Homotypic targeting of immunomodulatory nanoparticles for enhanced peripheral and central immunity

ObjectivesSynthetic oligodeoxynucleotides (ODNs) that contain unmethylated cytosine–phosphate–guanine (CpG) motifs serve as immune adjuvants in disease treatment. However, the poor cell permeability and safety concerns limit their medical applications, and biocompatible strategies for efficient delivery of functional CpG ODNs are highly desirable.Materials and MethodsSelf‐assembled, cell membrane‐coated CpG nanoparticles (NP) are prepared, and their physicochemical properties are characterized. The uncoated and membrane‐coated CpG NP are compared for their biocompatibility, cellular uptake kinetics, endocytic pathways, subcellular localization, and immunostimulatory activities in macrophages and microglia.ResultsMacrophage‐ or microglia‐derived cell membrane camouflaging alters the endocytic pathways of CpG NP, promotes their targeted delivery to the cells with homologous membrane, ensures their endosomal localization, and enhances their immunomodulatory effects.ConclusionsWe design a type of biomimetic NP consisting of self‐assembled CpG NP core and cell membrane shell, and demonstrate its advantages in the modulation of peripheral and central immune cells. Our study provides a new strategy for the application of CpG ODNs.

Cell membrane camouflage switches the endocytic pathways of self‐assembled CpG nanoparticles, promotes their delivery to macrophages or microglia via homotypic targeting, and enhances their immunostimulatory effects on cytokine production for the modulation of peripheral or central immune cells.     

Plasmodium sporozoite disintegration during skin passage limits malaria parasite transmission

During transmission of malaria‐causing parasites from mosquitoes to mammals, Plasmodium sporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here, we show that the membrane‐associated protein, concavin, is important for the maintenance of the Plasmodium sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin‐GFP localizes at the cytoplasmic periphery and concavin(−) sporozoites progressively round up upon entry of salivary glands. Rounded concavin(−) sporozoites fail to pass through the narrow salivary ducts and are rarely ejected by mosquitoes, while normally shaped concavin(−) sporozoites are transmitted. Strikingly, motile concavin(−) sporozoites disintegrate while migrating through the skin leading to parasite arrest or death and decreased transmission efficiency. Collectively, we suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex. Interfering with cell shape maintenance pathways might hence provide a new strategy to prevent a malaria infection.     

Calcium signaling induces a partial EMT

Epithelial plasticity, or epithelial‐to‐mesenchymal transition (EMT), is a well‐recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial–mesenchymal (E‐M) states and that cells exhibiting such partial EMT (P‐EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P‐EMT program operating in vivo by which carcinoma cells lose their epithelial state through post‐translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P‐EMT characterized by the internalization of membrane‐associated E‐cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq‐associated G‐protein‐coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin‐Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial–mesenchymal states in carcinoma cells.     

Linking transport and translation of mRNAs with endosomes and mitochondria

In eukaryotic cells, proteins are targeted to their final subcellular locations with precise timing. A key underlying mechanism is the active transport of cognate mRNAs, which in many systems can be linked intimately to membrane trafficking. A prominent example is the long‐distance endosomal transport of mRNAs and their local translation. Here, we describe current highlights of fundamental mechanisms of the underlying transport process as well as of biological functions ranging from endosperm development in plants to fungal pathogenicity and neuronal processes. Translation of endosome‐associated mRNAs often occurs at the cytoplasmic surface of endosomes, a process that is needed for membrane‐assisted formation of heteromeric protein complexes and for accurate subcellular targeting of proteins. Importantly, endosome‐coupled translation of mRNAs encoding mitochondrial proteins, for example, seems to be particularly important for efficient organelle import and for regulating subcellular mitochondrial activity. In essence, these findings reveal a new mechanism of loading newly synthesised proteins onto endocytic membranes enabling intimate crosstalk between organelles. The novel link between endosomes and mitochondria adds an inspiring new level of complexity to trafficking and organelle biology.     

Aromaticity at position 39 in α‐synuclein: A modulator of amyloid fibril assembly and membrane‐bound conformations

Recent studies revealed that molecular events related with the physiology and pathology of αS might be regulated by specific sequence motifs in the primary sequence of αS. The importance of individual residues in these motifs remains an important open avenue of investigation. In this work, we have addressed the structural details related to the amyloid fibril assembly and lipid‐binding features of αS through the design of site‐directed mutants at position 39 of the protein and their study by in vitro and in vivo assays. We demonstrated that aromaticity at position 39 of αS primary sequence influences strongly the aggregation properties and the membrane‐bound conformations of the protein, molecular features that might have important repercussions for the function and dysfunction of αS. Considering that aggregation and membrane damage is an important driver of cellular toxicity in amyloid diseases, future work is needed to link our findings with studies based on toxicity and neuronal cell death.Brief statement outlining significanceModulation by distinct sequential motifs and specific residues of αS on its physiological and pathological states is an active area of research. Here, we demonstrated that aromaticity at position 39 of αS modulates the membrane‐bound conformations of the protein, whereas removal of aromatic functionality at position 39 reduces strongly the amyloid assembly in vitro and in vivo. Our study provides new evidence for the modulation of molecular events related with the physiology and pathology of αS.     

High‐resolution analysis of the conformational transition of pro‐apoptotic Bak at the lipid membrane

Permeabilization of the outer mitochondrial membrane by pore‐forming Bcl2 proteins is a crucial step for the induction of apoptosis. Despite a large set of data suggesting global conformational changes within pro‐apoptotic Bak during pore formation, high‐resolution structural details in a membrane environment remain sparse. Here, we used NMR and HDX‐MS (Hydrogen deuterium exchange mass spectrometry) in lipid nanodiscs to gain important high‐resolution structural insights into the conformational changes of Bak at the membrane that are dependent on a direct activation by BH3‐only proteins. Furthermore, we determined the first high‐resolution structure of the Bak transmembrane helix. Upon activation, α‐helix 1 in the soluble domain of Bak dissociates from the protein and adopts an unfolded and dynamic potentially membrane‐bound state. In line with this finding, comparative protein folding experiments with Bak and anti‐apoptotic BclxL suggest that α‐helix 1 in Bak is a metastable structural element contributing to its pro‐apoptotic features. Consequently, mutagenesis experiments aimed at stabilizing α‐helix 1 yielded Bak variants with delayed pore‐forming activity. These insights will contribute to a better mechanistic understanding of Bak‐mediated membrane permeabilization.     

Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane‐adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel‐fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large‐scale lipid phase separation and protein segregation in intact, protein‐crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible.     

Berberine regulates mesangial cell proliferation and cell cycle to attenuate diabetic nephropathy through the PI3K/Akt/AS160/GLUT1 signalling pathway

High glucose (HG) is one of the basic factors of diabetic nephropathy (DN), which leads to high morbidity and disability. During DN, the expression of glomerular glucose transporter 1 (GLUT1) increases, but the relationship between HG and GLUT1 is unclear. Glomerular mesangial cells (GMCs) have multiple roles in HG‐induced DN. Here, we report prominent glomerular dysfunction, especially GMC abnormalities, in DN mice, which is closely related to GLUT1 alteration. In vivo studies have shown that BBR can alleviate pathological changes and abnormal renal function indicators of DN mice. In vitro, BBR (30, 60 and 90 μmol/L) not only increased the proportion of G1 phase cells but also reduced the proportion of S phase cells under HG conditions at different times. BBR (60 μmol/L) significantly reduced the expression of PI3K‐p85, p‐Akt, p‐AS160, membrane‐bound GLUT1 and cyclin D1, but had almost no effect on total protein. Furthermore, BBR significantly declined the glucose uptake and retarded cyclin D1‐mediated GMC cell cycle arrest in the G1 phase. This study demonstrated that BBR can inhibit the development of DN, which may be due to BBR inhibiting the PI3K/Akt/AS160/GLUT1 signalling pathway to regulate HG‐induced abnormal GMC proliferation and the cell cycle, supporting BBR as a potential therapeutic drug for DN.     

USP42 protects ZNRF3/RNF43 from R‐spondin‐dependent clearance and inhibits Wnt signalling

The tumour suppressors RNF43 and ZNRF3 play a central role in development and tissue homeostasis by promoting the turnover of the Wnt receptors LRP6 and Frizzled (FZD). The stem cell growth factor R‐spondin induces auto‐ubiquitination and membrane clearance of ZNRF3/RNF43 to promote Wnt signalling. However, the deubiquitinase stabilising ZNRF3/RNF43 at the plasma membrane remains unknown. Here, we show that the USP42 antagonises R‐spondin by protecting ZNRF3/RNF43 from ubiquitin‐dependent clearance. USP42 binds to the Dishevelled interacting region (DIR) of ZNRF3 and stalls the R‐spondin‐LGR4‐ZNRF3 ternary complex by deubiquitinating ZNRF3. Accordingly, USP42 increases the turnover of LRP6 and Frizzled (FZD) receptors and inhibits Wnt signalling. Furthermore, we show that USP42 functions as a roadblock for paracrine Wnt signalling in colon cancer cells and mouse small intestinal organoids. We provide new mechanistic insights into the regulation R‐spondin and conclude that USP42 is crucial for ZNRF3/RNF43 stabilisation at the cell surface.     

ORP2 couples LDL‐cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P2 exchange

Low‐density lipoprotein (LDL)‐cholesterol delivery from late endosomes to the plasma membrane regulates focal adhesion dynamics and cell migration, but the mechanisms controlling it are poorly characterized. Here, we employed auxin‐inducible rapid degradation of oxysterol‐binding protein‐related protein 2 (ORP2/OSBPL2) to show that endogenous ORP2 mediates the transfer of LDL‐derived cholesterol from late endosomes to focal adhesion kinase (FAK)‐/integrin‐positive recycling endosomes in human cells. In vitro, cholesterol enhances membrane association of FAK to PI(4,5)P₂‐containing lipid bilayers. In cells, ORP2 stimulates FAK activation and PI(4,5)P₂ generation in endomembranes, enhancing cell adhesion. Moreover, ORP2 increases PI(4,5)P₂ in NPC1‐containing late endosomes in a FAK‐dependent manner, controlling their tubulovesicular trafficking. Together, these results provide evidence that ORP2 controls FAK activation and LDL‐cholesterol plasma membrane delivery by promoting bidirectional cholesterol/PI(4,5)P₂ exchange between late and recycling endosomes.     

Cryptic association of B7‐2 molecules and its implication for clustering

T‐cell co‐stimulation through CD28/CTLA4:B7‐1/B7‐2 axis is one of the extensively studied pathways that resulted in the discovery of several FDA‐approved drugs for autoimmunity and cancer. However, many aspects of the signaling mechanism remain elusive, including oligomeric association and clustering of B7‐2 on the cell surface. Here, we describe the structure of the IgV domain of B7‐2 and its cryptic association into 1D arrays that appear to represent the pre‐signaling state of B7‐2 on the cell membrane. Super‐resolution microscopy experiments on heterologous cells expressing B7‐2 and B7‐1 suggest, B7‐2 form relatively elongated and larger clusters compared to B7‐1. The sequence and structural comparison of other B7 family members, B7‐1:CTLA4 and B7‐2:CTLA‐4 complex structures, support our view that the observed B7‐2 1D zipper array is physiologically important. This observed 1D zipper‐like array also provides an explanation for its clustering, and upright orientation on the cell surface, and avoidance of spurious signaling.     

Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM

Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane–cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the postfusion granule membrane–plasma membrane complex. Experiments on diI-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin.     

Response to stress in biological disorders: Implications of stress granule assembly and function

It is indispensable for cells to adapt and respond to environmental stresses, in order for organisms to survive. Stress granules (SGs) are condensed membrane‐less organelles dynamically formed in the cytoplasm of eukaryotes cells to cope with diverse intracellular or extracellular stress factors, with features of liquid‐liquid phase separation. They are composed of multiple constituents, including translationally stalled mRNAs, translation initiation factors, RNA‐binding proteins and also non‐RNA‐binding proteins. SG formation is triggered by stress stimuli, viral infection and signal transduction, while aberrant assembly of SGs may contribute to tissue degenerative diseases. Recently, a growing body of evidence has emerged on SG response mechanisms for cells facing high temperatures, oxidative stress and osmotic stress. In this review, we aim to summarize factors affecting SGs assembly, present the impact of SGs on germ cell development and other biological processes. We particularly emphasize the significance of recently reported RNA modifications in SG stress responses. In parallel, we also review all current perspectives on the roles of SGs in male germ cells, with a particular focus on the dynamics of SG assembly.     

Gamma frequency light flicker regulates amyloid precursor protein trafficking for reducing β‐amyloid load in Alzheimer's disease model

Inducing gamma oscillations with non‐invasive light flicker has been reported to impact Alzheimer''s disease‐related pathology. However, it is unclear which signaling pathways are involved in reducing amyloid load. Here, we found that gamma frequency light flicker increased anchoring of amyloid precursor protein (APP) to the plasma membrane for non‐amyloidogenic processing, and then physically interacted with KCC2, a neuron‐specific K⁺‐Cl⁻ cotransporter, suggesting that it is essential to maintain surface GABA_A receptor α1 levels and reduce β‐amyloid (Aβ) production. Stimulation with such light flicker limited KCC2 internalization and subsequent degradation via both tyrosine phosphorylation and ubiquitination, leading to an increase in surface‐KCC2 levels. Specifically, PKC‐dependent phosphorylation of APP on a serine residue was induced by gamma frequency light flicker, which was responsible for maintaining plasma membrane levels of full‐length APP, leading to its reduced trafficking to endosomes and inhibiting the β‐secretase cleavage pathway. The activated PKC from the gamma frequency light flicker subsequently phosphorylated serine of KCC2 and stabilized it onto the cell surface, which contributed to the upregulation of surface GABA_A receptor α1 levels. Together, these data indicate that enhancement of APP trafficking to the plasma membrane via light flicker plays a critical modulatory role in reduction of Aβ load in Alzheimer''s disease.     

Opposing activities of IFITM proteins in SARS‐CoV‐2 infection

Interferon‐induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS‐CoV‐2 Spike‐pseudotyped virus and genuine SARS‐CoV‐2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain‐ and loss‐of‐function approaches. Mechanistically, SARS‐CoV‐2 restriction occurred independently of IFITM3 S‐palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis‐promoting YxxФ motif converted human IFITM3 into an enhancer of SARS‐CoV‐2 infection, and cell‐to‐cell fusion assays confirmed the ability of endocytic mutants to enhance Spike‐mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS‐CoV‐2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro‐ and anti‐viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity‐based mechanisms used for endosomal SARS‐CoV‐2 restriction.     

TMPRSS2 expression dictates the entry route used by SARS‐CoV‐2 to infect host cells

SARS‐CoV‐2 is a newly emerged coronavirus that caused the global COVID‐19 outbreak in early 2020. COVID‐19 is primarily associated with lung injury, but many other clinical symptoms such as loss of smell and taste demonstrated broad tissue tropism of the virus. Early SARS‐CoV‐2–host cell interactions and entry mechanisms remain poorly understood. Investigating SARS‐CoV‐2 infection in tissue culture, we found that the protease TMPRSS2 determines the entry pathway used by the virus. In the presence of TMPRSS2, the proteolytic process of SARS‐CoV‐2 was completed at the plasma membrane, and the virus rapidly entered the cells within 10 min in a pH‐independent manner. When target cells lacked TMPRSS2 expression, the virus was endocytosed and sorted into endolysosomes, from which SARS‐CoV‐2 entered the cytosol via acid‐activated cathepsin L protease 40–60 min post‐infection. Overexpression of TMPRSS2 in non‐TMPRSS2 expressing cells abolished the dependence of infection on the cathepsin L pathway and restored sensitivity to the TMPRSS2 inhibitors. Together, our results indicate that SARS‐CoV‐2 infects cells through distinct, mutually exclusive entry routes and highlight the importance of TMPRSS2 for SARS‐CoV‐2 sorting into either pathway.     

Cell‐extracellular matrix interactions in the fluidic phase direct the topology and polarity of self‐organized epithelial structures

IntroductionIn vivo, cells are surrounded by extracellular matrix (ECM). To build organs from single cells, it is generally believed that ECM serves as scaffolds to coordinate cell positioning and differentiation. Nevertheless, how cells utilize cell‐ECM interactions for the spatiotemporal coordination to different ECM at the tissue scale is not fully understood.MethodsHere, using in vitro assay with engineered MDCK cells expressing H2B‐mCherry (nucleus) and gp135/Podocalyxin‐GFP (apical marker), we show in multi‐dimensions that such coordination for epithelial morphogenesis can be determined by cell‐soluble ECM interaction in the fluidic phase.ResultsThe coordination depends on the native topology of ECM components such as sheet‐like basement membrane (BM) and type I collagen (COL) fibres: scaffold formed by BM (COL) facilitates a close‐ended (open‐ended) coordination that leads to the formation of lobular (tubular) epithelium. Further, cells form apicobasal polarity throughout the entire lobule/tubule without a complete coverage of ECM at the basal side, and time‐lapse two‐photon scanning imaging reveals the polarization occurring early and maintained through the lobular expansion. During polarization, gp135‐GFP was converged to the apical surface collectively in the lobular/tubular structures, suggesting possible intercellular communications. Under suspension culture, the polarization was impaired with multi‐lumen formation in the tubules, implying the importance of ECM biomechanical microenvironment.ConclusionOur results suggest a biophysical mechanism for cells to form polarity and coordinate positioning at tissue scale, and in engineering epithelium through cell‐soluble ECM interaction and self‐assembly.     

Golgi maturation‐dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3

Glycosphingolipids are important components of the plasma membrane where they modulate the activities of membrane proteins including signalling receptors. Glycosphingolipid synthesis relies on competing reactions catalysed by Golgi‐resident enzymes during the passage of substrates through the Golgi cisternae. The glycosphingolipid metabolic output is determined by the position and levels of the enzymes within the Golgi stack, but the mechanisms that coordinate the intra‐Golgi localisation of the enzymes are poorly understood. Here, we show that a group of sequentially‐acting enzymes operating at the branchpoint among glycosphingolipid synthetic pathways binds the Golgi‐localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra‐Golgi retro‐transport, acting as a component of the cisternal maturation mechanism. Through these effects, GOLPH3 controls the sub‐Golgi localisation and the lysosomal degradation rate of specific enzymes. Increased GOLPH3 levels, as those observed in tumours, alter glycosphingolipid synthesis and plasma membrane composition thereby promoting mitogenic signalling and cell proliferation. These data have medical implications as they outline a novel oncogenic mechanism of action for GOLPH3 based on glycosphingolipid metabolism.     

Reverse fountain flow of phosphatidylinositol‐3,4‐bisphosphate polarizes migrating cells

The ability of cells to polarize and move toward external stimuli plays a crucial role in development, as well as in normal and pathological physiology. Migrating cells maintain dynamic complementary distributions of Ras activity and of the phospholipid phosphatidylinositol‐3,4‐bisphosphate (PI(3,4)P2). Here, we show that lagging‐edge component PI(3,4)P2 also localizes to retracting leading‐edge protrusions and nascent macropinosomes, even in the absence of phosphatidylinositol 3,4,5‐trisphosphate (PIP3). Once internalized, macropinosomes break up into smaller PI(3,4)P2‐enriched vesicles, which fuse with the plasma membrane at the rear of the cell. Subsequently, the phosphoinositide diffuses toward the front of the cell, where it is degraded. Computational modeling confirms that this cycle gives rise to stable back‐to‐front gradient. These results uncover a surprising “reverse‐fountain flow” of PI(3,4)P2 that regulates polarity.     

Lateral and Rotational Mobilities of Lipids in Specific Cellular Membranes of Eucalyptus gunnii Cultivars Exhibiting Different Freezing Tolerance

Two cell lines of Eucalyptus gunnii have been shown to keep their differential frost tolerance at the cellular level after long-term culture. They have been used to investigate the fluidity of specific cell membranes in relation with frost tolerance. Protoplasts and isolated vacuoles were obtained from both cell lines. In addition, purified plasma membrane and tonoplast (the vacuolar membrane) were separated from a crude microsomal fraction through free-flow electrophoresis. The lateral and rotational mobilities of lipids in these different membranes were studied by two biophysical techniques: fluorescence recovery after photobleaching (FRAP) and fluorescence polarization. After labeling the vacuoles isolated from the frost-sensitive cells with 1-oleoyl-2-(7-nitro-2,1,3-benz-oxadiazol-4-yl)aminocaproyl phosphatidylcholine, a single mobile component was observed with a diffusion coefficient of 2.4 × 10⁻⁹ cm² s⁻¹ and a mobile fraction close to 100% at a temperature of 23°C. When using isolated vacuoles from the frost tolerant line, a higher lateral diffusion of tonoplast lipids was found with a diffusion coefficient of 3.2 × 10⁻⁹ cm² s⁻¹, still with a mobile fraction close to 100%. No convincing data were obtained when performing fluorescence recovery after photobleaching experiments on protoplasts. Fluorescence polarization experiments confirmed the differential behavior of the two cell lines for tonoplast and also for plasma membrane. In addition, they showed that intrinsically tonoplast exhibited a higher fluidity than plasma membrane. Our results provide the first information on the fluidity of tonoplast and on the compared properties of two important plant membranes—tonoplast and plasma membrane—through the use of two complementary biophysical approaches. In addition, they suggest there is a correlation between membrane fluidity and cold tolerance. The potential interest of plant vacuole as a natural model system in membrane studies is emphasized.