Herein is reported a near-field microscopy based on electrochemiluminescence (ECL) which allows to image the plasma membrane of single cells at the interface with an electrode. By analyzing photoluminescence (PL), ECL and AFM images of mammalian CHO cells, we demonstrate that, in contrast to the wide-field fluorescence, ECL emission is confined to the immediate vicinity of the electrode surface and only the basal membrane of the cell becomes luminescent. The resulting ECL microscopy reveals details which are not resolved by classic fluorescence microscopy, without any light irradiation and specific setup. The thickness of the ECL-emitting regions is ~ 500 nm due to the unique ECL mechanism which involves short-lifetime electrogenerated radicals. In addition, the reported ECL microscopy is a dynamic technique which reflects the transport properties through the cell membranes and not only the specific labeling of the membranes. Finally, disposable transparent carbon nanotube (CNT)-based electrodes inkjet-printed on classic microscope glass coverslips, were used to image cells in both reflection and transmission configurations. Therefore, our approach opens new avenues for ECL as a near-field microscopy to develop single cell assays and to image the dynamics of biological entities in cells or in membranes. ASSOCIATED CONTENTSupporting Information. ECL-potential curves. PL images of the CHO cells recorded at high magnifications in different focal planes. ECL microscopy with DBAE. DPV. Scheme and photograph of the inkjet-printed CNT electrodes on glass coverslips. Optical effects on the PET and glass coverslips.
We report here the development of coreactant-based electrogenerated chemiluminescence (ECL) as a surface-confined microscopy to image single cells and their membrane proteins. Labeling the entire cell membrane allows one to demonstrate that, by contrast with fluorescence, ECL emission is only detected from fluorophores located in the immediate vicinity of the electrode surface (i.e., 1-2 μm). Then, to present the potential diagnostic applications of our approach, we selected carbon nanotubes (CNT)-based inkjet-printed disposable electrodes for the direct ECL imaging of a labeled plasma receptor overexpressed on tumor cells. The ECL fluorophore was linked to an antibody and enabled to localize the ECL generation on the cancer cell membrane in close proximity to the electrode surface. Such a result is intrinsically associated with the unique ECL mechanism and is rationalized by considering the limited lifetimes of the electrogenerated coreactant radicals. The electrochemical stimulus used for luminescence generation does not suffer from background signals, such as the typical autofluorescence of biological samples. The presented surface-confined ECL microscopy should find promising applications in ultrasensitive single cell imaging assays.
The transcription factor Rel/nuclear factor (NF)-kappaB is known for its fundamental role in regulating immune and inflammatory responses. In the brain, constitutive NF-kappaB activity has been detected exclusively in neurons, and a large diversity of stimuli have been reported to induce NF-kappaB activity. Yet the function of this transcription factor in the nervous system remains unclear, and its role in neuroprotection or neurodegeneration is open to debate. Recently it was suggested that kappaB-driven gene expression in neurons is controlled by Sp1-like factors. To clarify such controversy, we have characterized here a novel mouse model in which the entire NF-kappaB-dependent transcriptional response is abolished in the forebrain. Calcium-calmodulin-dependent kinase II alpha promoter-driven tetracycline transactivator was used for regulated expression of a transdominant negative mutant of inhibitor kappaBalpha (super-repressor) together with a green fluorescent protein tracer. Inhibition of expression of a kappaB-dependent lacZ transgene was shown in triple transgenic mice, which correlated with the loss of kappaB-specific DNA binding. In transgenic organotypic hippocampal slice cultures, expression of the super-repressor led to strong cell death after neurotoxic insults. These data demonstrate for the first time that neuron-restricted ablation of NF-kappaB-driven gene expression increases neurodegeneration. This might lead to the path for new treatments of neurodegenerative diseases.
Mutations of the human desmin gene on chromosome 2q35 cause a familial or sporadic form of skeletal myopathy frequently associated with cardiac abnormalities. Here, we report the pathogenic effects of a novel heterozygous R350P desmin missense mutation, which resides in the evolutionary highly conserved coil 2B domain of the alpha-helical coiled-coil desmin rod domain, on the assembly of desmin intermediate filaments (IF) in cultured cells and in vitro. By transfection experiments, we show that R350P desmin is incapable of de novo formation of a desmin IF network in vimentin-free BMGE+H, MCF7 and SW13 cells and that it disrupts the endogenous vimentin cytoskeleton in 3T3 fibroblast cells. Hence, transfected cells displayed abnormal cytoplasmic protein aggregates reminiscent of desmin-positive protein deposits seen in the immunohistochemical and ultrastructural analysis of skeletal muscle derived from the index patient of the affected family. To study the functional effects of the R350P desmin mutation at the protein level, we performed in vitro assembly studies with wild-type (WT) and mutant desmin protein. Our analysis revealed that the in vitro assembly process of R350P desmin is already disturbed at the unit length filament level and that further association reactions generate huge, tightly packed protein aggregates. On assessing the pathogenic effects of R350P desmin in various mixtures with WT desmin, we show that a ratio of 1 : 3 (R350P desmin/WT desmin) is sufficient to effectively block the normal polymerization process of desmin IFs. Our findings indicate that the heterozygous R350P desmin mutation exerts a dominant negative effect on the ordered lateral arrangement of desmin subunits. This disturbance of the lateral packing taking place in the first phase of assembly is ultimately leading to abnormal protein aggregation.
An electrochemiluminescent (ECL) swimmer driven by bipolar electrochemistry is reported for enzymatic glucose sensing. The chemo-mechanical motion is induced by localized hydrogen bubble generation. The concomitant oxidation of the luminophore and of the enzymatically-produced NADH leads to ECL emission with a direct glucose-dependent light intensity. We demonstrate herein the local sensing and reporting of glucose in a concentration gradient explored by the ECL swimmer. Such a dynamic sensing approach combines in a synergetic way the wireless propulsion with the enzymatic selectivity using ECL as a readout method at the level of moving objects.
Myofibrillar myopathies (MFMs) are an expanding and increasingly recognized group of neuromuscular disorders caused by mutations in DES, CRYAB, MYOT, and ZASP. The latest gene to be associated with MFM was FLNC; a p.W2710X mutation in the 24th immunoglobulin-like repeat of filamin C was shown to be the cause of a distinct type of MFM in several German families. We studied an International cohort of 46 patients from 39 families with clinically and myopathologically confirmed MFM, in which DES, CRYAB, MYOT, and ZASP mutations have been excluded. In an unrelated family a 12-nucleotide deletion (c.2997_3008del) in FLNC resulting in a predicted in-frame 4-residue deletion (p.Val930_Thr933del) in the 7th repeat of filamin C was identified. Both affected family members, mother and daughter, but not unrelated control individuals, carried the p.Val930_Thr933del mutation. The mutation is transcribed, and, based on myopathological features and immunoblot analysis, it leads to an accumulation of dysfunctional filamin C in the myocytes. The study results suggest that the novel p.Val930_Thr933del mutation in filamin C is the cause of myofibrillar myopathy but also indicate that filamin C mutations are a comparatively rare cause of MFM.
Recent studies in desmin (-/-) mice have shown that the targeted ablation of desmin leads to pathological changes of the extrasarcomeric intermediate filament cytoskeleton, as well as structural and functional abnormalities of mitochondria in striated muscle. Here, we report on a novel heterozygous single adenine insertion mutation (c.5141_5143insA) in a 40-year-old patient with a distal myopathy. The insertion mutation leads to a frameshift and a truncated desmin (K239fs242). Using transfection studies in SW13 and BHK21 cells, we show that the K239fsX242 desmin mutant is incapable of forming a desmin intermediate filament network. Furthermore, it induces the collapse of a pre-existing desmin cytoskeleton, alters the subcellular distribution of mitochondria and leads to abnormal cytoplasmic protein aggregates reminiscent of desmin-immunoreactive granulofilamentous material seen in the ultrastructural analysis of the patient's muscle. Analysis of mitochondrial function in isolated saponin-permeablized skeletal muscle fibres from our patient showed decreased maximal rates of respiration with the NAD-dependent substrate combination glutamate and malate, as well as a higher amytal sensitivity of respiration, indicating an in vivo inhibition of complex I activity. Our findings suggest that the heterozygous K239fsX242 desmin insertion mutation has a dominant negative effect on the polymerization process of desmin intermediate filaments and affects not only the subcellular distribution, but also biochemical properties of mitochondria in diseased human skeletal muscle. As a consequence, the intermediate filament pathology-induced mitochondrial dysfunction may contribute to the degeneration/regeneration process leading to progressive muscle dysfunction in human desminopathies.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.