Membrane Reorganization Research Articles

Septin Organization and Dynamics for Budding Yeast Cytokinesis.

Cytokinesis, the process by which the cytoplasm divides to generate two daughter cells after mitosis, is a crucial stage of the cell cycle. Successful cytokinesis must be coordinated with chromosome segregation and requires the fine orchestration of several processes, such as constriction of the actomyosin ring, membrane reorganization, and, in fungi, cell wall deposition. In Saccharomyces cerevisiae, commonly known as budding yeast, septins play a pivotal role in the control of cytokinesis by assisting the assembly of the cytokinetic machinery at the division site and controlling its activity. Yeast septins form a collar at the division site that undergoes major dynamic transitions during the cell cycle. This review discusses the functions of septins in yeast cytokinesis, their regulation and the implications of their dynamic remodelling for cell division.

Planarian Mucus: A Novel Source of Pleiotropic Cytotoxic and Cytostatic Agents against Cancer Cells.

Biological evolution has generated a vast array of natural compounds produced by organisms across all domains. Among these, secondary metabolites, selected to enhance an organism's competitiveness in its natural environment, make them a reservoir for discovering new compounds with cytotoxic activity, potentially useful as novel anticancer agents. Slime secretions, the first barrier between epithelial surfaces and the surrounding environment, frequently contain cytotoxic molecules to limit the growth of parasitic organisms. Planarians, freshwater Triclads, continuously secrete a viscous mucus with multiple physiological functions. The chemical composition of planarian mucus has been only partially elucidated, and there are no studies reporting its cytotoxic or cytostatic effects. In this study, we developed a protocol for collecting mucus from Dugesia japonica specimens and we demonstrated that it inhibits the growth of cancer cells by activating cytostatic and ROS-dependent cytotoxic mechanisms inducing lipid droplet accumulation and mitochondrial membrane reorganization. Although further research is needed to identify the specific chemicals responsible for the anticancer activity of planarian mucus, this work opens up numerous research avenues aimed at better understanding the mechanisms of action of this product for potential therapeutic applications.

Manganese deficiency alters photosynthetic electron transport in Marchantia polymorpha

Manganese (Mn) is considered as an essential element for plant growth. Mn starvation has been shown to affect photosystem II, the site of the Mn4CaO5 cluster responsible for water oxidation. Less is known on the effect of Mn starvation on photosystem I. Here we studied the effects of Mn deficiency in vivo on redox changes of P700 and plastocyanin (Pc) in the liverwort Marchantia polymorpha using the KLAS-NIR spectrophotometer. Far-red illumination is used to excite preferentially photosystem I, thus facilitating cyclic electron transport. Under Mn starvation, we observed slower oxidation of P700 and a decrease in the Pc signal relative to P700. The lower Pc content under Mn deficiency was confirmed by western blots. Re-reduction kinetics of P700+ and Pc+ were faster in Mn deficient thalli than in the control. The above findings show that the kinetics studied under Mn deficiency not only depend on the number of available reductants but also on how quickly electrons are transferred from stromal donors via the intersystem chain to Pc+ and P700+. We suggest that under Mn deficiency a structural reorganization of the thylakoid membrane takes place favoring the formation of supercomplexes between ferredoxin, cytochrome b6f complex, Pc and photosystem I, and thus an enhanced cyclic electron transport.

Resolving the interactions between hydrophilic CdTe quantum dots and positively charged membranes at the nanoscale

The use of quantum dot nanoparticles (QDs) in bio-applications has gained quite some interest and requires a deep understanding of their interactions with model cell membranes. This involves assessing the extent of nanoparticle disruption of the membrane and how it depends on both nanoparticle and membrane physicochemical properties. Surface charge plays an important role in nanoparticle adsorption, which is primarily driven by electrostatic interactions; yet, once adsorbed, most reported works overlook the subsequent spatial nanoparticle insertion and location within the membrane. There is therefore a need for studies to assess the mutual role of membrane and nanoparticle charge into membrane structure and stability at the nanoscale, with a view to better design and control the functionality of these nanomaterials. In this work, we have resolved the extent of the interactions between hydrophilic, negatively charged CdTe QDs and positively charged lipid bilayers. A multiscale combination of surface-sensitive techniques enabled probing how surface charge mediates QD adsorption and membrane reorganization. Increasing membrane surface charge results into a larger adsorption of oppositely charged QDs, concomitantly inducing structural changes. Hydration of the membrane hydrophobic parts by QDs goes deeper into the inner leaflet with increasing membrane charge, resulting in supported lipid bilayers with decreased nanomechanical stability.

Ratiometric Fluorescence Probes for In Situ Imaging of Membrane Tension in Live Cells.

Membrane tension is an important physical parameter of describing cellular homeostasis, and it is widely used in the study of cellular processes involving membrane deformation and reorganization, such as cell migration, cell spreading, and cell division. Despite the importance of membrane tension, direct measurement remains difficult. In this work, we developed a ratiometric fluorescent probe sensitive to membrane tension by adjusting the carbon chain structure based on polarity-sensitive fluorophores. The probe is sensitive to changes in membrane tension after cells were subjected to physical or chemical stimuli, such as osmotic shock, lipid peroxidation, and mechanical stress. When the polarity of the plasma membrane increases (the green/red ratio decreases) and the membrane tension increases, the relative magnitude of the membrane tension can be quantitatively calculated by fluorescence ratio imaging. Thus, the probe proved to be an efficient and sensitive membrane tension probe.

Arginine-Rich Cell-Penetrating Peptides Induce Lipid Rearrangements for Their Active Translocation across Laterally Heterogeneous Membranes.

Arginine-rich cell-penetrating peptides (CPPs) have emerged as valuable tools for the intracellular delivery of bioactive molecules, but their membrane perturbation during cell penetration is not fully understood. Here, nona-arginine (R9)-mediated membrane reorganization that facilitates the translocation of peptides across laterally heterogeneous membranes is directly visualized. The electrostatic binding of cationic R9 to anionic phosphatidylserine (PS)-enriched domains on a freestanding lipid bilayer induces lateral lipid rearrangements; in particular, in real-time it is observed that R9 fluidizes PS-rich liquid-ordered (Lo) domains into liquid-disordered (Ld) domains, resulting in the membrane permeabilization. The experiments with giant unilamellar vesicles (GUVs) confirm the preferential translocation of R9 through Ld domains without pore formation, even when Lo domains are more negatively charged. Indeed, whenever R9 comes into contact with negatively charged Lo domains, it dissolves the Lo domains first, promoting translocation across phase-separated membranes. Collectively, the findings imply that arginine-rich CPPs modulate lateral membrane heterogeneity, including membrane fluidization, as one of the fundamental processes for their effective cell penetration across densely packed lipid bilayers.

Photocleavable Polymer Cubosomes: Synthesis, Self-Assembly, and Photorelease.

Polymer cubosomes (PCs) are a recent class of self-assembled block copolymer (BCP) microparticles with an accessible periodic channel system. Most reported PCs consist of a polystyrene scaffold, which provides mechanical stability for templating but has a limited intrinsic functionality. Here, we report the synthesis of photocleavable BCPs with compositions suitable for PC formation. We analyze the self-assembly mechanism and study the model release of dyes during irradiation, where the transition of the BCPs from amphiphilic to bishydrophilic causes the rapid disassembly of the PCs. A combination of modeling and experiment shows that the evolution of PCs proceeds first via liquid-liquid phase separation into polymer-rich droplets, followed by microphase separation within this droplet confinement, and finally, membrane reorganization into high internal order. This insight may encourage exploration of alternative preparation strategies to better control the size and homogeneity of PCs.

How Does the Antibiotic Amphotericin B Enter Membranes and What Does It Do There?

Amphotericin B is a popular antifungal antibiotic, but the exact way it works is still a matter of debate. Here, we used monolayers composed of phosphatidylcholine with ergosterol as a model of fungal lipid membranes to study drug incorporation from the aqueous phase and analyze the molecular reorganization of membranes underlying the biological activity of the antibiotic. The results show that the internalization of antibiotic molecules into membranes occurs only in the presence of ergosterol in the lipid phase. Comparison of images of solid-supported monolayers obtained by atomic force microscopy and lifetime imaging fluorescence microscopy shows the formation of intramembrane clusters of various sizes in the lipid phase, consisting mainly of antibiotic dimers and relatively large membrane pores (∼15 nm in diameter). The results reveal multiple modes of action of amphotericin B, acting simultaneously, each of which adversely affects the structural properties of the lipid membranes and their physiological functionality.

Changes in lipid metabolism track with the progression of neurofibrillary pathology in tauopathies

BackgroundAccumulation of tau leads to neuroinflammation and neuronal cell death in tauopathies, including Alzheimer’s disease. As the disease progresses, there is a decline in brain energy metabolism. However, the role of tau protein in regulating lipid metabolism remains less characterized and poorly understood.MethodsWe used a transgenic rat model for tauopathy to reveal metabolic alterations induced by neurofibrillary pathology. Transgenic rats express a tau fragment truncated at the N- and C-terminals. For phenotypic profiling, we performed targeted metabolomic and lipidomic analysis of brain tissue, CSF, and plasma, based on the LC-MS platform. To monitor disease progression, we employed samples from transgenic and control rats aged 4, 6, 8, 10, 12, and 14 months. To study neuron-glia interplay in lipidome changes induced by pathological tau we used well well-established multicomponent cell model system. Univariate and multivariate statistical approaches were used for data evaluation.ResultsWe showed that tau has an important role in the deregulation of lipid metabolism. In the lipidomic study, pathological tau was associated with higher production of lipids participating in protein fibrillization, membrane reorganization, and inflammation. Interestingly, significant changes have been found in the early stages of tauopathy before the formation of high-molecular-weight tau aggregates and neurofibrillary pathology. Increased secretion of pathological tau protein in vivo and in vitro induced upregulated production of phospholipids and sphingolipids and accumulation of lipid droplets in microglia. We also found that this process depended on the amount of extracellular tau. During the later stages of tauopathy, we found a connection between the transition of tau into an insoluble fraction and changes in brain metabolism.ConclusionOur results revealed that lipid metabolism is significantly affected during different stages of tau pathology. Thus, our results demonstrate that the dysregulation of lipid composition by pathological tau disrupts the microenvironment, further contributing to the propagation of pathology.

Time dependence of membrane reorganization in antigen-stimulated mast cell signaling as monitored by boxcar imaging fluorescence correlation spectroscopy

Time dependence of membrane reorganization in antigen-stimulated mast cell signaling as monitored by boxcar imaging fluorescence correlation spectroscopy

Proximity to Photosystem II is necessary for activation of Plastid Terminal Oxidase (PTOX) for photoprotection

The Plastid Terminal Oxidase (PTOX) is a chloroplast localized plastoquinone oxygen oxidoreductase suggested to have the potential to act as a photoprotective safety valve for photosynthesis. However, PTOX overexpression in plants has been unsuccessful at inducing photoprotection, and the factors that control its activity remain elusive. Here, we show that significant PTOX activity is induced in response to high light in the model species Eutrema salsugineum and Arabidopsis thaliana. This activation correlates with structural reorganization of the thylakoid membrane. Over-expression of PTOX in mutants of Arabidopsis thaliana perturbed in thylakoid stacking also results in such activity, in contrast to wild type plants with normal granal structure. Further, PTOX activation protects against photoinhibition of Photosystem II and reduces reactive oxygen production under stress conditions. We conclude that structural re-arrangements of the thylakoid membranes, bringing Photosystem II and PTOX into proximity, are both required and sufficient for PTOX to act as a Photosystem II sink and play a role in photoprotection.

Structural Changes Induced by Resveratrol in Monounsaturated and Polyunsaturated Phosphatidylcholine-Enriched Model Membranes.

Resveratrol (Resv) is considered to exert a beneficial impact due to its radical scavenger, anti-microbial and anti-inflammatory properties through several mechanisms that could include its interaction with the cell plasma membrane. To address this issue, we investigated the influence of Resv on membrane lipid order and organization in large unilamellar vesicles composed of different lipids and ratios. The studied lipid membrane models were composed of phosphatidylcholine (PC) species (either palmitoyl-docosahexaenoyl phosphatidylcholine (PDPC) or palmitoyl-oleoyl phosphatidylcholine (POPC)), sphingomyelin (SM) and cholesterol (Chol). This study found that the addition of Resv resulted in complex membrane reorganization depending on the degree of fatty acid unsaturation at the sn-2 position, and the Lipid/Resv and SM/Chol ratios. Resv rigidified POPC-containing membranes and increased liquid-ordered (Lo) domain formation in 40/40/20 POPC/SM/Chol mixtures as this increase was lower at a 33/33/34 ratio. In contrast, Resv interacted with PDPC/SM/Chol mixtures in a bimodal manner by fluidizing/rigidifying the membranes in a dose-dependent way. Lo domain formation upon Resv addition occurred via the following bimodal mode of action: Lo domain size increased at low Resv concentrations; then, Lo domain size decreased at higher ones. To account for the variable effect of Resv, we suggest that it may act as a "spacer" at low doses, with a transition to a more "filler" position in the lipid bulk. We hypothesize that one of the roles of Resv is to tune the lipid order and organization of cell plasma membranes, which is closely linked to important cell functions such as membrane sorting and trafficking.

Encystation stimuli sensing is mediated by adenylate cyclase AC2-dependent cAMP signaling in Giardia

Protozoan parasites use cAMP signaling to precisely regulate the place and time of developmental differentiation, yet it is unclear how this signaling is initiated. Encystation of the intestinal parasite Giardia lamblia can be activated by multiple stimuli, which we hypothesize result in a common physiological change. We demonstrate that bile alters plasma membrane fluidity by reducing cholesterol-rich lipid microdomains, while alkaline pH enhances bile function. Through depletion of the cAMP producing enzyme Adenylate Cyclase 2 (AC2) and the use of a newly developed Giardia-specific cAMP sensor, we show that AC2 is necessary for encystation stimuli-induced cAMP upregulation and activation of downstream signaling. Conversely, over expression of AC2 or exogenous cAMP were sufficient to initiate encystation. Our findings indicate that encystation stimuli induce membrane reorganization, trigger AC2-dependent cAMP upregulation, and initiate encystation-specific gene expression, thereby advancing our understanding of a critical stage in the life cycle of a globally important parasite.

Arf6 as a therapeutic target: Structure, mechanism, and inhibitors.

ADP-ribosylation factor 6 (Arf6), a small G-protein of the Ras superfamily, plays pivotal roles in multiple cellular events, including exocytosis, endocytosis, actin remodeling, plasma membrane reorganization and vesicular transport. Arf6 regulates the progression of cancer through the activation of cell motility and invasion. Aberrant Arf6 activation is a potential therapeutic target. This review aims to understand the comprehensive function of Arf6 for future cancer therapy. The Arf6 GEFs, protein structure, and roles in cancer have been summarized. Comprehending the mechanism underlying Arf6-mediated cancer cell growth and survival is essential. The structural features of Arf6 and its efforts are discussed and may be contributed to the discovery of future novel protein-protein interaction inhibitors. In addition, Arf6 inhibitors and mechanism of action are listed in the table. This review further emphasizes the crucial roles in drug resistance and attempts to offer an outlook of Arf6 in cancer therapy.

ULK1 forms distinct oligomeric states and nanoscopic structures during autophagy initiation.

Autophagy induction involves extensive molecular and membrane reorganization. Despite substantial progress, the mechanism underlying autophagy initiation remains poorly understood. Here, we used quantitative photoactivated localization microscopy with single-molecule sensitivity to analyze the nanoscopic distribution of endogenous ULK1, the kinase that triggers autophagy. Under amino acid starvation, ULK1 formed large clusters containing up to 161 molecules at the endoplasmic reticulum. Cross-correlation analysis revealed that ULK1 clusters engaging in autophagosome formation require 30 or more molecules. The ULK1 structures with more than the threshold number contained varying levels of Atg13, Atg14, Atg16, LC3B, GEC1, and WIPI2. We found that ULK1 activity is dispensable for the initial clustering of ULK1, but necessary for the subsequent expansion of the clusters, which involves interaction with Atg14, Atg16, and LC3B and relies on Vps34 activity. This quantitative analysis at the single-molecule level has provided unprecedented insights into the behavior of ULK1 during autophagy initiation.

Fungal co-expression network analyses identify pathogen gene modules associated with host insect invasion.

The broad host range fungal insect pathogen, Beauveria bassiana, has been commercialized as an alternative to chemical insecticides for pest control worldwide. B. bassiana represents a unique model system with which to examine host-pathogen interactions, and a wide range of genes and processes have been studied. However, significant aspects of virulence, particularly on the genomic scale, remain poorly studied. Here, we have combined available transcriptomes with three newly generated data sets for a combined total analysis of 76 deep-sequenced samples covering growth, development, stress responses, and infection during the life cycle of B. bassiana. Co-expression network analyses resulted in the identification of gene modules enriched during two critical stages of the infection process, namely (i) cuticle penetration and (ii) in vivo hyphal body (dimorphic transition) growth capable of avoiding innate and humoral immune defenses. These analyses identify unique signatures of metabolism, signaling, secondary metabolite production, host defense suppression, membrane reorganization, effector production, and secretion for each stage, including genetic regulators and epigenetic patterns. These data provide a comprehensive framework for understanding and probing fungal adaptations to its pathogenic life cycle and expand the candidate repertoire for continued dissection of the host-pathogen interaction. IMPORTANCE Insect fungal pathogens have evolved unique strategies for overcoming host structural and immunological defenses that span from the sclerotized cuticle to innate and humoral cellular responses. Two critical stages of the infection process involve (i) cuticle penetration and (ii) immune evasion within the insect hemocoel. A set of 76 global transcriptomic data for B. bassiana that include the cuticle penetration and hemocoel growth stages were analyzed for patterns (gene modules) of expression, yielding unique insights into these different life stages. These analyses integrate gene networks involved in fungal development, stress response and pathogenesis to further the systematic understanding of the global processes integral to the unique adaptation employed by fungal pathogens of insects.

Atomic force microscopy of erythrocytes of patients with different severity of pancreatitis

Aim of the study was to prove the possibility of using red blood cell probe microscopy as a predictor of the development of pancreonecrosis.Material and methods. Atomic force microscopy was performed in 143 people (82 were male and 61 were female). The study was performed on an Integra Aura probe microscope (ZAO NT-MDT, Russia). Each erythrocyte was scanned for cell membrane and cell as a whole. A total of 940 red blood cells were examined. According to the severity of acute pancreatitis, patients are distributed in three groups. As a control group, 37 people without somatic pathology were examined. Diagnosis and treatment of patients was carried out in the public health institution “Design Bureau” Russian Railways-Medicine “Krasnoyarsk” from 2015–2019.Results. The study showed that the shape of erythrocyte does not depend on the severity of pancreatitis and does not change, however, structural changes of the membrane occur in the form of the formation of multiple erosions on its surface. The membrane is deformed, and its adhesion increases. These changes reduce the transport capacity of the blood, which, in our opinion, leads to an increase in the general intoxication of the body and to a deterioration in the condition of the patient.Conclusions. Apparently, pancreatic enzymes entering the free bloodstream in the first phase of pancreatitis cause not only oxidative stress, but also the reorganization and destruction of the erythrocyte membrane. The increase in membrane rigidity and deformation of the erythrocyte surface cytoskeleton against the background of increased adhesion seems to disrupt the gas transmission function of erythrocyte and microrheological properties of blood, which in turn disrupts the exchange of amino acids, lipids and detoxification capabilities of blood. The appearance of erythrocytes in the free bloodstream with the presence of erosion on the surface of the erythrocyte membrane can serve as a prediction of a non-favorable course of acute pancreatitis and a predictor of a possible transition of edema pancreatitis to pancreonecrosis.

Lipid composition modulates interactions of p7 viroporin during membrane insertion

Viral proteins interact with lipid membranes during various stages in the viral life cycle to propagate infection. p7 is an ion channel forming protein of Hepatitis C virus (HCV) that participates in viral assembly. Studies show that it has close ties to lipid metabolism in the cell and anionic phosphatidylserine (PS) lipids are suggested to be key for its permeabilizing function, but the mechanism of its interaction with the lipid environment is largely unknown. To begin unraveling the molecular processes of the protein, we evaluated the impact of lipid environment on the binding and insertion mechanism of p7 prior to channel formation and viral assembly using molecular dynamics simulations. It is seen that p7 is sensitive to its lipid environment and results in different remodeling patterns in membranes. Helix 1 (H1) is especially important for peptide insertion, with deeper entry taking place when the membrane contains phosphatidylserine (PS). Helix 2 (H2) and the adjacent loop connecting to Helix 3 (H3) prompts recruitment of phosphatidylethanolamine (PE) lipids to the protein binding site in membrane models with lower surface charge. This work provides perspectives on the interplay between protein-lipid dynamics and membrane composition, and insights on membrane reorganization in mechanisms of disease.

In Silico Identification and Analysis of Proteins Containing the Phox Homology Phosphoinositide-Binding Domain in Kinetoplastea Protists: Evolutionary Conservation and Uniqueness of Phox-Homology-Domain-Containing Protein Architectures.

Kinetoplastea are free living and parasitic protists with unique features among Eukaryota. Pathogenic Kinetoplastea parasites (i.e., Trypanosoma and Leishmania spp.) undergo several developmental transitions essential for survival in their hosts. These transitions require membrane and cytoskeleton reorganizations that involve phosphoinositides (PIs). Phospholipids like PIs are key regulators of vital functions in all eukaryotes including signal transduction, protein transport and sorting, membrane trafficking, and cytoskeleton and membrane remodeling. A large repertoire of PI-metabolizing enzymes and PI-binding proteins/effectors carrying distinct PI-binding modules like the PX (phox homology) module could play significant roles in the life and virulence of pathogenic Kinetoplastea. The aim of this study was to retrieve the entire spectrum of Kinetoplastea protein sequences containing the PX module (PX-proteins), predict their structures, and identify in them evolutionary conserved and unique traits. Using a large array of bioinformatics tools, protein IDs from two searches (based on PFam's pHMM for PX domain (PF00787)) were combined, aligned, and utilized for the construction of a new Kinetoplastea_PX pHMM. This three-step search retrieved 170 PX-protein sequences. Structural domain configuration analysis identified PX, Pkinase, Lipocalin_5, and Vps5/BAR3-WASP domains and clustered them into five distinct subfamilies. Phylogenetic tree and domain architecture analysis showed that some domain architectures exist in proteomes of all Kinetoplastea spp., while others are genus-specific. Finally, amino acid conservation logos of the Kinetoplastea spp. and Homo sapiens PX domains revealed high evolutionary conservation in residues forming the critical structural motifs for PtdIns3P recognition. This study highlights the PX-Pkinase domain architecture as unique within Trypanosoma spp. and forms the basis for a targeted functional analysis of Kinetoplastea PX-proteins as putative targets for a rational design of anti-parasitic drugs.

The bacterial lectin LecA from P. aeruginosa alters membrane organization by dispersing ordered domains

The dynamic reorganization of plasma membrane nanodomains (lipid rafts) plays a key role in host cell infection by human pathogens. Bacteria can induce the reorganization of lipid rafts as a result of the interactions between bacterial lectins and lipid raft glycosphingolipids (GSLs). However, it is still unclear how such interactions trigger the initial stage of the infection mechanism. Here, we study the GSL globotriaosylceramide (Gb3), a key receptor involved in the cellular uptake of the Gram-negative bacterium P. aeruginosa. The bacterial surface lectin LecA targets Gb3 and promotes bacterial invasion via a lipid zipper mechanism. We mimic the plasma membrane using supported lipid bilayers (SLBs), containing liquid-ordered and liquid-disordered lipid domains. We observe that the liquid-ordered domains in the SLBs disperse upon interaction with LecA, accompanied by a deformation of SLBs surface. Our results link the membrane reorganization triggered by LecA with the P. aeruginosa infections in host cells.