Search Results (48)

In epithelial cells, nonmuscle myosin-2B (NM2B) shows a cortical localization and is tethered to tight junctions (TJs) and adherens junctions (AJs) by the junctional adaptor proteins cingulin and paracingulin. MDCK cells knock-out (KO) for cingulin show decreased apical membrane cortex stiffness and decreased TJ membrane tortuosity, and the rescue of these phenotypes requires the myosin-binding region of cingulin. Here, we investigated whether NM2B contributes to these phenotypes independently of cingulin by generating and characterizing clonal lines of MDCK cells KO for NM2B. The loss of NM2B resulted in decreased stiffness and increased fluidity of the apical cortex and reduced accumulation of E-cadherin and phalloidin-labeled actin filaments at junctions but had no significant effect on TJ membrane tortuosity. Fluorescence recovery after photobleaching (FRAP) showed that the KO of NM2B increased the dynamics of the TJ scaffold protein ZO-1, correlating with decreased ZO-1 accumulation at TJs. Finally, the KO of NM2B increased cell size in cells grown both in 2D and 3D but did not alter lumen morphogenesis of cysts. These results extend our understanding of the functions of NM2B by describing its role in the regulation of the mechanical properties of the apical membrane cortex and cell size and validate our model about the role of cingulin–NM2B interaction in the regulation of ZO-1 dynamics. Full article

Capsella bursa-pastoris Medik. (CBP) is a species with antibacterial, anti-inflammatory, antioxidant, anticancer, and hepatoprotective effects. We have chosen to study this species because, although it is a common plant with a distinctive fruit appearance, its effects are not fully understood. The aim of this study was to characterize the histoanatomy of the vegetative, reproductive organs and to characterize CBP extracts in terms of bioactive compounds and its antioxidant capacity. This study investigated the quantitative chemical composition of this species using the HPLC method, revealing the total content in polyphenols, flavonoids, and anthocyanins, and investigated the antioxidant potential through fluorescence recovery after photobleaching (FRAP assay), cupric ion (Cu²⁺) reduction, (CUPRAC assay), and a free radical scavenging method (DPPH). Our results show that CBP is a rich source of flavonoids, mainly from the extract obtained from the fruits; it has an antioxidant capacity, with the highest values being obtained from mature flowers and ripe fruits. Of the active principles, the highest amounts, according to HPLC determinations, were obtained in flowers and are represented by hyperoside. Thus, we can recommend the studied species for phytopharmaceutical preparations. Full article

Advanced methods of treatment are needed to fight the threats of virus-transmitted diseases and pandemics. Often, they are based on an improved biophysical understanding of virus replication strategies and processes in their host cells. For instance, an essential component of the replication of the hepatitis C virus (HCV) proceeds under the influence of nonstructural HCV proteins (NSPs) that are anchored to the endoplasmatic reticulum (ER), such as the NS5A protein. The diffusion of NSPs has been studied by in vitro fluorescence recovery after photobleaching (FRAP) experiments. The diffusive evolution of the concentration field of NSPs on the ER can be described by means of surface partial differential equations (sufPDEs). Previous work estimated the diffusion coefficient of the NS5A protein by minimizing the discrepancy between an extended set of sufPDE simulations and experimental FRAP time-series data. Here, we provide a scaling analysis of the sufPDEs that describe the diffusive evolution of the concentration field of NSPs on the ER. This analysis provides an estimate of the diffusion coefficient that is based only on the ratio of the membrane surface area in the FRAP region to its contour length. The quality of this estimate is explored by a comparison to numerical solutions of the sufPDE for a flat geometry and for ten different 3D embedded 2D ER grids that are derived from fluorescence z-stack data of the ER. Finally, we apply the new data analysis to the experimental FRAP time-series data analyzed in our previous paper, and we discuss the opportunities of the new approach. Full article

Mutations in activin-like kinase 2 (ALK2), e.g., ALK2-R206H, induce aberrant signaling to SMAD1/5/8, leading to Fibrodysplasia Ossificans Progressiva (FOP). In spite of extensive studies, the underlying mechanism is still unclear. Here, we quantified the homomeric and heteromeric interactions of ACVR2A, ACVR2B, ALK2-WT, and ALK2-R206H by combining IgG-mediated immobilization of one receptor with fluorescence recovery after photobleaching (FRAP) measurements on the lateral diffusion of a co-expressed receptor. ACVR2B formed stable homomeric complexes that were enhanced by Activin A (ActA), while ACVR2A required ActA for homodimerization. ALK2-WT, but not ALK2-R206H, exhibited homomeric complexes unaffected by ActA. ACVR2B formed ActA-enhanced heterocomplexes with ALK2-R206H or ALK2-WT, while ACVR2A interacted mainly with ALK2-WT. The extent of the homomeric complex formation of ACVR2A or ACVR2B was reflected in their ability to induce the oligomerization of ALK2-R206H and ALK2-WT. Thus, ACVR2B, which forms dimers without ligand, induced ActA-independent ALK2-R206H clustering but required ActA for enhancing the oligomerization of the largely dimeric ALK2-WT. In contrast, ACVR2A, which undergoes homodimerization in response to ActA, required ActA to induce ALK2-R206H oligomerization. To investigate whether these interactions are translated into signaling, we studied signaling by the FOP-inducing hyperactive ALK2-R206H mutant, with ALK2-WT signaling as control. The activation of SMAD1/5/8 signaling in cells expressing ALK2-R206H alone or together with ACVR2A or ACVR2B was measured by blotting for pSMAD1/5/8 and by transcriptional activation assays using BRE-Luc reporter. In line with the biophysical studies, ACVR2B activated ALK2-R206H without ligand, while activation by ACVR2A was weaker and required ActA. We propose that the homodimerization of ACVR2B or ACVR2A dictates their ability to recruit ALK2-R206H into higher complexes, enabling the homomeric interactions of ALK2-R206H receptors and, subsequently, their activation. Full article

In recent years, the role of liquid–liquid phase separation (LLPS) and intrinsically disordered proteins (IDPs) in cellular molecular processes has received increasing attention from researchers. One such intrinsically disordered protein is TSPYL5, considered both as a marker and a potential therapeutic target for various oncological diseases. However, the role of TSPYL5 in intracellular processes remains unknown, and there is no clarity even in its intracellular localization. In this study, we characterized the intracellular localization and exchange dynamics with intracellular contents of TSPYL5 and its parts, utilizing TSPYL5 fusion proteins with EGFP. Our findings reveal that TSPYL5 can be localized in both the cytoplasm and nucleoplasm, including the nucleolus. The nuclear (nucleolar) localization of TSPYL5 is mediated by the nuclear/nucleolar localization sequences (NLS/NoLS) identified in the N-terminal intrinsically disordered region (4–27 aa), while its cytoplasmic localization is regulated by the ordered NAP-like domain (198–382 aa). Furthermore, our results underscore the significant role of the TSPYL5 N-terminal disordered region (1–198 aa) in the exchange dynamics with the nucleoplasm and its potential ability for phase separation. Bioinformatics analysis of the TSPYL5 interactome indicates its potential function as a histone and ribosomal protein chaperone. Taken together, these findings suggest a significant contribution of liquid–liquid phase separation to the processes involving TSPYL5, providing new insights into the role of this protein in the cell’s molecular life. Full article

Elucidating the dynamics of DNA repair proteins is essential to understanding the mechanisms that preserve genomic stability and prevent carcinogenesis. However, the measurement and modeling of protein dynamics at DNA lesions via currently available image analysis tools is cumbersome. Therefore, we developed CellTool—a stand-alone open-source software with a graphical user interface for the analysis of time-lapse microscopy images. It combines data management, image processing, mathematical modeling, and graphical presentation of data in a single package. Multiple image filters, segmentation, and particle tracking algorithms, combined with direct visualization of the obtained results, make CellTool an ideal application for the comprehensive analysis of DNA repair protein dynamics. This software enables the fitting of obtained kinetic data to predefined or custom mathematical models. Importantly, CellTool provides a platform for easy implementation of custom image analysis packages written in a variety of programing languages. Using CellTool, we demonstrate that the ALKB homolog 2 (ALKBH2) demethylase is excluded from DNA damage sites despite recruitment of its putative interaction partner proliferating cell nuclear antigen (PCNA). Further, CellTool facilitates the straightforward fluorescence recovery after photobleaching (FRAP) analysis of BRCA1 associated RING domain 1 (BARD1) exchange at complex DNA lesions. In summary, the software presented herein enables the time-efficient analysis of a wide range of time-lapse microscopy experiments through a user-friendly interface. Full article

The formation of supported lipid bilayers (SLBs) on hydrogels can act as a biocompatible anti-fouling interface. However, generating continuous and mobile SLBs on materials other than conventional glass or mica remains a significant challenge. The interaction between lipid membrane vesicles and a typical hydrogel is usually insufficient to induce membrane vesicle rupture and form a planar lipid membrane. In this study, we demonstrate that the water absorption ability of a dried polyacrylamide (PAAm) hydrogel could serve as a driving force to facilitate the formation of the hydrogel–SLBs. The absorption driving force vanishes after the hydrogels are fully hydrated, leaving no extra interaction hindering lipid lateral mobility in the formed SLBs. Our fluorescence recovery after photobleaching (FRAP) results show that SLBs only form on hydrogels with adequate absorption abilities. Moreover, we discovered that exposure to oxygen during drying could lead to the formation of an oxidized crust on the PAAm hydrogel surface, impeding SLB formation. Therefore, minimizing oxygen exposure during drying is crucial to achieving high-quality hydrogel surfaces for SLB formation. This water absorption method enables the straightforward fabrication of hydrogel–SLBs without the need for additional substrates or charges, thereby expanding their potential applications. Full article

HES1 (hairy and enhancer of split-1, effector of the NOTCH pathway) plays a role in oocyte maturation and has been detected so far mainly in somatic follicular cells. In this study, we aimed to investigate whether HES1 is present in both compartments of bovine cumulus oocyte complexes (COCs) and whether in vitro maturation itself has an effect on its distribution. We investigated the abundance of HES1 mRNA and protein in bovine COCs characterized by Brilliant-Cresyl-Blue (BCB) stainability by RT-PCR and immunofluorescence before and after in vitro maturation (IVM). To study the interaction of the compartments and the possible translocation of HES1, we injected GFP-HES1 mRNA into oocytes before maturation and analyzed fluorescence recovery after photobleaching (FRAP). The results showed that HES1 mRNA was detectable in oocytes but not in cumulus cells. The number of transcripts increased with maturation, especially in BCB-positive oocytes. In contrast, the protein was mainly visible in cumulus cells both before and after maturation. After GFP-HES1-mRNA injection into oocytes, a signal could be detected not only in the oocytes but also in cumulus cells. Our result shows a nearly exclusive distribution of HES1 mRNA and protein in oocytes and cumulus cells, respectively, that might be explained by the transfer of the protein from the oocyte into cumulus cells. Full article

Noble metal nanoparticles (NP) with intrinsic antiangiogenic, antibacterial, and anti-inflammatory properties have great potential as potent chemotherapeutics, due to their unique features, including plasmonic properties for application in photothermal therapy, and their capability to slow down the migration/invasion speed of cancer cells and then suppress metastasis. In this work, gold (Au), silver (Ag), and palladium (Pd) NP were synthesized by a green redox chemistry method with the reduction of the metal salt precursor with glucose in the presence of polyvinylpyrrolidone (PVP) as stabilizing and capping agent. The physicochemical properties of the PVP-capped NP were investigated by UV-visible (UV-vis) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopies, dynamic light scattering (DLS), and atomic force microscopy (AFM), to scrutinize the optical features and the interface between the metal surface and the capping polymer, the hydrodynamic size, and the morphology, respectively. Biophysical studies with model cell membranes were carried out by using laser scanning confocal microscopy (LSM) with fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET) techniques. To this purpose, artificial cell membranes of supported lipid bilayers (SLBs) made with 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) dye-labeled with 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD, FRET donor) and/or lissamine rhodamine B sulfonyl (Rh, FRET acceptor) were prepared. Proof-of-work in vitro cellular experiments were carried out with prostate cancer cells (PC-3 line) in terms of cytotoxicity, cell migration (wound scratch assay), NP cellular uptake, and cytoskeleton actin perturbation. Full article

The plasma membrane of mammalian cells is involved in a wide variety of cellular processes, including, but not limited to, endocytosis and exocytosis, adhesion and migration, and signaling. The regulation of these processes requires the plasma membrane to be highly organized and dynamic. Much of the plasma membrane organization exists at temporal and spatial scales that cannot be directly observed with fluorescence microscopy. Therefore, approaches that report on the membrane’s physical parameters must often be utilized to infer membrane organization. As discussed here, diffusion measurements are one such approach that has allowed researchers to understand the subresolution organization of the plasma membrane. Fluorescence recovery after photobleaching (or FRAP) is the most widely accessible method for measuring diffusion in a living cell and has proven to be a powerful tool in cell biology research. Here, we discuss the theoretical underpinnings that allow diffusion measurements to be used in elucidating the organization of the plasma membrane. We also discuss the basic FRAP methodology and the mathematical approaches for deriving quantitative measurements from FRAP recovery curves. FRAP is one of many methods used to measure diffusion in live cell membranes; thus, we compare FRAP with two other popular methods: fluorescence correlation microscopy and single-particle tracking. Lastly, we discuss various plasma membrane organization models developed and tested using diffusion measurements. Full article

Neurotrophins (NTs), which are crucial for the functioning of the nervous system, are also known to regulate vascularization. Graphene-based materials may drive neural growth and differentiation, and, thus, have great potential in regenerative medicine. In this work, we scrutinized the nano–biointerface between the cell membrane and hybrids made of neurotrophin-mimicking peptides and graphene oxide (GO) assemblies (pep−GO), to exploit their potential in theranostics (i.e., therapy and imaging/diagnostics) for targeting neurodegenerative diseases (ND) as well as angiogenesis. The pep−GO systems were assembled via spontaneous physisorption onto GO nanosheets of the peptide sequences BDNF(1-12), NT3(1-13), and NGF(1-14), mimicking the brain-derived neurotrophic factor (BDNF), the neurotrophin 3 (NT3), and the nerve growth factor (NGF), respectively. The interaction of pep−GO nanoplatforms at the biointerface with artificial cell membranes was scrutinized both in 3D and 2D by utilizing model phospholipids self-assembled as small unilamellar vesicles (SUVs) or planar-supported lipid bilayers (SLBs), respectively. The experimental studies were paralleled via molecular dynamics (MD) computational analyses. Proof-of-work in vitro cellular experiments with undifferentiated neuroblastoma (SH-SY5Y), neuron-like, differentiated neuroblastoma (dSH-SY5Y), and human umbilical vein endothelial cells (HUVECs) were carried out to shed light on the capability of the pep−GO nanoplatforms to stimulate the neurite outgrowth as well as tubulogenesis and cell migration. Full article

Cholesterol (Chol) is an essential component of animal cell membranes and is most abundant in plasma membranes (PMs) where its concentration typically ranges from 10 to 30 mol%. However, in red blood cells and Schwann cells, PMs Chol content is as high as 50 mol%, and in the PMs of the eye lens fiber cells, it can reach up to 66 mol%. Being amphiphilic, Chol molecules are easily incorporated into the lipid bilayer where they affect the membrane lateral organization and transmembrane physical properties. In the aqueous phase, Chol cannot form free bilayers by itself. However, pure Chol bilayer domains (CBDs) can form in lipid bilayer membranes with the Chol content exceeding 50 mol%. The range of Chol concentrations surpassing 50 mol% is less frequent in biological membranes and is consequently less investigated. Nevertheless, it is significant for the normal functioning of the eye lens and understanding how Chol plaques form in atherosclerosis. The most commonly used membrane models are unilamellar and multilamellar vesicles (MLVs) and supported lipid bilayers (SLBs). CBDs have been observed directly using confocal microscopy, X-ray reflectometry and saturation recovery electron paramagnetic resonance (SR EPR). Indirect evidence of CBDs has also been reported by using atomic force microscopy (AFM) and fluorescence recovery after photobleaching (FRAP) experiments. The overall goal of this review is to demonstrate the advantages and limitations of the various membrane models and experimental techniques suitable for the detection and investigation of the lateral organization, function and physical properties of CBDs. Full article

Odor detection and discrimination in mammals is known to be initiated by membrane-bound G-protein-coupled receptors (GPCRs). The role that the lipid membrane may play in odor discrimination, however, is less well understood. Here, we used model membrane systems to test the hypothesis that phospholipid bilayer membranes may be capable of odor discrimination. The effect of S-carvone, R-carvone, and racemic lilial on the model membrane systems was investigated. The odorants were found to affect the fluidity of supported lipid bilayers as measured by fluorescence recovery after photobleaching (FRAP). The effect of odorants on surface-supported lipid multilayer microarrays of different dimensions was also investigated. The lipid multilayer micro- and nanostructure was highly sensitive to exposure to these odorants. Fluorescently-labeled lipid multilayer droplets of 5-micron diameter were more responsive to these odorants than ethanol controls. Arrays of lipid multilayer diffraction gratings distinguished S-carvone from R-carvone in an artificial nose assay. Our results suggest that lipid bilayer membranes may play a role in odorant discrimination and molecular recognition in general. Full article

Medium Chain Triglyceride (MCT) oil was successfully combined with Glyceryl Monostearate (GMS) and Glyceryl Monoolein (GMO) to form oleogels that were subsequently whipped to form stable oleofoams. The co-crystallization of GMS and GMO at a ratio of 20:1, 20:2.5, and 20:5 within MCT oil was studied through Differential Scanning Calorimetry (DSC), X-ray Diffraction analysis (XRD), rheological analysis, Fluorescence Recovery after Photobleaching (FRAP), Fourier Transform Infrared Spectroscopy (FTIR), and polarized microscopy. The addition of 5% GMO resulted in the production of more stable oleogels in terms of crystal structure and higher peak melting point, rendering this formulation suitable for pharmaceutical applications that are intended to be used internally and those that require stability at temperatures close to 40 °C. All formulations were whipped to form oleofoams that were evaluated for their storage stability for prolonged period at different temperatures. The results show that oleofoams containing 5% MGO retained their foam characteristics even after 3 months of storage under different temperature conditions. Full article

The purposes of this investigatory study were to determine the chemical composition of the essential oils (EOs) of Origanum compactum from two Moroccan regions (Boulemane and Taounate), as well as the evaluation of their biological effects. Determining EOs’ chemical composition was performed by a gas chromatography–mass spectrophotometer (GC-MS). The antioxidant activity of EOs was evaluated using free radical scavenging ability (DPPH method), fluorescence recovery after photobleaching (FRAP), and lipid peroxidation inhibition assays. The anti-inflammatory effect was assessed in vitro using the 5-lipoxygenase (5-LOX) inhibition test and in vivo using the carrageenan-induced paw edema model. Finally, the antibacterial effect was evaluated against several strains using the disk-diffusion assay and the micro-dilution method. The chemical constituent of O. compactum EO (OCEO) from the Boulemane zone is dominated by carvacrol (45.80%), thymol (18.86%), and α-pinene (13.43%). However, OCEO from the Taounate zone is rich in 3-carene (19.56%), thymol (12.98%), and o-cymene (11.16%). OCEO from Taounate showed higher antioxidant activity than EO from Boulemane. Nevertheless, EO from Boulemane considerably inhibited 5-LOX (IC₅₀ = 0.68 ± 0.02 µg/mL) compared to EO from Taounate (IC₅₀ = 1.33 ± 0.01 µg/mL). A similar result was obtained for tyrosinase inhibition with Boulemane EO and Taounate EO, which gave IC_50s of 27.51 ± 0.03 μg/mL and 41.83 ± 0.01 μg/mL, respectively. The in vivo anti-inflammatory test showed promising effects; both EOs inhibit and reduce inflammation in mice. For antibacterial activity, both EOs were found to be significantly active against all strains tested in the disk-diffusion test, but O. compactum EO from the Boulemane region showed the highest activity. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) for O. compactum EO from the Boulemane region ranged from 0.06 to 0.25% (v/v) and from 0.15 to 0.21% (v/v) for O. compactum from the Taounate region. The MBC/MIC index revealed that both EOs exhibited remarkable bactericidal effects. Full article