Influence of the Charge Ratio of Guanine-Quadruplex Structure-Based CpG Oligodeoxynucleotides and Cationic DOTAP Liposomes on Cytokine Induction Profiles.

Cationic liposomes, specifically 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes, serve as successful carriers for guanine-quadruplex (G4) structure-based cytosine-guanine oligodeoxynucleotides (CpG ODNs). The combined benefits of CpG ODNs forming a G4 structure and a non-viral vector carrier endow the ensuing complex with promising adjuvant properties. Although G4-CpG ODN-DOTAP complexes show a higher immunostimulatory effect than naked G4-CpG ODNs, the effects of the complex composition, especially charge ratios, on the production of the pro-inflammatory cytokines interleukin (IL)-6 and interferon (IFN)-α remain unclear. Here, we examined whether charge ratios drive the bifurcation of cytokine inductions in human peripheral blood mononuclear cells. Linear CpG ODN-DOTAP liposome complexes formed micrometer-sized positively charged agglomerates; G4-CpG ODN-DOTAP liposome complexes with low charge ratios (0.5 and 1.5) formed ~250 nm-sized negatively charged complexes. Notably, low-charge-ratio (0.5 and 1.5) complexes induced significantly higher IL-6 and IFN-α levels simultaneously than high-charge-ratio (2 and 2.5) complexes. Moreover, confocal microscopy indicated a positive correlation between the cellular uptake of the complex and amount of cytokine induced. The observed effects of charge ratios on complex size, surface charge, and affinity for factors that modify cellular-uptake, intracellular-activity, and cytokine-production efficiency highlight the importance of a rational complex design for delivering and controlling G4-CpG ODN activity.

Antiviral Activity of Cell Membrane-Bound Amphiphilic Polymers.

We demonstrate that cholesterol-modified polyethylene glycol has antiviral activity, exerted by anchoring to plasma membranes and sterically inhibiting viruses from entering cells. These polymers distribute sparsely on cell membranes even at binding saturation. However, the polymers have sufficient elastic repulsion energy to repel various kinds of viruses with sizes larger than the mean distances between anchored polymers, including SARS-CoV-2 pseudoparticles. Our strategy can be applied to protect the epithelium from viruses. When these polymers are applied to the epithelium, they localize on the apical surface due to the tight junction barriers, resulting in surface-only coating. Therefore, these polymers can prevent the entry of viruses into cells of the epithelium with minimal disturbance to lateral cell-cell interactions and organizations.

Mechanochemical Regulation of Cell Adhesion by Incorporation of Synthetic Polymers to Plasma Membranes.

Chemical control of cell-cell interactions using synthetic materials is useful for a wide range of biomedical applications. Herein, we report a method to regulate cell adhesion and dispersion by introducing repulsive forces to live cell membranes. To induce repulsion, we tethered amphiphilic polymers, such as cholesterol-modified poly(ethylene glycol) (PEG-CLS), to cell membranes. We found that the repulsive forces introduced by these tethered polymers induced cell detachment from a substrate and allowed cell dispersion in a suspension, modulated the speed of cell migration, and improved the separation of cells from tissues. Our analyses showed that coating the cells with tethered polymers most likely generated two distinct repulsive forces, lateral tension and steric repulsion, on the surface, which were tuned by altering the polymer size and density. We modeled how these two forces are generated in kinetically distinctive manners to explain the various responses of cells to the coating. Collectively, our observations demonstrate mechanochemical regulation of cell adhesion and dispersion by simply adding polymers to cells without genetic manipulation or chemical synthesis in the cells, which may contribute to the optimization of chemical coating strategies to regulate various types of cell-cell interacting systems.

Regulations of T Cell Activation by Membrane and Cytoskeleton.

Among various types of membrane proteins that are regulated by cytoskeleton, the T cell receptor (TCR) greatly benefits from these cellular machineries for its function. The T cell is activated by the ligation of TCR to its target agonist peptide. However, the binding affinity of the two is not very strong, while the T cell needs to discriminate agonist from many nonagonist peptides. Moreover, the strength and duration of the activation signaling need to be tuned for immunological functions. Many years of investigations revealed that dynamic acto-myosin cytoskeletons and plasma membranes in T cells facilitate such regulations by modulating the spatiotemporal distributions of proteins in plasma membranes and by applying mechanical loads on proteins. In these processes, protein dynamics in multiple scales are involved, ranging from collective molecular motions and macroscopic molecular organizations at the cell-cell interface to microscopic changes in distances between receptor and ligand molecules. In this review, details of how cytoskeletons and membranes regulate these processes are discussed, with the emphasis on how all these processes are coordinated to occur within a single cell system.

Cytosolic Transport of Nanoparticles through Pressurized Plasma Membranes for Molecular Delivery and Amplification of Intracellular Fluorescence.

Transporting nanoparticles into live cells is important for drug delivery and other related applications. We found that cells exposed to hypoosmotic pressures can internalize substantial quantities of gold nanoparticles. Importantly, these nanoparticles can circumvent normal intracellular traffic and be transported directly into the cytosol, without the need for surface functionalization. In contrast, nanoparticles endocytosed at physiological osmolality are segregated inside endocytic organelles and are not able to reach the cytosol. Cytosolic internalization was observed for nanoparticles of various sizes and materials, with minimal short- or long-term damage induced by the internalized particles. Thus, our strategy can be used as a delivery platform for a range of applications from therapeutics to medical imaging. As examples, we demonstrated rapid delivery of membrane-impermeable molecules to the cytosol by using nanoparticles as carriers and the use of nanoparticles assembled within the cytosol as plasmonic nanoantenna to enhance intracellular fluorescence. We propose a model for the mechanisms behind nanoparticle internalization through pressurized plasma membranes via the release of lateral pressures. Such characterizations may constitute a foundation for developing new technologies, including nanoparticle-based drug delivery.

Direct Imaging of Carboxymethyl Cellulose-mediated Aggregation of Lactic Acid Bacteria Using Dark-field Microscopy.

The ecological functions of lactic acid bacteria (LAB) have been utilized in human life for food processing and probiotic therapy. Understanding the interaction mechanisms between LAB and food ingredients may help to clarify the fermentation process and physiological functions of LAB in the production of fermented foods made from plant materials and dairy products. However, the interaction mechanisms have yet to be fully clarified. Although laser diffraction was used for measuring the size changes of aggregates caused by the interaction between LAB and food ingredients, aggregate sizes could not be determined because of the precipitation of aggregates and its disruption from stirring. Therefore, a microscopy-based method for directly visualizing their interactions is required. We directly observed aggregation processes of LAB cells mediated by water-soluble polysaccharides, carboxymethyl cellulose (CMC), by dark-filed microscopy (DFM). DFM could visualize CMC-mediated cell aggregation with high contrast in real time, and revealed that the aggregates were formed by repeated collisions of LAB cells in a suspension. This suggests that our method can be used as a useful assay to directly visualize grain formation caused by interactions between LAB cells and various polysaccharides in food ingredients.

Binding of Lipopolysaccharide and Cholesterol-Modified Gelatin on Supported Lipid Bilayers: Effect of Bilayer Area Confinement and Bilayer Edge Tension.

Binding of amphiphilic molecules to supported lipid bilayers (SLBs) often results in lipid fibril extension from the SLBs. Previous studies proposed that amphiphiles with large and flexible hydrophilic regions trigger lipid fibril formation in SLBs by inducing membrane curvature via their hydrophilic regions. However, no experimental studies have verified this mechanism of fibril formation. In this work, we investigated the binding of lipopolysaccharide (LPS) and cholesterol-modified gelatin to SLBs using fluorescence microscopy. SLBs with restricted and unrestricted bilayer areas were employed to identify the mechanism of fibril generation. We show that the main cause of lipid fibril formation is an approximately 20% expansion in the bilayer area rather than increased membrane curvature. The data indicate that bilayer area confinement plays a critical role in morphological changes of SLBs even when bound amphiphilic molecules have a large hydrophilic domain. We also show that bilayer area change after LPS insertion is dependent on the patch shape of the SLB. When an SLB patch consists of a broad bilayer segment connected to a long thin streak, bilayer area expansion mainly occurs within the bilayer streak. The results indicate that LPS insertion causes net lipid flow from the broad bilayer region to the streak area. The differential increase in area is explained by the instability of planar bilayer streaks that originate from the large energetic contribution of line tension arising along the bilayer edge.

Induction of intermembrane adhesion by incorporation of synthetic adhesive molecules into cell membranes.

Modulation of cell adhesion by synthetic materials is useful for a wide range of biomedical applications. Here, we characterized cell adhesion mediated by a semisynthetic molecule, cholesteryl-modified gelatin (chol-gelatin). We found that this hybrid molecule facilitated cell adhesion by connecting two apposed membranes via multiple cholesterol moieties on the gelatin molecules, whereas unmodified gelatin did not bind to cell membranes. Analyses revealed that the rate of the formation of cell adhesions was increased by displaying more cholesterol moieties on the cell membrane. In contrast, the area of the cell adhesion site was unchanged by increasing the number of cholesterol molecules, suggesting that chol-gelatin may suppress cell spreading. Such restriction was not observed in cell adhesion mediated by the mutant of physiological adhesion protein CD2, which lacked its cytoplasmic domain and was unable to connect to cytoplasmic actin filaments, but had a similar affinity for its ligand compared with the chol-gelatin-cell membrane interaction. Further analysis suggested the restriction of cell spreading by chol-gelatin was largely independent of the modulation of the surface force, and thus we hypothesize that the restriction could be in part due to the modulation of cell membrane mechanics by membrane-incorporated chol-gelatin. Our study dissected the two roles of the hybrid molecule in cell adhesion, namely the formation of a molecular connection and the restriction of spreading, and may be useful for designing other novel synthetic agents to modulate various types of cell adhesions.

Phosphatase CD45 both positively and negatively regulates T cell receptor phosphorylation in reconstituted membrane protein clusters.

T cell receptor (TCR) phosphorylation requires the kinase Lck and phosphatase CD45. CD45 activates Lck by dephosphorylating an inhibitory tyrosine of Lck to relieve autoinhibition. However, CD45 also dephosphorylates the TCR, and the spatial exclusion of CD45 from TCR clustering in the plasma membrane appears to attenuate this negative effect of CD45. To further investigate the role of CD45 in signal initiation, we reconstituted membrane TCR clusters in vitro on supported lipid bilayers. Fluorescence microscopy of single clusters showed that incorporation of CD45 enhanced phosphorylation of TCR clusters, but only when Lck co-clustered with TCR. We found that clustered Lck autophosphorylated the inhibitory tyrosine and thus could be activated by CD45, whereas diffusive Lck molecules did not. In the TCR-Lck clusters and at low CD45 density, we speculate that the effect of Lck activation may overcome dephosphorylation of TCR, resulting in a net positive regulation. The CD45 density in physiological TCR clusters is also low because of the exclusion of CD45. Thus, we propose that the spatial organization of TCR/Lck/CD45 in T cell membranes is important not only for modulating the negative role of CD45 but also for creating conditions in which CD45 has a positive role in signal initiation.

Imaging of Fas-FasL membrane microdomains during apoptosis in a reconstituted cell-cell junction.

The Fas death receptor interacts with its ligand FasL and induces apoptosis. The Fas-FasL interaction occurs at the cell-cell interface in vivo, since both proteins are expressed in cell membranes. However, most studies on the Fas signal pathway have been performed in a nonphysiological manner by using soluble molecules (antibody or crosslinked FasL proteins) to stimulate Fas. The Fas-FasL interaction at the cell-cell contact site has only been studied recently, but the information derived from cell-cell interaction studies is still rather limited and not necessarily consistent with the past results obtained by using soluble Fas-stimulatory molecules. Therefore, we develop a novel reconstituted system that mimics the Fas-FasL interaction at cell-cell contact sites for further examination of the physiological Fas-FasL signaling system. By conjugating FasL extracellular domains to planar lipid bilayers, we created a model cell membrane to activate Fas-expressing cells. Using this system, we generated an image of Fas-FasL interactions at the cell-membrane interface at high resolution. We observed that the Fas-FasL interaction between two membranes creates submicron membrane microdomains. Shortly after microdomain formation, the cells exhibit various features of apoptosis. These results suggest that our reconstituted system provides a useful platform to dissect Fas-FasL apoptosis signaling at near physiological conditions.

Altered actin centripetal retrograde flow in physically restricted immunological synapses.

Antigen recognition by T cells involves large scale spatial reorganization of numerous receptor, adhesion, and costimulatory proteins within the T cell-antigen presenting cell (APC) junction. The resulting patterns can be distinctive, and are collectively known as the immunological synapse. Dynamical assembly of cytoskeletal network is believed to play an important role in driving these assembly processes. In one experimental strategy, the APC is replaced with a synthetic supported membrane. An advantage of this configuration is that solid structures patterned onto the underlying substrate can guide immunological synapse assembly into altered patterns. Here, we use mobile anti-CD3epsilon on the spatial-partitioned supported bilayer to ligate and trigger T cell receptor (TCR) in live Jurkat T cells. Simultaneous tracking of both TCR clusters and GFP-actin speckles reveals their dynamic association and individual flow patterns. Actin retrograde flow directs the inward transport of TCR clusters. Flow-based particle tracking algorithms allow us to investigate the velocity distribution of actin flow field across the whole synapse, and centripetal velocity of actin flow decreases as it moves toward the center of synapse. Localized actin flow analysis reveals that, while there is no influence on actin motion from substrate patterns directly, velocity differences of actin are observed over physically trapped TCR clusters. Actin flow regains its velocity immediately after passing through confined TCR clusters. These observations are consistent with a dynamic and dissipative coupling between TCR clusters and viscoelastic actin network.

The coreceptor CD2 uses plasma membrane microdomains to transduce signals in T cells.

The interaction between a T cell and an antigen-presenting cell (APC) can trigger a signaling response that leads to T cell activation. Prior studies have shown that ligation of the T cell receptor (TCR) triggers a signaling cascade that proceeds through the coalescence of TCR and various signaling molecules (e.g., the kinase Lck and adaptor protein LAT [linker for T cell activation]) into microdomains on the plasma membrane. In this study, we investigated another ligand-receptor interaction (CD58-CD2) that facilities T cell activation using a model system consisting of Jurkat T cells interacting with a planar lipid bilayer that mimics an APC. We show that the binding of CD58 to CD2, in the absence of TCR activation, also induces signaling through the actin-dependent coalescence of signaling molecules (including TCR-zeta chain, Lck, and LAT) into microdomains. When simultaneously activated, TCR and CD2 initially colocalize in small microdomains but then partition into separate zones; this spatial segregation may enable the two receptors to enhance signaling synergistically. Our results show that two structurally distinct receptors both induce a rapid spatial reorganization of molecules in the plasma membrane, suggesting a model for how local increases in the concentration of signaling molecules can trigger T cell signaling.

Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells.

T cells interacting with antigen-presenting cells (APCs) form an "immunological synapse" (IS), a bull's-eye pattern composed of a central supramolecular activation cluster enriched with T cell receptors (TCRs) surrounded by a ring of adhesion molecules (a peripheral supramolecular activation cluster). The mechanism responsible for segregating TCR and adhesion molecules remains poorly understood. Here, we show that immortalized Jurkat T cells interacting with a planar lipid bilayer (mimicking an APC) will form an IS, thereby providing an accessible model system for studying the cell biological processes underlying IS formation. We found that an actin-dependent process caused TCR and adhesion proteins to cluster at the cell periphery, but these molecules appeared to segregate from one another at the earliest stages of microdomain formation. The TCR and adhesion microdomains attached to actin and were carried centripetally by retrograde flow. However, only the TCR microdomains penetrated into the actin-depleted cell center, whereas the adhesion microdomains appeared to be unstable without an underlying actin cytoskeleton. Our results reveal that TCR and adhesion molecules spatially partition from one another well before the formation of a mature IS and that differential actin interactions help to shape and maintain the final bull's-eye pattern of the IS.

Hydrodynamic damping of membrane thermal fluctuations near surfaces imaged by fluorescence interference microscopy.

Hydrodynamic coupling between closely apposed lipid bilayer membranes can substantially slow the time scale of thermal fluctuations. These effects could dominate the kinetics of macromolecular interactions at membrane interfaces, and may exert important influences over the interactions between living cells. Here, we study a reconstituted membrane system consisting of two planar bilayers with average separation distances of 30-60 nm. This spacing range is comparable to that of natural intercellular junctions. Using real-time fluorescence interference contrast imaging, spatial and temporal characteristics of the thermal fluctuation spectra are monitored simultaneously. Strong hydrodynamic damping of the fluctuation time scale is observed whereas fluctuation spatial dimensions are unaltered by proximity to another surface. These observations provide experimental confirmation of recent theoretical models.

Synthesis of lipidated green fluorescent protein and its incorporation in supported lipid bilayers.

Herein we report a semisynthetic method of producing membrane-anchored proteins. Ligation of synthetic lipids with designed anchor structures to proteins was performed using native chemical ligation (NCL) of a C-terminal peptide thioester and an N-terminal cysteine lipid. This strategy mimics the natural glycosylphosphatidylinositol (GPI) linkage found in many natural membrane-associated proteins; however, the synthetic method utilizes simple lipid anchors without glycans. Synthetically lipidated recombinant green fluorescent protein (GFP) was shown to be stably anchored to the membrane, and its lateral fluidity was quantitatively characterized by direct fluorescence imaging in supported membranes. Circumventing the steps of purification from native cell membranes, this methodology facilitates the reconstitution of membrane-associated proteins.

Formation and spatio-temporal evolution of periodic structures in lipid bilayers.

Miscibility phase separation in lipid bilayers is widely implicated as an organizing principle in living cell membranes. However, the chemical and physical aspects of how membrane phase separation modulates protein activity remain obscure. Herein, we describe formation of ordered superstructures of coexisting liquid phases in bilayer membranes. Metastable stripe and hexagonal domain lattices are observed, as well as transitions between them. The high degree of order achieved by these methods facilitates statistical analysis of domain spatial distributions and enables measurement of domain interactions. Such long-range ordering principles may exist in more complicated membrane systems.

Structure and dynamics of supported intermembrane junctions.

Supported intermembrane junctions, formed by rupture of giant unilamellar vesicles onto conventional supported lipid membranes, have recently emerged as model systems for the study of biochemical processes at membrane interfaces. Using intermembrane fluorescence resonance energy transfer and optical standing wave fluorescence interferometry, we characterize the nanometer-scale topography of supported intermembrane junctions and find two distinct association states. In one state, the two membranes adhere in close apposition, with intermembrane separations of a few nanometers. In the second state, large intermembrane spacings of approximately 50 nm are maintained by a balance between Helfrich (entropic) repulsion and occasional sites of tight adhesion that pin the two membranes together. Reversible transitions between these two states can be triggered with temperature changes. We further examine the physical properties of membranes in each state using a membrane mixture near its miscibility phase transition temperature. Thermodynamic characteristics of the phase transition and diffusive mobility of individual lipids are comparable. However, collective Brownian motion of phase-separated domains and compositional fluctuations are substantially modulated by intermembrane spacing. The scaling properties of diffusion coefficient with particle size are determined from detailed analysis of domain motion in the different junction types. The results provide experimental verification of a theoretical model for two-dimensional mobility in membranes, which includes frictional coupling across an interstitial water layer.