Gotowa bibliografia na temat „Cryo-CLEM”

AbstractCorrelated light and electron microscopy (CLEM) has become a popular technique for combining the protein-specific labeling of fluorescence with electron microscopy, both at room and cryogenic temperatures. Fluorescence applications at cryo-temperatures have typically been limited to localization of tagged protein oligomers due to known issues of extended triplet state duration, spectral shifts, and reduced photon capture through cryo-CLEM objectives. Here, we consider fluorophore characteristics and behaviors that could enable more extended applications. We describe how dialkylcarbocanine DiD, and its autoquenching by resonant energy transfer (RET), can be used to distinguish the fusion state of a lipid bilayer at cryo-temperatures. By adapting an established fusion assay to work under cryo-CLEM conditions, we identified areas of fusion between influenza virus-like particles and fluorescently labeled lipid vesicles on a cryo-EM grid. This result demonstrates that cryo-CLEM can be used to localize functions in addition to tagged proteins, and that fluorescence autoquenching by RET can be incorporated successfully into cryo-CLEM approaches. In the case of membrane fusion applications, this method provides both an orthogonal confirmation of functional state independent of the morphological description from cryo-EM and a way to bridge room-temperature kinetic assays and the cryo-EM images.

Correlative light and electron cryo-microscopy (cryo-CLEM) combines information from the specific labeling of fluorescence cryo-microscopy (cryo-FM) with the high resolution in environmental context of electron cryo-microscopy (cryo-EM). Exploiting super-resolution methods for cryo-FM is advantageous, as it enables the identification of rare events within the environmental background of cryo-EM at a sensitivity and resolution beyond that of conventional methods. However, due to the need for relatively high laser intensities, current super-resolution cryo-CLEM methods require cryo-protectants or support films which can severely reduce image quality in cryo-EM and are not compatible with many samples, such as mammalian cells. Here, we introduce cryogenic super-resolution optical fluctuation imaging (cryo-SOFI), a low-dose super-resolution imaging scheme based on the SOFI principle. As cryo-SOFI does not require special sample preparation, it is fully compatible with conventional cryo-EM specimens, and importantly, it does not affect the quality of cryo-EM imaging. By applying cryo-SOFI to a variety of biological application examples, we demonstrate resolutions up to ∼135 nm, an improvement of up to three times compared with conventional cryo-FM, while maintaining the specimen in a vitrified state for subsequent cryo-EM. Cryo-SOFI presents a general solution to the problem of specimen devitrification in super-resolution cryo-CLEM. It does not require a complex optical setup and can easily be implemented in any existing cryo-FM system.

Abstract Correlative light and electron microscopy (CLEM) offers a means of guiding the search for the unique or rare events by fluorescence microscopy (FM) and allows electron microscopy (EM) to zoom in on them for subsequent EM examination in three-dimensions (3D) and with nanometer-scale resolution. FM visualizes the localization of specific antigens by using fluorescent tags or proteins in a large field-of-view to study their cellular function, whereas EM provides the high level of resolution for complex structures. And cryo CLEM combines the advantages of maintaining structural preservation in a near-native state throughout the entire imaging process and by avoiding potentially harmful pre-treatments, such as chemical fixation, dehydration and staining with heavy metals. Besides for frozen-hydrated biological samples, CLEM combines the advantages of a close-to-life preservation of biological materials by keeping them embedded in vitreous ice throughout the entire imaging process and the frozen-hydrated condition is very suitable to maintain fluorescent signals. In recent years, many new instruments and software which intended to optimize the workflow and to obtain better experimental results of CLEM have been presented or even commoditized. While, the specimen damage during transfer from FM to EM and the resolution of CLEM were still need to be improved. Here we set up a High-vacuum Optical Platform to develop CLEM imaging technology (HOPE), which was designed to realize high-vacuum optical ( fluorescent) imaging for cryo-sample on EM cryo-holder (e.g. Gatan 626). A non-integrated high-vacuum cryo-optical stage, which adapted to the EM cryo holder, was fixed on epi-fluorescence microscope (or super-resolution microscope) to obtain fluorescent images. And then the EM cryo holder would be transferred to EM for collection of EM data. This protocol was aimed to minimize the specimen damage during transfer from FM to EM and it was versatile to expend to different types of light microscopy or electron microscopy. Our HOPE had already passed correlative imaging test, and the results showed that it was convenient and effective.

Abstract:In correlative light and electron microscopy (CLEM) workflows, identifying the same sub-cellular features in tissue by both light (LM) and electron microscopy (EM) remains a challenge. Furthermore, use of cryo-fixation for EM is desirable to capture rapid biological phenomena. Here, we describe a workflow that incorporates cryo-confocal laser scanning microscopy into the CLEM process, mapping cells in brain slices to re-image them with serial section scanning electron microscopy (ssSEM) array tomography. The addition of Airyscan detection increased the signal-to-noise ratio (SNR), allowing individual spines in thick frozen tissue to be visualized at a sufficient spatial resolution, providing a new tool for a CLEM approach to capture biological dynamics.

Au cours de la mitose, la chromatine interphasique subit une compaction massive en structures en forme de bâtonnets. Les condensines sont des complexes protéiques dont on sait qu'ils jouent un rôle majeur dans l'organisation des chromosomes mitotiques. Les eucaryotes possèdent deux complexes de condensines conservés, à savoir les condensines 1 et 2. Des études in vitro sur des modèles d'ADN nus montrent que les condensines ont une activité d'extrusion de boucles dans l'organisation des chromosomes. Cependant, il reste encore beaucoup à explorer en ce qui concerne l'étude de la fonction des condensines dans l'environnement encombré de la chromatine. Nous avons utilisé la technologie halo tag où le domaine SMC2 des condensines est marqué par fluorescence à l'aide d'un ligand halo TMR. Cette approche nous aide à localiser les régions riches en condensines dans les chromosomes mitotiques partiellement décondensés en utilisant la cryo-microscopie en lumière à l'intérieur des chromosomes vitrifiés pour les études de cryo-tomographie électronique. Nos tomographies montrent les complexes de condensine dans l'environnement chromatinien. Cela ouvre une fenêtre sur l'étude de l'activité de liaison à l'ADN de la condensine, l'oligomérisation ou le regroupement de la condensine et son interaction avec d'autres composants non histoniques des chromosomes mitotiques
During mitosis, the interphase chromatin undergoes a massive round of compaction into rod-shaped structures. Condensins are protein complexes that have been known to play a major role in mitotic chromosome organization. Eukaryotes have two conserved condensin complexes, namely condensin 1 and 2. In vitro studies on naked DNA templates show evidence for loop extrusion activity of condensins in chromosome organization. However, there is still a lot to explore regarding the study of condensin function inside the crowded chromatin environment. We have used halo tag technology where the SMC2 domain of condensins is tagged to fluorescently label using a halo TMR ligand. This approach helps us to locate condensin-rich regions in partially decondensed mitotic chromosomes using cryo-light microscopy inside the vitrified chromosomes for cryo-electron tomography studies. Our tomograms show condensin complexes inside the chromatin environment. This opens up a window into the study of DNA binding activity of condensin, the oligomerization or clustering of condensin and its interaction with other non-histone components of mitotic chromosomes

La dopamine est un neurotransmetteur qui module l'activité neuronale et régule des fonctions essentielles telles que la prédiction de la récompense, la motivation et le contrôle moteur. Dans le striatum, la dopamine agit généralement sur un large volume via des récepteurs métabotropes à action lente. Cependant, des études récentes ont montré que la dopamine peut également agir localement dans des espaces mesurant quelques micromètres. De plus, il a été démontré que la dopamine renforce la force synaptique lorsque sa libération coïncide avec l’action du glutamate. Ces découvertes suggèrent que la dopamine pourrait être libérée dans des espaces confinés, favorisant ainsi la plasticité synaptique. Malgré ces avancées, les études moléculaires et ultrastructurales ont été limitées par des défis techniques. Durant mon doctorat, j'ai utilisé des techniques de pointe pour explorer les caractéristiques moléculaires et ultrastructurales des terminaisons dopaminergiques et leur relation avec les synapses dans le striatum de souris.Pour analyser la composition des terminaisons dopaminergiques (DA), nous avons combiné le tri cellulaire et l’immunomarquage. Nous avons spécifiquement marqué les neurones dopaminergiques avec une protéine fluorescente verte. Après fractionnement cellulaire du striatum, nous avons isolé les synaptosomes verts (synapses closes avec leurs partenaires) par le tri de synaptosomes par fluorescence activée (FASS). L’immunomarquage de ces synaptosomes isolés a révélé que 30 % des synaptosomes dopaminergiques étaient situés près des synapses cortico-striatales (GLU). Nous avons nommé ces structures synapses pivots de la dopamine (DHS). Nous avons démontrer que les protéines présynaptiques bassoon et VGLUT1, ainsi que la protéine d’échafaudage postsynaptique Homer1, étaient régulés à la hausse dans les DHS, tandis que PSD-95 était moins présente par rapport aux synapses classiques. Ces résultats indiquent que la présence de terminaisons dopaminergiques à proximité des synapses cortico-striatales induit une plasticité moléculaire. Les synapses pivot dopaminergiques pourraient ainsi servir de substrat structurel pour l'activité localisée de la dopamine dans le striatum et pourraient également augmenter la signalisation glutamatergique.Ensuite, nous avons utilisé la cryo-microscopie électronique corrélée à la lumière (cryo-CLEM) pour déterminer l'ultrastructure des terminaisons dopaminergiques et des DHS en trois dimensions. Par rapport aux présynapses cortico-striatales, les synaptosomes DA sont trois fois plus petits et contiennent dix fois moins de vésicules synaptiques (SV). La taille et la forme des SV dans les terminaisons DA est plus hétérogènes, elles sont généralement plus grandes et parfois allongées. Alors que les synapses GLU présentent des zones actives (AZ) et des densités postsynaptiques, les terminaisons DA manquent de groupement visibles de vésicules ou d’organisation synaptique claire. Seules 35 % des terminaisons dopaminergiques comportent au moins une vésicules arrimée nécessaire à l’exocytose. Dans ces terminaisons, les SV sont plus nombreuses et plus proches de la membrane plasmique, suggérant une activité plus élevée. Cependant, les SV en état de « priming » (arrimées à moins de 5 nm de la membrane plasmique) sont absentes des synaptosomes DA. De plus, les terminaisons GLU dans les DHS ont plus de SV en état de « priming », ce qui implique que la présence de terminaisons DA réorganise les SV dans les terminaisons glutamatergiques. Ces résultats suggèrent que l'interaction des terminaisons DA avec les synapses modifie les propriétés de libération du glutamate par un mécanisme local de plasticité dans le striatum.Étant donné que l'activité de la dopamine est dérégulée dans l’addiction, moduler artificiellement l’interaction entre les synapses DA et GLU in vivo pourrait fournir un moyen de rétablir une signalisation dopaminergique normale
Dopamine is a neurotransmitter that modulates neuronal activity and governs essentialfunctions such as reward prediction, motivation, and motor control. In the striatum, dopaminetypically acts over a large volume through slow-acting metabotropic receptors. However,recent studies have demonstrated that dopamine can also operate in localized hotspotsmeasuring a few cubic micrometers. Additionally, dopamine may trigger excitatory synapsepotentiation when released in synchrony with glutamate. Despite these advances, molecularand ultrastructural studies have been limited by technical challenges.During my doctoral training, I developed cutting-edge techniques to explore the molecular andultrastructural features of dopaminergic terminals and their relationship to synapses in themouse striatum.In a first piece of work, we aimed at analyzing the composition of dopaminergic (DA) terminals.To that end, we labeled dopaminergic neurons with a green fluorescent reporter under thecontrol of the Dopamine Transporter promoter (DAT-Cre line). Following subcellularfractionation of the striatum, we isolated green fluorescent synaptosomes (resealed terminalsbound to synaptic partners) with fluorescence-activated synaptosome sorting (FASS).Immunolabeling of these isolated DA synaptosomes confirmed the presence of genuinedopaminergic markers apposed to dopamine receptors. Surprisingly, 30% of dopaminesynaptosomes were bound to cortico-striatal excitatory synapses containing the type 1vesicular glutamate transporter (GLU). We termed these connections cortico-striataldopamine hub synapses (DHS). On these samples, I used 6 markers of the GLU pre- andpost-synapse to scrutinize the effect of the association in DHS and I could identify that theassociation in DHS corelates with a molecular remodeling of the cortico-striatal synapses.Dopamine hub synapses may thus serve as a structural substrate for localized dopamineactivity in the striatum and could further potentiate glutamatergic signaling.Next, I established a cryo-correlative light and electron microscopy (cryo-CLEM) protocol onlabeled synaptosomes to determine the ultrastructure of dopamine terminals and DHS in threedimensions. Compared to cortico-striatal pre-synapses, DA synaptosomes were three timessmaller and contained ten times fewer synaptic vesicles (SVs). The size and shape of SVs inDA terminals were more heterogeneous, they were generally larger and sometimes elongated.While GLU synapses exhibited active zones (AZ) and postsynaptic densities, DA terminalslacked distinct vesicle clusters or clear synaptic organization. Only 35% of dopamine terminalscontained at least one tethered SV required for exocytosis. Compared to synaptosomeswithout tethered vesicle, SVs were more abundant and closer to the plasma membrane,suggestive of a higher release activity. However, primed SVs (tethered within 5 nm ofthe plasma membrane) were absent. Interestingly, GLU terminals in DHS had more primedSVs compared to regular GLU synapses, implying that the presence of DA terminalsreorganizes SVs in glutamatergic terminals. These results suggest that the interaction of DAterminals with synapses modifies the release properties of GLU pre-synapses by a localdopamine-dependent plasticity.Given that dopamine dysregulation is implicated in various diseases as addiction, thediscovery of this multipartite structure responsible for specific dopamine activity onglutamatergic inputs represent a new conceptual framework for future studies in the field.Modulating the physical interaction between DA and GLU synapses in vivo could provide anew method to influence dopamine signaling in the striatum