SV2A controls the surface nanoclustering and endocytic recruitment of Syt1 during synaptic vesicle recycling.

Following exocytosis, the recapture of plasma membrane-stranded vesicular proteins into recycling synaptic vesicles (SVs) is essential for sustaining neurotransmission. Surface clustering of vesicular proteins has been proposed to act as a 'pre-assembly' mechanism for endocytosis that ensures high-fidelity retrieval of SV cargo. Here, we used single-molecule imaging to examine the nanoclustering of synaptotagmin-1 (Syt1) and synaptic vesicle protein 2A (SV2A) in hippocampal neurons. Syt1 forms surface nanoclusters through the interaction of its C2B domain with SV2A, which are sensitive to mutations in this domain (Syt1K326A/K328A) and SV2A knockdown. SV2A co-clustering with Syt1 is reduced by blocking SV2A's cognate interaction with Syt1 (SV2AT84A). Surprisingly, impairing SV2A-Syt1 nanoclustering enhanced the plasma membrane recruitment of key endocytic protein dynamin-1, causing accelerated Syt1 endocytosis, altered intracellular sorting and decreased trafficking of Syt1 to Rab5-positive endocytic compartments. Therefore, SV2A and Syt1 are segregated from the endocytic machinery in surface nanoclusters, limiting dynamin recruitment and negatively regulating Syt1 entry into recycling SVs.

Posterior approach to correct for focal plane offsets in lattice light-sheet structured illumination microscopy.

Significance: Lattice light-sheet structured illumination microscopy (latticeSIM) has proven highly effective in producing three-dimensional images with super resolution rapidly and with minimal photobleaching. However, due to the use of two separate objectives, sample-induced aberrations can result in an offset between the planes of excitation and detection, causing artifacts in the reconstructed images. Aim: We introduce a posterior approach to detect and correct the axial offset between the excitation and detection focal planes in latticeSIM and provide a method to minimize artifacts in the reconstructed images. Approach: We utilized the residual phase information within the overlap regions of the laterally shifted structured illumination microscopy information components in frequency space to retrieve the axial offset between the excitation and the detection focal planes in latticeSIM. Results: We validated our technique through simulations and experiments, encompassing a range of samples from fluorescent beads to subcellular structures of adherent cells. We also show that using transfer functions with the same axial offset as the one present during data acquisition results in reconstructed images with minimal artifacts and salvages otherwise unusable data. Conclusion: We envision that our method will be a valuable addition to restore image quality in latticeSIM datasets even for those acquired under non-ideal experimental conditions.

High-sensitivity whole-mount in situ Hybridization of Mouse Oocytes and Embryos Visualizes the Super-resolution Structures and Distributions of mRNA Molecules.

Mammalian oocytes accumulate more than ten thousand mRNAs, of which three to four thousand mRNAs are translationally repressed. The timings and sites of translational activation of these dormant mRNAs are crucial for promoting oocyte maturation and embryonic development. How these mRNAs are accumulated and distributed in oocytes is therefore a fundamental issue to be explored. A method that enables visualization of mRNA molecules with high resolution in a simple manner would be valuable for understanding how oocytes accumulate and regulate the dormant mRNAs. We have developed a highly sensitive whole-mount in situ hybridization method using in vitro-synthesized RNA probes and the tyramide signal amplification (TSA) system optimized for mouse oocytes and embryos. By using this method, Pou5f1/Oct4, Emi2, and cyclin B1 mRNAs were detected in immature oocytes and 2-cell stage embryos. Confocal microscopy showed that these mRNAs formed granular structures in the oocyte cytoplasm. The structures of Pou5f1/Oct4 and cyclin B1 mRNAs persisted in 2-cell stage embryos. Pou5f1/Oct4 RNA granules exhibited a solid-like property in immature oocytes and became liquid-like droplets in 2-cell stage embryos. Double-staining of cyclin B1 mRNA with Emi2 or Pou5f1/Oct4 mRNA revealed that these mRNAs were distributed as different RNA granules without overlapping each other and that the size of cyclin B1 RNA granules tended to be larger than that of Emi2 RNA granules. The structures and distribution patterns of these mRNAs were further analyzed by N-SIM super-resolution microscopy. This analysis revealed that the large-sized RNA granules consist of many small-sized granules, suggesting the accumulation and regulation of dormant mRNAs as basal-sized RNA granules. The method established in this study can easily visualize the structure and distribution of mRNAs accumulated in mammalian oocytes and embryos with high sensitivity and super-resolution. This method is useful for investigating the cellular and molecular mechanisms of translational control of mRNAs by which maturation and early developmental processes are promoted.

A non-toxic equinatoxin-II reveals the dynamics and distribution of sphingomyelin in the cytosolic leaflet of the plasma membrane.

Sphingomyelin (SM) is a major sphingolipid in mammalian cells. SM is enriched in the extracellular leaflet of the plasma membrane (PM). Besides this localization, recent electron microscopic and biochemical studies suggest the presence of SM in the cytosolic leaflet of the PM. In the present study, we generated a non-toxic SM-binding variant (NT-EqtII) based on equinatoxin-II (EqtII) from the sea anemone Actinia equina, and examined the dynamics of SM in the cytosolic leaflet of living cell PMs. NT-EqtII with two point mutations (Leu26Ala and Pro81Ala) had essentially the same specificity and affinity to SM as wild-type EqtII. NT-EqtII expressed in the cytosol was recruited to the PM in various cell lines. Super-resolution microscopic observation revealed that NT-EqtII formed tiny domains that were significantly colocalized with cholesterol and N-terminal Lyn. Meanwhile, single molecule observation at high resolutions down to 1 ms revealed that all the examined lipid probes including NT-EqtII underwent apparent fast simple Brownian diffusion, exhibiting that SM and other lipids in the cytosolic leaflet rapidly moved in and out of domains. Thus, the novel SM-binding probe demonstrated the presence of the raft-like domain in the cytosolic leaflet of living cell PMs.

Mild Hyperthermia-Induced Thermogenesis in the Endoplasmic Reticulum Defines Stress Response Mechanisms.

Previous studies reported that a mild, non-protein-denaturing, fever-like temperature increase induced the unfolded protein response (UPR) in mammalian cells. Our dSTORM super-resolution microscopy experiments revealed that the master regulator of the UPR, the IRE1 (inositol-requiring enzyme 1) protein, is clustered as a result of UPR activation in a human osteosarcoma cell line (U2OS) upon mild heat stress. Using ER thermo yellow, a temperature-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we detected significant intracellular thermogenesis in mouse embryonic fibroblast (MEF) cells. Temperatures reached at least 8 °C higher than the external environment (40 °C), resulting in exceptionally high ER temperatures similar to those previously described for mitochondria. Mild heat-induced thermogenesis in the ER of MEF cells was likely due to the uncoupling of the Ca2+/ATPase (SERCA) pump. The high ER temperatures initiated a pronounced cytosolic heat-shock response in MEF cells, which was significantly lower in U2OS cells in which both the ER thermogenesis and SERCA pump uncoupling were absent. Our results suggest that depending on intrinsic cellular properties, mild hyperthermia-induced intracellular thermogenesis defines the cellular response mechanism and determines the outcome of hyperthermic stress.

Identification of z-axis filopodia in growth cones using super-resolution microscopy.

A growth cone is a highly motile tip of an extending axon that is crucial for neural network formation. Three-dimensional-structured illumination microscopy, a type of super-resolution light microscopy with a resolution that overcomes the optical diffraction limitation (ca. 200 nm) of conventional light microscopy, is well suited for studying the molecular dynamics of intracellular events. Using this technique, we discovered a novel type of filopodia distributed along the z-axis ("z-filopodia") within the growth cone. Z-filopodia were typically oriented in the direction of axon growth, not attached to the substratum, protruded spontaneously without microtubule invasion, and had a lifetime that was considerably shorter than that of conventional filopodia. Z-filopodia formation and dynamics were regulated by actin-regulatory proteins, such as vasodilator-stimulated phosphoprotein, fascin, and cofilin. Chromophore-assisted laser inactivation of cofilin induced the rapid turnover of z-filopodia. An axon guidance receptor, neuropilin-1, was concentrated in z-filopodia and was transported together with them, whereas its ligand, semaphorin-3A, was selectively bound to them. Membrane domains associated with z-filopodia were also specialized and resembled those of lipid rafts, and their behaviors were closely related to those of neuropilin-1. The results suggest that z-filopodia have unique turnover properties, and unlike xy-filopodia, do not function as force-generating structures for axon extension.

Piezo1 ion channels are capable of conformational signaling.

Piezo1 is a mechanically activated ion channel that senses forces with short latency and high sensitivity. Piezos undergo large conformational changes, induce far-reaching deformation onto the membrane, and modulate the function of two-pore potassium (K2P) channels. Taken together, this led us to hypothesize that Piezos may be able to signal their conformational state to other nearby proteins. Here, we use chemical control to acutely restrict Piezo1 conformational flexibility and show that Piezo1 conformational changes, but not ion permeation through them, are required for modulating the K2P channel K2P2.1 (TREK1). Super-resolution imaging and stochastic simulations further reveal that both channels do not co-localize, which implies that modulation is not mediated through direct binding interactions; however, at high Piezo1 densities, most TREK1 channels are within the predicted Piezo1 membrane footprint, suggesting that the footprint may underlie conformational signaling. We speculate that physiological roles originally attributed to Piezo1 ionotropic function could, alternatively, involve conformational signaling.

Integrating bioengineering, super-resolution microscopy and mechanobiology in autophagy research: addendum to the guidelines (4th edition).

Recent key technological developments, such as super-resolution microscopy and microfabrication, enabled investigation of biological processes, including macroautophagy/autophagy, with unprecedented spatiotemporal resolution and control over experimental conditions. Such disruptive innovations deepened our capability to provide mechanistic understandings of the autophagic process and its causes. This addendum aims to expand the guidelines on autophagy in three key directions: optical methods enabling visualization of autophagic machinery beyond the diffraction-limited resolution; bioengineering enabling accurate designs and control over experimental conditions; and theoretical advances in mechanobiology connecting autophagy and mechanical processes of the cell. Abbreviation: 3D: three-dimensional; SIM: structured illumination microscopy; STORM: stochastic optical reconstruction microscopy.

Unveiling intracellular phase separation: advances in optical imaging of biomolecular condensates.

Intracellular biomolecular condensates, which form via phase separation, display a highly organized ultrastructure and complex properties. Recent advances in optical imaging techniques, including super-resolution microscopy and innovative microscopic methods that leverage the intrinsic properties of the molecules observed, have transcended the limitations of conventional microscopies. These advances facilitate the exploration of condensates at finer scales and in greater detail. The deployment of these emerging but sophisticated imaging tools allows for precise observations of the multiphasic organization and physicochemical properties of these condensates, shedding light on their functions in cellular processes. In this review, we highlight recent progress in methodological innovations and their profound implications for understanding the organization and dynamics of intracellular biomolecular condensates.

Improved Imaging Surface for Quantitative Single-Molecule Microscopy.

Preventing nonspecific binding is essential for sensitive surface-based quantitative single-molecule microscopy. Here we report a much-simplified RainX-F127 (RF-127) surface with improved passivation. This surface achieves up to 100-fold less nonspecific binding from protein aggregates compared to commonly used polyethylene glycol (PEG) surfaces. The method is compatible with common single-molecule techniques including single-molecule pull-down (SiMPull), super-resolution imaging, antibody-binding screening and single exosome visualization. This method is also able to specifically detect alpha-synuclein (α-syn) and tau aggregates from a wide range of biofluids including human serum, brain extracts, cerebrospinal fluid (CSF) and saliva. The simplicity of this method further allows the functionalization of microplates for robot-assisted high-throughput single-molecule experiments. Overall, this simple but improved surface offers a versatile platform for quantitative single-molecule microscopy without the need for specialized equipment or personnel.

High-resolution live cell imaging to define ultrastructural and dynamic features of the halotolerant yeast Debaryomyces hansenii.

Although some budding yeasts have proved tractable and intensely studied models, others are more recalcitrant. Debaryomyces hansenii, an important yeast species in food and biotechnological industries with curious physiological characteristics, has proved difficult to manipulate genetically and remains poorly defined. To remedy this, we have combined live cell fluorescent dyes with high-resolution imaging techniques to define the sub-cellular features of D. hansenii, such as the mitochondria, nuclei, vacuoles and the cell wall. Using these tools, we define biological processes like the cell cycle, organelle inheritance and various membrane trafficking pathways of D. hansenii for the first time. Beyond this, reagents designed to study Saccharomyces cerevisiae proteins were used to access proteomic information about D. hansenii. Finally, we optimised the use of label-free holotomography to image yeast, defining the physical parameters and visualising sub-cellular features like membranes and vacuoles. Not only does this work shed light on D. hansenii but this combinatorial approach serves as a template for how other cell biological systems, which are not amenable to standard genetic procedures, can be studied.

Single-Molecule Orientation Imaging Reveals the Nano-Architecture of Amyloid Fibrils Undergoing Growth and Decay.

Amyloid-beta (Aß42) aggregates are characteristic Alzheimer's disease signatures, but probing how their nanoscale architectures influence their growth and decay remains challenging using current technologies. Here, we apply time-lapse single-molecule orientation-localization microscopy (SMOLM) to measure the orientations and rotational "wobble" of Nile blue (NB) molecules transiently binding to Aß42 fibrils. We correlate fibril architectures measured by SMOLM with their growth and decay over the course of 5 to 20 min visualized by single-molecule localization microscopy (SMLM). We discover that stable Aß42 fibrils tend to be well-ordered and signified by well-aligned NB orientations and small wobble. SMOLM also shows that increasing order and disorder are signatures of growing and decaying fibrils, respectively. We also observe SMLM-invisible fibril remodeling, including steady growth and decay patterns that conserve ß-sheet organization. SMOLM reveals that increased fibril architectural heterogeneity is correlated with dynamic remodeling and that large-scale fibril remodeling tends to originate from strongly heterogeneous local regions.

Nanoscale insights into hematology: super-resolved imaging on blood cell structure, function, and pathology.

Fluorescence nanoscopy, also known as super-resolution microscopy, has transcended the conventional resolution barriers and enabled visualization of biological samples at nanometric resolutions. A series of super-resolution techniques have been developed and applied to investigate the molecular distribution, organization, and interactions in blood cells, as well as the underlying mechanisms of blood-cell-associated diseases. In this review, we provide an overview of various fluorescence nanoscopy technologies, outlining their current development stage and the challenges they are facing in terms of functionality and practicality. We specifically explore how these innovations have propelled forward the analysis of thrombocytes (platelets), erythrocytes (red blood cells) and leukocytes (white blood cells), shedding light on the nanoscale arrangement of subcellular components and molecular interactions. We spotlight novel biomarkers uncovered by fluorescence nanoscopy for disease diagnosis, such as thrombocytopathies, malignancies, and infectious diseases. Furthermore, we discuss the technological hurdles and chart out prospective avenues for future research directions. This review aims to underscore the significant contributions of fluorescence nanoscopy to the field of blood cell analysis and disease diagnosis, poised to revolutionize our approach to exploring, understanding, and managing disease at the molecular level.

PvdL Orchestrates the Assembly of the Nonribosomal Peptide Synthetases Involved in Pyoverdine Biosynthesis in Pseudomonas aeruginosa.

The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex coordination of four nonribosomal peptide synthetases (NRPSs), which are responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings reveal that PvdL differs from the three other NRPSs in terms of localization and mobility patterns. PvdL is predominantly located in the inner membrane, while the others also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function.

Unleashing the potential: super-resolution microscopy as the key to advanced mitochondrial research.

Investigating the fine structure of mitochondria and their dynamic interactions with other organelles is crucial for unraveling the mechanisms underlying mitochondrial-related diseases. The development of super-resolution techniques has provided powerful visualization tools for mitochondrial research, which is significant for investigating mitochondrial cristae structure, the localization of mitochondrial-related protein complex, and the interactions between mitochondria and other organelles. In this perspective, we introduce several advanced super-resolution techniques and their applications in mitochondrial research, and discuss the potential roles these techniques may play in future studies of mitochondria.

STORM Super-Resolution Visualization of Self-Assembled γPFD Chaperone Ultrastructures in Methanocaldococcus jannaschii.

Gamma-prefoldin (γPFD), a unique chaperone found in the extremely thermophilic methanogen Methanocaldococcus jannaschii, self-assembles into filaments in vitro, which so far have been observed using transmission electron microscopy and cryo-electron microscopy. Utilizing three-dimensional stochastic optical reconstruction microscopy (3D-STORM), here we achieve ∼20 nm resolution by precisely locating individual fluorescent molecules, hence resolving γPFD ultrastructure both in vitro and in vivo. Through CF647 NHS ester labeling, we first demonstrate the accurate visualization of filaments and bundles with purified γPFD. Next, by implementing immunofluorescence labeling after creating a 3xFLAG-tagged γPFD strain, we successfully visualize γPFD in M. jannaschii cells. Through 3D-STORM and two-color STORM imaging with DNA, we show the widespread distribution of filamentous γPFD structures within the cell. These findings provide valuable insights into the structure and localization of γPFD, opening up possibilities for studying intriguing nanoscale components not only in archaea but also in other microorganisms.

Immuno-surveillance and protection of the human cochlea.

Background: Despite its location near infection-prone areas, the human inner ear demonstrates remarkable resilience. This suggests that there are inherent instruments deterring the invasion and spread of pathogens into the inner ear. Here, we combined high-resolution light microscopy, super-resolution immunohistochemistry (SR-SIM) and synchrotron phase contrast imaging (SR-PCI) to identify the protection and barrier systems in the various parts of the human inner ear, focusing on the lateral wall, spiral ganglion, and endolymphatic sac. Materials and methods: Light microscopy was conducted on mid-modiolar, semi-thin sections, after direct glutaraldehyde/osmium tetroxide fixation. The tonotopic locations were estimated using SR-PCI and 3D reconstruction in cadaveric specimens. The sections were analyzed for leucocyte and macrophage activity, and the results were correlated with immunohistochemistry using confocal microscopy and SR-SIM. Results: Light microscopy revealed unprecedented preservation of cell anatomy and several macrophage-like cells that were localized in the cochlea. Immunohistochemistry demonstrated IBA1 cells frequently co-expressing MHC II in the spiral ganglion, nerve fibers, lateral wall, spiral limbus, and tympanic covering layer at all cochlear turns as well as in the endolymphatic sac. RNAscope assays revealed extensive expression of fractalkine gene transcripts in type I spiral ganglion cells. CD4 and CD8 cells occasionally surrounded blood vessels in the modiolus and lateral wall. TMEM119 and P2Y12 were not expressed, indicating that the cells labeled with IBA1 were not microglia. The round window niche, compact basilar membrane, and secondary spiral lamina may form protective shields in the cochlear base. Discussion: The results suggest that the human cochlea is surveilled by dwelling and circulating immune cells. Resident and blood-borne macrophages may initiate protective immune responses via chemokine signaling in the lateral wall, spiral lamina, and spiral ganglion at different frequency locations. Synchrotron imaging revealed intriguing protective barriers in the base of the cochlea. The role of the endolymphatic sac in human inner ear innate and adaptive immunity is discussed.

From Cell Populations to Molecular Complexes: Multiplexed Multimodal Microscopy to Explore p53-53BP1 Molecular Interaction.

Surpassing the diffraction barrier revolutionized modern fluorescence microscopy. However, intrinsic limitations in statistical sampling, the number of simultaneously analyzable channels, hardware requirements, and sample preparation procedures still represent an obstacle to its widespread diffusion in applicative biomedical research. Here, we present a novel pipeline based on automated multimodal microscopy and super-resolution techniques employing easily available materials and instruments and completed with open-source image-analysis software developed in our laboratory. The results show the potential impact of single-molecule localization microscopy (SMLM) on the study of biomolecules' interactions and the localization of macromolecular complexes. As a demonstrative application, we explored the basis of p53-53BP1 interactions, showing the formation of a putative macromolecular complex between the two proteins and the basal transcription machinery in situ, thus providing visual proof of the direct role of 53BP1 in sustaining p53 transactivation function. Moreover, high-content SMLM provided evidence of the presence of a 53BP1 complex on the cell cytoskeleton and in the mitochondrial space, thus suggesting the existence of novel alternative 53BP1 functions to support p53 activity.

Synapse-Specific Trapping of SNARE Machinery Proteins in the Anesthetized Drosophila Brain.

General anesthetics disrupt brain network dynamics through multiple pathways, in part through postsynaptic potentiation of inhibitory ion channels as well as presynaptic inhibition of neuroexocytosis. Common clinical general anesthetic drugs, such as propofol and isoflurane, have been shown to interact and interfere with core components of the exocytic release machinery to cause impaired neurotransmitter release. Recent studies however suggest that these drugs do not affect all synapse subtypes equally. We investigated the role of the presynaptic release machinery in multiple neurotransmitter systems under isoflurane general anesthesia in the adult female Drosophila brain using live-cell super-resolution microscopy and optogenetic readouts of exocytosis and neural excitability. We activated neurotransmitter-specific mushroom body output neurons and imaged presynaptic function under isoflurane anesthesia. We found that isoflurane impaired synaptic release and presynaptic protein dynamics in excitatory cholinergic synapses. In contrast, isoflurane had little to no effect on inhibitory GABAergic or glutamatergic synapses. These results present a distinct inhibitory mechanism for general anesthesia, whereby neuroexocytosis is selectively impaired at excitatory synapses, while inhibitory synapses remain functional. This suggests a presynaptic inhibitory mechanism that complements the other inhibitory effects of these drugs.