Super-resolution Confocal Microscopy Research Articles

Limited evidence for anatomical contacts between intestinal GLP-1 cells and vagal neurons in male mice

The communication between intestinal Glucagon like peptide 1 (GLP-1)-producing cells and the peripheral nervous system has garnered renewed interest considering the availability of anti-obesity and anti-diabetic approaches targeting GLP-1 signaling. While it is well-established that intestinal GLP-1 cells can exert influence through paracrine mechanisms, recent evidence suggests the possible existence of synaptic-like connections between GLP-1 cells and peripheral neurons, including those of the vagus nerve. In this study, using a reporter Phox2b-Cre-Tomato mouse model and super-resolution confocal microscopy, we demonstrated that vagal axons made apparent contacts with less than 0.5% of GLP-1 cells. Moreover, immunohistochemistry combined with super-resolution confocal microscopy revealed abundant post-synaptic density 95 (PSD-95) immunoreactivity within the enteric plexus of the lower intestines of C57/BL6 mice, with virtually none in its mucosa. Lastly, utilizing RNAScope in situ hybridization in the lower intestines of mice, we observed that GLP-1 cells expressed generic markers of secretory cells such as Snap25 and Nefm, but neither synaptic markers such as Syn1 and Nrxn2, nor glutamatergic markers such as Slc17a7. Through theoretical considerations and a critical review of the literature, we concluded that intestinal GLP-1 cells primarily communicate with vagal neurons through paracrine mechanisms, rather than synaptic-like contacts.

Unpaired data training enables super-resolution confocal microscopy from low-resolution acquisitions.

Supervised deep-learning models have enabled super-resolution imaging in several microscopic imaging modalities, increasing the spatial lateral bandwidth of the original input images beyond the diffraction limit. Despite their success, their practical application poses several challenges in terms of the amount of training data and its quality, requiring the experimental acquisition of large, paired databases to generate an accurate generalized model whose performance remains invariant to unseen data. Cycle-consistent generative adversarial networks (cycleGANs) are unsupervised models for image-to-image translation tasks that are trained on unpaired datasets. This paper introduces a cycleGAN framework specifically designed to increase the lateral resolution limit in confocal microscopy by training a cycleGAN model using low- and high-resolution unpaired confocal images of human glioblastoma cells. Training and testing performances of the cycleGAN model have been assessed by measuring specific metrics such as background standard deviation, peak-to-noise ratio, and a customized frequency content measure. Our cycleGAN model has been evaluated in terms of image fidelity and resolution improvement using a paired dataset, showing superior performance than other reported methods. This work highlights the efficacy and promise of cycleGAN models in tackling super-resolution microscopic imaging without paired training, paving the path for turning home-built low-resolution microscopic systems into low-cost super-resolution instruments by means of unsupervised deep learning.

Modulation of AIE and Intramolecular Charge Transfer of a Pyrene-Based Probe for Discriminatory Detection and Imaging of Oligomers and Amyloid Fibrils.

Oligomers and amyloid fibrils formed at different stages of protein aggregation are important biomarkers for a variety of neurodegenerative diseases including Alzheimer's and Parkinson's diseases. The development of probes for the sensitive detection of oligomeric species is important for early stage diagnosis of amyloidogenic diseases. Many small molecular dyes have been developed to probe the dynamic growth of amyloid fibrils. However, there is a lack of discriminatory detection strategies to monitor the dynamics of both oligomers and amyloid fibrils based on the differential modulation of the photophysical properties of a single dye. Here we report a pyrene-based intramolecular charge transfer (ICT) dye with large Stokes shifted red-emitting aggregation induced emission (AIE) for monitoring the dynamic populations of both oligomers and fibrils during the aggregation of hen egg white lysozyme (HEWL) protein. At the early stage of protein aggregation, the accumulation of HEWL oligomers results in a rapid and substantial increase in the red AIE intensity at 660 nm. Later, as the oligomers transform into mature fibrils, the dye exhibits a distinct photophysical change. Binding of the dye to HEWL fibrils strongly suppresses the red AIE and enhances ICT emission. This is evidenced by a gradual decrease in the AIE intensity (∼660 nm) and an increase in LE (∼490 nm) and ICT (∼540 nm) emission intensities during the later stages of protein aggregation. Thus, the dye provides simultaneous measurements of the population dynamics of both HEWL oligomers and fibrils during protein aggregation based on the discriminatory modulation of AIE and ICT of the dye. The dye also enables imaging of both HEWL oligomers and fibrils simultaneously using different emission channels in super-resolution confocal fluorescence microscopy.

Human neural stem cells derived from fetal human brain communicate with each other and rescue ischemic neuronal cells through tunneling nanotubes

Pre-clinical trials have demonstrated the neuroprotective effects of transplanted human neural stem cells (hNSCs) during the post-ischemic phase. However, the exact neuroprotective mechanism remains unclear. Tunneling nanotubes (TNTs) are long plasma membrane bridges that physically connect distant cells, enabling the intercellular transfer of mitochondria and contributing to post-ischemic repair processes. Whether hNSCs communicate through TNTs and their role in post-ischemic neuroprotection remains unknown. In this study, non-immortalized hNSC lines derived from fetal human brain tissues were examined to explore these possibilities and assess the post-ischemic neuroprotection potential of these hNSCs. Using Tau-STED super-resolution confocal microscopy, live cell time-lapse fluorescence microscopy, electron microscopy, and direct or non-contact homotypic co-cultures, we demonstrated that hNSCs generate nestin-positive TNTs in both 3D neurospheres and 2D cultures, through which they transfer functional mitochondria. Co-culturing hNSCs with differentiated SH-SY5Y (dSH-SY5Y) revealed heterotypic TNTs allowing mitochondrial transfer from hNSCs to dSH-SY5Y. To investigate the role of heterotypic TNTs in post-ischemic neuroprotection, dSH-SY5Y were subjected to oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/R) with or without hNSCs in direct or non-contact co-cultures. Compared to normoxia, OGD/R dSH-SY5Y became apoptotic with impaired electrical activity. When OGD/R dSH-SY5Y were co-cultured in direct contact with hNSCs, heterotypic TNTs enabled the transfer of functional mitochondria from hNSCs to OGD/R dSH-SY5Y, rescuing them from apoptosis and restoring the bioelectrical profile toward normoxic dSH-SY5Y. This complete neuroprotection did not occur in the non-contact co-culture. In summary, our data reveal the presence of a functional TNTs network containing nestin within hNSCs, demonstrate the involvement of TNTs in post-ischemic neuroprotection mediated by hNSCs, and highlight the strong efficacy of our hNSC lines in post-ischemic neuroprotection.Human neural stem cells (hNSCs) communicate with each other and rescue ischemic neurons through nestin-positive tunneling nanotubes (TNTs). A Functional mitochondria are exchanged via TNTs between hNSCs. B hNSCs transfer functional mitochondria to ischemic neurons through TNTs, rescuing neurons from ischemia/reperfusion ROS-dependent apoptosis.

Structural plasticity of pyramidal cell neurons measured after FLASH and conventional dose-rate irradiation.

Evidence shows that ultra-high dose-rate FLASH-radiotherapy (FLASH-RT) protects against normal tissue complications and functional decrements in the irradiated brain. Past work has shown that radiation-induced cognitive impairment, neuroinflammation and reduced structural complexity of granule cell neurons were not observed to the same extent after FLASH-RT (> MGy/s) compared to conventional dose-rate (CONV, 0.1 Gy/s) delivery. To explore the sensitivity of different neuronal populations to cranial irradiation and dose-rate modulation, hippocampal CA1 and medial prefrontal cortex (PFC) pyramidal neurons were analyzed by electron and confocal microscopy. Neuron ultrastructural analyses by electron microscopy after 10 Gy FLASH- or CONV-RT exposures indicated that irradiation had little impact on dendritic complexity and synapse density in the CA1, but did increase length and head diameter of smaller non-perforated synapses. Similarly, irradiation caused no change in PFC prelimbic/infralimbic axospinous synapse density, but reductions in non-perforated synapse diameters. While irradiation resulted in thinner myelin sheaths compared to controls, none of these metrics were dose-rate sensitive. Analysis of fluorescently labeled CA1 neurons revealed no radiation-induced or dose-rate-dependent changes in overall dendritic complexity or spine density, in contrast to our past analysis of granule cell neurons. Super-resolution confocal microscopy following a clinical dosing paradigm (3×10Gy) showed significant reductions in excitatory vesicular glutamate transporter 1 and inhibitory vesicular GABA transporter puncta density within the CA1 that were largely dose-rate independent. Collectively, these data reveal that, compared to granule cell neurons, CA1 and mPFC neurons are more radioresistant irrespective of radiation dose-rate.

P-558 Characterization of the morphology of the outer-kinetochore expandable module, aka fibrous corona, and its role in ensuring proper chromosome segregation in human and bovine oocytes

Abstract Study question How does the Fibrous Corona (FC) behave in human and bovine oocyte meiosis and what is its function in chromosome segregation during meiosis I? Summary answer In human and bovine oocytes, FC shows extreme plasticity expanding beyond the centromeric region of the chromosomes. Pharmacological inhibition of FC’s expansion induces chromosome mis-segregation. What is known already Embryonic aneuploidy of meiotic origin is one of the major causes of implantation failure and early pregnancy loss. Erroneous microtubule (MT)-kinetochore attachments in human meiosis are the main cause of meiotic chromosome segregation errors. In somatic cells, the FC, a multiprotein complex, assembles on kinetochores and expands around the centromere during prometaphase to facilitate MT interactions and stimulate correct MT-kinetochore attachment. We have shown that in bovine oocytes FC expand beyond the centrosomes and is a potent MTs nucleator (10.1016/j.cub.2023.01.010). However, it is still unknown how FC behaves and what are its functions in MT-kinetochore interactions in human oocytes. Study design, size, duration FC morphology was studied in immature human oocytes discarded during ICSI procedures and donated (informed consent) by the patients for research (Ethical permit TCBio.21-754) during the years 2021-2024. Because of ethical concerns and reduced availability of human oocytes, functional studies on FC were performed on bovine oocytes collected from slaughterhouse ovaries. Participants/materials, setting, methods 180 immature human oocytes were matured in vitro and either fixed at different meiosis I stages or treated 2h with colcemid, a MT depolymerizing drug, to induce maximum FC expansion and fixed. The oocytes were then immuno-stained for chromatin(DAPI), FC(ZW10), MT(alpha tubulin) and kinetochores(CREST) and imaged with super-resolution confocal microscopy. Bovine immature oocytes were matured in vitro with or without lonafarnib (inhibitor of FC expansion). After maturation chromosome spreads were performed to evaluate oocyte ploidy. Main results and the role of chance In human oocytes, after nuclear envelope breakdown, before microtubule appearance when the chromosomes appear compacted, FCs are visible on the top of the kinetochores. During MT nucleation, FCs form thick mesh in the center of the compacted chromosomes. During prometaphase I, FCs expand beyond the centromeric region of the chromosomes forming a cup around the sister kinetochores of the paired homologous chromosomes. When the chromosomes starts to align at the metaphase plate, FCs start to be stripped from the kinetochores and colocalize to the MTs. At metaphase I and II, only kinetochore localization is visible. When oocytes are treated with colcemid, FCs extend to partially cover chromosome arms. We observed a wide variability in FCs expansion between and within patients. Inhibition of FC expansion in bovine oocytes increase the incidence of oocyte aneuploidy (90% in lonafarnib treated vs 37% in control oocytes). Altogether, our data reveal that in human oocytes FCs expand more compared to human somatic cells. Considering that inhibition of FC expansion induces chromosome segregation errors, we propose that the large FC size in human oocytes may play a role in achieving correct chromosome-MT attachments within their extremely large cytoplasm thus ensuring proper chromosome segregation. Limitations, reasons for caution The limited availability of human oocytes requires the use of an animal model for in depth mechanistic studies. Although bovine oocytes have similar aneuploidy rate and spindle assembly mechanisms as human oocytes, they show morphological differences in FC expansion which could underlie intrinsic functional discrepancies between the two organisms. Wider implications of the findings A better understanding of the chromosome segregation mechanisms in human meiosis will help understanding what causes the high incidence of aneuploidies in human oocytes and possibly contribute to the development of interventions or of oocyte selection procedures to reduce the incidence of oocyte aneuploidy during in vitro maturation. Trial registration number Not applicable

The umbrella cell keratin network: organization as a tile-like mesh, formation of a girded layer in response to bladder filling, and dependence on the plectin cytolinker.

The keratin cytoskeleton and associated desmosomes contribute to the mechanical stability of epithelial tissues, but their organization in bladder umbrella cells and their responses to bladder filling are poorly understood. Using super-resolution confocal microscopy, along with 3D image reconstruction and platinum replica electron microscopy, we observed that the apical keratin network of umbrella cells was organized as a dense tile-like mesh comprised of tesserae bordered on their edges by cortical actin filaments, filled with woven keratin filaments, and crosslinked by plectin. A band of keratin was also observed at the cell periphery that was linked to the junction-associated actin ring by plectin. During bladder filling, the junction-localized desmosomal necklace expanded, and a subjacent girded layer was formed that linked the keratin network to desmosomes, including those at the umbrella cell-intermediate cell interface. Disruption of plectin led to focal keratin network dissolution, loss of the junction-associated band of keratin, perturbation of tight junction continuity, and loss of cell-cell cohesion. Our studies reveal a novel tile-like organization of the umbrella cell keratin cytoskeleton that is dependent on plectin, that reorganizes in response to bladder filling, and that likely serves to maintain umbrella cell continuity in the face of mechanical distension.

Central Amygdala Astrocyte Plasticity Underlies GABAergic Dysregulation in Ethanol Dependence.

Dependence is a hallmark of alcohol use disorder characterized by excessive alcohol intake and withdrawal symptoms. The central nucleus of the amygdala (CeA) is a key brain structure underlying the synaptic and behavioral consequences of ethanol dependence. While accumulating evidence suggests that astrocytes regulate synaptic transmission and behavior, there is a limited understanding of the role astrocytes play in ethanol dependence. The present study used a combination of viral labeling, super resolution confocal microscopy, 3D image analysis, and slice electrophysiology to determine the effects of chronic intermittent ethanol (CIE) exposure on astrocyte plasticity in the CeA. During withdrawal from CIE exposure, we observed increased GABA transmission, an upregulation in astrocytic GAT3 levels, and an increased proximity of astrocyte processes near CeA synapses. Furthermore, GAT3 levels and synaptic proximity were positively associated with voluntary ethanol drinking in dependent rats. Slice electrophysiology confirmed that the upregulation in astrocytic GAT3 levels was functional, as CIE exposure unmasked a GAT3-sensitive tonic GABA current in the CeA. A causal role for astrocytic GAT3 in ethanol dependence was assessed using viral-mediated GAT3 overexpression and knockdown approaches. However, GAT3 knockdown or overexpression had no effect on somatic withdrawal symptoms, dependence-escalated ethanol intake, aversion-resistant drinking, or post-dependent ethanol drinking in male or female rats. Moreover, intra-CeA pharmacological inhibition of GAT3 also did not alter dependent ethanol drinking. Together, these findings indicate that ethanol dependence induces GABAergic dysregulation and astrocyte plasticity in the CeA. However, astrocytic GAT3 does not appear necessary for the drinking related phenotypes associated with dependence.

Anti-EGFR Antibody-Drug Conjugate Carrying an Inhibitor Targeting CDK Restricts Triple-Negative Breast Cancer Growth.

Anti-EGFR antibodies show limited response in breast cancer, partly due to activation of compensatory pathways. Furthermore, despite the clinical success of cyclin-dependent kinase (CDK) 4/6 inhibitors in hormone receptor-positive tumors, aggressive triple-negative breast cancers (TNBC) are largely resistant due to CDK2/cyclin E expression, whereas free CDK2 inhibitors display normal tissue toxicity, limiting their therapeutic application. A cetuximab-based antibody drug conjugate (ADC) carrying a CDK inhibitor selected based on oncogene dysregulation, alongside patient subgroup stratification, may provide EGFR-targeted delivery. Expressions of G1/S-phase cell cycle regulators were evaluated alongside EGFR in breast cancer. We conjugated cetuximab with CDK inhibitor SNS-032, for specific delivery to EGFR-expressing cells. We assessed ADC internalization and its antitumor functions in vitro and in orthotopically grown basal-like/TNBC xenografts. Transcriptomic (6,173 primary, 27 baseline, and matched post-chemotherapy residual tumors), single-cell RNA sequencing (150,290 cells, 27 treatment-naïve tumors), and spatial transcriptomic (43 tumor sections, 22 TNBCs) analyses confirmed expression of CDK2 and its cyclin partners in basal-like/TNBCs, associated with EGFR. Spatiotemporal live-cell imaging and super-resolution confocal microscopy demonstrated ADC colocalization with late lysosomal clusters. The ADC inhibited cell cycle progression, induced cytotoxicity against high EGFR-expressing tumor cells, and bystander killing of neighboring EGFR-low tumor cells, but minimal effects on immune cells. Despite carrying a small molar fraction (1.65%) of the SNS-032 inhibitor, the ADC restricted EGFR-expressing spheroid and cell line/patient-derived xenograft tumor growth. Exploiting EGFR overexpression, and dysregulated cell cycle in aggressive and treatment-refractory tumors, a cetuximab-CDK inhibitor ADC may provide selective and efficacious delivery of cell cycle-targeted agents to basal-like/TNBCs, including chemotherapy-resistant residual disease.

Interfacial blending in co-axially electrospun polymer core-shell fibers and their interaction with cells via focal adhesion point analysis

Electrospun polymer scaffolds have gained prominence in biomedical applications, including tissue engineering, drug delivery, and wound dressings, due to their customizable properties. As the interplay between cells and materials assumes fundamental significance in biomaterials research, understanding the relationship between fiber properties and cell behaviour is imperative. Nevertheless, altering fiber properties introduces complexity by intertwining mechanical and surface chemistry effects, challenging the differentiation of their individual impacts on cell behaviour. Core-shell fibers present an appealing solution, enabling the control of mechanical properties of scaffolds, flexibility in material and drug selection, efficient encapsulation, strong protection of bioactive drugs against harsh environments, and controlled, prolonged drug release. This study addresses a key challenge in core-shell fiber design related to the blending effect between core and shell polymers. Two types of fibers, PMMA and core-shell PC-PMMA, were electrospun, and thorough analyses confirmed the desired core-shell structure in PC-PMMA fibers. Surface chemistry analysis revealed PC diffusion to the PMMA shell of the core-shell fiber during electrospinning, subsequently prompting an investigation of the fiber’s surface potential. Conducting cellular studies on osteoblasts by super-resolution confocal microscopy provided insights into the direct influence of interfacial polymer blending and, consequently, altered fiber surface and mechanical properties on cell focal adhesion points, bridging the gap between material attributes and cell responses in core-shell fibers.

STIM2 variants regulate Orai1/TRPC1/TRPC4-mediated store-operated Ca2+ entry and mitochondrial Ca2+ homeostasis in cardiomyocytes

The stromal interaction molecules (STIMs) are the sarcoplasmic reticulum (SR) Ca2+ sensors that trigger store-operated Ca2+ entry (SOCE) in a variety of cell types. While STIM1 isoform has been the focus of the research in cardiac pathophysiology, the function of the homolog STIM2 remains unknown. Using Ca2+ imaging and patch-clamp techniques, we showed that knockdown (KD) of STIM2 by siRNAs increased SOCE and the ISOC current in neonatal rat ventricular cardiomyocytes (NRVMs). Within this cardiomyocyte model, we identified the transcript expression of Stim2.1 and Stim2.2 splice variants, with predominance for Stim2.2. Using conventional and super-resolution confocal microscopy (STED), we found that exogenous STIM2.1 and STIM2.2 formed pre-clusters with a reticular organization at rest. Following SR Ca2+ store depletion, some STIM2.1 and STIM2.2 clusters were translocated to SR-plasma membrane (PM) junctions and co-localized with Orai1. The overexpression strategy revealed that STIM2.1 suppressed Orai1-mediated SOCE and the ISOC current while STIM2.2 enhanced SOCE. STIM2.2-enhanced SOCE was also dependent on TRPC1 and TRPC4. Even if STIM2 KD or splice variants overexpression did not affect cytosolic Ca2+ cycling, we observed, using Rhod-2/AM Ca2+ imaging, that Orai1 inhibition or STIM2.1 overexpression abolished the mitochondrial Ca2+ (mCa2+) uptake, as opposed to STIM2 KD. We also found that STIM2 was present in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) by interacting with the inositol trisphosphate receptors (IP3Rs), voltage-dependent anion channel (VDAC), mitochondrial Ca2+ uniporter (MCU), and mitofusin-2 (MNF2). Our results suggested that, in NRVMs, STIM2.1 constitutes the predominant functional variant that negatively regulates Orai1-generated SOCE. It participates in the control of mCa2+ uptake capacity possibly via the STIM2-IP3Rs-VDAC-MCU and MNF2 complex.

TGFβ1 Secretion in Megakaryocytes Is Autophagy-Dependent and Its Inhibition Ameliorates Myelofibrosis in Mice

Megakaryocytes (MKs) are large, polyploid platelet precursor cells that primarily reside within the bone marrow. Recent advances revealed that in addition to platelet production, MKs contribute to immunity and maintenance of bone marrow homeostasis, raising the question of whether these functions are hijacked in disease. One hallmark of primary and secondary myelofibrosis (MF), subsets of myeloproliferative neoplasms, is the accumulation of aberrant, immature MKs in the bone marrow. These release pro-fibrotic cytokines such as transforming growth factor β1 (TGFβ1) and interleukin 1β (IL1β) into the bone marrow, thus inducing fibrotic alterations that ultimately cause pancytopenia - the major cause of mortality in patients with MF. While current treatment strategies in MF aim to prevent MK proliferation and correct the differentiation defects, a direct targeting of cytokine release from MKs has not yet been explored. Recently, unconventional secretion of TGFβ1 and IL1β via secretory autophagy, a secretion pathway driven by the fusion of autophagosomes with the plasma membrane, has been shown to occur in other cell types. We therefore hypothesize that MKs may preferentially utilize secretory autophagy for cytokine release and that inhibiting autophagosome formation might therefore slow fibrosis progression in vivo. Previous studies suggested that TGFβ1 is stored within specialized α-granules in MKs. To test this, we cultured murine MKs and determined TGFβ1 co-localization with different α-granule proteins by super-resolution confocal microscopy. However, while we did not identify any correlation between TGFβ1 and α-granules, we found that TGFβ1 strongly co-localized with the autophagy marker light chain (LC) 3B, supporting a potential role for secretory autophagy. To investigate this, we disrupted secretory autophagy by inhibiting the GTPase RhoA and its downstream effector Rho kinase (ROCK), which were previously shown to promote autophagosome formation. Treatment of MKs with inhibitors of autophagy or RhoA/ROCK markedly reduced TGFβ1 secretion in vitro and led to intracellular accumulation of TGFβ1. Taken together, these data suggest that TGFβ1 is secreted by MKs via secretory autophagy. To examine whether secretory autophagy in MKs contributes to disease pathogenesis in vivo, we employed an MPL W515L-driven transplant model. Basal autophagy measured by LC3B conversion and RhoA protein levels were increased in platelets derived from mice with MPL W515L-driven MF. Conditional deletion of the essential autophagy gene Atg5 from the hematopoietic system ( Mx1-Cre+; Atg5fl/fl mice) prevented leukocytosis, MK clustering and aberrant TGFβ1 secretion in the MPL W515L-driven transplant model, indicating that autophagy promotes the initiation of MF. Furthermore, MPL W515L-transplanted mice treated with a low-dose ROCK inhibitor (ROCKi) - several of which are FDA-approved - reduced MK clustering and fibrosis in spleens and femurs of treated mice (Figure 1a, b). To further verify our data, we utilized a second preclinical mouse model of MF - a knockout mouse lacking the inhibitory MK receptor G6b-B ( Mpig6b -/-), which exhibits thrombocytopenia, MK clustering and MF. In line with our previous findings, conditional deletion of Rhoa in MK lineage cells in Pf4-Cre+; Rhoafl/fl; Mpig6b -/- mice normalized TGFβ1 levels within the bone marrow and protected against the development of fibrosis in bone marrow and spleen. In ongoing work, ROCK inhibitors are further being tested in higher doses alone or in combination with the JAK2 inhibitor ruxolitinib. Overall, our data suggest that TGFβ1 secretion is uncoupled from conventional granule secretion in MKs and is instead dependent on RhoA/autophagy signaling pathways. Targeting these pathways in aberrant MKs represents a new potential therapeutic target in the treatment of MF.

Abstract 15932: Lactobacillus Suppresses Glycoprotein-130-Mediated Microtubule Remodeling to Support Right Ventricular Function in Pulmonary Arterial Hypertension

Introduction: Microtubules (MTs) are crucial for cardiomyocyte function as they regulate t-tubule structure, nuclear morphology, and gap junction protein localization. Glycoprotein-130 (GP130) signaling promotes pathological MT remodeling and right ventricular (RV) dysfunction in pulmonary arterial hypertension (PAH), but the trigger for the inflammation-MT connection is unknown. Microbiome dysbiosis activates systemic inflammation, and levels of the anti-inflammatory bacteria Lactobacillus are associated with RV function in rodents. However, the direct effects of Lactobacillus on GP130 signaling, MT remodeling, and RV function in PAH are unknown. Methods: Rats were randomly allocated into 3 groups: control, MCT-water, and MCT rats given Lactobacillus (MCT- Lactobacillus ). Echocardiography and pressure-volume loops defined RV function. Next-generation sequencing evaluated the composition of the bacterial components of the fecal microbiome. SomaScan proteomics quantified the serum proteome. RV and jejunal morphology were assessed with light and super resolution confocal microscopy. The relationship between Lactobacillus abundance and RV response to afterload was assessed in 65 PAH patients. Results: Lactobacillus augmented RV function (A) and reduced RV hypertrophy and fibrosis without altering PAH severity. Lactobacillus shifted the serum proteomic signature towards control (B) and reduced circulating GP130 ligands, normalized RV t-tubule architecture (C), MT density (D), and nucleus size (E), and prevented connexin-43 lateralization in RV cardiomyocytes (F). Lactobacillus restructured the microbiome, and concomitantly improved jejunal epithelial cell morphology with increased villus/microvillus length and glycocalyx thickness. PAH patients with fecal Lactobacillus had superior RV adaptation as afterload increased (G). Conclusion: Lactobacillus combats GP130-mediated MT remodeling to support RV function.

BS-454195-1 ADRENERGIC MODULATION OF CONDUCTION DURING CARDIAC STRESS IS MEDIATED BY CALCIUM CHANNEL AUTOREGULATION OF INTERCALATED DISC NANODOMAINS

During stress, adrenergic signaling enhances cardiac conduction, although the mechanisms are not fully understood. Reducing extracellular calcium in the heart widens an intercalated disc (ID) nanodomain adjacent to gap junctions known as the perinexus. Under acute cardiac stress, the perinexus adaptively remodels; preventing this response by experimentally changing calcium levels slows conduction and increases arrhythmia risk. This raises the question of whether myocytes autoregulate calcium-mediated ID adhesion to stabilize conduction during cardiac stress. The cardiac voltage gated calcium channel (Cav1.2) is a primary source of calcium influx and a key target during adrenergic signaling. Expression of Cav1.2 within the ID has not been described. To test the hypothesis that Cav1.2 is expressed in the ID and autoregulates myocyte adhesion and conduction in the adrenergic response to cardiac stress. Ex vivo guinea pig hearts were Langendorff-perfused and ventricular tissue harvested for microscopy. Cav1.2 cellular localization was quantified by spinning disk confocal microscopy (n=5), conduction velocity (CV) was measured by optical mapping (n=9), and ID ultrastructure was quantified from transmission electron micrographs (n=17). Cav1.2 function was modulated by perfusion with the β-adrenergic agonist isoproterenol (Iso, 0.6μM), the Cav1.2 agonist BayK8644 (BayK, 0.5μM), or the Cav1.2 antagonist verapamil (Ver, 2μM). Super resolution confocal microscopy demonstrated that 2.5% of total Cav1.2 signal co-occurred with connexin43 and N-cadherin. At baseline, CV slowed at shorter CLs (150 vs 300ms = 4% slower), consistent with CV restitution. Treatment with Iso increased both Wp (20.2 vs 17.0 nm at baseline) and CV (300ms: 113% of baseline). Iso reversed CV restitution such that CV increased by 4%. Activation of Cav1.2 by BayK widened Wp to the same degree as Iso (20.1 vs 20.2 nm). Verapamil prevented both the Iso-induced increase in Wp (17.0 nm) and CV (93.9% of baseline), and exacerbated CV restitution. A subpopulation of Cav1.2 was identified within the ID. Adrenergic stimulation widened perinexal width, increased conduction, and reversed CV restitution. Blocking Cav1.2 prevented the adrenergic-mediated change in perinexal structure and conduction. Thus, Cav1.2 function regulates ID ultrastructure and may serve as an adaptive response to enhance conduction during conditions of acute cardiac stress.

OVERLOAD-INDUCED EXTRACELLULAR MITOCHONDRIA RELEASING FROM TENDON ORGANOID LEADS TO INFLAMMATION

Tendinopathy is the most frequent musculoskeletal disease that requires medical attention. Mechanical overload has been considered as a key driver of its pathology. However, the underline mechanism on how overload induces tendinopathy and inflammation is unclear. Extracellular mitochondria (EM) are newly identified as cell-to-cell communicators. The aim of this study is to elucidate the role of mitochondria in overload-induced inflammation.We performed three-dimensional uniaxial stretching to mouse tendon organoid in bioreactors. Cyclic strain of uniaxial loadings included underload, normal load, and overload, according to previous work. We then harvested microvesicles including EM, from the bioreactor by differential centrifugation and evaluated their characteristics by flow cytometry and super-resolution confocal microscopy. Raw 264.7 mouse macrophage cell line was used for chemotaxis assay in a Boyden Chamber System with Magnetic-Activated Cell Sorting Technology. EM induced cytokines secretion by macrophages was analyzed by a bead-based multiplex assay panel. N-Acetyl-L-cysteine (NAC) was used as the antioxidant to tendon organoid to regulate mitochondrial fitness.We showed mechanical load induced tendon organoid to release microvesicles including mitochondria. The size of microvesicles is mainly in the range from 220nm to 880nm. More than 75% of microvesicles could be stained by PKH26, confirming they were with lipophilic membrane. Super-resolution confocal microscopy identified two forms of mitochondria, including mitochondria encapsulated in vesicles and free mitochondria. Overload led to the degeneration of the organoid and induced microvesicles release containing most EM. Chemotaxis assay showed that EM from overloaded tendon organoid induced macrophages chemotaxis. In addition, microvesicles extracted from overloaded tendon organoid induced the production of proinflammatory cytokines including IL-6, KC (Keratinocyte-Derived Chemokine) and IL-18. NAC treatment to tendon cells could attenuate overload-induced macrophage chemotaxis.Overload induces EM releasing from tendon cells, which leads to chemotaxis of macrophages toward tendon, resulting in induction of inflammation.

Beyond the Limits of Light: An Application of Super-Resolution Confocal Microscopy (sCLSM) to Investigate Eocene Amber Microfossils.

Amber is known as one of the best sources of fossil organisms preserved with exceptional fidelity. Historically, different methods of imaging have been applied to amber, including optical microscopy and microtomography. These methods are sufficient to resolve millimeter-scaled fossils. However, microfossils, such as microarthropods, require another resolution. Here, we describe a non-destructive method of super resolution confocal microscopy (sCLSM) to study amber-preserved microfossils, using a novel astigmatid mite species (genus Histiogaster, Acaridae) from Eocene Rovno amber as a model. We show that the resolution obtained with sCLSM is comparable to that of scanning electron microscopy (SEM) routinely used to study modern mites. We compare sCLSM imaging to other methods that are used to study amber inclusions and emphasize its advantages in examination of unique fossil specimens. Furthermore, we show that the deterioration of amber, which manifests in its darkening, positively correlates with its increased fluorescence. Our results demonstrate a great potential of the sCLSM method for imaging of the tiniest organisms preserved in amber.

StaR Is a Positive Regulator of Topoisomerase I Activity Involved in Supercoiling Maintenance in Streptococcus pneumoniae

The DNA topoisomerases gyrase and topoisomerase I as well as the nucleoid-associated protein HU maintain supercoiling levels in Streptococcus pneumoniae, a main human pathogen. Here, we characterized, for the first time, a topoisomerase I regulator protein (StaR). In the presence of sub-inhibitory novobiocin concentrations, which inhibit gyrase activity, higher doubling times were observed in a strain lacking staR, and in two strains in which StaR was over-expressed either under the control of the ZnSO4-inducible PZn promoter (strain ΔstaRPZnstaR) or of the maltose-inducible PMal promoter (strain ΔstaRpLS1ROMstaR). These results suggest that StaR has a direct role in novobiocin susceptibility and that the StaR level needs to be maintained within a narrow range. Treatment of ΔstaRPZnstaR with inhibitory novobiocin concentrations resulted in a change of the negative DNA supercoiling density (σ) in vivo, which was higher in the absence of StaR (σ = -0.049) than when StaR was overproduced (σ = -0.045). We have located this protein in the nucleoid by using super-resolution confocal microscopy. Through in vitro activity assays, we demonstrated that StaR stimulates TopoI relaxation activity, while it has no effect on gyrase activity. Interaction between TopoI and StaR was detected both in vitro and in vivo by co-immunoprecipitation. No alteration of the transcriptome was associated with StaR amount variation. The results suggest that StaR is a new streptococcal nucleoid-associated protein that activates topoisomerase I activity by direct protein-protein interaction.

The balance between gyrase and topoisomerase I activities determines levels of supercoiling, nucleoid compaction, and viability in bacteria.

Two enzymes are responsible for maintaining supercoiling in the human pathogen Streptococcus pneumoniae, gyrase (GyrA2GyrB2) and topoisomerase I. To attain diverse levels of topoisomerase I (TopoI, encoded by topA), two isogenic strains derived from wild-type strain R6 were constructed: PZn topA, carrying an ectopic topA copy under the control of the ZnSO4-regulated PZn promoter and its derivative ΔtopAPZn topA, which carries a topA deletion at its native chromosomal location. We estimated the number of TopoI and GyrA molecules per cell by using Western-blot and CFUs counting, and correlated these values with supercoiling levels. Supercoiling was estimated in two ways. We used classical 2D-agarose gel electrophoresis of plasmid topoisomers to determine supercoiling density (σ) and we measured compaction of nucleoids using for the first time super-resolution confocal microscopy. Notably, we observed a good correlation between both supercoiling calculations. In R6, with σ = -0.057, the average number of GyrA molecules per cell (2,184) was higher than that of TopoI (1,432), being the GyrA:TopoI proportion of 1:0.65. In ΔtopAPZn topA, the number of TopoI molecules depended, as expected, on ZnSO4 concentration in the culture media, being the proportions of GyrA:TopoI molecules in 75, 150, and 300 μM ZnSO4 of 1:0.43, 1:0.47, and 1:0.63, respectively, which allowed normal supercoiling and growth. However, in the absence of ZnSO4, a higher GyrA:TopoI ratio (1:0.09) caused hyper-supercoiling (σ = -0.086) and lethality. Likewise, growth of ΔtopAPZn topA in the absence of ZnSO4 was restored when gyrase was inhibited with novobiocin, coincidentally with the resolution of hyper-supercoiling (σ change from -0.080 to -0.068). Given that TopoI is a monomer and two molecules of GyrA are present in the gyrase heterotetramer, the gyrase:TopoI enzymes proportion would be 1:1.30 (wild type R6) or of 1:1.26-0.86 (ΔtopAPZn topA under viable conditions). Higher proportions, such as 1:0.18 observed in ΔtopAPZn topA in the absence of ZnSO4 yielded to hyper-supercoiling and lethality. These results support a role of the equilibrium between gyrase and TopoI activities in supercoiling maintenance, nucleoid compaction, and viability. Our results shed new light on the mechanism of action of topoisomerase-targeting antibiotics, paving the way for the use of combination therapies.

Abstract 12004: Mitochondrial Fission Mediator MiD49 Regulates Proliferation via MYO1C in Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by pulmonary vascular obstruction that is partially due to hyperproliferation of apoptosis-resistant pulmonary arterial smooth muscle cells (PASMC). This “pseudoneoplastic phenotype” of PAH PASMC is due, in part, to excessive mitochondrial fission caused by activation of dynamin related protein-1 (Drp-1) and its binding partner MiD49. The mechanism by which MiD49 regulates the proliferation of PASMC is not completely understood. Here we investigate the possibility that MiD49 promotes cellular proliferation by regulating a non-filamentaous class-1 myosin protein, MYO1C. Hypothesis: MiD49 regulates cell proliferation via a MYO1C-dependent mechanism, which is pathologically upregulated in PAH. Methods: MiD49 and MYO1C expression was compared between normal (n=4) vs PAH (n=5) human PASMC using western blot analysis. The colocalization was visualized using super-resolution confocal microscopy. The effects of manipulating MYO1C on cell proliferation and apoptosis were assessed by flowcytometry. Results: MiD49 and MYO1C are upregulated and colocalized in PAH PASMC. The expression of MYO1C positively correlated with MiD49 in PAH. Silencing MiD49 downregulated MYO1C expression whilst augmenting MiD49 upregulated MYO1C. Silencing MYO1C, however, does not affect MiD49 expression, proving that MYO1C is downstream of MiD49. The downregulation of MYO1C by MiD49 silencing is rescued by a proteasomal blockade, MG132. Downregulating MYO1C using siRNA inhibited proliferation, prevented ERK1/2 activation, and induced apoptosis in PAH PASMC. Conclusion: Increased proliferation and apoptosis resistance of PAH PASMC are in part due to the dysregulation of MiD49-MYO1C pathway. Targeting this pathway may have therapeutic implications against PAH.

Abstract B012: Visualization of EWSR1’s colocalization with phosphorylated RNA-Polymerase II reveals its concentration at a subset of active regions of transcription in ewing sarcoma cells

Abstract EWSR1, a member of the FET protein family, contains low complexity and nucleic acid binding domains and functions in transcription regulation and RNA metabolism. Recent biochemical and EWSR1-depletion studies demonstrated that EWSR1 regulates critical phosphorylation events that control basal transcription. In Ewing sarcoma (EWS) cells, the interaction of EWSR1 and the fusion oncoprotein EWS-FLI1 results in EWSR1 no longer functioning as an effective regulator of transcription, which following DNA damage, enhances R-loop formation. To further elucidate EWSR1’s function in EWS and its contribution to EWS-FLI1’s deregulation of gene expression, we have generated reporter systems to visualize and quantify its endogenous expression within EWS cells. We used CRISPR-Cas9 and sgRNAs targeting sequences at the 5’ end of the first exon of EWSR1 to insert a fluorescent mNeonGreen (mNG) reporter gene into the EWSR1 loci of A673 or TC-32 EWS cell lines. We employed sequencing and RNAi-based analysis to identify and validate successfully modified clones. All modified clones harbored insertions into the unrearranged EWSR1 allele. We then employed super-resolution confocal microscopy to assess EWSR1’s localization in EWS cells. Analysis of modified EWS-cells showed mNG-EWSR1 forms puncta, restricted to the nucleoplasm, consistent with a nuclear protein in an active state. A subset of puncta exhibits a high density (HD) mNG-EWSR1 signal, defined by fluorescence at least twice the background signal. Our results show minimal colocalization of mNG-EWSR1 (total or HD) and a marker of chromatin accessibility, H3K27Ac. A small percentage (~5%) of total Ser5-phosphorylated RNA-pol II (pS5-RP-II), a marker of transcription initiation, colocalizes with total mNG-EWSR1, but critically the HD mNG-EWSR1 puncta all colocalize with pS5-RP-II. About 20% of total Ser2-phosphorylated RNA-pol II (pS2-RP-II), a marker of transcription elongation colocalizes with total mNG-EWSR1. As observed for pS5-RP-II, 100% of HD-mNG-EWSR1 puncta colocalize with pS2-RP-II. Finally, when we examined nuclear speckle structures (SC35/SRSF2 >200 µm), we observed a 20% overlap in their signals. We have seen comparable results using the mNG-EWSR1 expressing A673 cells. These findings demonstrate image-based quantification of endogenous EWSR1’s colocalization with RNA-polymerase II within EWS cells. Overall, EWSR1 colocalizes with about 20% of RNA-pol II in a state consistent with active transcription, and over 90% of high-density EWSR1 colocalizes with phosphorylated RNA pol II. Ongoing studies will assess changes in the distribution of EWSR1’s interactions with different proteins following inhibition of RNA-pol II phosphorylation, alterations in EWS-FLI1 expression, or the disruption of low complexity domain interactions. We anticipate findings from these studies will offer critical insights into the functional interactions that EWSR1 contributes to regulating gene expression in EWS cells. Citation Format: Natasha J. Caplen, Soumya Sundara Rajan, Vernon Ebegboni, Tamara L. Jones, Michael J. Kruhlak, Jan Wisniewski, Patricio Pichling, Katelyn R. Ludwig, Javed Khan, Raj Chari. Visualization of EWSR1’s colocalization with phosphorylated RNA-Polymerase II reveals its concentration at a subset of active regions of transcription in ewing sarcoma cells [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B012.