Lipid Sorting Research Articles

Hemifusomes and Interacting Proteolipid Nanodroplets: Formation of a Novel Cellular Organelle Complex.

Within cells, vesicle fusion, scission, and the formation of intraluminal vesicles are critical processes that facilitate traffic, degradation, and recycling of cellular components, and maintenance of cellular homeostasis. Despite significant advancements in elucidating the molecular mechanisms that drive these dynamic processes, the direct in situ visualization of membrane remodeling intermediates remains challenging. Here, through the application of cryo-electron tomography in mammalian cells, we have identified a previously undescribed vesicular organelle complex with unique membrane topology: heterotypic hemifused vesicles that share extended hemifusion diaphragms (HDs) with a 42 nm proteolipid nanodroplet (PND) at their rim. We have termed these organelle complexes "hemifusomes". The HDs of hemifusomes exhibit a range of sizes and curvatures, including the formation of lens-shaped compartments encapsulated within the membrane bilayer. The morphological diversity of the lens-shaped vesicle aligns with a step-wise process of their intraluminal budding, ultimately leading to their scission and the generation of intraluminal vesicles. We propose that hemifusomes function as versatile platforms for protein and lipid sorting and as central hubs for the biogenesis of intraluminal vesicles and the formation of multivesicular bodies.

Intrinsically Disordered Membrane Anchors of Rheb, RhoA, and DiRas3 Small GTPases: Molecular Dynamics, Membrane Organization, and Interactions.

Protein structure has been well established to play a key role in determining function; however, intrinsically disordered proteins and regions (IDPs and IDRs) defy this paradigm. IDPs and IDRs exist as an ensemble of structures rather than a stable 3D structure yet play essential roles in many cell-signaling processes. Nearly all Ras superfamily GTPases are tethered to membranes by a lipid tail at the end of a flexible IDR. The sequence of the IDR is a key determinant of membrane localization, and interaction between the IDR and the membrane has been shown to affect signaling in RAS proteins through the modulation of dynamic membrane organization. Here, we utilized atomistic molecular dynamics simulations to study the membrane interaction, conformational dynamics, and lipid sorting of three IDRs from small GTPases Rheb, RhoA, and DiRas3 in model membranes representing their physiological target membranes. We found that complementarity between the lipidated IDR sequence and target membrane lipid composition is a determinant of conformational plasticity. We also show that electrostatic interactions between anionic lipids and basic residues on IDRs are correlated with sampling of semistable conformational substates, and lack of these interactions is associated with greater conformational diversity. Finally, we show that small GTPase IDRs with a polybasic domain alter local lipid composition by segregating anionic lipids and, in some cases, excluding other lipids from their immediate vicinity in favor of anionic lipids.

Membranes on the move: The functional role of the extracellular vesicle membrane for contact-dependent cellular signalling.

Extracellular vesicles (EVs), lipid-enclosed structures released by virtually all life forms, have gained significant attention due to their role in intercellular and interorganismal communication. Despite their recognized importance in disease processes and therapeutic applications, fundamental questions about their primary function remain. Here, we propose a different perspective on the primary function of EVs, arguing that they serve as essential elements providing membrane area for long-distance, contact-dependent cellular communication based on protein-protein interaction. While EVs have been recognized as carriers of genetic information, additional unique advantages that they could provide for cellular communication remain unclear. Here, we introduce the concept that the substantial membrane area provided by EVs allows for membrane contact-dependent interactions that could be central to their function. This membrane area enables the lateral diffusion and sorting of membrane ligands like proteins, polysaccharides or lipids in two dimensions, promoting avidity-driven effects and assembly of co-stimulatory architectures at the EV-cell interface. The concept of vesicle-induced receptor sequestration (VIRS), for example, describes how EVs confine and focus receptors at the EV contact site, promoting a dense local concentration of receptors into signalosomes. This process can increase the signalling strength of EV-presented ligands by 10-1000-fold compared to their soluble counterparts. The speculations in this perspective advance our understanding of EV-biology and have critical implications for EV-based applications and therapeutics. We suggest a shift in perspective from viewing EVs merely as transporters of relevant nucleic acids and proteins to considering their unique biophysical properties as presentation platforms for long-distance, contact-dependent signalling. We therefore highlight the functional role of the EV membrane rather than their content. We further discuss how this signalling mechanism might be exploited by virus-transformed or cancer cells to enhance immune-evasive mechanisms.

ECM stiffness affects cargo sorting into MSC-EVs to regulate their secretion and uptake behaviors

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have garnered extensive attention as natural product-based nanomedicines and potential drug delivery vehicles. However, the specific mechanism for regulating MSC-EVs secretion and delivery remains unclear. Here, we demonstrate that extracellular matrix (ECM) stiffness regulates the secretion and delivery of EVs by affecting MSCs' cargo sorting mechanically. Using multi-omics analysis, we found that a decrease in ECM stiffness impeded the sorting of vesicular transport-related proteins and autophagy-related lipids into MSC-EVs, impairing their secretion and subsequent uptake by macrophages. Hence, MSC-EVs with different secretion and uptake behaviors can be produced by changing the stiffness of culture substrates. This study provides new insights into MSC-EV biology and establishes a connection between MSC-EV behaviors and ECM from a biophysical perspective, providing a basis for the rational design of biomedical materials.Graphical

Split but merge: Golgi fragmentation in physiological and pathological conditions.

The Golgi complex is a highly dynamic and tightly regulated cellular organelle with essential roles in the processing as well as the sorting of proteins and lipids. Its structure undergoes rapid disassembly and reassembly during normal physiological processes, including cell division, migration, polarization, differentiation, and cell death. Golgi dispersal or fragmentation also occurs in pathological conditions, such as neurodegenerative diseases, infectious diseases, congenital disorders of glycosylation diseases, and cancer. In this review, current knowledge about both structural organization and morphological alterations in the Golgi in physiological and pathological conditions is summarized together with the methodologies that help to reveal its structure.

Lipid rafts disruption by statins negatively impacts the interaction between SARS-CoV-2 S1 subunit and ACE2 in intestinal epithelial cells.

The causative agent of the COVID-19 pandemic, SARS-CoV-2, is a virus that targets mainly the upper respiratory tract. However, it can affect other systems such as the gastrointestinal (GI) tract. Therapeutic strategies for this virus are still inconclusive and understanding its entry mechanism is important for finding effective treatments. Cholesterol is an important constituent in the structure of cellular membranes that plays a crucial role in a variety of cellular events. In addition, it is important for the infectivity and pathogenicity of several viruses. ACE2, the main receptor of SARS-CoV-2, is associated with lipid rafts which are microdomains composed of cholesterol and sphingolipids. In this study, we investigate the role of statins, lipid-lowering drugs, on the trafficking of ACE2 and the impact of cholesterol modulation on the interaction of this receptor with S1 in Caco-2 cells. The data show that fluvastatin and simvastatin reduce the expression of ACE2 to variable extents, impair its association with lipid rafts and sorting to the brush border membrane resulting in substantial reduction of its interaction with the S1 subunit of the spike protein. By virtue of the substantial effects of statins demonstrated in our study, these molecules, particularly fluvastatin, represent a promising therapeutic intervention that can be used off-label to treat SARS-CoV-2.

Morphological determination of localization and function of Golgi proteins.

In animal cells, the Golgi apparatus serves as the central hub of the endomembrane secretory pathway. It is responsible for the processing, modification, and sorting of proteins and lipids. The unique stacking and ribbon-like architecture of the Golgi apparatus forms the foundation for its precise functionality. Under cellular stress or pathological conditions, the structure of the Golgi and its important glycosylation modification function may change. It is crucial to employ suitable methodologies to study the structure and function of the Golgi apparatus, particularly when assessing the involvement of a target protein in Golgi regulation. This article provides a comprehensive overview of the diverse microscopy techniques used to determine the specific location of the target protein within the Golgi apparatus. Additionally, it outlines methods for assessing changes in the Golgi structure and its glycosylation modification function following the knockout of the target gene.

Predicting lipid sorting in curved membranes.

Most biological membranes are curved, and both lipids and proteins play a role in generating curvature. For any given membrane shape and composition, it is not trivial to determine whether lipids are laterally distributed in a homogeneous or inhomogeneous way, and whether the inter-leaflet distribution is symmetric or not. Here we present a simple computational tool that allows to predict the preference of any lipid type for membranes with positive vs. negative curvature, for any given value of curvature. The tool is based on molecular dynamics simulations of tubular membranes with hydrophilic pores. The pores allow spontaneous, barrierless flip-flop of most lipids, while also preventing differences in pressure between the inner and outer water compartments and minimizing membrane asymmetric stresses. Specifically, we provide scripts to build and analyze the simulations. We test the tool by performing simulations on simple binary lipid mixtures, and we show that, as expected, lipids with negative intrinsic curvature distribute to the tubule inner leaflet, the more so when the radius of the tubular membrane is small. Compared to other existing computational methods, relying on membrane buckles and tethers, our method is based on spontaneous inter-leaflet transport of lipids, and therefore allows to explore lipid distribution in asymmetric membranes. The method can easily be adapted to work with any molecular dynamics code and any force field.

Intracellular sphingolipid sorting drives membrane phase separation in the yeast vacuole

The yeast vacuole membrane can phase separate into ordered and disordered domains, a phenomenon that is required for micro-lipophagy under nutrient limitation. Despite its importance as a biophysical model and physiological significance, it is not yet resolved if specific lipidome changes drive vacuole phase separation. Here we report that the metabolism of sphingolipids (SLs) and their sorting into the vacuole membrane can control this process. We first developed a vacuole isolation method to identify lipidome changes during the onset of phase separation in early stationary stage cells. We found that early stationary stage vacuoles are defined by increased abundance of putative raft components, including 40% higher ergosterol content and a nearly 3-fold enrichment in complex SLs. These changes were not found in the corresponding whole cell lipidomes, indicating that lipid sorting is associated with domain formation. Several facets of SL composition – headgroup stoichiometry, chain lengths, and increased hydroxylations – were also markers of phase-separated vacuole lipidomes. To test SL function in vacuole phase separation, we carried out a systematic genetic dissection of their biosynthetic pathway. The abundance of CSLs controlled the extent of domain formation and associated micro-lipophagy processes, while their headgroup composition altered domain morphology. These results suggest that lipid trafficking can drive membrane phase separation in vivo and identify SLs as key mediators of this process in yeast.

Conformational ensemble-dependent lipid recognition and segregation by prenylated intrinsically disordered regions in small GTPases

We studied diverse prenylated intrinsically disordered regions (PIDRs) of Ras and Rho family small GTPases using long timescale atomistic molecular dynamics simulations in an asymmetric model membrane of phosphatidylcholine (PC) and phosphatidylserine (PS) lipids. Here we show that conformational plasticity is a key determinant of lipid sorting by polybasic PIDRs and provide evidence for lipid sorting based on both headgroup and acyl chain structures. We further show that conformational ensemble-based lipid recognition is generalizable to all polybasic PIDRs, and that the sequence outside the polybasic domain (PBD) modulates the conformational plasticity, bilayer adsorption, and interactions of PIDRs with membrane lipids. Specifically, we find that palmitoylation, the ratio of basic to acidic residues, and the hydrophobic content of the sequence outside the PBD significantly impact the diversity of conformational substates and hence the extent of conformation-dependent lipid interactions. We thus propose that the PBD is required but not sufficient for the full realization of lipid sorting by prenylated PBD-containing membrane anchors, and that the membrane anchor is not only responsible for high affinity membrane binding but also directs the protein to the right target membrane where it participates in lipid sorting.

Lipidomic landscape of lipokines in adipose tissue derived extracellular vesicles.

Introduction: Adipose tissue-derived extracellular vesicles (EVs-AT) are recognized as critical mediators of metabolic alterations in obesity-related diseases. However, few studies have focused on the role of lipids within EVs-AT in the development of obesity-related diseases. Methods: In this study, we performed a targeted lipidomic analysis to compare the lipidome of EVs secreted by inguinal white adipose tissue (EVs-iWAT), epididymal white adipose tissue (EVs-eWAT), and interscapular brown adipose tissue (EVs-BAT) in lean and obese mice. Results: We uncovered a comprehensive lipidomic map, revealing the diversity and specific lipid sorting in EVs-iWAT, EVs-eWAT, and EVs-BAT in obesity. Biological function analyses suggested that lipids encapsulated within EVs-AT of obese individuals might correlate with metabolism, pro-inflammatory response, and insulin resistance. These effects were particularly pronounced in EVs-eWAT and EVs-BAT. Conclusion: Our findings indicated that EVs-AT serves as novel carriers for lipokines, thereby mediating the biological functions of EVs-AT. This study holds promise for the identification of new biomarkers for obesity-related diseases and the development of new strategies to combat metabolic diseases.

Lipid Sorting and Organelle Identity.

The sorting and trafficking of lipids between organelles gives rise to a dichotomy of bulk membrane properties between organelles of the secretory and endolysosome networks, giving rise to two "membrane territories" based on differences in lipid-packing density, net membrane charge, and bilayer leaflet asymmetries. The cellular organelle membrane dichotomy emerges from ER-to-PM anterograde membrane trafficking and the synthesis of sphingolipids and cholesterol flux at the trans-Golgi network, which constitutes the interface between the two membrane territories. Organelle homeostasis is maintained by vesicle-mediated retrieval of bulk membrane from the distal organelles of each territory to the endoplasmic reticulum or plasma membrane and by soluble lipid transfer proteins that traffic particular lipids. The concept of cellular membrane territories emphasizes the contrasting features of organelle membranes of the secretory and endolysosome networks and the essential roles of lipid-sorting pathways that maintain organelle function.

Clec12a inhibits MSU-induced immune activation through lipid raft expulsion.

Monosodium uric acid (MSU) crystal, the etiological agent of gout, has been shown to trigger innate immune responses via multiple pathways. It is known that MSU-induced lipid sorting on plasma membrane promotes the phosphorylation of Syk and eventually leads to the activation of phagocytes. However, whether this membrane lipid-centric mechanism is regulated by other processes is unclear. Previous studies showed that Clec12a, a member of the C-type lectin receptor family, is reported to recognize MSU and suppresses this crystalline structure-induced immune activation. How this scenario is integrated into the lipid sorting-mediated inflammatory responses by MSU, and particularly, how Clec12a intercepts lipid raft-originated signaling cascade remains to be elucidated. Here, we found that the ITIM motif of Clec12a is dispensable for its inhibition of MSU-mediated signaling; instead, the transmembrane domain of Clec12a disrupts MSU-induced lipid raft recruitment and thus attenuates downstream signals. Single amino acid mutagenesis study showed the critical role of phenylalanine in the transmembrane region for the interactions between C-type lectin receptors and lipid rafts, which is critical for the regulation of MSU-mediated lipid sorting and phagocyte activation. Overall, our study provides new insights for the molecular mechanisms of solid particle-induced immune activation and may lead to new strategies in inflammation control.

Golgi's Role in the Development of Possible New Therapies in Cancer.

The Golgi apparatus is an important organelle found in most eukaryotic cells. It plays a vital role in the processing and sorting of proteins, lipids and other cellular components for delivery to their appropriate destinations within the cell or for secretion outside of the cell. The Golgi complex also plays a role in the regulation of protein trafficking, secretion and post-translational modifications, which are significant in the development and progression of cancer. Abnormalities in this organelle have been observed in various types of cancer, although research into chemotherapies that target the Golgi apparatus is still in its early stages. There are a few promising approaches that are being investigated: (1) Targeting the stimulator of interferon genes protein: The STING pathway senses cytosolic DNA and activates several signaling events. It is regulated by numerous post-translational modifications and relies heavily on vesicular trafficking. Based on some observations which state that a decreased STING expression is present in some cancer cells, agonists for the STING pathway have been developed and are currently being tested in clinical trials, showing encouraging results. (2) Targeting glycosylation: Altered glycosylation, which refers to changes in the carbohydrate molecules that are attached to proteins and lipids in cells, is a common feature of cancer cells, and there are several methods that thwart this process. For example, some inhibitors of glycosylation enzymes have been shown to reduce tumor growth and metastasis in preclinical models of cancer. (3) Targeting Golgi trafficking: The Golgi apparatus is responsible for the sorting and trafficking of proteins within the cell, and disrupting this process may be a potential therapeutic approach for cancer. The unconventional protein secretion is a process that occurs in response to stress and does not require the involvement of the Golgi organelles. P53 is the most frequently altered gene in cancer, dysregulating the normal cellular response to DNA damage. The mutant p53 drives indirectly the upregulation of the Golgi reassembly-stacking protein 55kDa (GRASP55). Through the inhibition of this protein in preclinical models, the reduction of the tumoral growth and metastatic capacity have been obtained successfully. This review supports the hypothesis that the Golgi apparatus may be the target of cytostatic treatment, considering its role in the molecular mechanisms of the neoplastic cells.

IFITM3 blocks influenza virus entry by sorting lipids and stabilizing hemifusion

Interferon-induced transmembrane protein 3 (IFITM3) inhibits the entry of numerous viruses through undefined molecular mechanisms. IFITM3 localizes in the endosomal-lysosomal system and specifically affects virus fusion with target cell membranes. We found that IFITM3 induces local lipid sorting, resulting in an increased concentration of lipids disfavoring viral fusion at the hemifusion site. This increases the energy barrier for fusion pore formation and the hemifusion dwell time, promoting viral degradation in lysosomes. In situ cryo-electron tomography captured IFITM3-mediated arrest of influenza A virus membrane fusion. Observation of hemifusion diaphragms between viral particles and late endosomal membranes confirmed hemifusion stabilization as a molecular mechanism of IFITM3. The presence of the influenza fusion protein hemagglutinin in post-fusion conformation close to hemifusion sites further indicated that IFITM3 does not interfere with the viral fusion machinery. Collectively, these findings show that IFITM3 induces lipid sorting to stabilize hemifusion and prevent virus entry into target cells.

Lipidomic analysis of adipose-derived extracellular vesicles reveals specific EV lipid sorting informative of the obesity metabolic state

Adipose extracellular vesicles (AdEVs) transport lipids that could participate in the development of obesity-related metabolic dysfunctions. This study aims to define mouse AdEV lipid signature by a targeted LC-MS/MS approach in either healthy or obesity context. Distinct clustering of AdEV and visceral adipose tissue (VAT) lipidomes by principal component analysis reveals specific AdEV lipid sorting when compared with secreting VAT. Comprehensive analysis identifies enrichment of ceramides, sphingomyelins, and phosphatidylglycerols species in AdEVs compared with source VAT whose lipid content closely relates to the obesity status and is influenced by the diet. Obesity moreover impacts AdEV lipidome, mirroring lipid alterations retrieved in plasma and VAT. Overall, our study identifies specific lipid fingerprints for plasma, VAT, and AdEVs that are informative of the metabolic status. Lipid species enriched in AdEVs in the obesity context may constitute biomarker candidates or mediators of the obesity-associated metabolic dysfunctions.

Polarized cell plasma membrane composition and protein sorting.

Epithelial cell polarization is an essential biological process, serving many physiological roles including tissue morphogenesis and wound healing. A defining feature of polarization is the separation of cell plasma membrane lipids and proteins into apical and basolateral compartments between which molecular exchange is restricted. Decades ago, the apical membrane was found to be enriched in saturated lipids, glycolipids, and cholesterol. Mechanistic hypotheses to explain the biogenesis and unique composition of the apical membrane include self-assembling membrane domains (i.e., lipid rafts), specific protein sorting motifs, and post-translational modifications mediating protein sorting. However, neither the detailed composition nor the mechanisms of protein and lipid sorting between plasma membrane domains in epithelial cells have been resolved. Particularly, the lipid profile of the basolateral membrane remains unstudied, leaving doubts about the differentiation and lipid separation of the apical and basolateral membrane. We use advanced lipidomics and imaging techniques to characterize the changes in lipid organization, membrane composition, and membrane properties during the cellular polarization process. We observe that the apical membrane is enriched in highly saturated lipids and glycolipids relative to the basolateral membrane, with apical membrane biophysical properties reflecting a raft-enriched environment. Similar to lipids, the determinants of protein sorting between apical and basolateral domains are poorly understood, with past studies focusing on either specific proteins or trafficking machinery. Importantly, the apical membrane hosts an extensive extracellular glycocalyx consisting of glycolipids, glycoproteins, and polysaccharides. The role of these glycocalyx molecules on protein sorting has not been revealed, despite a major fraction of apical proteins being glycosylated. We systematically evaluate the structural determinants of apical versus basolateral sorting with a focus on transmembrane domain features, protein raft affinity, and glycosylation. We find that these protein features cooperatively direct protein sorting to the apical membrane.

Examining compositional variability of giant unilamellar vesicles via secondary ion mass spectrometry.

Giant unilamellar vesicles (GUVs) are a widely used model system to interrogate lipid phase behavior, study biomembrane mechanics, reconstitute membrane proteins, and provide a chassis for synthetic cells. However, there may be significant compositional variability between individual GUVs, either due to lipid sorting during film hydration or from vesicle budding following GUV formation. Although this compositional heterogeneity likely impacts phase behavior, the function and incorporation of membrane proteins, and the encapsulation of biochemical reactions, it has yet to be directly quantified. To assess heterogeneity, we use secondary ion mass spectrometry (SIMS) to probe the composition of individual GUVs using non-perturbing isotopic labels. Both 13C- and 2H-labeled lipids are incorporated into a ternary POPC/DSPC/CHOL mixture, which is then used to produce GUVs via gentle hydration or electroformation. Simultaneous detection of seven different ion species via SIMS allows for the concentration of 13C- and 2H-labeled lipids in a single GUVs to be quantified using calibration curves, which correlate ion intensity to composition. Additionally, the relative concentration of 13C- and 2H-labeled lipids is assessed for each GUV via the ion ratio 2H−/13C−, which is highly sensitive to compositional changes between individual GUVs and circumvents the need for calibration using standards. Both quantification methods suggest that gentle hydration produces GUVs with greater compositional variability than those formed by electroformation. However, both gentle hydration and electroformation display compositional variability on the order of several mole percent.

Nanoscale membrane curvature sorts lipid phases and alters lipid diffusion

The precise spatiotemporal control of nanoscale membrane shape and composition is the result of a complex interplay of individual and collective molecular behaviors. Here, we employed single-molecule localization microscopy and computational simulations to observe single-lipid diffusion and sorting in model membranes with varying compositions, phases, temperatures, and curvatures. Supported lipid bilayers were created over 50-nm-radius nanoparticles to mimic the size of naturally occurring membrane buds, such as endocytic pits and the formation of viral envelopes. The curved membranes recruited liquid-disordered lipid phases while altering the diffusion and sorting of tracer lipids. Disorder-preferring fluorescent lipids sorted to and experienced faster diffusion on the nanoscale curvature only when embedded in a membrane capable of sustaining lipid phase separation at low temperatures. The curvature-induced sorting and faster diffusion even occurred when the sample temperature was above the miscibility temperature of the planar membrane, implying that the nanoscale curvature could induce phase separation in otherwise homogeneous membranes. Further confirmation and understanding of these results are provided by continuum and coarse-grained molecular dynamics simulations with explicit and spontaneous curvature-phase coupling, respectively. The curvature-induced membrane compositional heterogeneity and altered dynamics were achieved only with a coupling of the curvature with a lipid phase separation. These cross-validating results demonstrate the complex interplay of lipid phases, molecular diffusion, and nanoscale membrane curvature that are critical for membrane functionality.

An Electron Tomographic Analysis of Giantin-Deficient Golgi Proposes a New Function of the Golgin Protein Family.

The Golgi apparatus is an organelle that mediates modifications, sorting, and transport of proteins and lipids. Golgins are a group of proteins with coiled-coil structures that localize to the Golgi and are thought to function as tethers to facilitate the docking of vesicles, Rab GTPases, and cytoskeleton components to the Golgi stack. Giantin is the longest golgin and has been thought to function as a tether for COPI vesicles along with other golgins, such as p115 and GM130. Contrary to our expectation that the loss of the tether will result in an increase in untethered COPI vesicles in the cytoplasm, our electron microscopy observations showed that the fenestrae normally present in Golgi cisternae were reduced upon Giantin knockdown. We also found that this structural change is accompanied by altered secretion of cargo proteins and cell surface glycosylation. These results indicate that there exists a correlation between Golgi structural changes caused by the loss of Giantin and Golgi function. Here, we describe electron tomography methods for the detection of structural changes in the Golgi.