Charged Lipids Research Articles

Effects of cholesterol on the anionic magnetite nanoparticle-induced deformation and poration of giant lipid vesicles.

We have investigated the effects of cholesterol on the deformation and poration of giant unilamellar vesicles (GUVs) induced by anionic magnetite nanoparticles (NPs). Negatively charged lipid, neutral lipid, and cholesterol were used to prepare the charged GUVs (surface charge density of membranes - 0.16 C m-2), while only neutral lipid and cholesterol were used to prepare the neutral GUVs. Cholesterol content varied from 0 to 40 mole% for preparing the biologically relevant membranes. The degree of deformation has been characterized by compactness, the value of which remains at 1.0 for spherical GUVs. The value of compactness increases with time for both membranes, but this increase depends on cholesterol content. The average compactness decreases with cholesterol content, and at 60 min, the values are 1.280 ± 0.002 and 1.131 ± 0.010 for 0 and 40 mole% cholesterol containing charged GUVs. The average compactness is relatively lower for neutral GUVs for the corresponding cholesterol. Membrane poration has been investigated by the leakage of calcein, which indicates a two-state transition model. The fraction of deformation is higher for charged GUVs than for neutral ones, while the fraction of poration shows the opposite result. Both the fractions decrease with cholesterol content.

Tryptophan, more than just an interfacial amino acid in the membrane activity of cationic cell-penetrating and antimicrobial peptides.

Trp is unique among the amino acids since it is involved in many different types of noncovalent interactions such as electrostatic and hydrophobic ones, but also in π-π, π-cation, π-anion and π-ion pair interactions. In membranotropic peptides and proteins, Trp locates preferentially at the water-membrane interface. In antimicrobial or cell-penetrating peptides (AMPs and CPPs respectively), Trp is well-known for its strong role in the capacity of these peptides to interact and affect the membrane organisation of both bacteria and animal cells at the level of the lipid bilayer. This essential amino acid can however be involved in other types of interactions, not only with lipids, but also with other membrane partners, that are crucial to understand the functional roles of membranotropic peptides. This review is focused on this latter less known role of Trp and describes in details, both in qualitative and quantitative ways: (i) the physico-chemical properties of Trp; (ii) its effect in CPP internalisation; (iii) its importance in AMP activity; (iv) its role in the interaction of AMPs with glycoconjugates or lipids in bacteria membranes and the consequences on the activity of the peptides; (v) its role in the interaction of CPPs with negatively charged polysaccharides or lipids of animal membranes and the consequences on the activity of the peptides. We intend to bring highlights of the physico-chemical properties of Trp and describe its extensive possibilities of interactions, not only at the well-known level of the lipid bilayer, but with other less considered cell membrane components, such as carbohydrates and the extracellular matrix. The focus on these interactions will allow the reader to reevaluate reported studies. Altogether, our review gathers dedicated studies to show how unique are Trp properties, which should be taken into account to design future membranotropic peptides with expected antimicrobial or cell-penetrating activity.

Role of polyplex charge density in lipopolyplexes.

Lipopolyplexes have received extensive attention lately in gene therapy delivery. However, the interactions between the polyplex and the liposome and their underlying molecular mechanisms remain to be elucidated. Here, we adopted a simple model, mainly to illustrate the impact of polyplex charge density on the self-assembly of liposomes (containing DOPE and CHEMS lipids) using coarse-grained molecular dynamics simulations. Our simulation results show that when the charge density increases in the polyplex, more lipids, especially CHEMS (a negatively charged helper lipid) lipids, are attracted to the polyplex (positively charged) surface, and meanwhile nearby water molecules are driven away from the polyplex, resulting in a less spherical liposome. Energy decomposition analyses further reveal that, at higher charge densities, the polyplex exhibits much stronger interactions with CHEMS lipids than with water molecules, with the majority contribution from electrostatic interactions. In addition, the mobility of lipids, especially CHEMS, is reduced as the polyplex charge density increases, indicating a more rigid liposome. Overall, our molecular dynamics simulations elucidate the influence of polyplex charge density on the liposome self-assembly process at the atomic level, which provides a complementary approach to experiments for a better understanding of this promising gene therapy delivery system.

Liposomal drug delivery: Recent developments and challenges

The spherical vesicles known as liposomes may contain one or many phospholipid bilayers. The first liposomes were found in the 1960s. One of the many distinctive drug delivery methods is the liposome, which offers a complex way to transfer active molecules to the site of action. Clinical trials are now testing a variety of formulations. Long-lasting second-generation liposomes are created by altering the vesicle’s lipid composition, size, and charge. Superficial vesicles have given way to liposome growth. Glycolipids and other substances have been used to make liposomes for the modification of outer surfaces through various types of targeting ligands and detecting agents or moiety. Now, the liposomes developed for the different market and it is flooded with cosmetics and, more crucially, medications. Three of the main applications of liposome technology include steric and environmental stabilization of loaded molecules, remote drug loading through pH and ion gradients approach, and simultaneously lipoplexes which is the complexes forms of cationic liposomes with anionic nucleic acids or proteins for the gene delivery or siRNA technology. The scope of liposome research was expanded, allowing for the production of various goods. The present review focuses on the different aspects of liposomal drug delivery concerning types, preparation, pros, and cons.

Hepatocyte-Directed Delivery of Lipid-Encapsulated Small Interfering RNA.

Lipid formulations for cell transfection are among the most efficient systems for nucleic acid delivery. During the COVID-19 pandemic, lipid-encapsulated RNA (lipid nanoparticles, LNP) has succeeded as a superior vaccine. Moreover, other similar lipid nanocarriers for siRNA are approved and many are on the pipelines. While lipid encapsulation required several devices for the mixing of components, lipoplex technology allows to rapidly mix nucleic acids and positively charged lipids for cell transfection. In vivo, hepatocytes are important target cells of lipid formulated RNAi. This chapter describes the state-of-the-art lipoplex and LPN manufacturing for treating primary hepatocytes with lipid formulations. Furthermore, protocols for isolating murine hepatocytes and for transfecting these cells with pharmaceutically relevant lipid formulations are provided and discussed.

Metal‐Supported Phospholipid Bilayers Formed by Redox Command

AbstractA surface‐confined redox reaction triggers the formation of single bilayer membranes from small unilamellar vesicles of anionic or zwitterionic phospholipids onto gold surfaces modified with a ferrocene‐terminated self‐assembled monolayer (SAM). The ion pairing association of the charged lipid headgroups with the electrogenerated cationic ferroceniums drives the assembly of the phospholipids on the SAM surface to produce solid‐supported bilayers of high surface coverage (≳90%) from gel‐ or fluid‐phase forming phospholipids within minutes at room temperature. The redox‐mediated strategy reported in this work is a conceptual advance in the preparation of solid‐supported lipid bilayers. It is fast, compatible with negatively‐charged and zwitterionic phospholipids, insensitive to the phase state of the phospholipid in the vesicle precursor, and not limited to hydrophilic surfaces.

Influence of Charge Lipid Head Group Structures on Electric Double Layer Properties.

In this study we derived a model for a multicomponent lipid monolayer in contact with an aqueous solution by means of a generalized classical density functional theory and Monte Carlo simulations. Some of the important biological lipid systems were studied as monolayers composed of head groups with different shapes and charge distributions. Starting from the free energy of the system, which includes the electrostatic interactions, additional internal degrees of freedom are included as positional and orientational entropic contributions to the free energy functional. The calculus of variation was used to derive Euler–Lagrange equations, which were solved numerically by the finite element method. The theory and Monte Carlo simulations predict that there are mainly two distinct regions of the electric double layer: (1) the interfacial region, with thickness less than or equal to the length of the fully stretched conformation of the lipid head group, and (2) the outside region, which follows the usual screening of the interface. In the interfacial region, the electric double layer is strongly perturbed, and electrostatic profiles and ion distributions have functionality distinct to classical mean-field theories. Based purely on Coulomb interactions, the theory suggests that the dominant effect on the lipid head group conformation is from the charge density of the interface and the structured lipid mole fraction in the monolayer, rather than the salt concentration in the system.

Role of Physicochemical Factors on the Efficacy and Safety of Lipid-Based Nanosystems as Potential Drug Carriers

The efficacy and safety are two challenging factors before trusting the lipid-based Nanosystems (LBNs) for diagnostic and therapeutic purposes. The aim of this paper is to evaluate the accessible in-vitro and in-vivo studies on the contribution of major physicochemical factors of LBNs for the effective and safe delivery of drugs and anticancer agents into the target site. These factors included lipid charge, particle size and size distribution, lipid composition and components, surface hydrophilicity and hydrophobicity, and surface coating. Here, reliable databases were searched for full-text, accessible and original articles to conduct this review. We included relevant studies since 1992, which were conducted on the development of LBNs for therapeutic and diagnostic purposes. Many studies have shown that using polyethylene glycol modification reduces the undesirable side effects and controls the efficacy and toxicity for in-vitro and in-vivo delivery of drugs or anticancer agents. Also, optimization of the size and composition of lipid nanoparticles minimizes the toxicity of the anticancer agents associated with a carrier. In addition, the presence of positively charged lipids in LBNs improves the efficacy of encapsulated drugs in spite of low blood circulation efficiency and high toxicity effects. The identification of important factors involved in the delivery of LBNs as potential drug carriers is crucial and contributes to the proper design of these interesting carriers for both therapeutic and diagnostic purposes.

Chemistry of Lipid Nanoparticles for RNA Delivery.

Lipid nanoparticles (LNPs) are a type of lipid vesicles that possess a homogeneous lipid core. These vesicles are widely used in small-molecule drug and nucleic acid delivery and recently gained much attention because of their remarkable success as a delivery platform for COVID-19 mRNA vaccines. Nonetheless, the utility of transient protein expression induced by mRNA extends far beyond vaccines against infectious diseases─they also hold promise as cancer vaccines, protein replacement therapies, and gene editing components for rare genetic diseases. However, naked mRNA is inherently unstable and prone to rapid degradation by nucleases and self-hydrolysis. Encapsulation of mRNA within LNPs protects mRNA from extracellular ribonucleases and assists with intracellular mRNA delivery.In this Account, we discuss the core features of LNPs for RNA delivery. We focus our attention on LNPs designed to deliver mRNA; however, we also include examples of siRNA-LNP delivery where appropriate to highlight the commonalities and the dissimilarities due to the nucleic acid structure. First, we introduce the concept of LNPs, the advantages and disadvantages of utilizing nucleic acids as therapeutic agents, and the general reasoning behind the molecular makeup of LNPs. We also briefly highlight the most recent clinical successes of LNP-based nucleic acid therapies. Second, we describe the theory and methods of LNP self-assembly. The common idea behind all of the preparation methods is inducing electrostatic interactions between the nucleic acid and charged lipids and promoting nanoparticle growth via hydrophobic interactions. Third, we break down the LNP composition with special attention to the fundamental properties and purposes of each component. This includes the identified molecular design criteria, commercial sourcing, impact on intracellular trafficking, and contribution to the properties of LNPs. One of the key components of LNPs is ionizable lipids, which initiate electrostatic binding with endosomal membranes and facilitate cytosolic release; however, the roles of other lipid components should not be disregarded, as they are associated with stability, clearance, and distribution of LNPs. Fourth, we review the attributes of LNP constructs as a whole that can heavily influence RNA delivery. These attributes are LNP size, charge, internal structure, lipid packing, lipid membrane hydration, stability, and affinity toward biomacromolecules. We also discuss the specific techniques used to examine these attributes and how they can be adjusted. Finally, we offer our perspective on the future of RNA therapies and some questions that remain in the realm of LNP formulation and optimization.

Self-Assembly of Graphene Oxide Nanoflakes in a Lipid Monolayer at the Air–Water Interface

The graphene family, especially graphene oxide (GO), has captured increasing prospects in the biomedical field due to its excellent physicochemical properties. Understanding the health and environmental impact of GO is of great importance for guiding future applications. Although their interactions with living organisms are omnipresent, the exact molecular mechanism is yet to be established. The cellular membrane is the first barrier for a foreign molecule to interact before entering into the cell. In the present study, a model system consisting of a lipid monolayer at the air-water interface represents one of the leaflets of this membrane. Surface pressure-area isotherms and advanced synchrotron X-ray scattering techniques have been employed to comprehend the interaction by varying the electrostatics of the membrane. The results depict a strong GO interaction with positively charged phospholipids, weak interaction with zwitterionic lipids, and interestingly negligible interaction with negatively charged lipids. GO flakes induce significant changes in the out-of-plane organization of a positively charged lipid monolayer with a minor influence on in-plane assembly of lipid chains. This interaction is packing-specific, and the influence of GO is much stronger at lower surface pressure. Even though for zwitterionic phospholipids, the GO flakes may partly insert into the lipid chains, the X-ray scattering results indicate that the flakes preferentially lie horizontally underneath the positively charged lipid monolayer. This in-depth structural description may pave new perspectives for the scientific community for the development of GO-based biosensors and biomedical materials.

Tug-of-War between Covalent Binding and Electrostatic Interaction Effectively Killing E. coli without Detectable Resistance

Antimicrobial resistance in Gram-negative bacteria has become one of the leading causes of morbidity and mortality and a serious worldwide public health concern due to the fact that Gram-negative bacteria have an additional outer membrane protecting them from an unwanted compound invading. It is still very difficult for antimicrobials to reach intracellular targets and very challenging to treat Gram-negative bacteria with the current strategies. Here, we found that (o-(bromomethyl)phenyl)boronic acid was incorporated into poly((2-N,N-diethyl)aminoethyl acrylate) (PDEA), forming a copolymer (poly(o-Bn-DEA)) having both phenylboronic acid (B) and ((2-N,N-diethyl)amino) (DEA) units. Poly(o-Bn-DEA) exhibits very strong intramolecular B-N coordination, which could highly promote the covalent binding of phenylboronic acid with lipopolysaccharide (LPS) on the outer membrane of E. coli and lodge poly(o-Bn-DEA) on the LPS layer on the surface of E. coli. Meanwhile, the strong electrostatic interaction between poly(o-Bn-DEA) and the negatively charged lipid preferred tugging the poly(o-Bn-DEA) into the lipid bilayer of E. coli. The combating interactions between covalent binding and electrostatic interaction form a tug-of-war action, which could trigger the lysis of the outer membrane, thereby killing Gram-negative E. coli effectively without detectable resistance.

Negatively charged phospholipids accelerate the membrane fusion activity of the plant-specific insert domain of an aspartic protease

Various plants use antimicrobial proteins/peptides to resist phytopathogens. In the potato, Solanum tuberosum, the plant-specific insert (PSI) domain of an aspartic protease performs this role by disrupting phytopathogen plasma membranes. However, the mechanism by which PSI selects target membranes has not been elucidated. Here, we studied PSI-induced membrane fusion, focusing on the effects of lipid composition on fusion efficiency. Membrane fusion by the PSI involves an intermediate state whereby adjacent liposomes share their bilayers. We found that increasing the concentration of negatively charged phosphatidylserine (PS) phospholipids substantially accelerated PSI-mediated membrane fusion. NMR data demonstrated that PS did not affect the binding between the PSI and liposomes but had seminal effects on the dynamics of PSI interaction with liposomes. In PS-free liposomes, the PSI underwent significant motion, which was suppressed on PS-contained liposomes. Molecular dynamics simulations showed that the PSI binds to PS-containing membranes with a dominant angle ranging from −31° to 30°, with respect to the bilayer, and is closer to the membrane surfaces. In contrast, PSI is mobile and exhibits multiple topological states on the surface of PS-free membranes. Taken together, our data suggested that PS lipids limit the motion of the anchored PSI, bringing it closer to the membrane surface and efficiently bridging different liposomes to accelerate fusion. As most phytopathogens have a higher content of negatively charged lipids as compared with host cells, these results indicate that the PSI selectively targets negatively charged lipids, which likely represents a way of distinguishing the pathogen from the host.

Efficient Treatment of Fish Intestinal Parasites Applying a Membrane-Penetrating Oral Drug Delivery Nanoparticle.

Nanodelivery of drugs aims to ensure drug stability in the face of adverse biochemical conditions in the course of administration, concomitant with appropriate pharmacological action provided by delivery at the targeted site. In this study, the application potential of a nanoparticle produced with biopolymers chitosan-N-arginine and alginate as an oral drug delivery material is evaluated. Both macromolecules being weak polyelectrolytes, the nanoparticle presents strong thermodynamic interactions with a biological model membrane consisting of a charged lipid liposome bilayer, leading to membrane disruption and membrane penetration of the nanoparticles in ideal conditions of pH corresponding to the oral route. The powder form of the nanoparticle was obtained by lyophilization and with a high percentage of entrapment of the anthelmintic drug praziquantel. In vivo studies were conducted with oral administration to Corydoras schwartzi fish with high intensity of intestinal parasites infection. The in vivo experiments confirmed the mucoadhesive and revealed membrane-penetrating properties of the nanoparticle by translocating the parasite cyst, which provided target drug release and reduction of over 97% of the fish intestinal parasites. Thus, it was evidenced that the nanoparticle was effective in transporting and releasing the drug to the target, providing an efficient treatment.

α-Synuclein interacts differently with membranes mimicking the inner and outer leaflets of neuronal membranes

The toxicity of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease, is likely related to its interaction with the asymmetric neuronal membrane. α-Syn exists as cytoplasmatic and as extracellular protein as well. To shed light on the different interactions occurring at the different α-syn localizations, we have here modelled the external and internal membrane leaflets of the neuronal membrane with two complex lipid mixtures, characterized by phase coexistence and with negative charge confined to either the ordered or the disordered phase, respectively. To this purpose, we selected a five-component (DOPC/SM/DOPE/DOPS/chol) and a four-component (DOPC/SM/GM1/chol) lipid mixtures, which contained the main membrane lipid constituents and exhibited a phase separation with formation of ordered domains. We have compared the action of α-syn in monomeric form and at different concentrations (1 nM, 40 nM, and 200 nM) with respect to lipid systems with different composition and shape by AFM, QCM-D, and vesicle leakage experiments. The experiments coherently showed a higher stability of the membranes composed by the internal leaflet mixture to the interaction with α-syn. Damage to membranes made of the external leaflet mixture was detected in a concentration-dependent manner. Interestingly, the membrane damage was related to the fluidity of the lipid domains and not to the presence of negatively charged lipids.

Vesicular and Stealth Vesicular Drug Delivery – A Review

Vesicular systems (liposomes), one of several potential novel drug delivery systems, provide an advanced technology for delivering active compounds to the site of action, and numerous formulations are currently in clinical use. Liposome technology has developed from typical vesicles to sterically stabilised vesicles, which produce long-circulating liposomes by varying the lipid content, size, and charge of the vesicle. Several compounds, such as glycolipids, have been used to create liposomes with changed surfaces. The addition of the synthetic polymer poly-(ethylene glycol) (PEG) in liposome composition was a crucial milestone in the creation of long-circulating liposomes. PEG on the liposomal carrier's surface has been found to increase blood circulation time while decreasing mononuclear phagocyte system uptake (Stealth Liposomes). As a consequence of this technique, a vast variety of liposome formulations encapsulating active compounds have been developed, all of which have excellent target efficiency and activity. Stealth liposomes can also be actively targeted with monoclonal antibodies or ligands thanks to a synthetic alteration of the terminal PEG molecule. This article focuses on vesicular drug delivery as well as stealth technology and presents preclinical and clinical data for the most common liposome formulations, as well as discussing developing developments in this promising technology.

Nontargeted Serum Lipid Profiling of Nonalcoholic Steatohepatitis by Multisegment Injection-Nonaqueous Capillary Electrophoresis-Mass Spectrometry: A Multiplexed Separation Platform for Resolving Ionic Lipids.

New methods are needed for global lipid profiling due to the complex chemical structures and diverse physicochemical properties of lipids. Herein we introduce a robust data workflow to unambiguously select lipid features from serum ether extracts by multisegment injection-nonaqueous capillary electrophoresis-mass spectrometry (MSI-NACE-MS). An iterative three-stage screening strategy is developed for nontargeted lipid analyses when using multiplexed electrophoretic separations coupled to an Orbitrap mass analyzer under negative ion mode. This approach enables the credentialing of 270 serum lipid features annotated based on their accurate mass and relative migration time, including 128 ionic lipids reliably measured (median CV ≈ 13%) in most serum samples (>75%) from nonalcoholic steatohepatitis (NASH) patients (n = 85). A mobility map is introduced to classify charged lipid classes over a wide polarity range with selectivity complementary to chromatographic separations, including lysophosphatidic acids, phosphatidylcholines, phosphatidylinositols, phosphatidylethanolamines, and nonesterified fatty acids (NEFAs). Serum lipidome profiles were also used to differentiate high- from low-risk NASH patients using a k-means clustering algorithm, where elevated circulating NEFAs (e.g., palmitic acid) were associated with increased glucose intolerance, more severe liver fibrosis, and greater disease burden. MSI-NACE-MS greatly expands the metabolome coverage of conventional aqueous-based CE-MS protocols and is a promising platform for large-scale lipidomic studies.

Staphylococcus aureus Lipase 3 (SAL3) is a surface-associated lipase that hydrolyzes short chain fatty acids.

Bacterial lipases play important roles during infection. The Staphylococcus aureus genome contains several genes that encode well-characterized lipases and several genes predicted to encode lipases or esterases for which the function has not yet been established. In this study, we sought to define the function of an uncharacterized S. aureus protein, and we propose the annotation S. aureus lipase 3 (SAL3) (SAUSA300_0641). We confirmed that SAL3 is a lipase and that it is surface associated and secreted through an unknown mechanism. We determined that SAL3 specifically hydrolyzes short chain (4-carbon and fewer) fatty acids and specifically binds negatively charged lipids including phosphatidic acid, phosphatidylinositol phosphate, and phosphatidylglycerol, which is the most abundant lipid in the staphylococcal cell membrane. Mutating the catalytic triad S66-A, D167-A, S168-A, and H301-A in the recombinant protein abolished lipase activity without altering binding to host lipid substrates. Taken together we report the discovery of a novel lipase from S. aureus specific to short chain fatty acids with yet to be determined roles in host pathogen interactions.

Chitosan effects on monolayers of zwitterionic, anionic and a natural lipid extract from E. coli at physiological pH

Langmuir monolayers are used to simulate the biological membrane environment, acting as a mimetic system of the outer or the inner membrane leaflet. Herein, we analyze the interaction of membrane models with a partially N-acetylated chitosan (Ch35%) possessing a quasi-ideal random pattern of acetylation, full water solubility up to pH ≈ 8.5 and unusually high weight average molecular weight. Lipid monolayers containing dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl ethalonamine (DPPE), dipalmitoyl phosphatidyl glycerol (DPPG) or E. coli total lipid extract were spread onto subphases buffered at pH 4.5 or 7.4. The incorporation of Ch35% chitosan caused monolayer expansion and a general trend of decreasing monolayer rigidity with Ch35% concentration. Due to its relatively high content of N-acetylglucosamine (GlcNAc) units, Ch35% interactions with negatively charged monolayers and with E. coli extract were weaker than those involving zwitterionic monolayers or lipid rafts. While the smaller interaction with negatively charged lipids was unexpected, this finding can be attributed to the degree of acetylation (35%) which imparts a small number of charged groups for Ch35% to interact. Chitosan properties are therefore determinant for interactions with model cell membranes, which explains the variability in chitosan bactericide activity in the literature. This is the first study on the effects from chitosans on realistic models of bacterial membranes under physiological pH.

Interaction of Amyloid-β-(1-42) Peptide and Its Aggregates with Lipid/Water Interfaces Probed by Vibrational Sum-Frequency Generation Spectroscopy.

In this study, we use surface-sensitive vibrational sum-frequency generation (VSFG) spectroscopy to investigate the interaction between model lipid monolayers and Aβ(1-42) in its monomeric and aggregated states. Combining VSFG with atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence measurements, we found that only small aggregates with probably a β-hairpin-like structure adsorbed to the zwitterionic lipid monolayer (DOPC). In contrast, larger aggregates with an extended β-sheet structure adsorbed to a negatively charged lipid monolayer (DOPG). The adsorption of small, initially formed aggregates strongly destabilized both monolayers, but only the DOPC monolayer was completely disrupted. We showed that the intensity of the amide-II' band in achiral (SSP) and chiral (SPP) polarization combinations increased in time when Aβ(1-42) aggregates accumulated at the DOPG monolayer. Nevertheless, almost no adsorption of preformed mature fibrils to DOPG monolayers was detected. By performing spectral VSFG calculations, we revealed a clear correlation between the amide-II' signal and the degree of amyloid aggregates (e.g., oligomers or (proto)fibrils) of various Aβ(1-42) structures. The calculations showed that only structures with a significant amyloid β-sheet content have a strong amide-II' intensity, in line with previous Raman studies. The combination of the presented results substantiates the amide-II(') band as a legitimate amyloid marker.

Phosphorylation of luminal region of the SUN-domain protein Mps3 promotes nuclear envelope localization during meiosis.

During meiosis, protein ensembles in the nuclear envelope (NE) containing SUN- and KASH-domain proteins, called linker nucleocytoskeleton and cytoskeleton (LINC) complex, promote the chromosome motion. Yeast SUN-domain protein, Mps3, forms multiple meiosis-specific ensembles on NE, which show dynamic localisation for chromosome motion; however, the mechanism by which these Mps3 ensembles are formed during meiosis remains largely unknown. Here, we showed that the cyclin-dependent protein kinase (CDK) and Dbf4-dependent Cdc7 protein kinase (DDK) regulate meiosis-specific dynamics of Mps3 on NE, particularly by mediating the resolution of Mps3 clusters and telomere clustering. We also found that the luminal region of Mps3 juxtaposed to the inner nuclear membrane is required for meiosis-specific localisation of Mps3 on NE. Negative charges introduced by meiosis-specific phosphorylation in the luminal region of Mps3 alter its interaction with negatively charged lipids by electric repulsion in reconstituted liposomes. Phospho-mimetic substitution in the luminal region suppresses the localisation of Mps3 via the inactivation of CDK or DDK. Our study revealed multi-layered phosphorylation-dependent regulation of the localisation of Mps3 on NE for meiotic chromosome motion and NE remodelling.