TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation.

The organization and dynamics of receptors and other molecules in the plasma membrane are not well understood. Here we analyzed the spatio-temporal dynamics of T cell antigen receptor (TCR) complexes and linker for activation of T cells (Lat), a key adaptor molecule in the TCR signaling pathway, in T cell membranes using high-speed photoactivated localization microscopy, dual-color fluorescence cross-correlation spectroscopy and transmission electron microscopy. In quiescent T cells, both molecules existed in separate membrane domains (protein islands), and these domains concatenated after T cell activation. These concatemers were identical to signaling microclusters, a prominent hallmark of T cell activation. This separation versus physical juxtapositioning of receptor domains and domains containing downstream signaling molecules in quiescent versus activated T cells may be a general feature of plasma membrane-associated signal transduction.

A chemical approach to unraveling the biological function of the glycosylphosphatidylinositol anchor.

The glycosylphosphatidylinositol (GPI) anchor is a C-terminal posttranslational modification found on many eukaryotic proteins that reside in the outer leaflet of the cell membrane. The complex and diverse structures of GPI anchors suggest a rich spectrum of biological functions, but few have been confirmed experimentally because of the lack of appropriate techniques that allow for structural perturbation in a cellular context. We previously synthesized a series of GPI anchor analogs with systematic deletions within the glycan core and coupled them to the GFP by a combination of expressed protein ligation and native chemical ligation [Paulick MG, Wise AR, Forstner MB, Groves JT, Bertozzi CR (2007) J Am Chem Soc 129:11543-11550]. Here we investigate the behavior of these GPI-protein analogs in living cells. These modified proteins integrated into the plasma membranes of a variety of mammalian cells and were internalized and directed to recycling endosomes similarly to GFP bearing a native GPI anchor. The GPI-protein analogs also diffused freely in cellular membranes. However, changes in the glycan structure significantly affected membrane mobility, with the loss of monosaccharide units correlating to decreased diffusion. Thus, this cellular system provides a platform for dissecting the contributions of various GPI anchor components to their biological function.

77Se NMR Probes the Protein Environment of Selenomethionine.

Sulfur is critical for the correct structure and proper function of proteins. Yet, lacking a sensitive enough isotope, nuclear magnetic resonance (NMR) experiments are unable to deliver for sulfur in proteins the usual wealth of chemical, dynamic, and structural information. This limitation can be circumvented by substituting sulfur with selenium, which has similar physicochemical properties and minimal impact on protein structures but possesses an NMR compatible isotope (77Se). Here we exploit the sensitivity of 77Se NMR to the nucleus' chemical milieu and use selenomethionine as a probe for its proteinaceous environment. However, such selenium NMR spectra of proteins currently resist a reliable interpretation because systematic connections between variations of system variables and changes in 77Se NMR parameters are still lacking. To start narrowing this knowledge gap, we report here on a biological 77Se magnetic resonance data bank based on a systematically designed library of GB1 variants in which a single selenomethionine was introduced at different locations within the protein. We recorded the resulting isotropic 77Se chemical shifts and relaxation times for six GB1 variants by solution-state 77Se NMR. For four of the GB1 variants we were also able to determine the chemical shift anisotropy tensor of SeM by solid-state 77Se NMR. To enable interpretation of the NMR data, the structures of five of the GB1 variants were solved by X-ray crystallography to a resolution of 1.2 Å, allowing us to unambiguously determine the conformation of the selenomethionine. Finally, we combine our solution- and solid-state NMR data with the structural information to arrive at general insights regarding the execution and interpretation of 77Se NMR experiments that exploit selenomethionine to probe proteins.

Noncovalent cell surface engineering: incorporation of bioactive synthetic glycopolymers into cellular membranes.

The controlled addition of structurally defined components to live cell membranes can facilitate the molecular level analysis of cell surface phenomena. Here we demonstrate that cell surfaces can be engineered to display synthetic bioactive polymers at defined densities by exogenous membrane insertion. The polymers were designed to mimic native cell-surface mucin glycoproteins, which are defined by their dense glycosylation patterns and rod-like structures. End-functionalization with a hydrophobic anchor permitted incorporation into the membranes of live cultured cells. We probed the dynamic behavior of cell-bound glycopolymers bearing various hydrophobic anchors and glycan structures using fluorescence correlation spectroscopy (FCS). Their diffusion properties mirrored those of many natural membrane-associated biomolecules. Furthermore, the membrane-bound glycopolymers were internalized into early endosomes similarly to endogenous membrane components and were capable of specific interactions with protein receptors. This system provides a platform to study cell-surface phenomena with a degree of chemical control that cannot be achieved using conventional biological tools.

Attractive membrane domains control lateral diffusion.

Lipid membranes play a fundamental role in vital cellular functions such as signal transduction. Many of these processes rely on lateral diffusion within the membrane, generally a complex fluid containing ordered microdomains. However, little attention has been paid to the alterations in transport dynamics of a diffusing species caused by long-range interactions with membrane domains. In this paper, we address the effect of such interactions on diffusive transport by studying lateral diffusion in a phase-separated Langmuir phospholipid monolayer via single-particle tracking. We find that attractive dipole-dipole interactions between condensed phase domains and diffusing probe beads lead to transient confinement at the phase boundaries, causing a transition from two- to one-dimensional diffusion. Using Brownian dynamics simulations, the long-term diffusion constant for such a system is found to have a sensitive, Boltzmann-like, dependence on the interaction strength. In addition, this interaction strength is shown to be a strong function of the ratio of domain to particle size. As similar interactions are expected in biological membranes, the modulation of diffusive transport dynamics by varying interaction strength and/or domain size may offer cells selective spatial and temporal control over signaling processes.

Investigation of the cytokine response to NF-κB decoy oligonucleotide coated polysaccharide based nanoparticles in rheumatoid arthritis in vitro models.

INTRODUCTION: The transcription factor nuclear factor-kappa B (NF-κB) is highly involved in regulation of a number of cellular processes, including production of inflammatory mediators. Thus, this transcription factor plays a role in pathology of many diseases, including rheumatoid arthritis, an autoimmune disease hallmarked by an imbalance of pro and anti-inflammatory cytokines. Small nucleic acids with sequences that mimic the native binding site of NF-κB have been proposed as treatment options for RA; however due to low cellular penetration and a high degree of instability, clinical applications of these therapeutics have been limited. METHODS: Here, we describe the use of N-trimethyl chitosan-polysialic acid (PSA-TMC) nanoparticles coated with decoy oligodeoxynucleotides (ODNs) specific to transcription factor NF-κB (PSA-TMC-ODN) as a method to enhance the stability of the nucleic acids and facilitate increased cellular penetration. In addition to decoy ODN, PSA-TMC nanoparticles were loaded with RA therapeutic methotrexate (MTX), to assess the anti-inflammatory efficacy of a combination therapy approach. Two different in vitro models, a cell line based model as well as a primary RA cell model were used to investigate anti-inflammatory activity. One way ANOVA followed by Holm-Sidak stepdown comparisons was used to determine statistical significance. RESULTS: In general, free ODN did not significantly affect secretion of pro-inflammatory cytokines interleukin-6 (IL-6) and interleukin-8, (IL-8) while free MTX had variable efficacy. However, PSA-TMC-ODN and PSA-TMC-ODN-MTX resulted in significant decreases in the inflammatory mediators IL-6 and IL-8 in both cell models. In addition, PSA-TMC exhibited sufficient cellular uptake, as observed through fluorescence microscopy. CONCLUSIONS: These results support our previous findings that PSA-TMC nanoparticles are an effective delivery vehicle for small nucleic acids, and effectively alter the pro-inflammatory state characteristic of RA.

Synthesis and evaluation of cyclosporine A-loaded polysialic acid-polycaprolactone micelles for rheumatoid arthritis.

Polysialic acid (PSA) has been identified as a natural, hydrophilic polymer that can be used to extend circulation time and improve therapeutic efficacy when used as the basis of drug carrier systems. Here, to further investigate the potential of PSA to alter the pharmacokinetic and pharmacodynamic profiles of associated therapeutics, PSA-based micelles were formed via self-assembly of PSA grafted with polycaprolactone (PCL) at a critical micelle concentration of 84.7±13.2 µg/ml. Cyclosporine A (CyA), a therapeutic used in the treatment of rheumatoid arthritis, was loaded into the PSA-PCL micelles with a loading capacity and loading efficiency of 0.09±0.02 mg CyA/mg PSA-PCL and 29.3±6.4%, respectively. CyA loading resulted in a size increase from 73.8±12.4 nm to 107.5±9.3 nm at 25 °C and from 138.4±40.7 nm to 195.3±52.1 nm at 37 °C, favorable size ranges for drug delivery to inflamed tissue characterized by leaky vasculature, as occurs during rheumatoid arthritis pathogenesis. As an indicator of the stealth nature the micelles are expected to exhibit in vivo, the fixed aqueous layer thickness of the PSA-PCL micelles was determined to be 0.63±0.02 nm, comparable to that obtained for traditionally utilized poly(ethylene glycol) coated liposomes. The PSA-PCL micelles had a negligible effect on the viability of the SW982 synovial fibroblast cell line. Fluorescent microscopy was utilized to demonstrate uptake by the synovial fibroblasts through a non-receptor mediated form of endocytosis and partitioning of CyA into the membrane.

Single molecule kinetics of ENTH binding to lipid membranes.

Transient recruitment of proteins to membranes is a fundamental mechanism by which the cell exerts spatial and temporal control over proteins' localization and interactions. Thus, the specificity and the kinetics of peripheral proteins' membrane residence are an attribute of their function. Here, we describe the membrane interactions of the interfacial epsin N-terminal homology (ENTH) domain with its target lipid phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)). The direct visualization and quantification of interactions of single ENTH molecules with supported lipid bilayers is achieved using total internal reflection fluorescence microscopy (TIRFM) with a time resolution of 13 ms. This enables the recording of the kinetic behavior of ENTH interacting with membranes with physiologically relevant concentrations of PtdIns(4,5)P(2) despite the low effective binding affinity. Subsequent single fluorophore tracking permits us to build up distributions of residence times and to measure ENTH dissociation rates as a function of membrane composition. Furthermore, due to the high time resolution, we are able to resolve details of the motion of ENTH associated with a simple, homogeneous membrane. In this case ENTH's diffusive transport appears to be the result of at least three different diffusion processes.

Fluorescence imaging of membrane dynamics.

Imaging membrane dynamics is an important goal, motivated by the abundance of biochemical and biophysical events that are orchestrated at, or by, cellular membranes. The short length scales, fast timescales, and environmental requirements of membrane phenomena present challenges to imaging experiments. Several technical advances offer means to overcome these challenges, and we describe here three powerful techniques applicable to membrane imaging: total internal reflection fluorescence (TIRF) microscopy, fluorescence interference contrast (FLIC) microscopy, and fluorescence correlation spectroscopy (FCS). For each, we discuss the physics underpinning the approach, its practical implementation, and recent examples highlighting its achievements in exploring the membrane environment.

Synthetic analogues of glycosylphosphatidylinositol-anchored proteins and their behavior in supported lipid bilayers.

Positioned at the C-terminus of many eukaryotic proteins, the glycosylphosphatidylinositol (GPI) anchor is a posttranslational modification that anchors the modified proteins in the outer leaflet of the plasma membrane. GPI-anchored proteins play vital roles in signal transduction, the vertebrate immune response, and the pathobiology of trypanosomal parasites. While many GPI-anchored proteins have been characterized, the biological functions of the GPI anchor have yet to be elucidated at a molecular level. We synthesized a series of GPI-protein analogues bearing modified anchor structures that were designed to dissect the contribution of various glycan components to the GPI-protein's membrane behavior. These anchor analogues were similar in length to native GPI anchors and included mimics of the native structure's three domains. A combination of expressed protein ligation and native chemical ligation was used to attach these analogues to the green fluorescent protein (GFP). These modified GFPs were incorporated in supported lipid bilayers, and their mobilities were analyzed using fluorescence correlation spectroscopy. The data from these experiments suggest that the GPI anchor is more than a simple membrane-anchoring device; it also may prevent transient interactions between the attached protein and the underlying lipid bilayer, thereby permitting rapid diffusion in the bilayer. The ability to generate chemically defined analogues of GPI-anchored proteins is an important step toward elucidating the molecular functions of this interesting post-translational modification.

Lipid lateral mobility and membrane phase structure modulation by protein binding.

Using a combination of fluorescence correlation and infrared absorption spectroscopies, we characterize lipid lateral diffusion and membrane phase structure as a function of protein binding to the membrane surface. In a supported membrane configuration, cholera toxin binding to the pentasaccharaide headgroup of membrane-incorporated GM1 lipid alters the long-range lateral diffusion of fluorescently labeled probe lipids, which are not involved in the binding interaction. This effect is prominently amplified near the gel-fluid transition temperature, Tm, of the majority lipid component. At temperatures near Tm, large changes in probe lipid diffusion are measured at average protein coverage densities as low as 0.02 area fraction. Spectral shifts of the methylene symmetric and asymmetric stretching modes in the lipid acyl chain confirm that protein binding alters the fraction of lipid in the gel phase.

Apparent subdiffusion inherent to single particle tracking.

Subdiffusion and its causes in both in vivo and in vitro lipid membranes have become the focus of recent research. We report apparent subdiffusion, observed via single particle tracking (SPT), in a homogeneous system that only allows normal diffusion (a DMPC monolayer in the fluid state). The apparent subdiffusion arises from slight errors in finding the actual particle position due to noise inherent in all experimental SPT systems. A model is presented that corrects this artifact, and predicts the time scales after which the effect becomes negligible. The techniques and results presented in this paper should be of use in all SPT experiments studying normal and anomalous diffusion.