RESUMO
Many plasma membrane (PM) functions depend on the cholesterol concentration in the PM in strikingly nonlinear, cooperative ways: fully functional in the presence of physiological cholesterol levels (35~45 mol%), and nonfunctional below 25 mol% cholesterol; namely, still in the presence of high concentrations of cholesterol. This suggests the involvement of cholesterol-based complexes/domains formed cooperatively. In this review, by examining the results obtained by using fluorescent lipid analogs and avoiding the trap of circular logic, often found in the raft literature, we point out the fundamental similarities of liquid-ordered (Lo)-phase domains in giant unilamellar vesicles, Lo-phase-like domains formed at lower temperatures in giant PM vesicles, and detergent-resistant membranes: these domains are formed by cooperative interactions of cholesterol, saturated acyl chains, and unsaturated acyl chains, in the presence of >25 mol% cholesterol. The literature contains evidence, indicating that the domains formed by the same basic cooperative molecular interactions exist and play essential roles in signal transduction in the PM. Therefore, as a working definition, we propose that raft domains in the PM are liquid-like molecular complexes/domains formed by cooperative interactions of cholesterol with saturated acyl chains as well as unsaturated acyl chains, due to saturated acyl chains' weak multiple accommodating interactions with cholesterol and cholesterol's low miscibility with unsaturated acyl chains and TM proteins. Molecules move within raft domains and exchange with those in the bulk PM. We provide a logically established collection of fluorescent lipid probes that preferentially partition into raft and non-raft domains, as defined here, in the PM.
Assuntos
Colesterol , Microdomínios da Membrana , Membrana Celular , Lipídeos , Lipossomas UnilamelaresRESUMO
Aqueous self-assembly of short peptides has attracted growing attention for the construction of supramolecular materials for various bioapplications. Herein, we describe how the thermolysin-assisted biocatalytic construction of a dipeptide hydrazide from an N-protected amino acid and an amino acid hydrazide leads to the formation of thermally stable supramolecular hydrogels. In addition, we demonstrate the post-assembly modification of the supramolecular architectures constructed inâ situ tethering hydrazide groups as a chemical handle by means of fluorescence imaging.
Assuntos
Dipeptídeos , Nanoestruturas , Hidrazinas , Hidrogéis , PeptídeosRESUMO
Exosomes are small extracellular vesicles (sEVs) involved in distal cell-cell communication and cancer migration by transferring functional cargo molecules. Membrane domains similar to lipid rafts are assumed to occur in exosome membranes and are involved in interactions with target cells. However, the bilayer membrane properties of these small vesicles have not been fully investigated. Therefore, we examined the fluidity, lateral domain separation, and transbilayer asymmetry of exosome membranes using fluorescence spectroscopy. Although there were some differences between the exosomes, TMA-DPH anisotropy showing moderate lipid chain order indicated that ordered phases comprised a significant proportion of exosome membranes. Selective TEMPO quenching of the TMA-DPH fluorescence in the liquid-disordered phase indicated that 40-50% of the exosome membrane area belonged to the ordered phase based on a phase-separated model. Furthermore, NBD-PC in the outer leaflet showed longer fluorescence lifetimes than those in the inner leaflets. Therefore, the exosome membranes maintained transbilayer asymmetry with a topology similar to that of the plasma membranes. In addition, the lateral and transbilayer orders of exosome membranes obtained from different cell lines varied, probably depending on the different membrane lipid components and compositions partially derived from donor cells. As these higher membrane orders and asymmetric topologies are similar to those of cell membranes with lipid rafts, raft-like functional domains are possibly enriched on exosome membranes. These domains likely play key roles in the biological functions and cellular uptake of exosomes by facilitating selective membrane interactions with target organs.
Assuntos
Exossomos , Fluidez de Membrana , Espectrometria de Fluorescência , Bicamadas Lipídicas/química , Exossomos/metabolismo , Membrana Celular/metabolismoRESUMO
Aqueous hybrid soft nanomaterials consisting of plural supramolecular architectures with a high degree of segregation (orthogonal coexistence) and precise hierarchy at the nano- and microscales, which are reminiscent of complex biomolecular systems, have attracted increasing attention. Remarkable progress has been witnessed in the construction of DNA nanostructures obtained by rational sequence design and supramolecular nanostructures of peptide derivatives through self-assembly under aqueous conditions. However, orthogonal self-assembly of DNA nanostructures and supramolecular nanostructures of peptide derivatives in a single medium has not yet been explored in detail. In this study, DNA microspheres, which can be obtained from three single-stranded DNAs, and three different supramolecular nanostructures (helical nanofibers, straight nanoribbons, and flowerlike microaggregates) of semi-artificial glycopeptides were simultaneously constructed in a single medium by a simple thermal annealing process, which gives rise to hybrid soft nanomaterials. Fluorescence imaging with selective staining of each supramolecular nanostructure uncovered the orthogonal coexistence of these structures with only marginal impact on their morphology. Additionally, the biostimuli-responsive degradation propensity of each supramolecular architecture is retained, and this may allow the construction of active soft nanomaterials exhibiting intelligent biofunctions.
Assuntos
DNA/química , Glicopeptídeos/química , Nanoestruturas/química , Peptídeos/química , Microesferas , Nanofibras/química , ÁguaRESUMO
Cholesterol distribution and dynamics in the plasma membrane (PM) are poorly understood. The recent development of Bodipy488-conjugated cholesterol molecule (Bdp-Chol) allowed us to study cholesterol behavior in the PM, using single fluorescent-molecule imaging. Surprisingly, in the intact PM, Bdp-Chol diffused at the fastest rate ever found for any molecules in the PM, with a median diffusion coefficient (D) of 3.4 µm²/second, which was â¼10 times greater than that of non-raft phospholipid molecules (0.33 µm²/second), despite Bdp-Chol's probable association with raft domains. Furthermore, Bdp-Chol exhibited no sign of entrapment in time scales longer than 0.5 milliseconds. In the blebbed PM, where actin filaments were largely depleted, Bdp-Chol and Cy3-conjugated dioleoylphosphatidylethanolamine (Cy3-DOPE) diffused at comparable Ds (medians = 5.8 and 6.2 µm²/second, respectively), indicating that the actin-based membrane skeleton reduces the D of Bdp-Chol only by a factor of â¼2 from that in the blebbed PM, whereas it reduces the D of Cy3-DOPE by a factor of â¼20. These results are consistent with the previously proposed model, in which the PM is compartmentalized by the actin-based membrane-skeleton fence and its associated transmembrane picket proteins for the macroscopic diffusion of all of the membrane molecules, and suggest that the probability of Bdp-Chol passing through the compartment boundaries, once it enters the boundary, is â¼10× greater than that of Cy3-DOPE. Since the compartment sizes are greater than those of the putative raft domains, we conclude that raft domains coexist with membrane-skeleton-induced compartments and are contained within them.
Assuntos
Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Compostos de Boro , Linhagem Celular , Colesterol/análogos & derivados , Difusão , Corantes Fluorescentes , Microdomínios da Membrana/efeitos dos fármacos , Fosfatidiletanolaminas/farmacologia , RatosRESUMO
Advanced single-molecule fluorescent imaging was applied to study the dynamic organization of raft-associated glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the plasma membrane and their stimulation-induced changes. In resting cells, virtually all of the GPI-APs are mobile and continually form transient (~200 ms) homodimers (termed homodimer rafts) through ectodomain protein interactions, stabilized by the presence of the GPI-anchoring chain and cholesterol. Heterodimers do not form, suggesting a fundamental role for the specific ectodomain protein interaction. Under higher physiological expression conditions , homodimers coalesce to form hetero- and homo-GPI-AP oligomer rafts through raft-based lipid interactions. When CD59 was ligated, it formed stable oligomer rafts containing up to four CD59 molecules, which triggered intracellular Ca(2+) responses that were dependent on GPI anchorage and cholesterol, suggesting a key part played by transient homodimer rafts. Transient homodimer rafts are most likely one of the basic units for the organization and function of raft domains containing GPI-APs.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Microdomínios da Membrana , Antígenos CD59/metabolismo , Dimerização , Transferência Ressonante de Energia de FluorescênciaRESUMO
Stimulator of interferon genes (STING) is critical for the type I interferon response to pathogen- or self-derived DNA in the cytosol. STING may function as a scaffold to activate TANK-binding kinase 1 (TBK1), but direct cellular evidence remains lacking. Here we show, using single-molecule imaging of STING with enhanced time resolutions down to 5 ms, that STING becomes clustered at the trans-Golgi network (about 20 STING molecules per cluster). The clustering requires STING palmitoylation and the Golgi lipid order defined by cholesterol. Single-molecule imaging of TBK1 reveals that STING clustering enhances the association with TBK1. We thus provide quantitative proof-of-principle for the signaling STING scaffold, reveal the mechanistic role of STING palmitoylation in the STING activation, and resolve the long-standing question of the requirement of STING translocation for triggering the innate immune signaling.
Assuntos
Lipoilação , Rede trans-Golgi , Rede trans-Golgi/metabolismo , Microscopia , Imagem Individual de Molécula , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Colesterol , Análise por Conglomerados , Imunidade InataRESUMO
Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small 'Golgi units' that have 1-3 µm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call 'zones'. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis.
Assuntos
Glicosaminoglicanos , Complexo de Golgi , Complexo de Golgi/metabolismo , Glicosilação , Humanos , Glicosaminoglicanos/metabolismo , Células HeLa , Sistemas CRISPR-Cas , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas da Matriz do Complexo de GolgiRESUMO
Small extracellular vesicles (sEVs) secreted from cancer cells play pivotal roles in cancer metastasis and malignancy by transferring biomolecules and conditioning future metastatic sites. Studies have elucidated structures and functions of glycans on sEVs; however, whether sEVs remodel glycans in recipient cells remains poorly understood. Here, we examined the enzyme activity of glycosyltransferases for complex N-glycan biosynthesis in cancer-derived sEVs and discovered that cancer-related glycosyltransferase, N-acetylglucosaminyltransferase-V (GnT-V, a.k.a. MGAT5), is selectively enriched in sEVs among various glycosyltransferases. GnT-V in sEVs is a cleaved form, and cleavage by SPPL3 protease is necessary for loading GnT-V in sEVs. Fractionation experiments and single-particle imaging further revealed that GnT-V was enriched in non-exosomal sEVs. Strikingly, we found that enzymatically active GnT-V in sEVs was transferred to recipient cells and the N-glycan structures of recipient cells were remodeled to express GnT-V-produced glycans. Our results suggest GnT-V-enriched sEVs' role in glycan remodeling in cancer metastasis.
RESUMO
Two very polarized views exist for understanding the cellular plasma membrane (PM). For some, it is the simple fluid described by the original Singer-Nicolson fluid mosaic model. For others, due to the presence of thousands of molecular species that extensively interact with each other, the PM forms various clusters and domains that are constantly changing and therefore, no simple rules exist that can explain the structure and molecular dynamics of the PM. In this article, we propose that viewing the PM from its two predominant components, cholesterol and actin filaments, provides an excellent and transparent perspective of PM organization, dynamics, and mechanisms for its functions. We focus on the actin-induced membrane compartmentalization and lipid raft domains coexisting in the PM and how they interact with each other to perform PM functions. This view provides an important update of the fluid mosaic model.
Assuntos
Actinas , Canto , Actinas/metabolismo , Aniversários e Eventos Especiais , Membrana Celular/metabolismo , Colesterol/metabolismoRESUMO
The artificial construction of multicomponent supramolecular materials comprising plural supramolecular architectures that are assembled orthogonally from their constituent molecules has attracted growing attention. Here, we describe the design and development of multicomponent supramolecular materials by combining peptide-based self-assembled fibrous nanostructures with globular DNA nanoflowers constructed by the rolling circle amplification reaction. The orthogonally constructed architectures were dissected by fluorescence imaging using the selective fluorescence staining procedures adapted to this study. The present, unique hybrid materials developed by taking advantage of each supramolecular architecture based on their peptide and DNA functions may offer distinct opportunities to explore their bioapplications as a soft matrix.
Assuntos
Nanofibras , Nanoestruturas , Nanofibras/química , Nanoestruturas/química , Peptídeos/química , DNA/química , Imagem ÓpticaRESUMO
Here, we describe the design and synthesis of a new reduction-cleavable spacer (RCS) based on a nitrobenzene scaffold for constructing reduction-responsive oligonucleotides according to standard phosphoramidite chemistry. In addition, we demonstrate that the introduction of the RCS in the middle of an oligonucleotide (30 nt) enables the construction of a self-assembled microsphere capable of exhibiting a reduction-responsive disassembly.
Assuntos
DNA , Oligonucleotídeos , Microesferas , NitrobenzenosRESUMO
Multicomponent supramolecular materials, which comprise plural supramolecular architectures that exhibit distinct self-sorting behaviors, are receiving increasing attention because they can be implemented with sophisticated functions and hierarchical structures, e.g., living cells. Nevertheless, the application of supramolecular system design to engineer self-sorting behaviors among plural supramolecular architectures remains challenging. Herein, we show that the thermal annealing-induced one-pot assembly of multiple single-stranded DNAs and a single semi-artificial glycopeptide (GP) results in the emergent formation of integrative self-sorted supramolecular nanostructures (ssSNs) consisting of a GP supramolecular nanoribbon surrounded by DNA tile-nanotubes. Fluorescence imaging revealed the formation of each supramolecular nanostructure through orthogonal molecular assembling processes. Moreover, the fluorescence recovery after photobleaching (FRAP) disclosed the presence of reversible attractive interactions between the DNA tile, prior to the formation of the tile-nanotube, and the GP supramolecular nanostructures at the mesoscale level, which are crucial for the formation of the integrative ssSNs. Further, we revealed that the integrative ssSNs retain their biostimuli responsiveness so that each supramolecular nanostructure can be selectively degraded. Finally, we successfully constructed a complex soft nanomaterial composed of ternary supramolecular architectures (a GP supramolecular nanostructure, DNA tile-nanotube, and DNA microsphere) based on the present as well as previous findings.
RESUMO
Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named "actin-pl-clusters". In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent "actin podosome-like clusters".
Assuntos
Actinas/metabolismo , Podossomos/metabolismo , Imagem Individual de Molécula/métodos , Animais , Células CultivadasRESUMO
Sphingomyelin (SM) has been proposed to form cholesterol-dependent raft domains and sphingolipid domains in the plasma membrane (PM). How SM contributes to the formation and function of these domains remains unknown, primarily because of the scarcity of suitable fluorescent SM analogs. We developed new fluorescent SM analogs by conjugating a hydrophilic fluorophore to the SM choline headgroup without eliminating its positive charge, via a hydrophilic nonaethylene glycol linker. The new analogs behaved similarly to the native SM in terms of their partitioning behaviors in artificial liquid order-disorder phase-separated membranes and detergent-resistant PM preparations. Single fluorescent molecule tracking in the live-cell PM revealed that they indirectly interact with each other in cholesterol- and sphingosine backbone-dependent manners, and that, for â¼10-50 ms, they undergo transient colocalization-codiffusion with a glycosylphosphatidylinositol (GPI)-anchored protein, CD59 (in monomers, transient-dimer rafts, and clusters), in CD59-oligomer size-, cholesterol-, and GPI anchoring-dependent manners. These results suggest that SM continually and rapidly exchanges between CD59-associated raft domains and the bulk PM.
Assuntos
Corantes Fluorescentes/metabolismo , Esfingomielinas/metabolismo , Animais , Antígenos CD59/metabolismo , Células CHO , Linhagem Celular , Membrana Celular/metabolismo , Colesterol/metabolismo , Cricetulus , Detergentes/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Microdomínios da Membrana/metabolismo , Esfingolipídeos/metabolismo , SuínosRESUMO
The impeccable photostability of fluorescent nanodiamonds (FNDs) is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the ß-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface.