Fluorescence Recovery After Photobleaching Research Articles

Probing Native CB2 Receptor Mobility in Plasma Membranes of Living Cells by Fluorescence Recovery After Photobleaching.

In this study, we employed a novel fluorescent probe, RO7304924-which selectively targets cannabinoid 2 receptor (CB2R)-to assess the lateral mobility of CB2R within the plasma membrane of Chinese hamster ovary cells stably expressing a functional, untagged receptor variant. Utilizing confocal fluorescence recovery after photobleaching (FRAP), we quantified the diffusion coefficient and mobile fraction of CB2R, thereby demonstrating the efficacy of RO7304924 as an innovative tool for elucidating the dynamics of this major endocannabinoid-binding G protein-coupled receptor. Our present findings highlight the potential of combining advanced ligand-based fluorescent probes with FRAP for future investigations into the biochemical details of CB2R mobility in living cells, and its impact on receptor-dependent cellular processes.

The Balbiani body is formed by microtubule-controlled molecular condensation of Buc in early oogenesis.

Vertebrate oocyte polarity has been observed for two centuries and is essential for embryonic axis formation and germline specification, yet its underlying mechanisms remain unknown. In oocyte polarization, critical RNA-protein (RNP) granules delivered to the oocyte's vegetal pole are stored by the Balbiani body (Bb), a membraneless organelle conserved across species from insects to humans. However, the mechanisms of Bb formation are still unclear. Here, we elucidate mechanisms of Bb formation in zebrafish through developmental biomolecular condensation. Using super-resolution microscopy, live imaging, biochemical, and genetic analyses invivo, we demonstrate that Bb formation is driven by molecular condensation through phase separation of the essential intrinsically disordered protein Bucky ball (Buc). Live imaging, molecular analyses, and fluorescence recovery after photobleaching (FRAP) experiments invivo reveal Buc-dependent changes in the Bb condensate's dynamics and apparent material properties, transitioning from liquid-like condensates to a solid-like stable compartment. Furthermore, we identify a multistep regulation by microtubules that controls Bb condensation: first through dynein-mediated trafficking of early condensing Buc granules, then by scaffolding condensed granules, likely through molecular crowding, and finally by caging the mature Bb to prevent overgrowth and maintain shape. These regulatory steps ensure the formation of a single intact Bb, which is considered essential for oocyte polarization and embryonic development. Our work offers insight into the long-standing question of the origins of embryonic polarity in non-mammalian vertebrates, supports a paradigm of cellular control over molecular condensation by microtubules, and highlights biomolecular condensation as a key process in female reproduction.

FRAP Assay to Trace Lipid Mixing of the Inner and Outer Leaflet of Yeast Vacuoles: Assessing the Fusion State in Live Cells.

Fluorescence recovery after photobleaching (FRAP) can be employed to investigate membrane lipid mixing of vacuoles in live budding yeast cells and distinguish the fused, hemi-fused or non-fused states of these organelles under physiological conditions. Here, we describe a protocol for labeling the outer and inner leaflets of vacuoles in live cells that allow to detect hemifusion intermediates and, thus, identify components necessary for fusion pore opening.

Live imaging of paracrine signaling: Advances in visualization and tracking techniques.

Live imaging techniques have revolutionized our understanding of paracrine signaling, a crucial form of cell-to-cell communication in biological processes. This review examines recent advances in visualizing and tracking paracrine factors through four key stages: secretion from producing cells, diffusion through extracellular space, binding to target cells, and activation of intracellular signaling within target cells. Paracrine factor secretion can be directly visualized by fluorescent protein tagging to ligand, or indirectly by visualizing the cleavage of the transmembrane pro-ligands or plasma membrane fusion of endosomes comprising the paracrine factors. Diffusion of paracrine factors has been studied using techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence decay after photoactivation (FDAP), and single-molecule tracking. Binding of paracrine factors to target cells has been visualized through various biosensors, including GPCR-activation-based (GRAB) sensors and Förster resonance energy transfer (FRET) probes for receptor tyrosine kinases. Finally, activation of intracellular signaling is monitored within the target cells by biosensors for second messengers, transcription factors, and so on. In addition to the imaging tools, the review also highlights emerging optogenetic and chemogenetic tools for triggering the release of paracrine factors, which is essential for associating the paracrine factor secretion to biological outcomes during the bioimaging of paracrine factor signaling.Key words: paracrine signaling, live imaging, biosensors, optogenetics, chemogenetics.

TFEB Phase Separation Mediates the Amelioration Effect of Intermittent Fasting on Inflammatory Colitis.

Intermittent fasting (IF) has been shown to ameliorate inflammation including DSS-induced colitis. It is well known that autophagy can limit inflammation and TFEB is a master transcriptional factor that regulates the processes of autophagy. However, whether TFEB is involved in the regulation of IF-mediated amelioration of inflammation and its mechanism remained unclear. In this study, we found that IF ameliorated DSS-induced colitis and induced TFEB. Nutrition deprivation induced TFEB puncta formation, which processes the characteristics of liquid-liquid phase separation (LLPS) showed by fluorescence recovery after photobleaching (FRAP) assay and 1,6-hexanediol treatment. We found the 24-33 amino acids of Coiled-Coil (CC) domain located in N terminus is essential for TFEB phase separation. Deletion of 24-33 amino acids within the CC domain inhibited TFEB-mediated target gene expression. In addition, we found transcription co-activators, EP300 and MED1, co-localized with TFEB condensate to formed a transcriptional hub that promotes the efficient expression of target genes. More importantly, TFEB inhibitor with ability to suppress TFEB puncta formation abolished the IF-mediated amelioration of DSS colitis. Together, these findings revealed a critical role of TFEB phase separation in the regulation of its transcriptional activity and anti-inflammatory functions induced by IF.

Super-enhancer Activates Master Transcription Factor NR3C1 Expression and Promotes 5-FU Resistance in Gastric Cancer.

Poor response to 5-fluorouracil (5-FU) remains an obstacle in the treatment of gastric cancer (GC). Super enhancers (SEs) are crucial for determining tumor cell survival under drug pressure. SE landscapes related to 5-FU-resistance are mapped to GC using chromatin immunoprecipitation-sequencing (ChIP-Seq). SiRNA transcription factors (TFs) screen determines master TF Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1) activated by SE. High NR3C1 expression driven by SE correlated with 5-FU resistance in patient-derived organoids (PDOs). Phase separation formed by NR3C1 is observed using fluorescence recovery after photobleaching (FRAP). NR3C1 protein and Mediator promoted SE-related gene transcription via phase separation. SEs and NR3C1 co-binding patterns are explored using Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing. 5-FU-related genes driven by NR3C1 are identified using epigenetic reader inhibitor JQ1 and NR3C1 specific inhibitor Cort108297. NR3C1 knockdown increases 5-FU sensitivity and alters the SE landscape through enhancer reprogramming, reducing downstream 5-FU-related target genes. JQ1 and Cort108297 both improve 5-FU efficacy in PDOs and patient-derived xenografts (PDXs) by destroying SEs or inhibiting NR3C1. In conclusion, SE-driven NR3C1 promotes 5-FU resistance in GC. SE destruction and NR3C1 inhibition lead to enhancer reconstruction and reduce 5-FU-related gene transcription, providing alternative therapeutic strategies for improving 5-FU sensitivity.

Enzymatically Polymerized Organic Conductors on Native Lipid Membranes.

The dual capability of conductive polymers to conduct ions and electrons, in combination with their flexible mechanical properties, makes them ideal for bioelectronic applications. This study explores the in situ enzymatic polymerization of water-soluble π-conjugated monomers on native lipid bilayers derived from the F11 cell line, mimicking mammalian neural membranes. Enzymatic polymerization was catalyzed using horseradish peroxidase (HRP) in the presence of oxidant hydrogen peroxide (H2O2) and monitored via electrochemical quartz crystal microbalance with dissipation (EQCM-D) and electrochemical impedance spectroscopy (EIS). Results showed polymerization with HRP. The structural properties of the formed polymer films were characterized ex situ using atomic force microscopy (AFM), while the quality of the F11 native lipid vesicles and bilayer was respectively assessed through dynamic light scattering (DLS) and fluorescence recovery after photobleaching (FRAP) techniques. This work demonstrates, for the first time, the feasibility of the in situ formation of conductive polymers on native lipid membranes, offering a promising approach for the development of minimally invasive neural electrodes to diagnose and treat neurological disorders.

Reversing protonation of weakly basic drugs greatly enhances intracellular diffusion and decreases lysosomal sequestration.

For drugs to be active they have to reach their targets. Within cells this requires crossing the cell membrane, and then free diffusion, distribution, and availability. Here, we explored the in-cell diffusion rates and distribution of a series of small molecular fluorescent drugs, in comparison to proteins, by microscopy and fluorescence recovery after photobleaching (FRAP). While all proteins diffused freely, we found a strong correlation between pKa and the intracellular diffusion and distribution of small molecule drugs. Weakly basic, small-molecule drugs displayed lower fractional recovery after photobleaching and 10- to-20-fold slower diffusion rates in cells than in aqueous solutions. As, more than half of pharmaceutical drugs are weakly basic, they, are protonated in the cell cytoplasm. Protonation, facilitates the formation of membrane impermeable ionic form of the weak base small molecules. This results in ion trapping, further reducing diffusion rates of weakly basic small molecule drugs under macromolecular crowding conditions where other nonspecific interactions become more relevant and dominant. Our imaging studies showed that acidic organelles, particularly the lysosome, captured these molecules. Surprisingly, blocking lysosomal import only slightly increased diffusion rates and fractional recovery. Conversely, blocking protonation by N-acetylated analogues, greatly enhanced their diffusion and fractional recovery after FRAP. Based on these results, N-acetylation of small molecule drugs may improve the intracellular availability and distribution of weakly basic, small molecule drugs within cells.

Characterization of Guest DNA Transport and Adsorption within Host Porous Protein Crystals.

Nucleic acid transport through protein-based pores is a well-characterized phenomenon due in part to advancements in nanopore sequencing. A less studied area is nucleic acid transport through extended protein-based channels, where the additional surface area and increased contact time allow for the study of prolonged binding interactions. Porous protein crystals composed of "CJ", a putative polyisoprenoid-binding protein from Campylobacter jejuni, represent a favorable, highly ordered material for studying DNA transport and binding/unbinding along protein-based channels. These crystals adopt a hexagonal prism habit and contain a densely packed hexagonal array of 13 nm diameter axial nanopores that run from the top to the bottom of the crystal. After cross-linking, the crystals are easily manipulated for experimentation. An adsorption isotherm between host crystals and guest double-stranded 8 base pair DNA (8mer) revealed a high equilibrium adsorption constant of 206 ± 30 L/g. Fluorescence confocal microscopy tracked the loading of guest DNA into host crystals predominately along the major axial crystal nanopores. Four different computational models based on the finite volume (FV) method were assessed to model the transport process for guest 8mer and 15mer dsDNA loading into empty host crystals in terms of fundamental parameters, such as the intrapore diffusion constant. Fitting the models to the data revealed that the most basic FV model sufficed to describe the observed loading behavior, characterized by a single effective diffusion coefficient. Leveraging Fick's first law, we more directly fit a numerical range for the observed intrapore diffusion coefficient as a function of time, position within the crystal, and relative guest concentration. This new transport analysis strategy was applied to both out-of-equilibrium loading and fluorescence recovery after photobleaching (FRAP) experiments. The intrapore diffusion constants are comparable between 8mer and 15mer dsDNA and were found to be 2 orders of magnitude faster for DNA loading into empty crystals than that observed in FRAP experiments, which averaged (10 ± 4) × 10-11 cm2/s.

Effect of cigarette smoke exposure and cessation on regional diffusion properties in rat intervertebral discs.

Cigarette smoking is a recognized risk factor for orthopedic disorders, particularly intervertebral disc (IVD) degenerative disease. However, the IVD pathophysiology, especially the spatial-temporal remodeling progression in the context of cigarette smoking, remains unclear. This study aimed to address this knowledge gap through a quantitative assessment of IVD structural composition and diffusion properties using a Sprague-Dawley rat model. Twenty-four rats were divided into control and smoke exposure cohorts, each with two sub-groups of six rats. One smoke exposure sub-group was sacrificed after 2 months of daily cigarette smoke exposure in a custom smoking apparatus, while the other was sacrificed after an additional 5 months of smoke cessation. The control groups were age-matched to the smoke exposure groups. A fluorescent recovery after photobleaching (FRAP) technique was used to determine solute diffusivities and multi-photon excitation (MPE) imaging was performed to characterize structural changes in the annulus fibrosus (AF), nucleus pulposus (NP), and cartilage endplate (CEP). A decrease in diffusivity was observed in the CEP and the AF (radial direction only) after 2 months of smoke exposure. MPE imaging showed aberrant CEP calcification and reduced AF radial collagen fiber bundle diameter, suggesting that the IVD exhibits regionally dependent structural remodeling due to smoke exposure. Furthermore, the smoke cessation group showed deteriorating alterations of structure and diffusivities in all three-disc regions, including the NP, indicating that five-month smoke cessation alone didn't reverse the progression of IVD degenerative remodeling during aging. This study advances the understanding of IVD pathophysiology in the context of cigarette smoke exposure and cessation, laying the groundwork for potential earlier diagnosis and optimized interventions.

Knockdown of RUVBL2 improves hnRNPA2/B1-stress granules dynamics to inhibit perioperative neurocognitive disorders in aged mild cognitive impairment rats.

Perioperative neurocognitive disorders (PND) is common in aged mild cognitive impairment (MCI) patients and can accelerate the progression to dementia. This process involves heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1)-mediated aggregates of stress granules (SGs), while RUVBL2 influences the dynamics of these SGs. Our research explored a new target for modulating hnRNAPA2/B1-SGs dynamics to accelerate their disassembly and potentially delay MCI progression due to PND. We assessed the effect of hippocampal RUVBL2 knockdown on hnRNPA2/B1-SGs in aged MCI rats through behavioral studies, biochemical experiments and MRI. We also examined hnRNPA2/B1-SGs dynamics using immunofluorescence staining and fluorescence recovery after photobleaching (FRAP) in rat primary hippocampal neurons. Our results revealed that hnRNPA2/B1 in the hippocampus of aged MCI rats translocates to the cytoplasm to form SGs following anesthesia. RUVBL2 knockdown promotes the disappearance of hnRNPA2/B1-SGs, allowing hnRNPA2/B1 to return to the nucleus and enhancing functional activity in the brain regions of aged MCI rats. In primary hippocampal neurons, RUVBL2 deletion facilitated hnRNPA2/B1-SGs transition from hydrogel to liquid, promoting disassembly. We compared three commonly used general anesthetics-3% sevoflurane, 40 mg·kg-1·h-1 propofol, and 9% desflurane. Sevoflurane upregulated RUVBL2, which decreased the intraneuronal pH and disrupted energy metabolism. These changes resulted in greater stabilization of hnRNPA2/B1- SGs. In conclusion, our findings indicated that the knockdown of RUVBL2 expression contributes to the transition of hnRNPA2/B1-SGs from the hydrogel phase to the liquid phase. Targeted interference with RUVBL2 may represent a novel approach to delay the progression to dementia due to PND in aged MCI patients.

Two-directional trafficking of the IFT25 protein in the developing mouse sperm flagella.

Intraflagellar transport 25 (IFT25) is a component of the IFT-B complex. In mice, even though this IFT component is not required for cilia formation in somatic cells, it is essential for sperm formation. However, the intracellular localization of this protein in male germ cells is not known given no reliable antibodies are available for histologic studies, and the dynamic trafficking in the developing sperm flagella is not clear. To examine localization of the protein in male germ cells and further investigate the mechanism of IFT in sperm formation, particularly to look into the dynamic trafficking of the protein, we generated a mouse IFT25-GFP knock-in (KI) mouse model using the CRISPR/cas9 system, with the mouse IFT25 protein fused with a GFP tag in the C-terminus. Three independent lines were analyzed. Western blotting using both anti-IFT25 and anti-GFP antibodies showed that the IFT25-GFP fusion protein was highly abundant only in the testis, which is consistent with the endogenous IFT25 protein. Examination of localization of the IFT25-GFP in isolated germ cells revealed that the fusion protein was present in the cytoplasm of spermatocytes and round spermatids and a strong signal was present in the developing sperm flagellar. The homozygous KI mice had normal spermatogenesis, fertility and sperm parameters. Diffusion analysis of IFT25 within the developing flagellar revealed the presence of both mobile and immobile fractions as revealed by fluorescence recovery after photobleaching (FRAP). Kymograph and FRAP analyses demonstrate the transport of IFT25-GFP within the developing tail demonstrate no apparent preference for trafficking towards and away from the cell body. The speed of trafficking depends on the stage of sperm development, ranging from highly mobile unrestricted diffusion initially, mobile punctate structures in developing sperm, and immobile punctate structures in mature sperm. Our studies demonstrate that mouse IFT25 travels along the developing sperm flagella in two directions that might be essential for functional sperm formation.

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles.

Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of Gram-negative bacteria limits the efficacy of many existing antibiotics and adds complexity to antibiotic development by limiting transport of antibiotics to the bacterial cytosol. New methods to mimic this barrier would enable high-throughput studies for antibiotic development. In this study, we introduce an innovative approach to modify outer membrane vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, to generate planar supported lipid bilayer membranes. Our method first involves the incorporation of synthetic lipids into OMVs using a rapid freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when in contact with SiO2 surfaces to form a planar outer membrane supported bilayer (OM-SB). We assessed the formation of OM-Hybrids using dynamic light scattering and a fluorescence quenching assay. To analyze the formation of OM-SBs from OM-Hybrids we used quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP). Additionally, we conducted assays to detect surface-associated DNA and proteins on OM-SBs. The interaction of an antimicrobial peptide, polymyxin B, with the OM-SBs was also assessed. These findings emphasize the capability of our platform to produce planar surfaces of bacterial outer membranes, which in turn, could function as a valuable tool for streamlining the development of antibiotics.

Nucleolar dynamics are determined by the ordered assembly of the ribosome.

Ribosome biogenesis occurs in the nucleolus, a nuclear biomolecular condensate that exhibits dynamic biophysical properties thought to be important for function. However, the relationship between ribosome assembly and nucleolar dynamics is incompletely understood. Here, we present a platform for high-throughput fluorescence recovery after photobleaching (HiT-FRAP), which we use to screen hundreds of genes for their impact on dynamics of the nucleolar scaffold nucleophosmin (NPM1). We find that scaffold dynamics and nucleolar morphology respond to disruptions in key stages of ribosome biogenesis. Accumulation of early ribosomal intermediates leads to nucleolar rigidification while late intermediates lead to increased fluidity. We map these biophysical changes to specific ribosomal intermediates and their affinity for NPM1. We also discover that disrupting mRNA processing impacts nucleolar dynamics and ribosome biogenesis. This work mechanistically ties ribosome assembly to the biophysical features of the nucleolus and enables study of how dynamics relate to function across other biomolecular condensates.

Human mesenchymal stem/stromal cell-derived extracellular vesicle transport in meniscus fibrocartilage.

Extracellular vesicles (EVs) derived from endometrial-derived mesenchymal stem/stromal cells (eMSC) play a crucial role in tissue repair due to their immunomodulatory and reparative properties. Given these properties, eMSC EVs may offer potential benefits for meniscal repair. The meniscus, being partly vascularized, relies on diffusivity for solute trafficking. This study focuses on EVs transport properties characterization within fibrocartilage that remains unknown. Specifically, EVs were isolated from Crude and CD146+ eMSC populations. Green fluorescence-labeled EVs transport properties were investigated in three structurally distinct layers (core, femoral, and tibial surfaces) of porcine meniscus. Diffusivity was measured via custom fluorescence recovery after photobleaching (FRAP) technique. Light spectrometry was used to determine EVs solubility. Both Crude and CD146+ eMSC EVs exhibited high purity (>90% CD63CD9 marker expression) and an average diffusivity of 10.924 (±4.065) µm²/s. Importantly, no significant difference was observed between Crude and CD146+ eMSC EV diffusivity on the meniscal layer (p > 0.05). The mean partitioning coefficient was 0.2118 (±0.1321), with Crude EVs demonstrating significantly higher solubility than CD146+ EVs (p < 0.05). In conclusion, this study underscores the potential of both Crude and CD146+ eMSC EVs to traverse all layers of the meniscus, supporting their capacity to enhance delivery of orthobiologics for cartilaginous tissue healing.

Characterization of a Charged Biomimetic Lipid Membrane for Unique Antifouling Effects against Clinically Relevant Matrices in Biosensing.

Clinically relevant matrices such as human blood and serum can cause substantial interference in biosensing measurements, severely compromising the effectiveness of the sensors. We report the characterization of a positively charged lipid membrane that has demonstrated unique features to suppress the nonspecific signal for antifouling effects by using SPR, fluorescence recovery after photobleaching (FRAP), and MALDI-TOF-MS. The ethylphosphocholine (EPC) lipid membrane proved to be exceptionally effective at reducing irreversible interactions from human serum on a Protein A surface. The membrane formation conditions and their effects on membrane fluidity and mobility were characterized for understanding the antifouling functions when various capture molecules were immobilized. Specifically, EPC lipid membranes on a Protein A substrate appear to exhibit a strong interaction, likely through the electrostatic effect with the negatively charged proteins that resulted in a stable hydration layer. The strong interaction also limited lipid mobility, contributing to a robust, protective interface that remained undamaged in undiluted serum. Tailoring a surface with antifouling lipid membranes allows for a range of biosensing applications in highly complex biological media.

Dissecting SOX9 dynamics reveals its differential regulation in osteoarthritis.

The transcription factor SOX9 is integral to tissue homeostasis and is implicated in skeletal malformation, campomelic dysplasia, and osteoarthritis (OA). Despite extensive research, the complete regulatory landscape of SOX9 transcriptional activity, interconnected with signaling pathways (TGFβ, WNT, BMP, IHH, NFκB, and HIF), remains challenging to decipher. This study focuses on elucidating SOX9 signaling in OA pathology using Fluorescence Recovery After Photobleaching (FRAP) to assess SOX9 activity directly in live human primary chondrocytes (hPCs). Single cell FRAP data revealed two distinct subpopulations with differential SOX9 dynamics, showing varied distribution between healthy and OA hPCs. Moreover, inherently elevated SOX9-DNA binding was observed in healthy hPCs compared to preserved and OA counterparts. Anabolic factors (BMP7 and GREM1) and catabolic inhibitors (DKK1 and FRZb) were found to modulate SOX9 transcriptional activity in OA-hPCs. These findings provide valuable insights into the intricate regulation of SOX9 signaling in OA, suggesting potential therapeutic avenues for modulating SOX9 activity in diseased states.

Mechanosensitive recruitment of Vinculin maintains junction integrity and barrier function at epithelial tricellular junctions

Apical cell-cell junctions, including adherens junctions and tight junctions, adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing the embryonic epithelium of gastrula-stage Xenopus laevis embryos, we define a mechanism by which the mechanosensitive protein Vinculin helps anchor the actomyosin network at TCJs, thus maintaining TCJ integrity and barrier function. Using an optogenetic approach to acutely increase junctional tension, we find that Vinculin is mechanosensitively recruited to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that Vinculin KD reduces actin stability at TCJs and destabilizes Angulin-1, a key tricellular tight junction protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJ integrity is not maintained, filamentous actin (F-actin) morphology at TCJs is disrupted, and breaks in the signal of the tight junction protein ZO-1 signal are detected. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs.

The Alphabet Soup of Microscopy: An Introduction to Advanced Imaging Techniques. Part II: What the FLIP, FLAP, FRAP, FRET, and FLIM is Going On?

Abstract The use of fluorescence in imaging has been a pivotal factor in advancing our scientific understanding. As microscopy continues to evolve, the terminology used to describe these techniques becomes increasingly complex, often resulting in a bewildering array of acronyms that resemble alphabet soup. Among the most prominent acronyms are those associated with advanced fluorescence microscopy techniques: Fluorescence Loss in Photobleaching (FLIP), Fluorescence Localization after Photobleaching (FLAP), Fluorescence Recovery after Photobleaching (FRAP), Förster Resonance Energy Transfer (FRET), and Fluorescence Lifetime Imaging Microscopy (FLIM). Each of these methods provides invaluable insights into molecular dynamics and interactions within living cells and tissues. This article, Part 2 of the Alphabet Soup of Microscopy series, aims to clarify these widely used fluorescence microscopy techniques and to illuminate their contributions to our understanding of cellular processes.

Reversible in situ Control over Monolayer Organization.

Cr2+ and Cr3+ ions are shown to mediate the formation, morphology, and organization of arachidic acid (AA) Langmuir-Blodgett (LB) monolayers. This finding, based on cyclic voltammetry (CV), linear sweep voltammetry (LSV) and fluorescence recovery after photobleaching (FRAP) measurements, show that Langmuir monolayer formation depends on subphase pH and metal ion concentration. Following monolayer deposition on indium tin oxide (ITO), the LB monolayer organization can be modified reversibly through control of the Cr oxidation state, which has not been shown before by other monolayers formed with other divalent metal ions. The dynamics and the mobility of a chromophore (perylene) incorporated into the monolayer sense changes in Cr oxidation state-dependent organization of the LB monolayer. Demonstrating reversible changes in monolayer organization provides an opportunity to control chemical and electron access to the interface support.