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-CD3ε 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. © 2010 Yu et al.published_or_final_versio

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-ζ 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

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

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 ∼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

Formation and Spatio-Temporal Evolution of Periodic Structures in Lipid

bution functions of the domain lattices. Such analysis is employed to estimate domain interaction forces. The morphology and dynamics of superstructures in lipid bilayers were monitored in giant unilamellar vesicles (GUVs), composed of an equimolar ternary mixture of sphingomyelin, cholesterol, and dioleoylphosphatidylcholine (DOPC). This composition is widely used as a model raft system based on the constituents of the cell membrane detergent insoluble fraction. Cholesterol and sphingolipids interact favorably, creating a liquid-ordered phase in which mobility is reduced relative to the DOPC-enriched phase. Strong adhesion of closed-form GUVs to a silica substrate, coated with a conventional supported lipid bilayer, leads to asymmetric shapes with relatively high surface area-to-volume ratios, similar to shapes observed previously under thermal expansion and osmotic deflation. 10,11 After adhesion, the bound shape gradually transforms to a lowest-energy shape under the adhesio

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

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

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