Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids allows access to masked epitopes in live neurons
TL;DR: In this article, a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in primary neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices.
Abstract: Progress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution – typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in primary neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allowed us to image the differential localization of two glutamate receptor auxiliary proteins in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy.
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TL;DR: In this article , the authors demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used to determine the number and distance even of spatially unresolvable fluorophores in the sub-10-nm range.
Abstract: Advances in super-resolution microscopy have demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10-nm spatial resolution in biological samples remains challenging. Here we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10-nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used to determine the number and distance even of spatially unresolvable fluorophores in the sub-10-nm range. In combination with genetic code expansion with unnatural amino acids and bioorthogonal click labeling with small fluorophores, photoswitching fingerprint analysis can be used advantageously to reveal information about the number of fluorophores present and their distances in the sub-10-nm range in cells.
23 citations
TL;DR: In this paper , the role of AMPAR lateral diffusion in long-term potentiation (LTP) was investigated and it was shown that there are multiple solutions for achieving the diffusional trapping of AmPAR during LTP, mainly mediated by the interaction between interchangeable AMPAR auxiliary subunits and cell-adhesion molecules containing PDZ-binding domains.
6 citations
TL;DR: A review of the trends in MS-based neuroproteomics for the study of synaptic protein-protein interactions and protein signaling dynamics, with a focus on sample types, different labeling and enrichment approaches, and data analysis and interpretation is provided in this article.
5 citations
TL;DR: It is demonstrated that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used advantageously to determine the number and distance even of spatially unresolvable fluorophores in the sub-10 nm range.
Abstract: Advances in superresolution microscopy demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10 nm spatial resolution in biological samples remains challenging. Here, we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10 nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used advantageously to determine the number and distance even of spatially unresolvable fluorophores in the sub-10 nm range. In combination with genetic code expansion (GCE) with unnatural amino acids and bioorthogonal click-labeling with small fluorophores photoswitching fingerprint analysis enables sub-10 nm resolution fluorescence imaging in cells.
3 citations
TL;DR: In this paper, site-specific labeling of the extracellular domain of GABA-A receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine-dyes in HEK-293-T cells and primary cultured neurons was reported.
Abstract: Fluorescence labeling of difficult to access protein sites, e.g. in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of GABA-A receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine-dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine-dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in crowded environment, e.g. extracellular protein domains in confined compartments such as the synaptic cleft.
1 citations
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TL;DR: Hippocampal slices prepared from 2-23-day-old neonates were maintained in culture at the interface between air and a culture medium and yielded thin slices which remain 1-4 cell layers thick and are characterized by a well preserved organotypic organization.
3,086 citations
1,692 citations
TL;DR: The development of new orthogonal aminoacyl-tRNA synthetase/tRNA pairs has led to the addition of approximately 70 unnatural amino acids to the genetic codes of Escherichia coli, yeast, and mammalian cells, which provide new opportunities to generate proteins with enhanced or novel properties and probes of protein structure and function.
Abstract: The development of new orthogonal aminoacyl-tRNA synthetase/tRNA pairs has led to the addition of approximately 70 unnatural amino acids (UAAs) to the genetic codes of Escherichia coli, yeast, and mammalian cells. These UAAs represent a wide range of structures and functions not found in the canonical 20 amino acids and thus provide new opportunities to generate proteins with enhanced or novel properties and probes of protein structure and function.
1,554 citations
TL;DR: This protocol provides protocols for preparing low-density dissociated-cell cultures of hippocampal neurons from embryonic rats or mice that become appropriately polarized, develop extensive axonal and dendritic arbors and form numerous, functional synaptic connections with one another.
Abstract: We provide protocols for preparing low-density dissociated-cell cultures of hippocampal neurons from embryonic rats or mice. The neurons are cultured on polylysine-treated coverslips, which are suspended above an astrocyte feeder layer and maintained in serum-free medium. When cultured according to this protocol, hippocampal neurons become appropriately polarized, develop extensive axonal and dendritic arbors and form numerous, functional synaptic connections with one another. Hippocampal cultures have been used widely for visualizing the subcellular localization of endogenous or expressed proteins, for imaging protein trafficking and for defining the molecular mechanisms underlying the development of neuronal polarity, dendritic growth and synapse formation. Preparation of glial feeder cultures must begin 2 weeks in advance, and it takes 5 d to prepare coverslips as a substrate for neuronal growth. Dissecting the hippocampus and plating hippocampal neurons takes 2-3 h.
1,452 citations
TL;DR: A bioorthogonal reaction that proceeds with unusually fast reaction rates without need for catalysis: the cycloaddition of s-tetrazine and trans-cyclooctene derivatives, which enables protein modification at low concentration.
Abstract: Described is a bioorthogonal reaction that proceeds with unusually fast reaction rates without need for catalysis: the cycloaddition of s-tetrazine and trans-cyclooctene derivatives. The reactions tolerate a broad range of functionality and proceed in high yield in organic solvents, water, cell media, or cell lysate. The rate of the ligation between trans-cyclooctene and 3,6-di-(2-pyridyl)-s-tetrazine is very rapid (k2 2000 M−1 s−1). This fast reactivity enables protein modification at low concentration.
1,257 citations