Assessment of Viability of Listeria monocytogenes by Flow Cytometry.

In food industry, Listeria monocytogenes contamination can occur accidentally despite the quality control of raw materials and factory. Decontamination processes or inhibitory effects of ingredients/additives in food products are set up to ensure compliance with hygiene and microbiological criteria. These actions represent stresses for the pathogenic agent, causing fluctuations in its physiological states. Moreover, during these environmental stresses, Listeria monocytogenes can enter in a viable but nonculturable (VBNC) state which is not detected by plate counting but by flow cytometry. This technique coupled with cell staining by fluorescent dyes offers the possibility to assess different physiological states based on different cellular parameters: enzymatic activity, transmembrane integrity, membrane potential, and respiratory activity. In this chapter, we present a method to assess the viability of foodborne pathogens using a double-staining principle based on the assessment of membrane integrity and intracellular esterase activity.

Characterization of Bacterial Membrane Fatty Acid Profiles for Biofilm Cells.

When submitted to environmental stresses, bacteria can modulate its fatty acid composition of membrane phospholipids in order to optimize membrane fluidity. Characterization of bacterial membrane fatty acid profiles is thus an interesting indicator of cellular physiological state. The methodology described here aims to improve the recovering of biofilm cells for the characterization of their fatty acid profiles. The saponification reagent is directly applied on the whole biofilm before the removal of cells from the inert surface. In this way, maximum of the cells and their fatty acids can be recovered from the deepest layers of the biofilm.

Astroglial membrane camouflaged Ptbp1 siRNA delivery hinders glutamate homeostasis via SDH/Nrf2 pathway.

Polypyrimidine tract-binding protein 1 (PTBP1) regulates numerous alternative splicing events during tumor progression and neurogenesis. Previously, PTBP1 downregulation was reported to convert astrocytes into functional neurons; however, how PTBP1 regulates astrocytic physiology remains unclear. In this study, we revealed that PTBP1 modulated glutamate uptake via ATP1a2, a member of Na+/K+-ATPases, and glutamate transporters in astrocytes. Ptbp1 knockdown altered mitochondrial function and energy metabolism, which involved PTBP1 regulating mitochondrial redox homeostasis via the succinate dehydrogenase (SDH)/Nrf2 pathway. The malfunction of glutamate transporters following Ptbp1 knockdown resulted in enhanced excitatory synaptic transmission in the cortex. Notably, we developed a biomimetic cationic triblock polypeptide system, i.e., polyethylene glycol44-polylysine30-polyleucine10 (PEG44-PLL30-PLLeu10) with astrocytic membrane coating to deliver Ptbp1 siRNA in vitro and in vivo, which approach allowed Ptbp1 siRNA to efficiently cross the blood-brain barrier and target astrocytes in the brain. Collectively, our findings suggest a framework whereby PTBP1 serves as a modulator in glutamate transport machinery, and indicate that biomimetic methodology is a promising route for in vivo siRNA delivery.

Insights into phosphoethanolamine cellulose synthesis and secretion across the Gram-negative cell envelope.

Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the catalytically active BcsA enzyme, a hexameric semicircle of the periplasmic BcsB protein, and the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a periplasmic cellulase of unknown biological function. While cellulose synthesis and translocation by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three BcsG copies near the nascent cellulose polymer. Further, the semicircle's terminal BcsB subunit tethers the N-terminus of a single BcsC protein in a trans-envelope secretion system. BcsC's TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of translocation incompetent cellulose. Lastly, protein engineering introduces cellulose pEtN modification in orthogonal cellulose biosynthetic systems. These findings advance our understanding of pEtN cellulose modification and secretion.

Novel phosphatidylinositol flippases contribute to phosphoinositide homeostasis in the plasma membrane.

Phosphatidylinositol is a precursor of various phosphoinositides, which play crucial roles in intracellular signaling and membrane dynamics and have impact on diverse aspects of cell physiology. Phosphoinositide synthesis and turnover occur in the cytoplasmic leaflet of the organellar and plasma membranes. P4-ATPases (lipid flippases) are responsible for translocating membrane lipids from the exoplasmic (luminal) to the cytoplasmic leaflet, thereby regulating membrane asymmetry. However, the mechanism underlying phosphatidylinositol translocation across cellular membranes remains elusive. Here, we discovered that the phosphatidylcholine flippases ATP8B1, ATP8B2, and ATP10A can also translocate phosphatidylinositol at the plasma membrane. To explore the function of these phosphatidylinositol flippases, we used cells depleted of CDC50A, a protein necessary for P4-ATPase function and ATP8B1 and ATP8B2, which express in HeLa cells. Upon activation of the Gq-coupled receptor, depletion of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was accelerated in CDC50A knockout (KO) and ATP8B1/8B2 double KO cells compared with control cells, suggesting a decrease in PtdIns(4,5)P2 levels within the plasma membrane of the KO cells upon stimulation. These findings highlight the important role of P4-ATPases in maintaining phosphoinositide homeostasis and suggest a mechanism for asymmetry of phosphatidylinositol in the cytoplasmic leaflet of the plasma membrane.

Toxoplasma FER1 is a versatile and dynamic mediator of differential microneme trafficking and microneme exocytosis.

Toxoplasma gondii is a polarized cell concentrating several secretory organelles at the apical pole. The secretory micronemes come in two sub-populations differentiated by dependence on Rab5A/C in their biogenesis. Calcium-dependent exocytosis of micronemes occurs at the very apical tip and is critical for parasite egress from its host cell, adhesion and invasion of the next cell. Ferlins represent a protein family with roles in exocytosis containing multiple Ca2+-sensing C2 domains. We determined that T. gondii's ferlin 1 (FER1) localized dynamically to the parasite's secretory pathway. FER1 function was dissected by dominant negative overexpression strategies. We demonstrated that FER1 traffics microneme organelles along the following trajectories: (1) Along the cortex to the apical end; (2) To the apical tip for fusion with the plasma membrane; (3) Differential microneme sub-population traffic, and that FER1 could putatively be responsible for microneme protein trafficking. (4) From the trans-Golgi-endosomal network to the subpellicular cortex; (5) Retrograde transport allowing microneme recycling from mother to daughter. Finally, FER1 overexpression triggers a microneme exocytosis burst, supporting the notion that the radially organized micronemes at the apical tip comprise a readily-releasable microneme pool. In summary, FER1 is pivotal for dynamic microneme trafficking, acts differently on the two microneme subpopulations, and acts on the plasma membrane fusion step during microneme exocytosis.

Lemon Juice and Peel Constituents Potently Stabilize Rat Peritoneal Mast Cells.

BACKGROUND/AIMS: Lemons (Citrus limon ) contain various nutrients and are among the most popular citrus fruit. Besides their antioxidant, anticancer, antibacterial, and anti-inflammatory properties, clinical studies have indicated their anti-allergic properties. METHODS: Using the differential-interference contrast (DIC) microscopy, we examined the effects of lemon juice and peel constituents, such as citric acid, ascorbic acid, hesperetin and eriodictyol, on the degranulation from rat peritoneal mast cells. Using fluorescence imaging with a water-soluble dye, Lucifer Yellow, we also examined their effects on the deformation of the plasma membrane. RESULTS: Lemon juice dose-dependently decreased the number of degranulated mast cells. At concentrations equal to or higher than 0.25 mM, citric acid, hesperetin, and eriodictyol significantly reduced the number of degranulating mast cells in a dose-dependent manner, while ascorbic acid required much higher doses to exert significant effects. At 1 mM, citric acid, hesperetin, and eriodictyol almost completely inhibited exocytosis and washed out the Lucifer Yellow trapped on the mast cell surface, while ascorbic acid did not. CONCLUSION: This study provides in vitro evidence for the first time that lemon constituents, such as citric acid, hesperetin, and eriodictyol, potently exert mast cell-stabilizing properties. These properties are attributable to their inhibitory effects on plasma membrane deformation in degranulating mast cells.

Membrane remodeling via ubiquitin-mediated pathways.

The dynamic process of membrane shaping and remodeling plays a vital role in cellular functions, with proteins and cellular membranes interacting intricately to adapt to various cellular needs and environmental cues. Ubiquitination-a posttranslational modification-was shown to be essential in regulating membrane structure and shape. It influences virtually all pathways relying on cellular membranes, such as endocytosis and autophagy by directing protein degradation, sorting, and oligomerization. Ubiquitin is mostly known as a protein modifier; however, it was reported that ubiquitin and ubiquitin-like proteins can associate directly with lipids, affecting membrane curvature and dynamics. In this review, we summarize some of the current knowledge on ubiquitin-mediated membrane remodeling in the context of endocytosis, autophagy, and ER-phagy.

Macrophage membrane-camouflaged biomimetic nanoparticles for rheumatoid arthritis treatment via modulating macrophage polarization.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic joint inflammation and cartilage damage. Current therapeutic strategies often result in side effects, necessitating the development of targeted and safer treatment options. This study introduces a novel nanotherapeutic system, 2-APB@DGP-MM, which utilizes macrophage membrane (MM)-encapsulated nanoparticles (NPs) for the targeted delivery of 2-Aminoethyl diphenylborinate (2-APB) to inflamed joints more effectively. The NPs are designed with a matrix metalloproteinase (MMP)-cleavable peptide, allowing for MMP-responsive drug release within RA microenvironment. Comprehensive in vitro and in vivo assays confirmed the successful synthesis and loading of 2-APB into the DSPE-GPLGVRGC-PEG (DGP) NPs, as well as their ability to repolarize macrophages from a pro-inflammatory M1 to an anti-inflammatory M2 phenotype. The NPs demonstrated high biocompatibility, low cytotoxicity, and enhanced cellular uptake. In a collagen-induced arthritis (CIA) mouse model, intra-articular injection of 2-APB@DGP-MM significantly reduced synovial inflammation and cartilage destruction. Histological analysis corroborated these findings, demonstrating marked improvements in joint structure and delayed disease progression. Above all, the 2-APB@DGP-MM nanotherapeutic system offers a promising and safe approach for RA treatment by modulating macrophage polarization and delivering effective agents to inflamed joints.

ER-plasma membrane contact sites deliver ER lipids and proteins for rapid cell surface expansion.

As a consequence of hypoosmotic shock, yeast cells swell rapidly and increase the surface area by ∼20% in 20 s. Approximately, 35% of this surface increase is mediated by the ER-plasma membrane contact sites, specifically the tricalbins, which are required for the delivery of both lipids and the GPI-anchored protein Crh2 from the cortical ER to the plasma membrane. Therefore, we propose a new function for the tricalbins: mediating the fusion of the ER to the plasma membrane at contact sites. This proposed fusion is triggered by calcium influx via the stretch-gated channel Cch1 and is supported by the anoctamin Ist2.

Cancer cell stiffening via CoQ10 and UBIAD1 regulates ECM signaling and ferroptosis in breast cancer.

CoQ10 (Coenzyme Q10) is an essential fat-soluble metabolite that plays a key role in cellular metabolism. A less-known function of CoQ10 is whether it may act as a plasma membrane-stabilizing agent and whether this property can affect cancer development and progression. Here, we show that CoQ10 and its biosynthetic enzyme UBIAD1 play a critical role in plasmamembrane mechanical properties that are of interest for breast cancer (BC) progression and treatment. CoQ10 and UBIAD1 increase membrane fluidity leading to increased cell stiffness in BC. Furthermore, CoQ10 and UBIAD1 states impair ECM (extracellular matrix)-mediated oncogenic signaling and reduce ferroptosis resistance in BC settings. Analyses on human patients and mouse models reveal that UBIAD1 loss is associated with BC development and progression and UBIAD1 expression in BC limits CTCs (circulating tumor cells) survival and lung metastasis formation. Overall, this study reveals that CoQ10 and UBIAD1 can be further investigated to develop therapeutic interventions to treat BC patients with poor prognosis.

Cell swelling enhances ligand-driven ß-adrenergic signaling.

G protein-coupled receptors' conformational landscape can be affected by their local, microscopic interactions within the cell plasma membrane. We employ here a pleiotropic stimulus, namely osmotic swelling, to alter the cortical environment within intact cells and monitor the response in terms of receptor function and downstream signaling. We observe that in osmotically swollen cells the ß2-adrenergic receptor, a prototypical GPCR, favors an active conformation, resulting in cAMP transient responses to adrenergic stimulation that have increased amplitude. The results are validated in primary cell types such as adult cardiomyocytes, a model system where swelling occurs upon ischemia-reperfusion injury. Our results suggest that receptors' function is finely modulated by their biophysical context, and specifically that osmotic swelling acts as a potentiator of downstream signaling, not only for the ß2-adrenergic receptor, but also for other receptors, hinting at a more general regulatory mechanism.

Uptake of substances into living mammalian cells by microwave induced perturbation of the plasma membrane.

Delivering foreign molecules and genetic material into cells is a crucial process in life sciences and biotechnology, resulting in great interest in effective cell transfection methods. Importantly, physical transfection methods allow delivery of molecules of different chemical composition and are, thus, very flexible. Here, we investigated the influence of microwave radiation on the transfection and survival of mammalian cells. We made use of an optimized microwave-poration device and analyzed its performance (frequency and electric field strength) in comparison with simulations. We, then, tested the effect of microwave irradiation on cells and found that 18 GHz had the least impact on cell survival, viability, cell division and genotoxicity while 10 GHz drastically impacted cell physiology. Using live-cell fluorescence microscopy and image analysis, we tested the uptake of small chemical substances, which was most efficient at 18 GHz and correlated with electric field strength and frequency. Finally, we were able to obtain cellular uptake of molecules of very different chemical composition and sizes up to whole immunoglobulin antibodies. In conclusion, microwave-induced poration enables the uptake of widely different substances directly into mammalian cells growing as adherent cultures and with low physiological impact.

Insights into Alphaproteobacterial regulators of cell envelope remodeling.

The cell envelope is at the center of many processes essential for bacterial lifestyles. In addition to giving bacteria shape and delineating it from the environment, it contains macromolecules important for energy transduction, cell division, protection against toxins, biofilm formation, or virulence. Hence, many systems coordinate different processes within the cell envelope to ensure function and integrity. Two-component systems have been identified as crucial regulators of cell envelope functions over the last few years. In this review, we summarize the new information obtained on the regulation of cell envelope biosynthesis and homeostasis in α-proteobacteria, as well as newly identified targets that coordinate the processes in the cell envelope.

Plasma membrane damage limits cytoplasmic delivery by conventional cell penetrating peptides.

Intracellular delivery of large molecule cargo via cell penetrating peptides (CPPs) is an inefficient process and despite intense efforts in past decades, improvements in efficiency have been marginal. Utilizing a standardized and comparative analysis of the delivery efficiency of previously described cationic, anionic, and amphiphilic CPPs, we demonstrate that the delivery ceiling is accompanied by irreparable plasma membrane damage that is part of the uptake mechanism. As a consequence, intracellular delivery correlates with cell toxicity and is more efficient for smaller peptides than for large molecule cargo. The delivery of pharmaceutically relevant cargo quantities with acceptable toxicity thus seems hard to achieve with the CPPs tested in our study. Our results suggest that any engineered intracellular delivery system based on conventional cationic or amphiphilic CPPs, or the design principles underlying them, needs to accept low delivery yields due to toxicity limiting efficient cytoplasmic uptake. Novel peptide designs based on detailed study of uptake mechanisms are required to overcome these limitations.

Cell-Int: a cell-cell interaction assay to identify native membrane protein interactions.

Intercellular protein-protein interactions (PPIs) have pivotal roles in biological functions and diseases. Membrane proteins are therefore a major class of drug targets. However, studying such intercellular PPIs is challenging because of the properties of membrane proteins. Current methods commonly use purified or modified proteins that are not physiologically relevant and hence might mischaracterize interactions occurring in vivo. Here, we describe Cell-Int: a cell interaction assay for studying plasma membrane PPIs. The interaction signal is measured through conjugate formation between two populations of cells each expressing either a ligand or a receptor. In these settings, membrane proteins are in their native environment thus being physiologically relevant. Cell-Int has been applied to the study of diverse protein partners, and enables to investigate the inhibitory potential of blocking antibodies, as well as the retargeting of fusion proteins for therapeutic development. The assay was also validated for screening applications and could serve as a platform for identifying new protein interactors.

Singlet oxygen detection in vivo is hindered by nonspecific SOSG staining.

Singlet oxygen is considered an important cell damaging agent due to its propensity to react with organic compounds. This drives the interest in developing methods for determination of 1O2. Simplicity of application and high sensitivity makes fluorescent probes a popular choice for in vivo 1O2 detection. Despite its proclaimed cell-impermeability, the commercially available Singlet Oxygen Sensor Green (SOSG) is widely applied to support assertions of 1O2 involvement in cell and tissue damage. Our investigation, however, demonstrate that different microbial species and cancer cells become fluorescent when exposed to SOSG under conditions which exclude generation of 1O2. Cells, permeabilized with chlorhexidine or by heat exposure under anaerobic conditions, exhibited SOSG fluorescence. Permeabilized cells could be stained with SOSG even 24 h post-permeabilization. Since SOSG is cell impermeable, the main factor that led to fluorescent staining was plasma membrane damage. Spectral analyses of different batches of SOSG revealed that SOSG endoperoxide (SOSG-EP) did not increase even after prolonged storage under the recommended conditions. The commercial preparations of SOSG, however, were not SOSG-EP free, which can produce erroneous results when SOSG staining is used as a proof of singlet oxygen production in vivo.

Cell-Penetrating Peptides Translocate across the Plasma Membrane by Inducing Vesicle Budding and Collapse.

Cell-penetrating peptides (CPPs) enter the cell by two different mechanisms-endocytosis followed by endosomal escape and direct translocation at the plasma membrane. The mechanism of direct translocation remains unresolved. In this work, the direct translocation of nonaarginine (R9) and two cyclic CPPs (CPP12 and CPP17) into Jurkat cells was monitored by time-lapse confocal microscopy. Our results provide direct evidence that all three CPPs translocate across the plasma membrane by a recently discovered vesicle budding-and-collapse (VBC) mechanism. Membrane translocation is preceded by the formation of nucleation zones. Up to four different types of nucleation zones and three variations of the VBC mechanism were observed. The VBC mechanism reconciles the enigmatic and conflicting observations in the literature.

MeCP2 is a naturally supercharged protein with cell membrane transduction capabilities.

The intrinsically disordered protein MeCP2 is a global transcriptional regulator encoded by the MECP2 gene. Although the structured domains of MeCP2 have been the subject of multiple studies, its unstructured regions have not been that extensively characterized. In this work, we show that MeCP2 possesses properties akin to those of supercharged proteins. By utilizing its unstructured portions, MeCP2 can successfully transduce across cell membranes and localize to heterochromatic foci in the nuclei, displaying uptake levels a third lower than a MeCP2 construct fused to the cell-penetrating peptide TAT. MeCP2 uptake can further be enhanced by the addition of compounds that promote endosomal escape following cellular trafficking by means of macropinocytosis. Using a combination of in silico prediction algorithms and live-cell imaging experiments, we mapped the sequence in MeCP2 responsible for its cellular incorporation, which bears a striking resemblance to TAT itself. Transduced MeCP2 was shown to interact with HDAC3. These findings provide valuable insight into the properties of MeCP2 and may be beneficial for devising future protein-based treatment strategies.

Endomembrane trafficking driven by microtubule growth regulates stomatal movement in Arabidopsis.

Microtubule-based vesicle trafficking usually relies upon kinesin and dynein motors and few reports describe microtubule polymerisation driving directional vesicle trafficking. Here we show that Arabidopsis END BINDING1b (EB1b), a microtubule plus-end binding protein, directly interacts with SYP121, a SNARE protein that mediates the trafficking of the K+ channel KAT1 and its distribution to the plasma membrane (PM) in Arabidopsis guard cells. Knockout of AtEB1b and its homologous proteins results in a modest but significant change in the distribution of KAT1 and SYP121 in guard cells and consequently delays light-induced stomatal opening. Live-cell imaging reveals that a portion of SYP121-associated endomembrane compartments co-localise with AtEB1b at the growing ends of microtubules, trafficking along with the growth of microtubules for targeting to the PM. Our study reveals a mechanism of vesicle trafficking driven by microtubule growth, which is involved in the redistribution of PM proteins to modulate guard cell movement.