1,721,037 research outputs found
Tools and limitations to study the molecular composition of synapses by fluorescence microscopy
No full textThe synapse is densely packed with proteins involved in various highly regulated processes. Synaptic protein copy numbers and their stoichiometric distribution have a drastic influence on neuronal integrity and function. Therefore, the molecular analysis of synapses is a key element to understand their architecture and function. The overall structure of the synapse has been revealed with an exquisite amount of details by electron microscopy. However, the molecular composition and the localization of proteins are more easily addressed with fluorescence imaging, especially with the improved resolution achieved by super-resolution microscopy techniques. Notably, the fast improvement of imaging instruments has not been reflected in the optimization of biological sample preparation. During recent years, large efforts have been made to generate affinity probes smaller than conventional antibodies adapted for fluorescent super-resolution imaging. In this review, we briefly discuss the current views on synaptic organization and necessary key technologies to progress in the understanding of synaptic physiology. We also highlight the challenges faced by current fluorescent super-resolution methods, and we describe the prerequisites for an ideal study of synaptic organization.The synapse is densely packed with proteins involved in various highly regulated processes. Synaptic protein copy numbers and their stoichiometric distribution have a drastic influence on neuronal integrity and function. Therefore, the molecular analysis of synapses is a key element to understand their architecture and function. The overall structure of the synapse has been revealed with an exquisite amount of details by electron microscopy. However, the molecular composition and the localization of proteins are more easily addressed with fluorescence imaging, especially with the improved resolution achieved by super-resolution microscopy techniques. Notably, the fast improvement of imaging instruments has not been reflected in the optimization of biological sample preparation. During recent years, large efforts have been made to generate affinity probes smaller than conventional antibodies adapted for fluorescent super-resolution imaging. In this review, we briefly discuss the current views on synaptic organization and necessary key technologies to progress in the understanding of synaptic physiology. We also highlight the challenges faced by current fluorescent super-resolution methods, and we describe the prerequisites for an ideal study of synaptic organization
The fate of synaptic vesicle components upon fusion
No full textNeurotransmitter release relies on the fusion of synaptic vesicles with the plasma membrane of synaptic boutons, which is followed by the recycling of vesicle components and formation of new vesicles. It is not yet clear whether upon fusion the vesicles persist as multimolecular patches in the plasma membrane, or whether they segregate into individual components. Evidence supporting each of these two models has been suggested in recent years. Using diffraction-unlimited imaging (stimulated emission depletion, or STED) of native synaptic vesicle proteins, we have proposed that vesicle proteins remain in clusters on the neuronal surface. These clusters do not appear to intermix. We discuss here these findings in the context of previous studies on synaptic vesicle fusion, and we propose a recycling model which accounts for most of the recent findings on the post-fusion fate of synaptic vesicle components
Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes
No full textThe fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K(+)). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1A(i-iii;)). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1A(iv-v;)), process that can be followed by monitoring the fluorescence intensity decrease (destaining). Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles
Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons.
No full textSynaptic vesicle fusion (exocytosis) is a precisely regulated process that entails the formation of SNARE complexes between the vesicle protein synaptobrevin 2 (VAMP2) and the plasma membrane proteins Syntaxin 1 and SNAP-25. The sub-cellular localization of the latter two molecules remains unclear, although they have been the subject of many recent investigations. To address this, we generated two novel camelid single domain antibodies (nanobodies) specifically binding to SNAP-25 and Syntaxin 1A. These probes penetrated more easily into samples and detected their targets more efficiently than conventional antibodies in crowded regions. When investigated by super-resolution imaging, the nanobodies revealed substantial extra-synaptic populations for both SNAP-25 and Syntaxin 1A, which were poorly detected by antibodies. Moreover, extra-synaptic Syntaxin 1A molecules were recruited to synapses during stimulation, suggesting that these are physiologically-active molecules. We conclude that nanobodies are able to reveal qualitatively and quantitatively different organization patterns, when compared to conventional antibodies
Quantum Defects As a Toolbox for the Covalent Functionalization of Carbon Nanotubes with Peptides and Proteins
No full textFluorescent In Situ Hybridization of Synaptic Proteins Imaged With Super-Resolution STED Microscopy
No full textSuper-resolution fluorescence microscopy is still a developing field. One of the limitations has been that standard labeling assays, which had been developed for conventional imaging, must be adjusted and optimized for each super-resolution method. These methods are more sensitive to noise, and require more intense labeling than conventional microscopy, which is not always trivial to achieve. Here, we describe the use of stimulation-emission depletion (STED) microscopy to locate messenger RNAs (mRNAs) in single neurons with high spatial precision. We address several technical difficulties we encountered in using fluorescent in situ hybridization (FISH) for STED imaging. We optimized the experimental protocol to detect mRNAs and proteins simultaneously, by performing FISH and immunostaining on the same samples. We tested our imaging approach in primary hippocampal neurons, studying the mRNAs of three important presynaptic proteins (synaptobrevin, synaptotagmin, and synaptophysin). Our approach allowed us to relate changes in mRNA levels and localization to neuronal physiology, under different activity regimes and also during neuronal development. We conclude that FISH can be performed efficiently using super-resolution techniques. This should contribute significantly to the clarification of the molecular mechanisms that govern mRNA distribution and dynamics within cells. (C) 2014 Wiley Periodicals, Inc
Super‐resolution imaging for cell biologists
No full textThe recent 2014 Nobel Prize in chemistry honored an era of discoveries and technical advancements in the field of super-resolution microscopy. However, the applications of diffraction-unlimited imaging in biology have a long road ahead and persistently engage scientists with new challenges. Some of the bottlenecks that restrain the dissemination of super-resolution techniques are tangible, and include the limited performance of affinity probes and the yet not capillary diffusion of imaging setups. Likewise, super-resolution microscopy has introduced new paradigms in the design of projects that require imaging with nanometer-resolution and in the interpretation of biological images. Besides structural or morphological characterization, super-resolution imaging is quickly expanding towards interaction mapping, multiple target detection and live imaging. Here we review the recent progress of biologists employing super-resolution imaging, some pitfalls, implications and new trends, with the purpose of animating the field and spurring future developments
The extracellular membrane-proximal domain of membrane-bound IgE restricts B cell activation by limiting B cell antigen receptor surface expression
No full textAbstract Immunoglobulin E (IgE) antibodies are key mediators of allergic reactions. Due to their potentially harmful anaphylactic properties, their production is tightly regulated. The membrane‐bound isoform of IgE (mIgE), which is an integral component of the B cell antigen receptor, has been shown to be critical for the regulation of IgE responses in mice. In primate species including humans, mIgE can be expressed in two isoforms that are produced by alternative splicing of the primary ε Ig heavy chain transcript, and differ in the absence or presence of an extracellular membrane‐proximal domain (EMPD) consisting of 52 amino acids. However, the function of the EMPD remains unclear. Here, we demonstrate that the EMPD restricts surface expression of mIgE‐containing BCRs in human and murine B cells. The EMPD does not interfere with BCR assembly but acts as an autonomous endoplasmic reticulum retention domain. Limited surface expression of EMPD‐containing mIgE‐BCRs caused impaired activation of intracellular signaling cascades and hence represents a regulatory mechanism that may control the production of potentially anaphylactic IgE antibodies in primate species
Quantendefekte als Werkzeugkasten für die kovalente Funktionalisierung von Kohlenstoffnanoröhren mit Peptiden und Proteinen
No full textVolkswagen Foundation (DE)Niedersächsisches Ministerium für Wissenschaft und Kultur http://dx.doi.org/10.13039/50110001057
Staining of Membrane Receptors with Fluorescently-labeled DNA Aptamers for Super-resolution Imaging
No full textOne of the most prominent applications of fluorescent super-resolution microscopy is the study of nanodomain arrangements of receptors and the endocytic pathway. Staining methods are becoming crucial for answering questions on the nanoscale, therefore, the use of small and monovalent affinity probes is of great interest in super-resolution microscopy with biological samples. One kind of affinity probe is the aptamer. Aptamers are single DNA or RNA sequences that bind with high affinity to their targets and due to their small size they are able to (i) place the fluorophore in close proximity to the protein of interest and (ii) bind to most of the protein of interest overcoming the steric hindrance effect, resulting in better staining density. Here we describe a detailed protocol with which to stain live cells using aptamers and to image them with Stimulated Emission Depletion (STED) microscopy. In this protocol, the stainings were performed with commercially available aptamers that target the epidermal growth factor receptor (EGFR), the human epidermal growth factor receptor 2 (HER2 or ErbB2) and the ephrin type-A receptor 2 (Epha2). Since aptamers can be coupled to most of the popular fluorophores, we believe that the procedure presented here can be extended to the large majority of the current super-resolution microscopy techniques
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