a homologue of the juvenile myoclonic gene EFHC1, modulates architecture and basal activity of the neuromuscular junction in Drosophila.
Hum. Mol. Genet.
PUBLISHED: 08-11-2011
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Mutations in the EFHC1 gene have been linked to juvenile myoclonic epilepsy. To understand EFHC1 function in vivo, we generated knockout Drosophila for the fly homolog Defhc1.1. We found that the neuromuscular junction synapse of Defhc1.1 mutants displays an increased number of satellite boutons resulting in increased spontaneous neurotransmitter release. Defhc1.1 binds to microtubules in vitro and overlaps in vivo with axonal and synaptic microtubules. Elimination of Defhc1.1 from synaptic terminals reduces the number of microtubule loops, suggesting that Defhc1.1 is a negative regulator of microtubule dynamics. In fact, pharmacological treatment of Defhc1.1 mutants with vinblastine, an inhibitor of microtubule dynamics, suppresses the satellite bouton phenotype. Furthermore, Defhc1.1 mutants display overgrowth of the dendritic arbor and Defhc1.1 overexpression reduces dendrite elaboration. These results suggest that Defhc1.1 functions as an inhibitor of neurite growth by finely tuning the microtubule cytoskeleton dynamics and that EFHC1-dependent juvenile myoclonic epilepsy may result from augmented spontaneous neurotransmitter release due to overgrowth of neuronal processes.
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The modulation of myogenic cells differentiation using a semiconductor-muscle junction.
Biomaterials
PUBLISHED: 02-12-2011
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The present study is aimed to design a prototype of hybrid silicon-muscle cell junction, analog to an artificial neuromuscular junction prototype and relevant to the development of advanced neuro-prostheses and bionic systems. The device achieves focal Electric Capacitive Stimulation (ECS) by coupling of single cells and semiconductors, without electrochemical reaction with the substrate. A voltage change applied to a stimulation spot beneath an electrogenic cell leads to a capacitive current (charge accumulation) that opens voltage-gated ion channels in the membrane and generates an action potential. The myo-electronic junction was employed to chronically stimulate muscle cells via ECS and to induce cytosolic calcium transients in myotubes, fibers isolated from mouse FDB (fast [Ca(2+)](i) transients) and surprisingly also in undifferentiated myoblasts (slow [Ca(2+)](i) waves). The hybrid junction elicited, via chronic ECS, a differential reprogramming of single muscle cells by inducing early muscle contraction maturation and plasticity effects, such as NFAT-C3 nuclear translocation. In addition, in the presence of agrin, chronic ECS induced a modulation of AchR clustering which simulates in vitro synaptogenesis. This methodology can coordinate the myogenic differentiation, thus offering direct but non-invasive single cell/wiring, providing a platform for regenerative medicine strategies.
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Arg206 of SNAP-25 is essential for neuroexocytosis at the Drosophila melanogaster neuromuscular junction.
J. Cell. Sci.
PUBLISHED: 09-07-2010
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An analysis of SNAP-25 isoform sequences indicates that there is a highly conserved arginine residue (198 in vertebrates, 206 in the genus Drosophila) within the C-terminal region, which is cleaved by botulinum neurotoxin A, with consequent blockade of neuroexocytosis. The possibility that it may play an important role in the function of the neuroexocytosis machinery was tested at neuromuscular junctions of Drosophila melanogaster larvae expressing SNAP-25 in which Arg206 had been replaced by alanine. Electrophysiological recordings of spontaneous and evoked neurotransmitter release under different conditions as well as testing for the assembly of the SNARE complex indicate that this residue, which is at the P(1) position of the botulinum neurotoxin A cleavage site, plays an essential role in neuroexocytosis. Computer graphic modelling suggests that this arginine residue mediates protein-protein contacts within a rosette of SNARE complexes that assembles to mediate the fusion of synaptic vesicles with the presynaptic plasma membrane.
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Ca2+ hot spots on the mitochondrial surface are generated by Ca2+ mobilization from stores, but not by activation of store-operated Ca2+ channels.
Mol. Cell
PUBLISHED: 02-03-2010
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Although it is widely accepted that mitochondria in living cells can efficiently uptake Ca(2+) during stimulation because of their vicinity to microdomains of high [Ca(2+)], the direct proof of Ca(2+) hot spots existence is still lacking. Thanks to a GFP-based Ca(2+) probe localized on the cytosolic surface of the outer mitochondrial membrane, we demonstrate that, upon Ca(2+) mobilization, the [Ca(2+)] in small regions of the mitochondrial surface reaches levels 5- to 10-fold higher than in the bulk cytosol. We also show that the [Ca(2+)] to which mitochondria are exposed during capacitative Ca(2+) influx is similar between near plasma membrane mitochondria and organelles deeply located in the cytoplasm, whereas it is 2- to 3-fold higher in subplasma membrane mitochondria upon activation of voltage-gated Ca(2+) channels. These results demonstrate that mitochondria are exposed to Ca(2+) hot spots close to the ER but are excluded from the regions where capacitative Ca(2+) influx occurs.
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A Drosophila mutant of LETM1, a candidate gene for seizures in Wolf-Hirschhorn syndrome.
Hum. Mol. Genet.
PUBLISHED: 12-21-2009
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Human Wolf-Hirschhorn syndrome (WHS) is a multigenic disorder resulting from a hemizygous deletion on chromosome 4. LETM1 is the best candidate gene for seizures, the strongest haploinsufficiency phenotype of WHS patients. Here, we identify the Drosophila gene CG4589 as the ortholog of LETM1 and name the gene DmLETM1. Using RNA interference approaches in both Drosophila melanogaster cultured cells and the adult fly, we have assayed the effects of down-regulating the LETM1 gene on mitochondrial function. We also show that DmLETM1 complements growth and mitochondrial K(+)/H(+) exchange (KHE) activity in yeast deficient for LETM1. Genetic studies allowing the conditional inactivation of LETM1 function in specific tissues demonstrate that the depletion of DmLETM1 results in roughening of the adult eye, mitochondrial swelling and developmental lethality in third-instar larvae, possibly the result of deregulated mitophagy. Neuronal specific down-regulation of DmLETM1 results in impairment of locomotor behavior in the fly and reduced synaptic neurotransmitter release. Taken together our results demonstrate the function of DmLETM1 as a mitochondrial osmoregulator through its KHE activity and uncover a pathophysiological WHS phenotype in the model organism D. melanogaster.
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Mechanisms underlying the attachment and spreading of human osteoblasts: from transient interactions to focal adhesions on vitronectin-grafted bioactive surfaces.
Acta Biomater
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The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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