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When nutrient starvation sets in, the tubular endoplasmic reticulum (ER) within the morphological domain transforms into sheet-like ER structures. Motile early endosomes and lysosomes, which establish membrane contact sites (MCS) with the tubular ER, exert force to organize the tubular ER network in fed cells at steady-state, a phenomenon known as hitchhiking. During early stages of starvation, MCS are reconfigured, leading to conversion of the tubular ER network into sheet-like ER. Lipid metabolism involving phosphatidylinositol 3-phosphate [PI(3)P] at MCS between the tubular ER and motile endolysosomes has an important role in early starvation-induced ER remodeling. ER-phagy is activated during prolonged starvation and may selectively remove specific ER subdomains enriched in (oxidized) polyunsaturated fatty acid phospholipids. Unlike most other organelles found in multiple copies, the endoplasmic reticulum (ER) is a unique singular organelle within eukaryotic cells. Despite its continuous membrane structure, encompassing more than half of the cellular endomembrane system, the ER is subdivided into specialized sub-compartments, including morphological, membrane contact site (MCS), and de novo organelle biogenesis domains. In this review, we discuss recent emerging evidence indicating that, in response to nutrient stress, cells undergo a reorganization of these sub-compartmental ER domains through two main mechanisms: non-destructive remodeling of morphological ER domains via regulation of MCS and organelle hitchhiking, and destructive remodeling of specialized domains by ER-phagy. We further highlight and propose a critical role of membrane lipid metabolism in this ER remodeling during starvation. [ABSTRACT FROM AUTHOR]

Cryo-electron tomography enables capture of 3D images of membrane fusion intermediate states with resolution of fusion proteins and membrane leaflet remodeling. Studies of class I, II, and III viral membrane fusion proteins are revealing general features as well as differences in stages of membrane reorganization, leading to complete fusion and pore formation. Viral fusion machinery is comprised of fusion proteins, viral membrane, and internal structural components such as matrix proteins that regulate fusion protein conformation and function. Tightly docked membrane contact zones with closely apposed proximal headgroup layers appear to be a near universal intermediate state along the fusion pathway. Computational modeling that builds upon observed structural information from cryo-electron tomography may offer a path towards a detailed mechanistic understanding of the forces and energetics that drive protein-mediated membrane fusion. Protein-mediated membrane fusion is the dynamic process where specialized protein machinery undergoes dramatic conformational changes that drive two membrane bilayers together, leading to lipid mixing and opening of a fusion pore between previously separate membrane-bound compartments. Membrane fusion is an essential stage of enveloped virus entry that results in viral genome delivery into host cells. Recent studies applying cryo-electron microscopy techniques in a time-resolved fashion provide unprecedented glimpses into the interaction of viral fusion proteins and membranes, revealing fusion intermediate states from the initiation of fusion to release of the viral genome. In combination with complementary structural, biophysical, and computation modeling approaches, these advances are shedding new light on the mechanics and dynamics of protein-mediated membrane fusion. [ABSTRACT FROM AUTHOR]

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. [Display omitted] In their review, Jacomin and Dikic discuss the role of ubiquitination in regulating membrane structure and its impact on pathways such as endocytosis and autophagy. Besides protein modification, ubiquitin also interacts with lipids, directly influencing membrane dynamics. The review covers ubiquitin-mediated membrane remodeling in endocytosis, autophagy, and ER-phagy. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are central regulators of the immune response by detecting inflammatory signals, aberrant cells, or pathogens. DC‐mediated immune surveillance requires morphology changes to adapt to the physical and biochemical cues of the external environment. These changes are assisted by a dynamic actin cytoskeleton–membrane interface connected to surface receptors that will trigger signaling cascades. In recent years, the development of synthetic immune environments has allowed to investigate the impact of the external environment in the immune cell response. In this direction, the bioengineering of functional topographical features should make it possible to establish how membrane morphology modulates specific cellular functions in DCs. Herein, the engineering of one‐dimensional nanostructured SiO2 surfaces by soft‐nanoimprint lithography to manipulate the membrane morphology of ex vivo human DCs is reported. Super‐resolution microscopy and live‐cell imaging studies show that vertical pillar topographies promote the patterning and stabilization of adhesive actin‐enriched structures in DCs. Furthermore, vertical topographies stimulate the spatial organization of innate immune receptors and regulate the Syk‐ and ERK‐mediated signaling pathways across the cell membrane. In conclusion, engineered SiO2 surface topographies can modulate the cellular response of ex vivo human immune cells by imposing local plasma membrane nano‐deformations. [ABSTRACT FROM AUTHOR]

High-throughput clustered regularly interspaced short palindromic repeats (CRISPR) screens identified pro- and antiviral host factors. SARS-CoV-2 subverts the membrane trafficking system at multiple levels. The virus recruits host proteins to modulate membrane remodeling for replication, virus particle formation, and propagation. Different SARS-CoV-2 egress routes have been proposed. The molecular mechanisms underlying SARS-CoV-2 host cell invasion and life cycle have been studied extensively in recent years, with a primary focus on viral entry and internalization with the aim of identifying antiviral therapies. By contrast, our understanding of the molecular mechanisms involved in the later steps of the coronavirus life cycle is relatively limited. In this review, we describe what is known about the host factors and viral proteins involved in the replication, assembly, and egress phases of SARS-CoV-2, which induce significant host membrane rearrangements. We also discuss the limits of the current approaches and the knowledge gaps still to be addressed. [ABSTRACT FROM AUTHOR]

The endosomal sorting complexes required for transport (ESCRT) machinery is an evolutionarily conserved cytosolic protein complex that plays a crucial role in membrane remodeling and scission events across eukaryotes. Initially discovered for its function in multivesicular body (MVB) formation, the ESCRT complex has since been implicated in a wide range of membrane-associated processes, including endocytosis, exocytosis, cytokinesis, and autophagy. Recent advances have elucidated the ESCRT assembly pathway and highlighted the distinct functions of the various ESCRT complexes and their associated partners. Among the ESCRT complexes, ESCRT-III stands out as a critical player in membrane remodeling, with its subunits assembled into higher-order multimers capable of bending and severing membranes. This review focuses on the ESCRT-III complex, exploring its diverse functions in cellular processes beyond MVB biogenesis. We delve into the molecular mechanisms underlying ESCRT-III-mediated membrane remodeling and highlight its emerging roles in processes such as viral budding, autophagosome closure, and cytokinetic abscission. We also discuss the implications of ESCRT-III dysregulation in neurodegenerative diseases. The versatile membrane remodeling capabilities of ESCRT-III across diverse cellular processes underscore its importance in maintaining proper cellular function. Furthermore, we highlight the promising potential of ESCRT-III as a therapeutic target for neurodegenerative diseases, offering insights into the treatments of the diseases and the technical applications in related research fields.

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. [ABSTRACT FROM AUTHOR]

Many microbes accumulate energy storage molecules such as triglycerides (TAG) and starch during nutrient limitation. In eukaryotic green algae grown under nitrogen-limiting conditions, triglyceride accumulation is coupled with chlorosis and growth arrest. In this study, we show that reactive oxygen species (ROS) actively accumulate during nitrogen limitation in the microalga Chlorella sorokiniana. Accumulation of ROS is mediated by the downregulation of genes encoding ROS-quenching enzymes, such as superoxide dismutases, catalase, peroxiredoxin, and glutathione peroxidase-like, and by the upregulation of enzymes involved in generating ROS, such as NADPH oxidase, xanthine oxidase, and amine oxidases. The expression of genes involved in ascorbate and glutathione metabolism is also affected under this condition. ROS accumulation contributes to the degradation of monogalactosyl diacylglycerol (MGDG) and thylakoid membrane remodeling, leading to chlorosis. Quenching ROS under nitrogen limitation reduces the degradation of MGDG and the accumulation of TAG. This work shows that ROS accumulation, membrane remodeling, and TAG accumulation under nitrogen limitation are intricately linked in the microalga C. sorokiniana. [ABSTRACT FROM AUTHOR]

Membrane remodeling is a fundamental cellular process that is crucial for physiological functions such as signaling, membrane fusion and cell migration. Tetraspanins (TSPANs) are transmembrane proteins of central importance to membrane remodeling events. During these events, TSPANs are known to interact with themselves and other proteins and lipids; however, their mechanism of action in controlling membrane dynamics is not fully understood. Since these proteins span the membrane, membrane properties such as rigidity, curvature and tension can influence their behavior. In this Review, we summarize recent studies that explore the roles of TSPANs in membrane remodeling processes and highlight the unique structural features of TSPANs that mediate their interactions and localization. Further, we emphasize the influence of membrane curvature on TSPAN distribution and membrane domain formation and describe how these behaviors affect cellular functions. This Review provides a comprehensive perspective on the multifaceted function of TSPANs in membrane remodeling processes and can help readers to understand the intricate molecular mechanisms that govern cellular membrane dynamics. [ABSTRACT FROM AUTHOR]

During infection, positive-stranded RNA causes a rearrangement of the host cell membrane, resulting in specialized membrane structure formation aiding viral genome replication. Double-membrane vesicles (DMVs), typical structures produced by virus-induced membrane rearrangements, are platforms for viral replication. Nidoviruses, one of the most complex positive-strand RNA viruses, have the ability to infect not only mammals and a few birds but also invertebrates. Nidoviruses possess a distinctive replication mechanism, wherein their nonstructural proteins (nsps) play a crucial role in DMV biogenesis. With the participation of host factors related to autophagy and lipid synthesis pathways, several viral nsps hijack the membrane rearrangement process of host endoplasmic reticulum (ER), Golgi apparatus, and other organelles to induce DMV formation. An understanding of the mechanisms of DMV formation and its structure and function in the infectious cycle of nidovirus may be essential for the development of new and effective antiviral strategies in the future. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are central regulators of the immune response by detecting inflammatory signals, aberrant cells, or pathogens. DC‐mediated immune surveillance requires morphology changes to adapt to the physical and biochemical cues of the external environment. These changes are assisted by a dynamic actin cytoskeleton–membrane interface connected to surface receptors that will trigger signaling cascades. In recent years, the development of synthetic immune environments has allowed to investigate the impact of the external environment in the immune cell response. In this direction, the bioengineering of functional topographical features should make it possible to establish how membrane morphology modulates specific cellular functions in DCs. Herein, the engineering of one‐dimensional nanostructured SiO2 surfaces by soft‐nanoimprint lithography to manipulate the membrane morphology of ex vivo human DCs is reported. Super‐resolution microscopy and live‐cell imaging studies show that vertical pillar topographies promote the patterning and stabilization of adhesive actin‐enriched structures in DCs. Furthermore, vertical topographies stimulate the spatial organization of innate immune receptors and regulate the Syk‐ and ERK‐mediated signaling pathways across the cell membrane. In conclusion, engineered SiO2 surface topographies can modulate the cellular response of ex vivo human immune cells by imposing local plasma membrane nano‐deformations.

A recent study in our group reports a new “condensates to VPS41-associated phagic vacuole (VAPVs) conversion pathway” that is essential for macroautophagy/autophagy degradation in plant cells. Here, we compare the autophagy process between plants and other eukaryotic systems and discuss the potential roles of biomolecular condensates and synaptic-soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in plant autophagy. [ABSTRACT FROM AUTHOR]

Mitochondria are essential organelles performing important cellular functions ranging from bioenergetics and metabolism to apoptotic signaling and immune responses. They are highly dynamic at different structural and functional levels. Mitochondria have been shown to constantly undergo fusion and fission processes and dynamically interact with other organelles such as the endoplasmic reticulum, peroxisomes, and lipid droplets. The field of mitochondrial dynamics has evolved hand in hand with technological achievements including advanced fluorescence super-resolution nanoscopy. Dynamic remodeling of the cristae membrane within individual mitochondria, discovered very recently, opens up a further exciting layer of mitochondrial dynamics. In this review, we discuss mitochondrial dynamics at the following levels: (a) within an individual mitochondrion, (b) among mitochondria, and (c) between mitochondria and other organelles. Although the three tiers of mitochondrial dynamics have in the past been classified in a hierarchical manner, they are functionally connected and must act in a coordinated manner to maintain cellular functions and thus prevent various human diseases. [ABSTRACT FROM AUTHOR]

Biomolecular condensates are highly versatile membraneless organelles involved in a plethora of cellular processes. Recent years have witnessed growing evidence of the interaction of these droplets with membrane-bound cellular structures. Condensates' adhesion to membranes can cause their mutual molding and regulation, and their interaction is of fundamental relevance to intracellular organization and communication, organelle remodeling, embryogenesis, and phagocytosis. In this article, we review advances in the understanding of membrane–condensate interactions, with a focus on in vitro models. These minimal systems allow the precise characterization and tuning of the material properties of both membranes and condensates and provide a workbench for visualizing the resulting morphologies and quantifying the interactions. These interactions can give rise to diverse biologically relevant phenomena, such as molecular-level restructuring of the membrane, nano- to microscale ruffling of the condensate–membrane interface, and coupling of the protein and lipid phases. [ABSTRACT FROM AUTHOR]

As for all single-stranded, positive-sense RNA (+RNA) viruses, intracellular RNA synthesis relies on extensive remodeling of host cell membranes that leads to the formation of specialized structures. In the case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus causing COVID-19, endoplasmic reticulum membranes are modified, resulting in the formation of double-membrane vesicles (DMVs), which contain the viral dsRNA intermediate and constitute membrane-bound replication organelles. The non-structural and transmembrane protein nsp3 is a key player in the biogenesis of DMVs and, therefore, represents an interesting antiviral target. However, as an integral transmembrane protein, it is challenging to express for structural biology. The C-terminus of nsp3 encompasses all the membrane-spanning, -interacting, and -remodeling elements. By using a cell-free expression system, we successfully produced the C-terminal region of nsp3 (nsp3C) and reconstituted puri fied nsp3C into phospholipid nanodiscs, opening the way for structural studies. Negativestain transmission electron microscopy revealed the presence of nsp3C oligomers very similar to the region abutting and spanning the membrane on the cytosolic side of DMVs in a recent subtomogram average of the SARS-CoV-2 nsp3-4 pore (1). AlphaFoldpredicted structural models fit particularly well with our experimental data and support a pore-forming hexameric assembly. Altogether, our data give unprecedented clues to understand the structural organization of nsp3, the principal component that shapes the molecular pore that spans the DMVs and is required for the export of RNA in vivo. IMPORTANCE Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies. [ABSTRACT FROM AUTHOR]

Abstract 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.

Many microbes accumulate energy storage molecules such as triglycerides (TAG) and starch during nutrient limitation. In eukaryotic green algae grown under nitrogen-limiting conditions, triglyceride accumulation is coupled with chlorosis and growth arrest. In this study, we show that reactive oxygen species (ROS) actively accumulate during nitrogen limitation in the microalga Chlorella sorokiniana. Accumulation of ROS is mediated by the downregulation of genes encoding ROS-quenching enzymes, such as superoxide dismutases, catalase, peroxiredoxin, and glutathione peroxidase-like, and by the upregulation of enzymes involved in generating ROS, such as NADPH oxidase, xanthine oxidase, and amine oxidases. The expression of genes involved in ascorbate and glutathione metabolism is also affected under this condition. ROS accumulation contributes to the degradation of monogalactosyl diacylglycerol (MGDG) and thylakoid membrane remodeling, leading to chlorosis. Quenching ROS under nitrogen limitation reduces the degradation of MGDG and the accumulation of TAG. This work shows that ROS accumulation, membrane remodeling, and TAG accumulation under nitrogen limitation are intricately linked in the microalga C. sorokiniana.

Plasma membranes not only serve as physical barriers to separate the cell or organelle from extracellular or intracellular environments, but also play important roles in many cellular processes, e.g., cell adhesion, cell migration, endocytosis as well as membrane budding, whose correct executions rely on locally highly curved membrane shaping. Mechanically, these membranes are soft fluid interfaces exhibiting extremely dynamic remodeling processes in response to mechanobiological stimulus from their surrounding complex intra/extra-membranous circumstances containing thermal fluctuations, protein binding, protein-protein interaction on the membrane surface forming protein superstructures and active cytoskeletal networks. Correlating these dynamic membrane shaping involving characteristic membrane mechanical properties with cellular functions is essential to improving fundamental understandings in cell physiology and cell biomechanics. The challenge here is to explicitly describe the dynamics of membrane remodeling under the complex biological situations. Interestingly, the developed mesoscopic Monte Carlo (MC) method has the capacity to concurrently capture the elasticity and fluidity of fluid membranes well on large time and length scales, as well as to successfully reproduce fluctuating membrane morphology as observed in experiments. In this review, we focus on this mesoscopic MC method used to depict the thermodynamics of fluctuating fluid membranes and further explore how diverse biophysical factors drive large membrane curvature generation. We also discuss the current efforts of the roles of membrane morphology on the regulation of biological processes on the basis of this mesoscopic MC method, provide the insights into the known biomechanical mechanisms of effect of membrane shape on cellular functions, and point out the potential opportunities where this mesoscopic dynamic MC method can be modified to investigate more intricate biological processes, such as membrane fusion and adhesion.

During infection, positive-stranded RNA causes a rearrangement of the host cell membrane, resulting in specialized membrane structure formation aiding viral genome replication. Double-membrane vesicles (DMVs), typical structures produced by virus-induced membrane rearrangements, are platforms for viral replication. Nidoviruses, one of the most complex positive-strand RNA viruses, have the ability to infect not only mammals and a few birds but also invertebrates. Nidoviruses possess a distinctive replication mechanism, wherein their nonstructural proteins (nsps) play a crucial role in DMV biogenesis. With the participation of host factors related to autophagy and lipid synthesis pathways, several viral nsps hijack the membrane rearrangement process of host endoplasmic reticulum (ER), Golgi apparatus, and other organelles to induce DMV formation. An understanding of the mechanisms of DMV formation and its structure and function in the infectious cycle of nidovirus may be essential for the development of new and effective antiviral strategies in the future.

The endosomal sorting complex required for transport (ESCRT) machinery is centrally involved in the repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale, and Ca2+ has been proposed to play a key signaling role in the process. Here, we show that the Ca2+-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca2+-dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to the membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, and CHMP2A subunits along with the AAA+ ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open an avenue for the mechanistic understanding of endolysosomal membrane repair.

HIV-1 budding as well as many other cellular processes require the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Understanding the architecture of the native ESCRT-III complex at HIV-1 budding sites is limited due to spatial resolution and transient ESCRT-III recruitment. Here, we developed a drug-inducible transient HIV-1 budding inhibitory tool to enhance the ESCRT-III lifetime at budding sites. We generated autocleavable CHMP2A, CHMP3, and CHMP4B fusion proteins with the hepatitis C virus NS3 protease. We characterized the CHMP-NS3 fusion proteins in the absence and presence of protease inhibitor Glecaprevir with regard to expression, stability, localization, and HIV-1 Gag VLP budding. Immunoblotting experiments revealed rapid and stable accumulation of CHMP-NS3 fusion proteins. Notably, upon drug administration, CHMP2A-NS3 and CHMP4B-NS3 fusion proteins substantially decrease VLP release while CHMP3-NS3 exerted no effect but synergized with CHMP2A-NS3. Localization studies demonstrated the relocalization of CHMP-NS3 fusion proteins to the plasma membrane, endosomes, and Gag VLP budding sites. Through the combined use of transmission electron microscopy and video-microscopy, we unveiled drug-dependent accumulation of CHMP2A-NS3 and CHMP4B-NS3, causing a delay in HIV-1 Gag-VLP release. Our findings provide novel insight into the functional consequences of inhibiting ESCRT-III during HIV-1 budding and establish new tools to decipher the role of ESCRT-III at HIV-1 budding sites and other ESCRT-catalyzed cellular processes. [ABSTRACT FROM AUTHOR]

ABSTRACT Endosomal sorting complexes required for transport (ESCRT) play key roles in protein sorting between membrane-bounded compartments of eukaryotic cells. Homologs of many ESCRT components are identifiable in various groups of archaea, especially in Asgardarchaeota, the archaeal phylum that is currently considered to include the closest relatives of eukaryotes, but not in bacteria. We performed a comprehensive search for ESCRT protein homologs in archaea and reconstructed ESCRT evolution using the phylogenetic tree of Vps4 ATPase (ESCRT IV) as a scaffold and using sensitive protein sequence analysis and comparison of structural models to identify previously unknown ESCRT proteins. Several distinct groups of ESCRT systems in archaea outside of Asgard were identified, including proteins structurally similar to ESCRT-I and ESCRT-II, and several other domains involved in protein sorting in eukaryotes, suggesting an early origin of these components. Additionally, distant homologs of CdvA proteins were identified in Thermoproteales which are likely components of the uncharacterized cell division system in these archaea. We propose an evolutionary scenario for the origin of eukaryotic and Asgard ESCRT complexes from ancestral building blocks, namely, the Vps4 ATPase, ESCRT-III components, wH (winged helix-turn-helix fold) and possibly also coiled-coil, and Vps28-like domains. The last archaeal common ancestor likely encompassed a complex ESCRT system that was involved in protein sorting. Subsequent evolution involved either simplification, as in the TACK superphylum, where ESCRT was co-opted for cell division, or complexification as in Asgardarchaeota. In Asgardarchaeota, the connection between ESCRT and the ubiquitin system that was previously considered a eukaryotic signature was already established.IMPORTANCEAll eukaryotic cells possess complex intracellular membrane organization. Endosomal sorting complexes required for transport (ESCRT) play a central role in membrane remodeling which is essential for cellular functionality in eukaryotes. Recently, it has been shown that Asgard archaea, the archaeal phylum that includes the closest known relatives of eukaryotes, encode homologs of many components of the ESCRT systems. We employed protein sequence and structure comparisons to reconstruct the evolution of ESCRT systems in archaea and identified several previously unknown homologs of ESCRT subunits, some of which can be predicted to participate in cell division. The results of this reconstruction indicate that the last archaeal common ancestor already encoded a complex ESCRT system that was involved in protein sorting. In Asgard archaea, ESCRT systems evolved toward greater complexity, and in particular, the connection between ESCRT and the ubiquitin system that was previously considered a eukaryotic signature was established.

Docosahexaenoic acid (DHA, C22:6 ω3) may be involved in various neuroprotective mechanisms that could prevent Alzheimer’s disease (AD). Its influence has still been little explored regarding the dysfunction of the endolysosomal pathway, known as an early key event in the physiopathological continuum triggering AD. This dysfunction could result from the accumulation of degradation products of the precursor protein of AD, in particular the C99 fragment, capable of interacting with endosomal proteins and thus contributing to altering this pathway from the early stages of AD. This study aims to evaluate whether neuroprotection mediated by DHA can also preserve the endolysosomal function. AD-typical endolysosomal abnormalities were recorded in differentiated human SH-SY5Y neuroblastoma cells expressing the Swedish form of human amyloid precursor protein. This altered phenotype included endosome enlargement, the reduced secretion of exosomes, and a higher level of apoptosis, which confirmed the relevance of the cellular model chosen for studying the associated deleterious mechanisms. Second, neuroprotection mediated by DHA was associated with a reduced interaction of C99 with the Rab5 GTPase, lower endosome size, restored exosome production, and reduced neuronal apoptosis. Our data reveal that DHA may influence protein localization and interactions in the neuronal membrane environment, thereby correcting the dysfunction of endocytosis and vesicular trafficking associated with AD.

Mitochondria in mammalian cardiomyocytes display considerable structural heterogeneity, the significance of which is not currently understood. We use electron microscopic tomography to analyze a dataset of 68 mitochondrial subvolumes to look for correlations among mitochondrial size and shape, crista morphology and membrane density, and organelle location within rat cardiac myocytes. A tomographic analysis guided the definition of four classes of crista morphology: lamellar, tubular, mixed and transitional, the last associated with remodeling between lamellar and tubular cristae. Correlations include an apparent bias for mitochondria with lamellar cristae to be located in the regions between myofibrils and a two-fold larger crista membrane density in mitochondria with lamellar cristae relative to mitochondria with tubular cristae. The examination of individual cristae inside mitochondria reveals local variations in crista topology, such as extent of branching, alignment of fenestrations and progressive changes in membrane morphology and packing density. The findings suggest both a rationale for the interfibrillar location of lamellar mitochondria and a pathway for crista remodeling from lamellar to tubular morphology.

Endosomal sorting complex required for transport-III (ESCRT-III) complexes are involved in membrane remodeling. Recently, ESCRT-III homologs were identified in bacteria and the structures of three bacterial representatives were solved. A set of minimal structural features is common to all pro- and eukaryotic ESCRT-IIIs. Conserved interactions stabilize bacterial and eukaryotic oligomeric ESCRT-III assemblies. Annotation and numbering of ESCRT-III helices has not been consistent in the past; a common nomenclature applicable across the ESCRT-III superfamily is suggested. Structural and evolutionary studies of cyanobacterial phage shock protein A (PspA) and inner membrane-associated protein of 30 kDa (IM30) have revealed that these proteins belong to the endosomal sorting complex required for transport-III (ESCRT-III) superfamily, which is conserved across all three domains of life. PspA and IM30 share secondary and tertiary structures with eukaryotic ESCRT-III proteins, whilst also oligomerizing via conserved interactions. Here, we examine the structures of bacterial ESCRT-III-like proteins and compare the monomeric and oligomerized forms with their eukaryotic counterparts. We discuss conserved interactions used for self-assembly and highlight key hinge regions that mediate oligomer ultrastructure versatility. Finally, we address the differences in nomenclature assigned to equivalent structural motifs in both the bacterial and eukaryotic fields and suggest a common nomenclature applicable across the ESCRT-III superfamily. [ABSTRACT FROM AUTHOR]

Recently, the prevalence of atopic dermatitis has increased drastically, especially in urban populations. This multifactorial skin disease is caused by complex interactions between various factors including genetics, environment, lifestyle, and diet. In eczema, apart from using an elimination diet, the adequate content of fatty acids from foods (saturated, monounsaturated, and polyunsaturated fatty acids) plays an important role as an immunomodulatory agent. Different aspects regarding atopic dermatitis include connections between lipid metabolism in atopic dermatitis, with the importance of the MUFA levels, as well as of the omega-6/omega-3 balance that affects the formation of long-chain (C20 eicosanoic and C22 docosaenoic) fatty acids and bioactive lipids from them (such as prostaglandins). Impair/repair of the functioning of epidermal barrier is influenced by these fatty acid levels. The purpose of this review is to drive attention to membrane fatty acid composition and its involvement as the target of fatty acid supplementation. The membrane-targeted strategy indicates the future direction for dermatological research regarding the use of nutritional synergies, in particular using red blood cell fatty acid profiles as a tool for checking the effects of supplementations to reach the target and influence the inflammatory/anti-inflammatory balance of lipid mediators. This knowledge gives the opportunity to develop personalized strategies to create a healthy balance by nutrition with an anti-inflammatory outcome in skin disorders. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy is a conserved pathway allowing the cell to clear and recycle unwanted materials. While decades of research have revealed molecular players and their hierarchical relationships in autophagy, the detailed mechanism by which these molecules function remains largely unknown. In a recent study, Jagan et al. revealed the membrane remodeling ability of two important proteins, MAP1LC3B/LC3B and ATG16L1, in autophagy. LC3B and the ATG12–ATG5-ATG16L1 complex function synergically to induce the formation of phagophore-like membrane cups on membranes both in vitro and in vivo. In addition, the authors showed that the recently characterized C-terminal membrane-binding domain of ATG16L1 is required for the cup formation and the subsequent transition to autophagic vesicles. Together this research provides more insight into the molecular function of LC3B and ATG16L1, as well as a possible mechanism for phagophore biogenesis. [ABSTRACT FROM AUTHOR]

Red blood cell (RBC) deformability, expressing their ability to change their shape, allows them to minimize their resistance to flow and optimize oxygen delivery to the tissues. RBC with reduced deformability may lead to increased vascular resistance, capillary occlusion, and impaired perfusion and oxygen delivery. A reduction in deformability, as occurs during RBC physiological aging and under blood storage, is implicated in the pathophysiology of diverse conditions with circulatory disorders and anemias. The change in RBC deformability is associated with metabolic and structural alterations, mostly uncharacterized. To bridge this gap, we analyzed the membrane protein levels, using mass spectroscopy, of RBC with varying deformability determined by image analysis. In total, 752 membrane proteins were identified. However, deformability was positively correlated with the level of only fourteen proteins, with a highly significant inter-correlation between them. These proteins are involved in membrane rafting and/or the membrane–cytoskeleton linkage. These findings suggest that the reduction of deformability is a programmed (not arbitrary) process of remodeling and shedding of membrane fragments, possibly mirroring the formation of extracellular vesicles. The highly significant inter-correlation between the deformability-expressing proteins infers that the cell deformability can be assessed by determining the level of a few, possibly one, of them. [ABSTRACT FROM AUTHOR]

Since decades plant lipid droplets (LDs) are described as storage organelles accumulated in seeds to provide energy for seedling growth after germination. Indeed, LDs are the site of accumulation for neutral lipids, predominantly triacylglycerols (TAGs), one of the most energy-dense molecules, and sterol esters. Such organelles are present in the whole plant kingdom, from microalgae to perennial trees, and can probably be found in all plant tissues. Several studies over the past decade have revealed that LDs are not merely simple energy storage compartments, but also dynamic structures involved in diverse cellular processes like membrane remodeling, regulation of energy homeostasis and stress responses. In this review, we aim to highlight the functions of LDs in plant development and response to environmental changes. In particular, we tackle the fate and roles of LDs during the plant post-stress recovery phase. [ABSTRACT FROM AUTHOR]

Over the last few decades, in vitro single-molecule manipulation techniques have enabled the use of force and displacement as controlled variables in biochemistry. Measuring the effect of mechanical force on the real-time kinetics of a biological process gives us access to the rates, equilibrium constants and free-energy landscapes of the mechanical steps of the reaction; this information is not accessible by ensemble assays. Optical tweezers are the current method of choice in single-molecule manipulation due to their versatility, high force and spatial and temporal resolutions. The aim of this review is to describe the contributions of our lab in the single-molecule manipulation field. We present here several optical tweezers assays refined in our laboratory to probe the dynamics and mechano-chemical properties of biological molecular motors and synthetic molecular devices at the single-molecule level.

Phafins are PH (Pleckstrin Homology) and FYVE (Fab1, YOTB, Vac1, and EEA1) domain-containing proteins. The Phafin protein family is classified into two groups based on their sequence homology and functional similarity: Phafin1 and Phafin2. This protein family is unique because both the PH and FYVE domains bind to phosphatidylinositol 3-phosphate [PtdIns(3)P], a phosphoinositide primarily found in endosomal and lysosomal membranes. Phafin proteins act as PtdIns(3)P effectors in apoptosis, endocytic cargo trafficking, and autophagy. Additionally, Phafin2 is recruited to macropinocytic compartments through coincidence detection of PtdIns(3)P and PtdIns(4)P. Membrane-associated Phafins serve as adaptor proteins that recruit other binding partners. In addition to the phosphoinositide-binding domains, Phafin proteins present a poly aspartic acid motif that regulates membrane binding specificity. In this review, we summarize the involvement of Phafins in several cellular pathways and their potential physiological functions while highlighting the similarities and differences between Phafin1 and Phafin2. Besides, we discuss research perspectives for Phafins. [ABSTRACT FROM AUTHOR]

Since decades plant lipid droplets (LDs) are described as storage organelles accumulated in seeds to provide energy for seedling growth after germination. Indeed, LDs are the site of accumulation for neutral lipids, predominantly triacylglycerols (TAGs), one of the most energy-dense molecules, and sterol esters. Such organelles are present in the whole plant kingdom, from microalgae to perennial trees, and can probably be found in all plant tissues. Several studies over the past decade have revealed that LDs are not merely simple energy storage compartments, but also dynamic structures involved in diverse cellular processes like membrane remodeling, regulation of energy homeostasis and stress responses. In this review, we aim to highlight the functions of LDs in plant development and response to environmental changes. In particular, we tackle the fate and roles of LDs during the plant post-stress recovery phase.

The Sar1 GTPase initiates coat protein II (COPII)-mediated protein transport by generating membrane curvature at subdomains on the endoplasmic reticulum, where it is activated by the guanine nucleotide exchange factor (GEF) Sec12. Crystal structures of GDP- and GTP-bound forms of Sar1 suggest that it undergoes a conformational switch in whichGTPbinding enhances the exposure of an amino-terminal amphipathic helix necessary for efficient membrane penetration. However, key residues in the amino terminus were not resolved in crystal structures, and experimental studies have suggested that the amino terminus of Sar1 is solvent-exposed in the absence of a membrane, even in the GDP-bound state. Therefore, the molecular mechanism by which GTP binding activates the membrane-remodeling activity of Sar1 remains unclear. Using atomistic molecular dynamics simulations, we compare the membranebinding and curvature generation activities of Sar1 in its GDP- and GTP-bound states. We show that in the GTP-bound state, Sar1 inserts into the membrane with its complete (residues 1 to 23) amphipathic amino-terminal helix, while Sar1-GDP binds to the membrane only through its first 12 residues. Such differential membranebinding modes translate into significant differences in the protein volume inserted into the membrane. As a result, Sar1-GTP generates positive membrane curvature 10 to 20 times higher than Sar1-GDP. Dimerization of the GTP-bound form of Sar1 further amplifies curvature generation. Taken together, our results present a detailed molecular mechanism for how the nucleotide-bound state of Sar1 regulates its membrane-binding and remodeling activities in a concentration-dependent manner, paving the way toward a better understanding COPII-mediated membrane transport. [ABSTRACT FROM AUTHOR]

Specialized classes of proteins, working together in a tightly orchestrated manner, induce and maintain highly curved cellular and organelles membrane morphology. Due to the various experimental constraints, including the resolution limits of imaging techniques, it is non-trivial to accurately elucidate interactions among the various components involved in membrane deformation. The spatial and temporal scales of the systems also make it formidable to investigate them using simulations with molecular details. Interestingly, mechanics-based mesoscopic models have been used with great success in recapitulating the membrane deformations observed in experiments. In this review, we collate together and discuss the various mechanics-based mesoscopic models for protein-mediated membrane deformation studies. In particular, we provide an elaborate description of a mesoscopic model where the membrane is modeled as a triangulated sheet and proteins are represented as either nematics or filaments. This representation allows us to explore the various aspects of protein–protein and protein–membrane interactions as well as examine the underlying mechanistic pathways for emergent behavior such as curvature-mediated protein localization and membrane deformation. We also put forward current efforts in the field towards back-mapping these mesoscopic models to finer-grained particle-based models—a framework that could be used to explore how molecular interactions propagate to physical scales and vice-versa. We end the review with an integrative-modeling-based road map where experimental imaging micrograph and biochemical data are combined with mesoscopic and molecular simulations methods in a theoretically consistent manner to faithfully recapitulate the multiple length and time scales in the membrane remodeling processes. [ABSTRACT FROM AUTHOR]

Optical tweezers allow precise measurement of forces and distances with piconewton and nanometer precision, and have thus been instrumental in elucidating the mechanistic details of various biological processes. Some examples include the characterization of motor protein activity, studies of protein–DNA interactions, and characterizing protein folding trajectories. The use of optical tweezers (OT) to study membranes is, however, much less abundant. Here, we review biophysical studies of membranes that utilize optical tweezers, with emphasis on various assays that have been developed and their benefits and limitations. First, we discuss assays that employ membrane-coated beads, and overview protein–membrane interactions studies based on manipulation of such beads. We further overview a body of studies that make use of a very powerful experimental tool, the combination of OT, micropipette aspiration, and fluorescence microscopy, that allow detailed studies of membrane curvature generation and sensitivity. Finally, we describe studies focused on membrane fusion and fission. We then summarize the overall progress in the field and outline future directions. [ABSTRACT FROM AUTHOR]

The plasma membrane (PM) of cells is a dynamic structure whose morphology and composition is in constant flux. PM morphologic changes are particularly relevant for the assembly and disassembly of signaling platforms involving surface-bound signaling proteins, as well as for many other mechanochemical processes that occur at the PM surface. Surface-bound membrane proteins (SBMP) require efficient association with the PM for their function, which is often achieved by the coordinated interactions of intrinsically disordered regions (IDRs) and globular domains with membrane lipids. This review focuses on the role of IDR-containing SBMPs in remodeling the composition and curvature of the PM. The ability of IDR-bearing SBMPs to remodel the Gaussian and mean curvature energies of the PM is intimately linked to their ability to sort subsets of phospholipids into nanoclusters. We therefore discuss how IDRs of many SBMPs encode lipid-binding specificity or facilitate cluster formation, both of which increase their membrane remodeling capacity, and how SBMP oligomers alter membrane shape by monolayer surface area expansion and molecular crowding. [ABSTRACT FROM AUTHOR]

Over the last few decades, in vitro single-molecule manipulation techniques have enabled the use of force and displacement as controlled variables in biochemistry. Measuring the effect of mechanical force on the real-time kinetics of a biological process gives us access to the rates, equilibrium constants and free-energy landscapes of the mechanical steps of the reaction; this information is not accessible by ensemble assays. Optical tweezers are the current method of choice in single-molecule manipulation due to their versatility, high force and spatial and temporal resolutions. The aim of this review is to describe the contributions of our lab in the single-molecule manipulation field. We present here several optical tweezers assays refined in our laboratory to probe the dynamics and mechano-chemical properties of biological molecular motors and synthetic molecular devices at the single-molecule level. [ABSTRACT FROM AUTHOR]

Syn DLP, a dynamin-like protein (DLP) encoded in the cyanobacterium Synechocystis sp. PCC 6803, has recently been identified to be structurally highly similar to eukaryotic dynamins. To elucidate structural changes during guanosine triphosphate (GTP) hydrolysis, we solved the cryoelectron microscopy (cryo-EM) structures of oligomeric full-length Syn DLP after addition of guanosine diphosphate (GDP) at 4.1 Å and GTP at 3.6-Å resolution as well as a GMPPNP-bound dimer structure of a minimal G-domain construct of Syn DLP at 3.8-Å resolution. In comparison with what has been seen in the previously resolved apo structure, we found that the G-domain is tilted upward relative to the stalk upon GTP hydrolysis and that the G-domain dimerizes via an additional extended dimerization domain not present in canonical G-domains. When incubated with lipid vesicles, we observed formation of irregular tubular Syn DLP assemblies that interact with negatively charged lipids. Here, we provide the structural framework of a series of different functional Syn DLP assembly states during GTP turnover. [Display omitted] • Cryo-EM structures of Syn DLP with GDP and GTP • Cryo-EM structure of GMPPNP-bound Syn DLP minimal GD construct • Syn DLP GDs dimerize via an unique extended dimerization domain • Syn DLP remodels membranes Syn DLP is a cyanobacterial dynamin-like protein. Junglas et al. show the structures of Syn DLP incubated with nucleotides and the structure of a GMPPNP-bound minimal GD construct that dimerizes via an unique extended dimerization domain not present in canonical G-domains. [ABSTRACT FROM AUTHOR]

Recently, the prevalence of atopic dermatitis has increased drastically, especially in urban populations. This multifactorial skin disease is caused by complex interactions between various factors including genetics, environment, lifestyle, and diet. In eczema, apart from using an elimination diet, the adequate content of fatty acids from foods (saturated, monounsaturated, and polyunsaturated fatty acids) plays an important role as an immunomodulatory agent. Different aspects regarding atopic dermatitis include connections between lipid metabolism in atopic dermatitis, with the importance of the MUFA levels, as well as of the omega-6/omega-3 balance that affects the formation of long-chain (C20 eicosanoic and C22 docosaenoic) fatty acids and bioactive lipids from them (such as prostaglandins). Impair/repair of the functioning of epidermal barrier is influenced by these fatty acid levels. The purpose of this review is to drive attention to membrane fatty acid composition and its involvement as the target of fatty acid supplementation. The membrane-targeted strategy indicates the future direction for dermatological research regarding the use of nutritional synergies, in particular using red blood cell fatty acid profiles as a tool for checking the effects of supplementations to reach the target and influence the inflammatory/anti-inflammatory balance of lipid mediators. This knowledge gives the opportunity to develop personalized strategies to create a healthy balance by nutrition with an anti-inflammatory outcome in skin disorders.

Lipid droplets are fat storage organelles ubiquitously distributed across the eukaryotic kingdom. They have a central role in regulating lipid metabolism and undergo a dynamic turnover of biogenesis and breakdown to meet cellular requirements for fatty acids, including polyunsaturated fatty acids. Polyunsaturated fatty acids esterified in membrane phospholipids define membrane fluidity and can be released by the activity of phospholipases A2 to act as ligands for nuclear receptors or to be metabolized into a wide spectrum of lipid signaling mediators. Polyunsaturated fatty acids in membrane phospholipids are also highly susceptible to lipid peroxidation, which if left uncontrolled leads to ferroptotic cell death. On the one hand, lipid droplets act as antioxidant organelles that control polyunsaturated fatty acid storage in triglycerides in order to reduce membrane lipid peroxidation, preserve organelle function and prevent cell death, including ferroptosis. On the other hand, lipid droplet breakdown fine-tunes the delivery of polyunsaturated fatty acids into metabolic and signaling pathways, but unrestricted lipid droplet breakdown may also lead to the release of lethal levels of polyunsaturated fatty acids. Precise regulation of lipid droplet turnover is thus essential for polyunsaturated fatty acid distribution and cellular homeostasis. In this review, we focus on emerging aspects of lipid droplet-mediated regulation of polyunsaturated fatty acid trafficking, including the management of membrane lipid peroxidation, ferroptosis and lipid mediator signaling.

The endosomal sorting complexes required for transport (ESCRT) pathway mediates cellular membrane remodeling and fission reactions. The pathway comprises five core complexes: ALIX, ESCRT-I, ESCRT-II, ESCRT-III, and Vps4. These soluble complexes are typically recruited to target membranes by site-specific adaptors that bind one or both of the early-acting ESCRT factors: ALIX and ESCRT-I/ESCRT-II. These factors, in turn, nucleate assembly of ESCRT-III subunits into membrane-bound filaments that recruit the AAA ATPase Vps4. Together, ESCRT-III filaments and Vps4 remodel and sever membranes. Here, we review recent advances in our understanding of the structures, activities, and mechanisms of the ESCRT-III and Vps4 machinery, including the first high-resolution structures of ESCRT-III filaments, the assembled Vps4 enzyme in complex with an ESCRT-III substrate, the discovery that ESCRT-III/Vps4 complexes can promote both inside-out and outside-in membrane fission reactions, and emerging mechanistic models for ESCRT-mediated membrane fission.

A recent study by Beeman et al. exploring disease-related missense mutations in TAOK1 revealed a self-regulating association of the kinase with the plasma membrane that is critical for neuronal morphogenesis. Using a combination of in vitro approaches and elegant in silico modeling, the authors describe an aberrant membrane protrusions phenotype in kinase-deficient mutants reminiscent of TAOK2's indirect regulation of neuronal morphology, thus providing a converging patho-mechanism across several neurodevelopmental disorders. [ABSTRACT FROM AUTHOR]

Chloride is the major free anion in the extracellular space (>100 mM) and within the cytoplasm in eukaryotes (10 ∼ 20 mM). Cytoplasmic Cl- level is dynamically regulated by Cl- channels and transporters. It is well established that movement of Cl- across the cell membrane is coupled with cell excitability through changes in membrane potential and with water secretion. However, whether cytoplasmic Cl- plays additional roles in animal development and tissue homeostasis is unknown. Here we use genetics, cell biological and pharmacological tools to demonstrate that TMEM16A, an evolutionarily conserved calcium-activated chloride channel (CaCC), regulates cytoplasmic Cl- homeostasis and promotes plasma membrane remodeling required for mammalian epithelial morphogenesis. We demonstrate that TMEM16A-mediated control of cytoplasmic Cl- regulates the organization of the major phosphoinositide species PtdIns(4,5)P2 into microdomains on the plasma membrane, analogous to processes that cluster soluble and membrane proteins into phase-separated droplets. We further show that an adequate cytoplasmic Cl- level is required for proper endocytic trafficking and membrane supply during early stages of ciliogenesis and adherens junction remodeling. Our study thus uncovers a critical function of CaCC-mediated cytoplasmic Cl- homeostasis in controlling the organization of PtdIns(4,5)P2 microdomains and membrane remodeling. This newly defined role of cytoplasmic Cl- may shed light on the mechanisms of intracellular Cl- signaling events crucial for regulating tissue architecture and organelle biogenesis during animal development.

[Display omitted] Rhamnolipids (RLs) are biosurfactants with significant tensioactive and emulsifying properties. They are mainly composed by mono-RL and di-RL components. Although there are numerous studies concerning their molecular properties, information is scarce regarding the mechanisms by which each of the two components interacts with cell membranes. Herein, we performed phase-contrast and fluorescence microscopy experiments on plasma membrane models represented by giant-unilamellar-vesicles (GUVs) composed of 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC), 2-[[(E ,2 S ,3 R)-1,3-dihydroxy-2-(octadecanoylamino) octadec-4-enyl]peroxy-hydroxyphosphoryl]oxyethyl-trimethylazanium (sphingomyelin, SM) and (3 β)-cholest-5-en-3-ol (cholesterol, CHOL) (1:1:1 M ratio), which present liquid-order ( L o ) liquid-disorder ( L d ) phase coexistence, in the presence of either mono-RL or di-RL in 0.06–0.25 mM concentration range. A new method has been developed to determine area and volume of GUVs with asymmetrical shape and a kinetic model describing GUV-RL interaction in terms of two mechanisms, RL-insertion and pore formation, has been worked out. Results show that the insertion of mono-RL in the membrane outer leaflet is the dominant process with no pore formation and a negligible effect in modifying membrane permeability, but induces lipid mixing. Conversely, the di-RL-GUV interaction begins with the insertion mechanism and, as the time passes by, the pore formation process occurs. The analyses of di-RL show that the whole process is only relevant in the L d phase with a higher extent to 0.25 mM than to 0.06 mM. [ABSTRACT FROM AUTHOR]