Search Results (93)

Transmembrane channel-like (TMC) proteins are essential for hearing and balance; however, the molecular mechanisms that regulate their proper folding and membrane targeting remain poorly understood. Here, we establish Caenorhabditis elegans as a genetically tractable model to dissect TMC-1 trafficking by combining CRISPR knock-in strains, super-resolution microscopy, and genome-wide forward genetic screening. We show that TMC-1 robustly localizes to the plasma membrane in both neurons and muscle cells and identify a conserved valine (V803) in transmembrane domain 9 (TM9) as critical for its biogenesis and trafficking. Structural analyses guided by AlphaMissense and AlphaFold uncover two evolutionarily conserved functional hotspots, one in the extracellular loop adjacent to TM9 and the other in the TMC signature motif, which are interconnected by an evolutionarily conserved disulfide bond. Disrupting this bond in worm TMC-1 abolishes its cell-surface localization and destabilizes the mechanotransduction channel complex. Together, these findings provide a structural framework for interpreting deafness-causing mutations in human TMC1 and highlight disulfide-bond-linked hotspots as key molecular determinants of TMC protein biogenesis and trafficking. Full article

Retinal ribbon synapses are continuously active chemical synapses. The eponymous synaptic ribbon is anchored to the active zone neurotransmitter release sites of ribbon synapses, recruits synaptic vesicles and guides ribbon-associated synaptic vesicles to the release sites. RIBEYE is the major protein component of synaptic ribbons. But likely, additional proteins contribute to ribbon synapse function. The synaptic ribbon of photoreceptor synapses is embedded into a highly polarized microtubule cytoskeleton. Interestingly, proteins of the photoreceptor primary cilium, such as NPHP4 and other ciliary proteins, including KIF3A, were shown to be localized to photoreceptor synaptic ribbons. Previous studies demonstrated that the microtubule motor protein KIF13B catalyzes secretory vesicle transport to the plus ends of microtubules and identified an interaction of KIF13B with NPHP4 at primary cilia. However, the localization of KIF13B, a kinesin-3 family motor protein, in the retina is still unknown. In the present study, we used two different antibodies against KIF13B and high-resolution confocal microscopy, super-resolution structured illumination microscopy (SR-SIM), and post-embedding immunogold electron microscopy to determine the localization of KIF13B in retinal photoreceptors. Apart from its localization at the primary photoreceptor cilium, we found a strong enrichment of KIF13B at photoreceptor synaptic ribbons. The synaptic ribbon is needed for the synaptic enrichment of KIF13B as shown by analyses of synaptic ribbon-deficient RIBEYE knockout mice. These findings suggest that KIF13B performs vesicle trafficking functions at the photoreceptor synaptic ribbon complex at the interface between the synaptic ribbon and the presynaptic microtubule transport system. Full article

Extracellular vesicles (EVs), secreted from most cells, are small lipid membranes of vesicles of 30 to 1000 nm in diameter and contain nucleic acids, proteins, and intracellular organelles originating from donor cells. EVs play pivotal roles in intercellular communication, particularly in forming niches for cancer cell metastasis. However, EVs derived from donor cells exhibit significant heterogeneity, complicating the investigation of EV subtypes using ensemble averaging methods. In this context, we highlight recent studies that characterize individual EVs using advanced techniques, including single-fluorescent-particle tracking, single-metal-nanoparticle tracking, single-non-label-particle tracking, super-resolution microscopy, and atomic force microscopy. These techniques have facilitated high-throughput analyses of the properties of individual EV particles such as their sizes, compositions, and physical properties. Finally, we address the challenges that need to be resolved via single-particle (-molecule) imaging and super-resolution microscopy in future research. Full article

The rate of spontaneous action potentials (APs) generated by sinoatrial node cells (SANC) is regulated by local Ca²⁺ release (LCR) from the sarcoplasmic reticulum via Ca²⁺ release channels (ryanodine receptors, RyRs). LCR events propagate and self-organize within the network of RyR clusters (Ca release units, CRUs) via Ca-induced-Ca-release (CICR) that depends on CRU sizes and locations: While larger CRUs generate stronger release signals, the network’s topology governs signal diffusion and propagation. This study used super-resolution structured illumination microscopy to image the 3D network of CRUs in rabbit SANC. The peripheral CRUs formed a spatial mesh, reflecting the cell surface geometry. Two distinct subpopulations of CRUs were identified within each cell, with size distributions conforming to a two-component Gamma mixture model. Furthermore, neighboring CRUs exhibited repulsive behavior. Functional properties of the CRU network were further examined in a novel numerical SANC model developed using our experimental data. Model simulations revealed that heterogeneities in both CRU sizes and locations facilitate CICR and increase the AP firing rate in a cooperative manner. However, these heterogeneities reduce the effect of β-adrenergic stimulation in terms of its relative change in AP firing rate. The presence of heterogeneities in both sizes and locations allows SANC to reach higher absolute AP firing rates during β-adrenergic stimulation. Thus, the CICR facilitation by heterogeneities in CRU sizes and locations regulates and optimizes cardiac pacemaker cell operation under various physiological conditions. Dysfunction of this optimization could be a key factor in heart rate reserve decline in aging and disease. Full article

Super-resolution single-molecule localization microscopy (SMLM) of presynaptic active zones (AZs) and postsynaptic densities contributed to the observation of protein nanoclusters that are involved in defining functional characteristics and in plasticity of synaptic connections. Among SMLM techniques, direct stochastic optical reconstruction microscopy (dSTORM) depends on organic fluorophores that exert high brightness and reliable photoswitching. While multicolor imaging is highly desirable, the requirements necessary for high-quality dSTORM make it challenging to identify combinations of equally performing, spectrally separated dyes. Red-excited carbocyanine dyes, e.g., Alexa Fluor 647 (AF647) or Cy5, are currently regarded as “gold standard” fluorophores for dSTORM imaging. However, a recent study introduced a set of chemically modified rhodamine dyes, including CF583R, that promise to display similar performance in dSTORM. In this study, we defined CF583R’s performance compared to AF647 and CF568 based on a nanoscopic analysis of Bruchpilot (Brp), a nanotopologically well-characterized scaffold protein at Drosophila melanogaster AZs. We demonstrate equal suitability of AF647, CF568 and CF583R for basal AZ morphometry, while in Brp subcluster analysis CF583R outperforms CF568 and is on par with AF647. Thus, the AF647/CF583R combination will be useful in future dSTORM-based analyses of AZs and other subcellularly located marker molecules and their role in physiological and pathophysiological contexts. Full article

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that is expressed in several brain locations encompassing the hypothalamus and the brainstem, where the receptor controls several body functions, including metabolism. In a well-defined pathway to decrease appetite, hypothalamic proopiomelanocortin (POMC) neurons localized in the arcuate nucleus (Arc) project to MC4R neurons in the paraventricular nuclei (PVN) to release the natural MC4R agonist α-melanocyte-stimulating hormone (α-MSH). Arc neurons also project excitatory glutamatergic fibers to the MC4R neurons in the PVN for a fast synaptic transmission to regulate a satiety pathway potentiated by α-MSH. By using super-resolution microscopy, we found that in hypothalamic neurons in a primary culture, postsynaptic density protein 95 (PSD95) colocalizes with GluN1, a subunit of the ionotropic N-methyl-D-aspartate receptor (NMDAR). Thus, hypothalamic neurons form excitatory postsynaptic specializations. To study the MC4R distribution at these sites, tagged HA-MC4R under the synapsin promoter was expressed in neurons by adeno-associated virus (AAV) gene transduction. HA-MC4R immunofluorescence peaked at the center and in proximity to the PSD95- and NMDAR-expressing sites. These data provide morphological evidence that MC4R localizes together with glutamate receptors at postsynaptic and peri-postsynaptic sites. Full article

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. Full article

Intracellular cargo delivery via distinct transport routes relies on vesicle carriers. A key trafficking route distributes cargo taken up by clathrin-mediated endocytosis (CME) via early endosomes. The highly dynamic nature of the endosome network presents a challenge for its quantitative analysis, and theoretical modelling approaches can assist in elucidating the organization of the endosome trafficking system. Here, we introduce a new computational modelling approach for assessment of endosome distributions. We employed a model of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with inherited mutations causing dilated cardiomyopathy (DCM). In this model, vesicle distribution is defective due to impaired CME-dependent signaling, resulting in plasma membrane-localized early endosomes. We recapitulated this in iPSC-CMs carrying two different mutations, TPM1-L185F and TnT-R141W (MUT), using 3D confocal imaging as well as super-resolution STED microscopy. We computed scaled distance distributions of EEA1-positive vesicles based on a spherical approximation of the cell. Employing this approach, 3D spherical modelling identified a bi-modal segregation of early endosome populations in MUT iPSC-CMs, compared to WT controls. Moreover, spherical modelling confirmed reversion of the bi-modal vesicle localization in RhoA II-treated MUT iPSC-CMs. This reflects restored, homogeneous distribution of early endosomes within MUT iPSC-CMs following rescue of CME-dependent signaling via RhoA II-dependent RhoA activation. Overall, our approach enables assessment of early endosome distribution in cell-based disease models. This new method may provide further insight into the dynamics of endosome networks in different physiological scenarios. Full article

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. Full article

The accumulation of lipid droplets (LDs) and ceramides (Cer) is linked to non-alcoholic fatty liver disease (NAFLD), regularly co-existing with type 2 diabetes and decreased immune function. Chronic inflammation and increased disease severity in viral infections are the hallmarks of the obesity-related immunopathology. The upregulation of neutral sphingomyelinase-2 (NSM2) has shown to be associated with the pathology of obesity in tissues. Nevertheless, the role of sphingolipids and specifically of NSM2 in the regulation of immune cell response to a fatty acid (FA) rich environment is poorly studied. Here, we identified the presence of the LD marker protein perilipin 3 (PLIN3) in the intracellular nano-environment of NSM2 using the ascorbate peroxidase APEX2-catalyzed proximity-dependent biotin labeling method. In line with this, super-resolution structured illumination microscopy (SIM) shows NSM2 and PLIN3 co-localization in LD organelles in the presence of increased extracellular concentrations of oleic acid (OA). Furthermore, the association of enzymatically active NSM2 with isolated LDs correlates with increased Cer levels in these lipid storage organelles. NSM2 enzymatic activity is not required for NSM2 association with LDs, but negatively affects the LD numbers and cellular accumulation of long-chain unsaturated triacylglycerol (TAG) species. Concurrently, NSM2 expression promotes mitochondrial respiration and fatty acid oxidation (FAO) in response to increased OA levels, thereby shifting cells to a high energetic state. Importantly, endogenous NSM2 activity is crucial for primary human CD4⁺ T cell survival and proliferation in a FA rich environment. To conclude, our study shows a novel NSM2 intracellular localization to LDs and the role of enzymatically active NSM2 in metabolic response to enhanced FA concentrations in T cells. Full article

Glutamate receptors at the postsynaptic side translate neurotransmitter release from presynapses into postsynaptic excitation. They play a role in many forms of synaptic plasticity, e.g., homeostatic scaling of the receptor field, activity-dependent synaptic plasticity and the induction of presynaptic homeostatic potentiation (PHP). The latter process has been extensively studied at Drosophila melanogaster neuromuscular junctions (NMJs). The genetic removal of the glutamate receptor subunit IIA (GluRIIA) leads to an induction of PHP at the synapse. So far, mostly imprecise knockouts of the GluRIIA gene have been utilized. Furthermore, mutated and tagged versions of GluRIIA have been examined in the past, but most of these constructs were not expressed under endogenous regulatory control or involved the mentioned imprecise GluRIIA knockouts. We performed CRISPR/Cas9-assisted gene editing at the endogenous locus of GluRIIA. This enabled the investigation of the endogenous expression pattern of GluRIIA using tagged constructs with an EGFP and an ALFA tag for super-resolution immunofluorescence imaging, including structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). All GluRIIA constructs exhibited full functionality and PHP could be induced by philanthotoxin at control levels. By applying hierarchical clustering algorithms to analyze the dSTORM data, we detected postsynaptic receptor cluster areas of ~0.15 µm². Consequently, our constructs are suitable for ultrastructural analyses of GluRIIA. Full article

Coordinated events of calcium (Ca²⁺) released from the endoplasmic reticulum (ER) are key second messengers in excitable cells. In pain-sensing dorsal root ganglion (DRG) neurons, these events can be observed as Ca²⁺ sparks, produced by a combination of ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptors (IP3R1). These microscopic signals offer the neuronal cells with a possible means of modulating the subplasmalemmal Ca²⁺ handling, initiating vesicular exocytosis. With super-resolution dSTORM and expansion microscopies, we visualised the nanoscale distributions of both RyR and IP3R1 that featured loosely organised clusters in the subplasmalemmal regions of cultured rat DRG somata. We adapted a novel correlative microscopy protocol to examine the nanoscale patterns of RyR and IP3R1 in the locality of each Ca²⁺ spark. We found that most subplasmalemmal sparks correlated with relatively small groups of RyR whilst larger sparks were often associated with larger groups of IP3R1. These data also showed spontaneous Ca²⁺ sparks in <30% of the subplasmalemmal cell area but consisted of both these channel species at a 3.8–5 times higher density than in nonactive regions of the cell. Taken together, these observations reveal distinct patterns and length scales of RyR and IP3R1 co-clustering at contact sites between the ER and the surface plasmalemma that encode the positions and the quantity of Ca²⁺ released at each Ca²⁺ spark. Full article

β-actin belongs to cytoskeletal structures that change dynamically in cells according to various stimuli. Human skin can be considered as an organ that is very frequently exposed to various stress factors, of which light plays an important role. The present study focuses on adult human fibroblasts exposed to two types of light stress. Orange light with a wavelength of 590 nm was used here to stimulate the photosensitizer localized in the cells as a residual dose of photodynamic therapy (PDT). On the other hand, near-infrared light with a wavelength of 808 nm was considered for photobiomodulation (PBM), which is often used in healing processes. Confocal fluorescence microscopy was used to observe changes in intercellular communication, mitochondrial structures, and cytoskeletal dynamics defined by the remodulation of β-actin of fibroblasts. The number of β-actin bundles forming spherical structures was detected after light exposure. These structures as β-actin oligomers were confirmed with super-resolution microscopy. While PDT led to the disintegration of actin oligomers, PBM increased their number. The interaction of β-actin with mitochondria was observed. The combination of PDT and PBM treatments is important to minimize the side effects of cancer treatment with PDT on healthy cells, as shown by the cell metabolism assay in this work. In this work, β-actin is presented as an important parameter that changes and is involved in the response of cells to PDT and PBM. Full article

Optical nanoscopy, also known as super-resolution optical microscopy, has provided scientists with the means to surpass the diffraction limit of light microscopy and attain new insights into nanoscopic structures and processes that were previously inaccessible. In recent decades, numerous studies have endeavored to enhance super-resolution microscopy in terms of its spatial (lateral) resolution, axial resolution, and temporal resolution. In this review, we discuss recent efforts to push the resolution limit of stimulated emission depletion (STED) optical nanoscopy across multiple dimensions, including lateral resolution, axial resolution, temporal resolution, and labeling precision. We introduce promising techniques and methodologies building on the STED concept that have emerged in the field, such as MINSTED, isotropic STED, and event-triggered STED, and evaluate their respective strengths and limitations. Moreover, we discuss trade-off relationships that exist in far-field optical microscopy and how they come about in STED optical nanoscopy. By examining the latest developments addressing these aspects, we aim to provide an updated overview of the current state of STED nanoscopy and its potential for future research. Full article

Nuclear pore complexes (NPCs) are highly dynamic macromolecular protein structures that facilitate molecular exchange across the nuclear envelope. Aberrant NPC functioning has been implicated in neurodegeneration. The translocated promoter region (Tpr) is a critical scaffolding nucleoporin (Nup) of the nuclear basket, facing the interior of the NPC. However, the role of Tpr in adult neural stem/precursor cells (NSPCs) in Alzheimer’s disease (AD) is unknown. Using super-resolution (SR) and electron microscopy, we defined the different subcellular localizations of Tpr and phospho-Tpr (P-Tpr) in NSPCs in vitro and in vivo. Elevated Tpr expression and reduced P-Tpr nuclear localization accompany NSPC differentiation along the neurogenic lineage. In 5xFAD mice, an animal model of AD, increased Tpr expression in DCX+ hippocampal neuroblasts precedes increased neurogenesis at an early stage, before the onset of amyloid-β plaque formation. Whereas nuclear basket Tpr interacts with chromatin modifiers and NSPC-related transcription factors, P-Tpr interacts and co-localizes with cyclin-dependent kinase 1 (Cdk1) at the nuclear chromatin of NSPCs. In hippocampal NSPCs in a mouse model of AD, aberrant Tpr expression was correlated with altered NPC morphology and counts, and Tpr was aberrantly expressed in postmortem human brain samples from patients with AD. Thus, we propose that altered levels and subcellular localization of Tpr in CNS disease affect Tpr functionality, which in turn regulates the architecture and number of NSPC NPCs, possibly leading to aberrant neurogenesis. Full article