Positive-sense RNA viruses, including SARS-CoV-1 and -2, DENV, and CHIKV, replicate in curved membrane compartments within host cells. Non-structural proteins (nsPs) critically regulate these nanoscale membrane structures, yet their curvature-dependent assembly remains elusive due to the challenges of imaging nanoscale interaction on curved surfaces. Using vertically aligned nanostructures to generate pre-defined membrane curvatures, we here investigate the impact of curvature on nsPs assembly. Taking CHIKV as a model, we reveal that nsP1 preferentially binds and stabilizes on positively curved membranes, with stronger accumulation at radii ≤150 nm. This is driven by hydrophobic residues in the membrane association (MA) loops of individual nsP1. Molecular dynamics simulations further confirm the improved binding stability of nsP1 on curved membranes, particularly when it forms a dodecamer ring. Together, nsP1 supports a strong saddle curvature association, with flexible MA loops sensing a range of positive curvatures in the x-z plane while the rigid dodecamer stabilizing fixed negative curvature in the x-y plane - crucial for constraining the membrane spherule neck during replication progression. Moreover, CHIKV replication enriches on patterned nanoring structures, underscoring the curvature-guided assembly of the viral replication complex. Our findings highlight membrane curvature as a key regulator of viral nsPs organization, opening new avenues for studying membrane remodeling in viral replication.

Previous studies have confirmed ambient fine particulate matter (PM2.5) as a major environmental risk factor for cardiovascular diseases (CVDs), yet the specific molecular pathways remain poorly understood. Furthermore, while physical activity benefits cardiovascular health, its protective effects against PM2.5-induced damage need further explored. We aimed to investigate the relationship between long-term PM2.5 exposure, physical activity, and cardiovascular health, and explore the potential molecular mechanisms. This research combined epidemiological and experimental approaches. The epidemiological study analyzed data from the China Health and Retirement Longitudinal Study (CHARLS) to investigate the associations among long-term PM2.5 exposure, physical activity, and CVDs. For the experimental study, C57BL/6 male mice were assigned to either regular physical activity or sedentary behavior, and were exposed to PM2.5 or filtered air (FA) for 2, 4, and 6 months. We observed that long-term PM2.5 exposure significantly increased cardiovascular disease risk, while physical activity exhibited protective effects and can partially mitigate the adverse impacts of PM2.5 on heart disease and dyslipidemia. In animal study, mice with long-term exposure to PM2.5 demonstrated elevated blood pressure, disrupted adipokine levels, altered lipid profiles, and mitochondrial damage. Regular physical activity partially mitigated these adverse effects. Lipidomics and proteomics analyses revealed that PM2.5 exposure disrupted lipid metabolism networks and protein regulatory pathways, while regular physical activity mitigated these perturbations through the modulation of lipid metabolism, the coagulation cascade, and immune responses. These findings underscore the importance of regular physical activity in public health strategies, while prioritizing PM2.5 reduction measures for cardiovascular disease prevention.

The dynamic addition and removal of long-chain fatty acids modulate protein function and localization. The α/β hydrolase domain-containing (ABHD) 17 enzymes remove acyl chains from membrane-localized proteins such as the oncoprotein NRas, but how the ABHD17 proteins are regulated is unknown. Here, we used cell-based studies and molecular dynamics simulations to show that ABHD17 activity is controlled by two mobile elements-an S-acylated N-terminal helix and a loop-that flank the putative substrate-binding pocket. Multiple S-acylation events anchor the N-terminal helix in the membrane, enabling hydrophobic residues in the loop to engage with the bilayer. This stabilizes the conformation of both helix and loop, alters the conformation of the binding pocket, and optimally positions the enzyme for substrate engagement. S-acylation may be a general feature of acyl-protein thioesterases. By providing a mechanistic understanding of how the lipid modification of a lipid-removing enzyme promotes its enzymatic activity, this work contributes to our understanding of cellular S-acylation cycles.

The COVID-19 pandemic offered an unprecedented glimpse into the evolution of its causative virus, SARS-CoV-2. It has been estimated that since its outbreak in late 2019, the virus has explored all possible alternatives in terms of missense mutations for all sites of its polypeptide chain. Spike protein of the virus exhibits the largest sequence variation in particular, with many individual mutations impacting target recognition, cellular entry, and endosomal escape of the virus. Moreover, recent studies unveiled a significant increase in the total charge on the spike protein during the evolution of the virus in the initial period of the pandemic. While this trend has recently come to a halt, we perform a sequence-based analysis of the spike protein of 2665 SARS-CoV-2 variants which shows that mutations in ionizable amino acids continue to occur with the newly emerging variants, with notable differences between lineages from different clades. What is more, we show that within mutations of amino acids which can acquire positive charge, the spike protein of SARS-CoV-2 exhibits a prominent preference for lysine residues over arginine residues. This lysine-to-arginine ratio increased at several points during spike protein evolution, most recently with BA.2.86 and its sublineages, including the recently dominant JN.1, KP.3, and XEC variants. The increased ratio is a consequence of mutations in different structural regions of the spike protein and is now among the highest among viral species in the Coronaviridae family. The impact of high lysine-to-arginine ratio in the spike proteins of BA.2.86 and its daughter lineages on viral fitness remains unclear; we discuss several potential mechanisms that could play a role and that can serve as a starting point for further studies.

Antimicrobial peptides (AMPs) have emerged as a promising solution to the escalating public health threat caused by multidrug-resistant bacteria. Although ongoing research efforts have established AMP's role in membrane permeabilization and leakage, the precise mechanisms driving these disruption patterns remain unclear. We leverage molecular dynamics (MD) simulations enhanced by membrane mimetic (HMMM) to systematically investigate how the physiochemical properties of magainin (+3) and pexiganan (+9) affect their localization, insertion, curvature perturbation, and membrane binding ensemble. Building on existing microbiology, NMR, circular dichroism, and fluorescence data, our analysis reveals that the lipid makeup is a key determinant in the binding dynamics and structural conformation of AMPs. We find that phospholipid type is crucial for peptide localization, demonstrated through magainin's predominant interaction with lipid tails and pexiganan's with polar headgroups in POPC/POPS membranes. The membrane curvature changes induced by pexiganan relative to magainin suggest that AMPs with larger charges have more potential in modulating bilayer bending. These insights advance our understanding of AMP-membrane interactions at the molecular level, offering guidance for the design of targeted antimicrobial therapies.

Membranes are molecular interfaces that compartmentalize cells to control the flow of nutrients and information. These functions are facilitated by diverse collections of lipids, nearly all of which are distributed asymmetrically between the two bilayer leaflets. Most models of biomembrane structure and function include the implicit assumption that these leaflets have similar abundances of phospholipids. Here, we show that this assumption is generally invalid and investigate the consequences of lipid abundance imbalances in mammalian plasma membranes (PMs). Using lipidomics, we report that cytoplasmic leaflets of human erythrocyte membranes have >50% overabundance of phospholipids compared with exoplasmic leaflets. This imbalance is enabled by an asymmetric interleaflet distribution of cholesterol, which regulates cellular cholesterol homeostasis. These features produce unique functional characteristics, including low PM permeability and resting tension in the cytoplasmic leaflet that regulates protein localization.

Cells contain a plethora of structurally diverse lipid species, which are unevenly distributed across the different cellular membrane compartments. Some of these lipid species require vesicular trafficking to reach their subcellular destinations. Here, we review recent advances made in the field that contribute to understanding lipid sorting during endomembrane trafficking.

The evolution of eukaryotes is a fundamental event in the history of life. The closest prokaryotic lineage to eukaryotes, the Asgardarchaeota, encode proteins previously found only in eukaryotes, providing insight into their archaeal ancestor. Eukaryotic cells are characterized by endomembrane organelles, and the Arf family GTPases regulate organelle dynamics by recruiting effector proteins to membranes upon activation. The Arf family is ubiquitous among eukaryotes, but its origins remain elusive. Here we report a group of prokaryotic GTPases, the ArfRs, which are widely present in Asgardarchaeota. Phylogenetic analyses reveal that eukaryotic Arf family proteins arose from the ArfR group. Expression of representative Asgardarchaeota ArfR proteins in yeast and X-ray crystallographic studies show that ArfR GTPases possess the mechanism of membrane binding and structural features unique to Arf family proteins. Our results indicate that Arf family GTPases originated in the archaeal ancestor of eukaryotes, consistent with aspects of the endomembrane system evolving early in eukaryogenesis.

α-Synuclein (αSyn), an intrinsically disordered protein implicated in Parkinson's disease, is potentially thought to initiate aggregation through binding to cellular membranes. Previous studies have suggested that anionic membrane charge is necessary for this binding. However, these studies largely focus on unmodified αSyn, while nearly all αSyn in the body is N-terminally acetylated (NTA). NTA dramatically shifts the narrative by diminishing αSyn's reliance on anionic charge for membrane binding. Instead, we demonstrate that membrane packing defects are the dominant forces driving NTA-αSyn interactions, challenging the long-standing paradigm that anionic membranes are essential for αSyn binding. Using fluorescence microscopy and circular dichroism spectroscopy, we monitored the binding of NTA-αSyn to reconstituted membrane surfaces with different lipid compositions. Phosphatidylcholine and phosphatidylserine concentrations were varied to control surface charge, while phospholipid tail unsaturation and methylation were varied to control lipid packing. All-atom molecular dynamics simulations of lipid bilayers supported the observation that membrane packing defects are necessary for NTA-αSyn binding and that defect-rich membranes are sufficient for NTA-αSyn binding regardless of membrane charge. We further demonstrated that this affinity for membrane defects persisted in reconstituted, cholesterol-containing membranes that mimicked the physiological lipid composition of synaptic vesicles. Increasing phospholipid unsaturation in these mimics led to more membrane packing defects and a corresponding increase in NTA-αSyn binding. Altogether, our results point to a mechanism for the regulation of NTA-αSyn binding in biological membranes that extends beyond phospholipid charge to the structural properties of the lipids themselves.

Amphipathic helices (AHs) are ubiquitous protein motifs that modulate targeting to organellar membranes by sensing differences in bulk membrane properties. However, the adaptation between membrane-targeting AHs and the nuclear membrane environment that surrounds the genome is poorly understood. Here, we computationally screened for candidate AHs in a curated list of characterized and putative human inner nuclear membrane (INM) proteins. Cell biological and in vitro experimental assays combined with computational calculations demonstrated that AHs detect lipid packing defects over electrostatics to bind to the INM, indicating that the INM is loosely packed under basal conditions. Membrane tension resulting from hypotonic shock further promoted AH binding to the INM, whereas cell-substrate stretch did not enhance recruitment of membrane tension-sensitive AHs. Together, our work demonstrates the rules driving lipid-protein interactions at the INM, and its implications in the response of the nucleus to different stimuli.

Membranes are molecular interfaces that compartmentalize cells to control the flow of nutrients and information. These functions are facilitated by diverse collections of lipids, nearly all of which are distributed asymmetrically between the two bilayer leaflets. Most models of biomembrane structure and function often include the implicit assumption that these leaflets have similar abundances of phospholipids. Here, we show that this assumption is generally invalid and investigate the consequences of lipid abundance imbalances in mammalian plasma membranes (PM). Using quantitative lipidomics, we discovered that cytoplasmic leaflets of human erythrocyte membranes have >50% overabundance of phospholipids compared to exoplasmic leaflets. This imbalance is enabled by an asymmetric interleaflet distribution of cholesterol, which regulates cellular cholesterol homeostasis. These features produce unique functional characteristics, including low PM permeability and resting tension in the cytoplasmic leaflet that regulates protein localization. These largely overlooked aspects of membrane asymmetry represent an evolution of classic paradigms of biomembrane structure and physiology.

This review examines the relationships between membrane chemistry, curvature-sensing proteins, and cellular morphogenesis. Curvature-sensing proteins are often orders of magnitude smaller than the membrane curvatures they localize to. How are nanometer-scale proteins used to sense micrometer-scale membrane features? Here, we trace the journey of curvature-sensing proteins as they engage with lipid membranes through a combination of electrostatic and hydrophobic interactions. We discuss how curvature sensing hinges on membrane features like lipid charge, packing, and the directionality of membrane curvature. Once bound to the membrane, many curvature sensors undergo self-assembly (i.e., they oligomerize or form higher-order assemblies that are key for initiating and regulating cell shape transformations). Central to these discussions are the micrometer-scale curvature-sensing proteins' septins. By discussing recent literature surrounding septin membrane association, assembly, and their many functions in morphogenesis with support from other well-studied curvature sensors, we aim to synthesize possible mechanisms underlining cell shape sensing.

Perilipins (PLINs), the most abundant proteins on lipid droplets (LDs), display similar domain organization including amphipathic helices (AH). However, the five human PLINs bind different LDs, suggesting different modes of interaction. We established a minimal system whereby artificial LDs covered with defined polar lipids were transiently deformed to promote surface tension. Binding of purified PLIN3 and PLIN4 AH was strongly facilitated by tension but was poorly sensitive to phospholipid composition and to the presence of diacylglycerol. Accordingly, LD coverage by PLIN3 increased as phospholipid coverage decreased. In contrast, PLIN1 bound readily to LDs fully covered by phospholipids; PLIN2 showed an intermediate behavior between PLIN1 and PLIN3. In human adipocytes, PLIN3/4 were found in a soluble pool and relocated to LDs upon stimulation of fast triglyceride synthesis, whereas PLIN1 and PLIN2 localized to pre-existing LDs, consistent with the large difference in LD avidity observed in vitro. We conclude that the PLIN repertoire is adapted to handling LDs with different surface properties.

Most of the biological interfaces are curved. Understanding the organizational structures and interaction patterns at such curved biointerfaces is therefore crucial not only for deepening our comprehension of the principles that govern life processes but also for designing and developing targeted drugs aimed at diseased cells and tissues. Despite the considerable efforts dedicated to this area of research, our understanding of curved biological interfaces is still limited. Many aspects of these interfaces remain elusive, presenting both challenges and opportunities for further exploration. In this review, we summarize the structural characteristics of biological interfaces found in nature, the current research status of materials associated with curved biointerfaces, and the theoretical advancements achieved to date. Finally, we outline future trends and challenges in the theoretical and technological development of curved biointerfaces. By addressing these challenges, people could bridge the knowledge gap and unlock the full potential of curved biointerfaces for scientific and technological advancements, ultimately benefiting various fields and improving human health and well-being.

The Golgi apparatus is essential for protein sorting, yet its quality control mechanisms are poorly understood. Here we show that the Dsc ubiquitin ligase complex uses its rhomboid pseudo-protease subunit, Dsc2, to assess the hydrophobic length of α-helical transmembrane domains (TMDs) at the Golgi. Thereby the Dsc complex likely interacts with orphaned ER and Golgi proteins that have shorter TMDs and ubiquitinates them for targeted degradation. Some Dsc substrates will be extracted by Cdc48 for endosome and Golgi associated proteasomal degradation (EGAD), while others will undergo ESCRT dependent vacuolar degradation. Some substrates are degraded by both, EGAD- or ESCRT pathways. The accumulation of Dsc substrates entails a specific increase in glycerophospholipids with shorter and asymmetric fatty acyl chains. Hence, the Dsc complex mediates the selective degradation of orphaned proteins at the sorting center of cells, which prevents their spreading across other organelles and thereby preserves cellular membrane protein and lipid composition.

α-Synuclein (α-syn), a crucial molecule in Parkinson's disease (PD), is known for its interaction with lipid membranes, which facilitates vesicle trafficking and modulates its pathological aggregation. Deciphering the complexity of the membrane-binding behavior of α-syn is crucial to understand its functions and the pathology of PD. Here, we used single-molecule imaging to show that α-syn forms multimers on lipid membranes with huge intermultimer distances. The multimers are characterized by self-limiting growth, manifesting in concentration-dependent exchanges of monomers, which are fast at micromolar concentrations and almost stop at nanomolar concentrations. We further uncovered movement patterns of α-syn's occasional trapping on membranes, which may be attributed to sparse lipid packing defects. Mutations such as E46K and E35K may disrupt the limit on the growth, resulting in larger multimers and accelerated amyloid fibril formation. This work emphasizes sophisticated regulation of α-syn multimerization on membranes as a critical underlying factor in the PD pathology.

Statins are drugs used in cardiovascular pharmacotherapy to decrease hypercholesterolemia and lower the risk of atherosclerosis. Statins also increase the risk of rhabdomyolysis, which is often minimized in comparison with large relative risk reductions of cardiovascular disease reported in clinical trials. By contrast, absolute risk reductions of cardiovascular disease are often clinically insignificant and unreported in statin clinical trials. Additionally, cytotoxic effects of statins inhibit cancer cell proliferation and reduce cancer risk, but other studies found that statins are carcinogenic. Due to an inverse association between incidence of cancer and atherosclerosis, the indication to prescribe statins likely biases the association of statins with cancer prevention. Dietary patterns associated with atherosclerosis and cancer contain inverse amounts of cholesterol and phosphate, an essential mineral that stimulates tumorigenesis. Accordingly, lower cancer risk is associated with high dietary cholesterol intake and increased risk of atherosclerosis. Furthermore, serum is exposed to excessive inorganic phosphate that could increase cancer risk as rhabdomyolysis induced by statins releases phosphate from skeletal muscle breakdown. Increased risk of comorbid conditions associated with statins may share the mediating factor of phosphate toxicity. More research is warranted on statins in the cause and prevention of cancer.

The carnitine/acylcarnitine carrier (CAC) is a crucial protein for cellular energy metabolism, facilitating the exchange of acylcarnitines and free carnitine across the mitochondrial membrane, thereby enabling fatty acid β-oxidation and oxidative phosphorylation (OXPHOS). Although CAC has not been crystallised, structural insights are derived from the mitochondrial ADP/ATP carrier (AAC) structures in both cytosolic and matrix conformations. These structures underpin a single binding centre-gated pore mechanism, a common feature among mitochondrial carrier (MC) family members. The functional implications of this mechanism are well-supported, yet the structural organization of the CAC, particularly the formation of dimeric or oligomeric assemblies, remains contentious. Recent investigations employing biochemical techniques on purified and reconstituted CAC, alongside molecular modelling based on crystallographic AAC dimeric structures, suggest that CAC can indeed form dimers. Importantly, this dimerization does not alter the transport mechanism, a phenomenon observed in various other membrane transporters across different protein families. This observation aligns with the ping-pong kinetic model, where the dimeric form potentially facilitates efficient substrate translocation without necessitating mechanistic alterations. The presented findings thus contribute to a deeper understanding of CAC's functional dynamics and its structural parallels with other MC family members.

Phosphatidylcholine (PC) is the major membrane phospholipid in most eukaryotic cells. Bi-allelic loss of function variants in CHKB, encoding the first step in the synthesis of PC, is the cause of a rostrocaudal muscular dystrophy in both humans and mice. Loss of sarcolemma integrity is a hallmark of muscular dystrophies; however, how this occurs in the absence of choline kinase function is not known. We determine that in Chkb -/- mice there is a failure of the α7β1 integrin complex that is specific to affected muscle. We observed that in Chkb -/- hindlimb muscles there is a decrease in sarcolemma association/abundance of the PI(4,5)P2 binding integrin complex proteins vinculin, and α-actinin, and a decrease in actin association with the sarcolemma. In cells, pharmacological inhibition of choline kinase activity results in internalization of a fluorescent PI(4,5)P2 reporter from discrete plasma membrane clusters at the cell surface membrane to cytosol, this corresponds with a decreased vinculin localization at plasma membrane focal adhesions that was rescued by overexpression of CHKB.

The endoplasmic reticulum (ER) is the key organelle for membrane biogenesis. Most lipids are synthesized in the ER, and most membrane proteins are first inserted into the ER membrane before they are transported to their target organelle. The composition and properties of the ER membrane must be carefully controlled to provide a suitable environment for the insertion and folding of membrane proteins. The unfolded protein response (UPR) is a powerful signaling pathway that balances protein and lipid production in the ER. Here, we summarize our current knowledge of how aberrant compositions of the ER membrane, referred to as lipid bilayer stress, trigger the UPR.

Deformation of the cell membrane is well understood from the viewpoint of protein interactions and free energy balance. However, the various dynamic properties of the membrane, such as lipid packing and hydrophobicity, and their relationship with cell membrane deformation are unknown. Therefore, the deformation of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and oleic acid (OA) giant unilamellar vesicles (GUVs) was induced by heating and cooling cycles, and time-lapse analysis was conducted based on the membrane hydrophobicity and physical parameters of "single-parent" and "daughter" vesicles. Fluorescence ratiometric analysis by simultaneous dual-wavelength detection revealed the variation of different hydrophilic GUVs and enabled inferences of the "daughter" vesicle composition and the "parent" membrane's local composition during deformation; the "daughter" vesicle composition of OA was lower than that of the "parents", and lateral movement of OA was the primary contributor to the formation of the "daughter" vesicles. Thus, our findings and the newly developed methodology, named in situ quantitative membrane property-morphology relation (QmPMR) analysis, would provide new insights into cell deformation and accelerate research on both deformation and its related events, such as budding and birthing.

Cells depend on their endolysosomal system for nutrient uptake and downregulation of plasma membrane proteins. These processes rely on endosomal maturation, which requires multiple membrane fusion steps. Early endosome fusion is promoted by the Rab5 GTPase and its effector, the hexameric CORVET tethering complex, which is homologous to the lysosomal HOPS. How these related complexes recognize their specific target membranes remains entirely elusive. Here, we solve the structure of CORVET by cryo-electron microscopy and revealed its minimal requirements for membrane tethering. As expected, the core of CORVET and HOPS resembles each other. However, the function-defining subunits show marked structural differences. Notably, we discover that unlike HOPS, CORVET depends not only on Rab5 but also on phosphatidylinositol-3-phosphate (PI3P) and membrane lipid packing defects for tethering, implying that an organelle-specific membrane code enables fusion. Our data suggest that both shape and membrane interactions of CORVET and HOPS are conserved in metazoans, thus providing a paradigm how tethering complexes function.

Brain lipid homeostasis is an absolute requirement for proper functionality of nerve cells and neurological performance. Current evidence demonstrates that lipid alterations are linked to neurodegenerative diseases, especially Alzheimer's disease (AD). The complexity of the brain lipidome and its metabolic regulation has hampered the identification of critical processes associated with the onset and progression of AD. While most experimental studies have focused on the effects of known factors on the development of pathological hallmarks in AD, e.g., amyloid deposition, tau protein and neurofibrillary tangles, neuroinflammation, etc., studies addressing the causative effects of lipid alterations remain largely unexplored. In the present study, we have used a multifactor approach combining diets containing different amounts of polyunsaturated fatty acids (PUFAs), estrogen availabilities, and genetic backgrounds, i.e., wild type (WT) and APP/PS1 (FAD), to analyze the lipid phenotype of the frontal cortex in middle-aged female mice. First, we observed that severe n-3 PUFA deficiency impacts the brain n-3 long-chain PUFA (LCPUFA) composition, yet it was notably mitigated by hepatic de novo synthesis. n-6 LCPUFAs, ether-linked fatty acids, and saturates were also changed by the dietary condition, but the extent of changes was dependent on the genetic background and hormonal condition. Likewise, brain cortex phospholipids were mostly modified by the genotype (FAD>WT) with nuanced effects from dietary treatment. Cholesterol (but not sterol esters) was modified by the genotype (WT>FAD) and dietary condition (higher in DHA-free conditions, especially in WT mice). However, the effects of estrogen treatment were mostly observed in relation to phospholipid remodeling in a genotype-dependent manner. Analyses of lipid-derived variables indicate that nerve cell membrane biophysics were significantly affected by the three factors, with lower membrane microviscosity (higher fluidity) values obtained for FAD animals. In conclusion, our multifactor analyses revealed that the genotype, diet, and estrogen status modulate the lipid phenotype of the frontal cortex, both as independent factors and through their interactions. Altogether, the outcomes point to potential strategies based on dietary and hormonal interventions aimed at stabilizing the brain cortex lipid composition in Alzheimer's disease neuropathology.

Subcellular membranes have complex lipid and protein compositions, which give rise to organelle-specific membrane packing, fluidity, and permeability. Due to its exquisite solvent sensitivity, the lipophilic fluorescence dye Nile Red has been used extensively to study membrane packing and polarity. Further improvement of Nile Red can be achieved by introducing electron-donating or withdrawing functional groups. Here, we compare the potential of derivatives of Nile Red with such functional substitutions for super-resolution fluorescence microscopy of lipid packing in model membranes and living cells. All studied Nile Red derivatives exhibit cholesterol-dependent fluorescence changes in model membranes, as shown by spectrally resolved stimulated emission depletion (STED) microscopy. STED imaging of Nile Red probes in cells reveals lower membrane packing in fibroblasts from healthy subjects compared to those from patients suffering from Niemann Pick type C1 (NPC1) disease, a lysosomal storage disorder with accumulation of cholesterol and sphingolipids in late endosomes and lysosomes. We also find small but consistent changes in the fluorescence lifetime of the Nile Red derivatives in NPC1 cells, suggesting altered hydrogen-bonding capacity in their membranes. All Nile Red derivatives are essentially non-fluorescent in water but increase their brightness in membranes, allowing for their use in MINFLUX single molecule tracking experiments. Our study uncovers the potential of Nile Red probes with functional substitutions for nanoscopic membrane imaging.

During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.

The essential Golgi protein Sly1 is a member of the Sec1/mammalian Unc-18 (SM) family of SNARE chaperones. Sly1 was originally identified through remarkable gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory but also acts as a positive effector. An amphipathic lipid packing sensor (ALPS)-like helix within the loop directly binds high-curvature membranes. Membrane binding is required for relief of Sly1 autoinhibition and also allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 mutation bypasses requirements for diverse tethering factors but loses this ability if the tethering activity is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to initiate trans-SNARE complex assembly and fusion in the early secretory pathway.

Lipids are the most abundant but poorly explored components of the human brain. Here, we present a lipidome map of the human brain comprising 75 regions, including 52 neocortical ones. The lipidome composition varies greatly among the brain regions, affecting 93% of the 419 analyzed lipids. These differences reflect the brain's structural characteristics, such as myelin content (345 lipids) and cell type composition (353 lipids), but also functional traits: functional connectivity (76 lipids) and information processing hierarchy (60 lipids). Combining lipid composition and mRNA expression data further enhances functional connectivity association. Biochemically, lipids linked with structural and functional brain features display distinct lipid class distribution, unsaturation extent, and prevalence of omega-3 and omega-6 fatty acid residues. We verified our conclusions by parallel analysis of three adult macaque brains, targeted analysis of 216 lipids, mass spectrometry imaging, and lipidome assessment of sorted murine neurons.

Tools for accessing and studying organelles remain underdeveloped. Here, we present a method by which giant organelle vesicles (GOVs) are generated by submitting cells to a hypotonic medium followed by plasma membrane breakage. By this means, GOVs ranging from 3 to over 10 µm become available for micromanipulation. GOVs are made from organelles such as the endoplasmic reticulum, endosomes, lysosomes and mitochondria, or in contact with one another such as giant mitochondria-associated ER membrane vesicles. We measure the mechanical properties of each organelle-derived GOV and find that they have distinct properties. In GOVs procured from Cos7 cells, for example, bending rigidities tend to increase from the endoplasmic reticulum to the plasma membrane. We also found that the mechanical properties of giant endoplasmic reticulum vesicles (GERVs) vary depending on their interactions with other organelles or the metabolic state of the cell. Lastly, we demonstrate GERVs' biochemical activity through their capacity to synthesize triglycerides and assemble lipid droplets. These findings underscore the potential of GOVs as valuable tools for studying the biophysics and biology of organelles.

Lipid binding domains and protein lipidations are essential features to recruit proteins to intracellular membranes, enabling them to function at specific sites within the cell. Membrane association can also be exploited to answer fundamental and applied research questions, from obtaining insights into the understanding of lipid metabolism to employing them for metabolic engineering to redirect fluxes. This review presents a broad catalog of membrane binding strategies focusing on the plasma membrane of Saccharomyces cerevisiae. Both lipid binding domains (pleckstrin homology, discoidin-type C2, kinase associated-1, basic-rich and bacterial phosphoinositide-binding domains) and co- and post-translational lipidations (prenylation, myristoylation and palmitoylation) are introduced as tools to target the plasma membrane. To provide a toolset of membrane targeting modules, respective candidates that facilitate plasma membrane targeting are showcased including their in vitro and in vivo properties. The relevance and versatility of plasma membrane targeting modules are further highlighted by presenting a selected set of use cases.

Pluripotent stem cells (PSCs), comprising embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), offer immense potential for regenerative medicine due to their ability to differentiate into all cell types of the adult body. A critical aspect of harnessing this potential is understanding their metabolic requirements during derivation, maintenance, and differentiation in vitro. Traditional culture methods using fetal bovine serum often lead to issues such as heterogeneous cell populations and diminished pluripotency. Although the chemically-defined 2i/LIF medium has provided solutions to some of these challenges, prolonged culturing of these cells, especially female ESCs, raises concerns related to genome integrity. This review discusses the pivotal role of lipids in genome stability and pluripotency of stem cells. Notably, the introduction of lipid-rich albumin, AlbuMAX, into the 2i/LIF culture medium offers a promising avenue for enhancing the genomic stability and pluripotency of cultured ESCs. We further explore the unique characteristics of lipid-induced pluripotent stem cells (LIP-ESCs), emphasizing their potential in regenerative medicine and pluripotency research.

Plasma membranes are flexible and can exhibit numerous shapes below the optical diffraction limit. The shape of cell periphery can either induce or be a product of local protein density changes, encoding numerous cellular functions. However, quantifying membrane curvature and the ensuing sorting of proteins in live cells remains technically demanding. Here, we demonstrate the use of simple widefield fluorescence microscopy to study the geometrical properties (i.e., radius, length, and number) of thin membrane protrusions. Importantly, the quantification of protrusion radius establishes a platform for studying the curvature preferences of membrane proteins.

The specific recognition of peripheral membrane-binding proteins for their target membranes is mediated by a complex constellation of various lipid contacts. Despite the inherent complexities of the heterogeneous protein-membrane interface, the binding dependence of such proteins is, surprisingly, often reliably described by simple models such as the Langmuir Adsorption Isotherm or the Hill equation. However, these models were not developed to describe associations with two-dimensional, highly concentrated heterogeneous ligands such as lipid membranes. In particular, these models fail to capture the dependence on the lipid composition, a significant determinant of binding that distinguishes target from non-target membranes. In this work, we present a model that describes the dependence of peripheral proteins on lipid composition through an analytic expression for their association. The resulting membrane-binding equation retains the features of these simple models but completely describes the binding dependence on multiple relevant variables in addition to the lipid composition, such as protein and vesicle concentration. Implicit in this lipid composition dependence is a new form of membrane-based cooperativity that significantly differs from traditional solution-based cooperativity. We introduce the Membrane-Hill number as a measure of this cooperativity and describe its unique properties. We illustrate the utility and interpretational power of our model by analyzing previously published data on two peripheral proteins that associate with phosphatidylserine-containing membranes: The transmembrane immunoglobulin and mucin domain-containing protein 3 (TIM3) that employs calcium in its association, and milk fat globulin epidermal growth factor VIII (MFG-E8) which is completely insensitive to calcium. We also provide binding equations for systems that exhibit more complexity in their membrane-binding.

Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work has provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remains insufficiently characterized. Here, we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics, and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress.

An analysis of the membrane organization and intracellular trafficking of lipids often relies on multiphoton (MP) and super-resolution microscopy of fluorescent lipid probes. A disadvantage of particularly intrinsically fluorescent lipid probes, such as the cholesterol and ergosterol analogue, dehydroergosterol (DHE), is their low MP absorption cross-section, resulting in a low signal-to-noise ratio (SNR) in live-cell imaging. Stimulated emission depletion (STED) microscopy of membrane probes like Nile Red enables one to resolve membrane features beyond the diffraction limit but exposes the sample to a lot of excitation light and suffers from a low SNR and photobleaching. Here, dynamic mode decomposition (DMD) and its variant, higher-order DMD (HoDMD), are applied to efficiently reconstruct and denoise the MP and STED microscopy data of lipid probes, allowing for an improved visualization of the membranes in cells. HoDMD also allows us to decompose and reconstruct two-photon polarimetry images of TopFluor-cholesterol in model and cellular membranes. Finally, DMD is shown to not only reconstruct and denoise 3D-STED image stacks of Nile Red-labeled cells but also to predict unseen image frames, thereby allowing for interpolation images along the optical axis. This important feature of DMD can be used to reduce the number of image acquisitions, thereby minimizing the light exposure of biological samples without compromising image quality. Thus, DMD as a computational tool enables gentler live-cell imaging of fluorescent probes in cellular membranes by MP and STED microscopy.

VPS37A, an ESCRT-I complex component, is required for recruiting a subset of ESCRT proteins to the phagophore for autophagosome closure. However, the mechanism by which VPS37A is targeted to the phagophore remains obscure. Here, we demonstrate that the VPS37A N-terminal domain exhibits selective interactions with highly curved membranes, mediated by two membrane-interacting motifs within the disordered regions surrounding its Ubiquitin E2 variant-like (UEVL) domain. Site-directed mutations of residues in these motifs disrupt ESCRT-I localization to the phagophore and result in defective phagophore closure and compromised autophagic flux in vivo, highlighting their essential role during autophagy. In conjunction with the UEVL domain, we postulate that these motifs guide a functional assembly of the ESCRT machinery at the highly curved tip of the phagophore for autophagosome closure. These results advance the notion that the distinctive membrane architecture of the cup-shaped phagophore spatially regulates autophagosome biogenesis.

Oxysterol-binding protein-related proteins (ORPs) play key roles in the distribution of lipids in eukaryotic cells by exchanging sterol or phosphatidylserine for PI4P between the endoplasmic reticulum (ER) and other cell regions. However, it is unclear how their exchange capacity is coupled to PI4P metabolism. To address this question quantitatively, we analyze the activity of a representative ORP, Osh4p, in an ER/Golgi interface reconstituted with ER- and Golgi-mimetic membranes functionalized with PI4P phosphatase Sac1p and phosphatidylinositol (PI) 4-kinase, respectively. Using real-time assays, we demonstrate that upon adenosine triphosphate (ATP) addition, Osh4p creates a sterol gradient between these membranes, relying on the spatially distant synthesis and hydrolysis of PI4P, and quantify how much PI4P is needed for this process. Then, we develop a quantitatively accurate kinetic model, validated by our data, and extrapolate this to estimate to what extent PI4P metabolism can drive ORP-mediated sterol transfer in cells. Finally, we show that Sec14p can support PI4P metabolism and Osh4p activity by transferring PI between membranes. This study establishes that PI4P synthesis drives ORP-mediated lipid exchange and that ATP energy is needed to generate intermembrane lipid gradients. Furthermore, it defines to what extent ORPs can distribute lipids in the cell and reassesses the role of PI-transfer proteins in PI4P metabolism.

The envelope (E) protein of SARS-CoV-2 participates in virion encapsulation and budding at the membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC). The positively curved membrane topology required to fit an 80 nm viral particle is energetically unfavorable; therefore, viral proteins must facilitate ERGIC membrane curvature alteration. To study the possible role of the E protein in this mechanism, we examined the structural modification of the host lipid membrane by the SARS-CoV-2 E protein using synchrotron-based X-ray methods. Our reflectometry results on solid-supported planar bilayers show that E protein markedly condenses the surrounding lipid bilayer. For vesicles, this condensation effect differs between the two leaflets such that the membrane becomes asymmetric and increases its curvature. The formation of such a curved and condensed membrane is consistent with the requirements to stably encapsulate a viral core and supports a role for E protein in budding during SARS-CoV-2 virion assembly.

Although the structural rearrangement of the membrane-bound matrix (MA) protein trimers upon HIV-1 maturation has been reported, the consequences of MA maturation on the MA-lipid interactions are not well understood. Long-timescale molecular dynamics simulations of the MA multimeric assemblies of immature and mature virus particles with our realistic asymmetric membrane model have explored MA-lipid interactions and lateral organization of lipids around MA complexes. The number of stable MA-phosphatidylserine and MA-phosphatidylinositol 4,5-bisphosphate (PIP2) interactions at the trimeric interface of the mature MA complex is observed to be greater compared to that of the immature MA complex. Our simulations identified an alternative PIP2-binding site in the immature MA complex where the multivalent headgroup of a PIP2 lipid with a greater negative charge binds to multiple basic amino acid residues such as ARG3 residues of both the MA monomers at the trimeric interface and highly basic region (HBR) residues (LYS29, LYS31) of one of the MA monomers. Our enhanced sampling simulations have explored the conformational space of phospholipids at different binding sites of the trimer-trimer interface of MA complexes that are not accessible by conventional unbiased molecular dynamics. Unlike the immature MA complex, the 2' acyl tail of two PIP2 lipids at the trimeric interface of the mature MA complex is observed to sample stable binding pockets of MA consisting of helix-4 residues. Together, our results provide molecular-level insights into the interactions of MA trimeric complexes with membrane and different lipid conformations at the specific binding sites of MA protein before and after viral maturation.

Hepatic steatosis is the leading cause of discarded liver grafts. Defatting steatotic liver grafts using drug combinations during ex vivo normothermic machine perfusion (NMP) has been reported. However, the effectiveness of NMP in reducing fat content using epigallocatechin gallate (EGCG) as a single defatting agent and its effect on lipid metabolism are poorly investigated.

In this study, an NMP system was set up to perfuse a steatotic liver from a rat model with 10 mM EGCG. Livers without EGCG served as NMP controls, whereas static cold-preserved livers in the University of Wisconsin medium were used as static cold storage controls. Liver enzyme, reactive oxygen species (ROS), histology, and lipid content assessments were conducted post-perfusion, complemented by lipidomics, RNA sequencing, and western blotting to determine the lipid metabolism changes.

EGCG during NMP reduced hepatocellular injury markers and defatted steatotic liver grafts. Additionally, we observed a significant increase in triglyceride (TG) content in the perfusate post-NMP in the NMP + EGCG group, suggesting TG output from the liver. Furthermore, lipidomics analysis revealed that EGCG primarily affected metabolites involved in glycerophospholipid (GP) and glycerolipid (GL) metabolism. Further, the RNA sequencing indicated the modulation of these metabolic pathways via ECGC, which was associated with the downregulated Lpin1 and Gpat3 expression.

EGCG defats steatotic livers as a single defatting agent during NMP by promoting GL and GP metabolism via decreasing Lpin1 and Agpat9 levels.

Identification of the phosphatidylserine (PS) discrepancies occurring on the cellular membrane during apoptotic processes is of the utmost importance. However, monitoring the quantity of PS molecules in real-time at a single-cell level currently remains a challenging task. Here, we demonstrate this objective by leveraging the specific binding and reversible interaction exhibited by the zinc(II) dipyridinamine complex (ZnDPA) with PS. Lipoic acid-functionalized ZnDPA (LP-ZnDPA) was subsequently immobilized onto the surface of an atomic force microscopy cantilever to form a force probe, ALP-ZnDPA, enabling a PS-specific dynamic imaging and detection mode. By utilizing this technique, we can not only create a heat map of the expression level of PS with submicron resolution but also quantify the number of molecules present on a single cell's surface with a detection limit of 1.86 × 104 molecules. The feasibility of the proposed method is demonstrated through the analysis of PS expression levels in different cancer cell lines and at various stages of paclitaxel-induced apoptosis. This study represents the first application of a force probe to quantify PS molecules on the surface of individual cells, providing insight into dynamic changes in PS content during apoptosis at the molecular level and introducing a novel dimension to current detection methodologies.

A number of intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms, such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic-abundant heat-soluble proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. In this study, we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 shows enhanced tolerance to hyperosmotic stress under nonfermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such dynamic and weak protein-membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represent a general biophysical solution for stress tolerance across the domains of life.