Christian G Specht- PhD
- Senior Researcher at French Institute of Health and Medical Research
Christian G Specht
- PhD
- Senior Researcher at French Institute of Health and Medical Research
About
98
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Introduction
My research explores the regulation of molecular interactions in axons and at synapses using quantitative and dynamic single molecule imaging strategies.
Current institution
Publications
Publications (98)
Glycine receptors (GlyRs) are heteropentameric chloride channels that mediate fast inhibitory neurotransmission in the brainstem and spinal cord, where they regulate motor and sensory processes. GlyRs are clustered at the post-synaptic membrane by a strong interaction of the beta subunit with the scaffold protein gephyrin. Even though GlyRbeta mRNA...
Glycine and GABA receptors are ligand-gated chloride channels that mediate inhibitory neurotransmission throughout the central nervous system. The receptors co-localise widely at inhibitory synapses in the spinal cord and in the brainstem due to their interaction with an overlapping binding site of the synaptic scaffold protein gephyrin, pointing t...
Startle disease is due to the disruption of recurrent inhibition in the spinal cord. Most common causes are genetic variants in genes ( GLRA1 , GLRB ) encoding inhibitory glycine receptor (GlyR) subunits. The adult GlyR is a heteropentameric complex composed of α1 and β subunits that localizes at postsynaptic sites and replaces embryonically expres...
Single-molecule localization microscopy (SMLM) enables crucial insights into cellular structures and processes to be revealed at the single-molecule level. However, SMLM is often hampered by limited temporal resolution and the fixed frame rate of the acquisition. Here we present a new approach to SMLM data acquisition and processing based on an aff...
Numerous models have been developed to account for the complex properties of the random walks of biomolecules. However, when analysing experimental data, conditions are rarely met to ensure model identification. The dynamics may simultaneously be influenced by spatial and temporal heterogeneities of the environment, out-of-equilibrium fluxes and co...
Single-molecule localization microscopy (SMLM) applications are often hampered by the fixed frame rate at which data acquisition is performed. Here, we present an alternative new approach of acquiring and processing SMLM data based on an event-based (or neuromorphic vision) sensor. This type of sensor reacts to light intensity changes rather than i...
Sylite, eine kurze, dimerisierte Fluoreszenzsonde auf Peptidbasis, bindet Gephyrin, ein charakteristisches Protein der inhibitorischen Synapse. Wie Christian G. Specht, Hans M. Maric et al. in ihrem Forschungsartikel (DOI: 10.1002/ange.202202078) berichten, macht Sylite inhibitorische Synapsen sowohl in Neuronenkulturen als auch in Hirngewebe sicht...
Sylite, a short, dimerized peptide‐based fluorescent probe, binds gephyrin, a hallmark protein of the inhibitory synapse. As reported by Christian G. Specht, Hans M. Maric et al. in their Research Article (DOI: 10.1002/anie.202202078), Sylite visualizes inhibitory synapses both in neuron cultures and brain tissue and can be flexibly applied for wid...
Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short pep...
Numerous models have been developed to account for the complex properties of the random walks of biomolecules. However, when analysing experimental data, conditions are rarely met to ensure model identification. The dynamics may simultaneously be influenced by spatial and temporal heterogeneities of the environment, out-of-equilibrium fluxes and co...
Visualization of inhibitory synapses requires protocols tailored to different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short pept...
Precise quantitative information about the molecular architecture of synapses is essential to understanding the functional specificity and downstream signaling processes at specific populations of synapses. Glycine receptors (GlyRs) are the primary fast inhibitory neurotransmitter receptors in the spinal cord and brainstem. These inhibitory glycine...
The function of synapses depends on spatially and temporally controlled molecular interactions between synaptic components that can be described in terms of copy numbers, binding affinities, and diffusion properties. To understand the functional role of a given synaptic protein, it is therefore crucial to quantitatively characterise its biophysical...
Precise quantitative information about the molecular architecture of synapses is essential to understanding the functional specificity and downstream signaling processes at specific populations of synapses. Glycine receptors (GlyRs) are the primary fast inhibitory neurotransmitter receptors in the spinal cord and brain stem. These inhibitory glycin...
Super-resolution imaging has revealed that key synaptic proteins are dynamically organized within sub-synaptic domains (SSDs). To examine how different inhibitory receptors are regulated, we carried out dual-color direct stochastic optical reconstruction microscopy (dSTORM) of GlyRs and GABAARs at mixed inhibitory synapses in spinal cord neurons. W...
Supplement: Expanded View Figures EV1-5
We introduce Sylites - small and versatile fluorogenic affinity probes for high-contrast visualization of inhibitory synapses. Having stoichiometric labeling and exceptional selectivity for neuronal gephyrin, a hallmark protein of the inhibitory post-synapse, Sylites enable superior synapse staining compared with antibodies. Combined with super-res...
Signaling at nerve cell synapses is a key determinant of proper brain function, and synaptic defects - or synaptopathies - are at the basis of many neurological and psychiatric disorders. Collybistin (CB), a brain-specific guanine nucleotide exchange factor (GEF), is essential for the formation of γ-aminobutyric acidergic (GABAergic) postsynapses i...
Postsynaptic scaffold proteins immobilise neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptor...
Postsynaptic scaffold proteins immobilise neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptor...
Postsynaptic scaffold proteins immobilise neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptor...
Postsynaptic scaffold proteins immobilise neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptor...
Super-resolution imaging of synapses has revealed that key synaptic proteins are dynamically organized within sub-synaptic domains (SSDs). At mixed inhibitory synapses in spinal cord neurons, both GlyRs and GABA A Rs reside at the same post-synaptic density (PSD). To examine how the different inhibitory receptors are organized and regulated, we car...
The application of single-molecule localization microscopy (SMLM) to the study of synaptic proteins has shown that the postsynaptic density (PSD) is organized heterogeneously in subsynaptic domains (SSDs) that are thought to play important roles in neurotransmission and synaptic plasticity. However, the dense packing of neurotransmitter receptors a...
We introduce junctured-DNA (J-DNA) forceps as a generic platform for real-time observation, at the single-molecule level, of biomolecular interactions. The tool is based on a modular double-strand DNA construct to which proteins of interest can be attached using various tagging strategies. When combined with magnetic tweezers, J-DNA allows us to si...
Supplemental information including Transparent Methods, Figures S1-S5 and Tables S1 and S2.
GABAA and glycine receptors are thought to compete for gephyrin binding sites at mixed inhibitory synapses. Changes in the occupancy of one receptor type are therefore expected to have opposite effects on the clustering of the other receptors. This does not explain, however, whether different receptors can be regulated independently from one anothe...
The application of super-resolution optical microscopy to investigating synaptic structures has revealed a highly heterogeneous and variable intra-synaptic organization. Dense subsynaptic protein assemblies named subsynaptic domains or SSDs have been proposed as structural units that regulate the efficacy of neuronal transmission. However, an in-de...
The efficacy of synaptic transmission is determined by the number of neurotransmitter receptors at synapses. Their recruitment depends upon the availability of postsynaptic scaffolding molecules that interact with specific binding sequences of the receptor. At inhibitory synapses, gephyrin is the major scaffold protein that mediates the accumulatio...
Accumulation of glycine receptors at synapses requires the interaction between the beta subunit of the receptor and the scaffold protein gephyrin. Here, we questioned whether different alpha subunits could modulate the receptors’ diffusion and propensity to cluster at spinal cord synapses. Using quantitative photoactivated localisation microscopy w...
The ability to count molecules is essential to elucidating cellular mechanisms, as these often depend on the absolute numbers and concentrations of molecules within specific compartments. Such is the case at chemical synapses, where the transmission of information from presynaptic to postsynaptic terminals requires complex interactions between smal...
This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-c...
The dynamic exchange of neurotransmitter receptors at synapses relies on their lateral diffusion in the plasma membrane. At synapses located on dendritic spines this process is limited by the geometry of the spine neck that restricts the passage of membrane proteins. Biochemical compartmentalisation of the spine is believed to underlie the input-sp...
Figure A. Distribution of lentivirus expressed dendra-SP. Figure B. SP distribution within the spine neck. Figure C. Overlapping localisation of mGluR5 and SP in dendritic spines. Figure D. Role of neck width and SP for membrane protein diffusion in the spine neck. Figure E. Organisation of the actin cytoskeleton in SP+ and SP- spines. Table A. Flu...
Significance
We developed a small protein tag enabling fluorescent labeling of proteins in living cells and in multicellular organisms through the specific binding and activation of a cell-permeant and nontoxic fluorogenic ligand. This tag, called Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), was engineered by directed evolut...
The scaffold protein gephyrin plays a critical regulatory role in the transmission of nerve signals in inhibitory synapses.. Its interactions with receptors of inhibitory neurotransmitters, such as glycine or GABA, are postulated to be a key molecular mechanism of synaptic formation and plasticity. Previous studies have shown that glycine receptors...
Synapses, although seemingly stable, undergo constant rearrangements and exhibit a high level of dynamic movement as revealed by molecular imaging. This apparent biological paradox has emerged as a key element enabling synaptic plasticity. The development of super-resolution imaging combined with theoretical modelling has advanced our understanding...
Development of the nervous system requires extensive axonal and dendritic growth during which neurons massively increase their surface area. Here we report that the endoplasmic reticulum (ER)-resident SNARE Sec22b has a conserved non-fusogenic function in plasma membrane expansion. Sec22b is closely apposed to the plasma membrane SNARE syntaxin1. S...
There are many differences between what can be extracted from biomolecules motion using small numbers of long trajectories (Qdots, Nanoparticles, fluorophores) or large numbers of short, dense trajectories (PALM, uPaint, Storm). Long time recordings allow various estimators to converge towards mostly accurate values, but don't allow access to the s...
Protein mobility is conventionally analyzed in terms of an effective diffusion. Yet, this description often fails to properly distinguish and evaluate the physical parameters (such as the membrane friction) and the biochemical interactions governing the motion. Here, we present a method combining high-density single-molecule imaging and statistical...
Nanoscopic imaging techniques provide a powerful set of tools for static and dynamic fluorescence microscopy below the diffraction limit of light. Among these super-resolution techniques, photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) are based on the detection of single fluorophores, whose si...
The strength of synaptic transmission is controlled by the number and activity of neurotransmitter receptors. However, little is known about absolute numbers and densities of receptor and scaffold proteins and the stoichiometry of molecular interactions at synapses. Here, we conducted three-dimensional and quantitative nanoscopic imaging based on s...
The movement of proteins in the cell membrane is governed by the local friction and their interactions with molecular partners. Yet, most experimental descriptions fail to unequivocally distinguish these effects; instead, they combine the diffusive and energetic contributions into an effective diffusion coefficient or anomalous exponent. Here, we s...
Little is known about the origin, supply pattern and production technology of Byzantine glass mosaic tesserae. In this study, we have analysed forty-eight glass tesserae from Sagalassos (Asia Minor) of different colours and from two archaeological contexts that were stratigraphically dated to the sixth century CE. The main aim was to identify the r...
Single-molecule tracking (SMT) experiments have shown that post-synaptic receptors (e.g. AMPA, NMDA, Glycine or GABA receptors) can be described as being in a dynamic equilibrium between free extrasynaptic diffusion and confined motion at synapses where they are transiently stabilized by molecular scaffolds. Although these experiments have been use...
Localization of single molecules in microscopy images is a key step in quantitative single particle data analysis. Among them, single molecule based super-resolution optical microscopy techniques require high localization accuracy as well as computation of large data sets in the order of 10⁵ single molecule detections to reconstruct a single image....
The structure of the centrosome was resolved by EM many years ago to reveal a pair of centrioles embedded in a dense network of proteins. More recently, the molecular composition of the centrosome was catalogued by mass spectroscopy and many novel components were identified. Determining precisely where a novel component localizes to within the cent...
Glycine receptors (GlyRs) can dynamically exchange between synaptic and extrasynaptic locations through lateral diffusion within the plasma membrane. Their accumulation at inhibitory synapses depends on the interaction of the β-subunit of the GlyR with the synaptic scaffold protein gephyrin. An alteration of receptor-gephyrin binding could thus shi...
SAP97 is a multidomain scaffold protein implicated in the forward trafficking and synaptic localization of NMDA- and AMPA-type glutamate receptors. Alternative splicing of SAP97 transcripts gives rise to palmitoylated αSAP97 and L27-domain containing βSAP97 isoforms that differentially regulate the subsynaptic localization of GluR1 subunits of AMPA...
Sample images of dendritic protrusions, showing the morphological variety of spines described in [42], as seen by PALM imaging (A). In panel (B), neck length and width of dendritic spines show no correlation with the spine head diameter (for quantification see Fig. 2E).
(TIF)
Simultaneous PALM imaging and QD tracking. ABP-tdEosFP (top) and QD signals (bottom) were simultaneously imaged in a hippocampal neuron at DIV 27. The detected ABP-tdEosFP fluorophores were used to reconstruct the actin cytoskeleton of the dendritic spine (cumulative movie, middle panel); the QD trajectory (bottom) served to outline the shape of th...
Pointillist representation of live PALM imaging of a dendritic segment of an immature hippocampal neuron (DIV 9) at time 0 (black points), 12 min (blue) and 25 min (red) under continuous illumination and recording. The 405 nm laser power was continuously adjusted to yield a constant number of single molecule events. The chosen time-window for image...
Live PALM imaging of a dendritic segment of a mature hippocampal neuron (DIV 27) under baseline conditions. The total length of the movie is 12.5 minutes. Each PALM frame was reconstructed from 2000 image frames of 25 ms, hence the temporal resolution is 50 s. The movie is rendered with a temporal sliding window of a step of 2.5 s. The width of the...
Live PALM imaging of a large dendritic segment of a mature hippocampal neuron (DIV 28), treated with 10 µM AMPA, as indicated in the movie. Movie rendered with a sliding window of 2000 frames of 25 ms ( = 50 s) and a step of 2.5 s. Field of view, 35 µm×16 µm.
(AVI)
Movie of an individual spine during 10 µM AMPA treatment (detail from Movie S3, see also Fig. 6). Field of view, 8 µm×5 µm.
(AVI)
The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great...
https://www.the-scientist.com/?articles.view/articleNo/29252/title/Opinion--Mutations-of-citations/
The transmission of signals across synapses requires the precise interaction of a large number of different synaptic proteins such as neurotransmitter receptors, adhesion, scaffold, signaling and cytoskeletal proteins. In small central excitatory synapses, this molecular machinery is contained in specialized cellular compartments called dendritic s...
The NMDA receptor (NMDAR) subunit GluN1 is an obligatory component of NMDARs without a known functional homolog and is expressed in almost every neuronal cell type. The NMDAR system is a coincidence detector with critical roles in spatial learning and synaptic plasticity. Its coincidence detection property is crucial for the induction of hippocampa...
Synaptic plasticity is dependent on the differential sorting, delivery and retention of neurotransmitter receptors, but the mechanisms underlying these processes are poorly understood. We found that differential sorting of glutamate receptor subtypes began in the endoplasmic reticulum of rat hippocampal neurons. As AMPA receptors (AMPARs) were traf...
High local concentrations of glycine receptors (GlyRs) at inhibitory postsynaptic sites are achieved through their binding to the scaffold protein gephyrin. The N- and C-terminal domains of gephyrin are believed to trimerize and dimerize, respectively, thus contributing to the formation of submembranous gephyrin clusters at synapses. GlyRs are asso...
The synaptic insertion of GluR1-containing AMPA-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, mechanisms responsible for GluR1 insertion and retention at the synapse are unclear. The synapse-associated protein SAP97 directly binds GluR1 and participates in its forward trafficking from the Golgi network to the plasm...
The development of novel physical tools to image biological samples at a resolution in the nanometer range is likely to revolutionize our current understanding of the spatial organization and compartmentalization of cells. The high-resolution analysis of biological macromolecular assemblies has long remained widely inaccessible by conventional opti...
Complex functions of the central nervous system such as learning and memory are believed to result from the modulation of the synaptic transmission between neurons. The sequence of events leading to the fusion of synaptic vesicles at the presynaptic active zone and the detection of this signal at the postsynaptic density involve the activity of ion...
The activity of neurotransmitter receptors determines the strength of synaptic transmission. Therefore, the clustering of receptors at synapses is an important mechanism underlying synaptic plasticity. The dynamic exchange of receptors between synaptic and extrasynaptic membranes is dependent on their interaction with synaptic scaffold proteins. He...
The postsynaptic density (PSD) signaling machinery contains proteins with diverse functions. Brain region-specific variations in PSD components mediate distinct physiological responses to synaptic activation. We have developed mass spectrometry-based methods to comprehensively compare both relative protein expression and phosphorylation status from...
In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which inc...
O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited...
N-Methyl-D-aspartate (NMDA) receptor (NMDAR) activity regulates the net number of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR) at the cell surface by modulating the balance between AMPAR membrane insertion and endocytosis. In this study, we addressed the role of NMDAR subtypes and of NMDAR-mediated Ca2+ influx i...
The postsynaptic density (PSD) is an electron-dense structure located at the synaptic contacts between neurons. Its considerable complexity includes cytoskeletal and scaffold proteins, receptors, ion channels and signaling molecules, in line with the role of PSDs in signal transduction and processing. The phosphorylation state of components of the...
alpha-Synuclein, a protein implicated in neurodegenerative diseases and of elusive physiological function owes its name to an observed presence in presynaptic and nuclear compartments. However, its nuclear localisation has remained controversial. We expressed synuclein-eGFP fusion proteins in organotypic rat hippocampal slice cultures and murine hi...
alpha-Synuclein, a protein implicated in neurodegenerative diseases and of elusive physiological function owes its name to an observed presence in presynaptic and nuclear compartments. However, its nuclear localisation has remained controversial. We expressed synuclein-eGFP fusion proteins in organotypic rat hippocampal slice cultures and murine hi...
alpha-Synuclein, a protein implicated in neurodegenerative diseases and of elusive physiological function owes its name to an observed presence in presynaptic and nuclear compartments. However, its nuclear localisation has remained controversial. We expressed synuclein-eGFP fusion proteins in organotypic rat hippocampal slice cultures and murine hi...
Diamond has a number of unique properties that make it an attractive electronic and bio-electronic material. Here we show the ordered growth of mammalian neurons, the principal electrogenic cells of the nervous system, on diamond. Proteins were specifically patterned on diamond surfaces by micro-contact printing. Mouse cortical neurons were then cu...
A deletion of the murine Snca gene has been discovered in C57BL/6JOlaHsd, a population of the inbred strain C57BL/6J. We now characterize the exact nature of this deletion, Del(6)Snca1Slab. Detailed mapping and sequencing of the breakpoint revealed the absence of 365 kb, encompassing the Mmrn1 gene in addition to Snca. Despite the lack of alpha-syn...
Alpha-synuclein belongs to a family of structurally related proteins expressed highly in the brain and is the major component of filamentous deposits present in a range of neurodegenerative diseases (synucleinopathies). It has been implicated in learning and memory, yet the physiological role of this protein is still unclear. It was recently found...
mRNA expression profiling was performed on a knock-in mouse model with a mutation in the NMDA receptor subunit NRl (N598R) that affects the receptors' function as coincidence detector. This approach was aimed at identifying downstream effects produced by the Ca2+ influx through NMDA receptors at the level of gene expression. cDNA array technology r...
The presynaptic protein alpha-synuclein is involved in a range of neurodegenerative diseases. Here we analyze potential compensatory mechanisms in alpha-synuclein null mutant mice. Furthermore, the findings reveal problems that may be associated with inbred mouse strains.
Expression profiling by cDNA array technology in a transgenic mouse model rev...
Questions
Question (1)
Is it right that editors decide which articles are sent out to peer review, or does this distort what type of research gets published?
It is understandable that editors apply certain criteria to select from a large number of articles, including the scope of the journal, the quality of the data, or the formal structure of the manuscript. However, it appears that in many cases, the editors' decisions are based on criteria that are less well defined, such as the perceived relevance of the study. Should this not be judged by the reviewers who are experts in their field?
