Glycosphingolipids in microdomain formation and their spatial organization

Plasma membranes regulate the influx and efflux of molecules across themselves and are also responsible for primary signal transduction between cells or within the same cell. Presence of lateral heterogeneity and the ability of reorganization are essential requirements for effective functioning of biomembranes. Lipid rafts are small, heterogeneous, dynamic domains enriched in glycosphingolipids, sphingomyelin and cholesterol, and profoundly influence membrane organization. Glycosphingolipids are inclined towards formation of liquid-ordered phases in membranes, both with and without cholesterol; they are therefore prime players in domain formation. Here, we discuss the role of glycosphingolipids in microdomain formation and their spatial organization within these rafts.

Constitutive plasma membrane targeting and microdomain localization of Dok5 studied by single-molecule microscopy

In the present study, single-molecule fluorescence microscopy was used to examine the characteristics of plasma membrane targeting and microdomain localization of enhanced yellow fluorescent protein (eYFP)-tagged wild-type Dok5 and its variants in living Chinese hamster ovary (CHO) cells. We found that Dok5 can target constitutively to the plasma membrane, and the PH domain is essential for this process. Furthermore, single-molecule trajectories analysis revealed that Dok5 can constitutively partition into microdomain on the plasma membrane. Finally, the potential mechanism of microdomain localization of Dok5 was discussed. This study provided insights into the characteristics of plasma membrane targeting and microdomain localization of Dok5 in living CHO cells. (C) 2008 Elsevier B.V. All rights reserved.

Square lamellar structure having phase-separated microdomain in H-shaped block copolymer thin film

The morphology of a H-shaped block copolymer (poly(ethylene glycol) backbone and polystyrene branches (PS)(2)PEG(PS)(2)) in a thin film has been investigated. A peculiar square lamella that has a phase-separated microdomain at its surface is obtained after spin coating. The experimental temperature plays a critical role in the lamellar formation. The copolymer first self-assembles into square lamellar micelles with an incomplete crystalline core due to the crystallizability of PEG.

N-Terminal microdomain peptide from human dihydroorotate dehydrogenase: structure and model membrane interactions

The N-terminus of the human dihydroorotate dehydrogenase (HsDHODH) has been described as important for the enzyme attachment in the inner mitochondrial membrane and possibly to regulate enzymatic activity. In this study, we synthesized the peptide acetyl-GDERFYAEHLMPTLQGLLDPESAHRL AVRFTSLGamide, comprising the residues 33-66 of HsDHODH N-terminal conserved microdomain. Langmuir monolayers and circular dichroism (CD) were employed to investigate the interactions between the peptide and membrane model, as micelles and monolayers of the lipids phosphatidylcholine (PC), 3-phosphatidylethanolamine (PE) and cardiolipin (CL). These lipids represent the major constituents of inner mitochondrial membranes. According to CD data, the peptide adopted a random structure in water, whereas it acquired α-helical structures in the presence of micelles. The π–A isotherms and polarization- modulated infrared reflection-absorption spectroscopy on monolayers showed that the peptide interacted with all lipids, but in different ways. In DPPC monolayers, the peptide penetrated into the hydrophobic region. The strongest initial interaction occurred with DPPE, but the peptide was expelled from this monolayer at high surface pressures. In CL, the peptide could induce a partial dissolution of the monolayer, leading to shorter areas at the monolayer collapse. These results corroborate the literature, where the HsDHODH microdomain is anchored into the inner mitochondrial membrane. Moreover, the existence of distinct conformations and interactions with the different membrane lipids indicates that the access to the enzyme active site may be controlled not only by conformational changes occurring at the microdomain of the protein, but also by some lipid-protein synergetic mechanism, where the HsDHODH peptide would be able to recognize lipid domains in the membrane. - See more at: http://www.eurekaselect.com/122062/article#sthash.1ZZbc7E0.dpuf

Conformational changes of the HsDHODH N-terminal microdomain via DEER spectroscopy

The human enzyme dihydroorotate dehydrogenase (HsDHODH) has been studied for being a target for development of new antineoplasic and antiproliferative drugs. The synthetic peptide N-t(DH) represents the N-terminal microdomain of this enzyme, responsible for anchoring it to the inner mitochondrial membrane. Also, it is known to harbor quinones that are essential for enzyme catalysis. Here we report structural features of the peptide/membrane interactions obtained by using CD and DEER spectroscopic techniques, both in micelles and in lipid vesicles. The data revealed different peptide conformational states in micelles and liposomes, which could suggest that this microdomain acts in specific regions or areas of the mitochondria, which can be related with the control of the quinone access to the HsDHODH active site. This is the first study to report on conformational changes of the HsDHODH N-terminal microdomain through a combination of CD and DEER spectroscopic techniques.

Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.

Identifying optimal lipid raft characteristics required to promote nanoscale protein-protein interactions on the plasma membrane

The dynamic lateral segregation of signaling proteins into microdomains is proposed to facilitate signal transduction, but the constraints on microdomain size, mobility, and diffusion that might realize this function are undefined. Here we interrogate a stochastic spatial model of the plasma membrane to determine how microdomains affect protein dynamics. Taking lipid rafts as representative microdomains, we show that reduced protein mobility in rafts segregates dynamically partitioning proteins, but the equilibrium concentration is largely independent of raft size and mobility. Rafts weakly impede small-scale protein diffusion but more strongly impede long-range protein mobility. The long-range mobility of raft-partitioning and raft-excluded proteins, however, is reduced to a similar extent. Dynamic partitioning into rafts increases specific interprotein collision rates, but to maximize this critical, biologically relevant function, rafts must be small (diameter, 6 to 14 nm) and mobile. Intermolecular collisions can also be favored by the selective capture and exclusion of proteins by rafts, although this mechanism is generally less efficient than simple dynamic partitioning. Generalizing these results, we conclude that microdomains can readily operate as protein concentrators or isolators but there appear to be significant constraints on size and mobility if microdomains are also required to function as reaction chambers that facilitate nanoscale protein-protein interactions. These results may have significant implications for the many signaling cascades that are scaffolded or assembled in plasma membrane microdomains.

CD151 is associated with prostate cancer cell invasion and lymphangiogenesis in vivo

CD151, a member of the tetraspanin family, is associated with regulation of migration of normal and tumour cells via cell surface microdomain formation. CD151 was found in our laboratory to have a prognostic value in prostate cancer and is a promoter of prostate cancer migration and invasion. These roles involve association with integrins on both cell-cell and cell-stroma levels. Furthermore, CD151 plays a role in endothelial cell motility. CD151 expression was examined in three commonly used prostate cancer cell lines. We investigated CD151 expression, angiogenesis (microvessel density; MVD) and lymphangiogenesis (lymphatic vessel density; LVD) in an orthotopic xenograft model of prostate cancer in matched tumours from primary and secondary sites. CD151 was found to be heterogeneously expressed across different prostate cancer cell lines and the levels of CD151 expression were significantly higher in the highly tumorigenic, androgen-insensitive cells PC-3 and DU-145 compared to the androgen-sensitive cell line LNCaP (P<0.05). The majority of in vivo xenografts developed pelvic lymph node metastases. Importantly, primary tumours that developed metastasis had significantly higher CD151 expression and MVD compared to those which did not develop metastasis (P<0.05). We identified, for the first time, that CD151 expression is associated with LVD in prostate cancer. These findings underscore the potential role of CD151 and angiogenesis in the metastatic potential of prostate cancer. CD151 has a prognostic value in this mouse model of prostate cancer and may play a role in lymphangiogenesis. CD151 is likely an important regulator of cancer cell communication with the surrounding microenvironment.

Modeling growth of hydrogen bubbles in aluminum castings using the level-set method

A new approach is proposed to solve for the growth as well as the movement of hydrogen bubbles during solidification in aluminum castings. A level-set methodology has been adopted to handle this multiphase phenomenon. A microscale domain is considered and the growth and movement of hydrogen bubbles in this domain has been studied. The growth characteristics of hydrogen bubbles have been evaluated under free growth conditions in a melt having a hydrogen input caused b solidification occurring around the microdomain.

Characterization of the mitochondrial active-site serine protein LACTB: filaments in the mitochondrial intermembrane space

Mitochondria have evolved from endosymbiotic alpha-proteobacteria. During the endosymbiotic process early eukaryotes dumped the major component of the bacterial cell wall, the peptidoglycan layer. Peptidoglycan is synthesized and maintained by active-site serine enzymes belonging to the penicillin-binding protein and the β-lactamase superfamily. Mammals harbor a protein named LACTB that shares sequence similarity with bacterial penicillin-binding proteins and β-lactamases. Since eukaryotes lack the synthesis machinery for peptidoglycan, the physiological role of LACTB is intriguing. Recently, LACTB has been validated in vivo to be causative for obesity, suggesting that LACTB is implicated in metabolic processes. The aim of this study was to investigate the phylogeny, structure, biochemistry and cell biology of LACTB in order to elucidate its physiological function. Phylogenetic analysis revealed that LACTB has evolved from penicillin binding-proteins present in the bacterial periplasmic space. A structural model of LACTB indicates that LACTB shares characteristic features common to all penicillin-binding proteins and β-lactamases. Recombinat LACTB protein expressed in E. coli was recovered in significant quantities. Biochemical and cell biology studies showed that LACTB is a soluble protein localized in the mitochondrial intermembrane space. Further analysis showed that LACTB preprotein underwent proteolytic processing disclosing an N-terminal tetrapeptide motif also found in a set of cell death-inducing proteins. Electron microscopy structural studies revealed that LACTB can polymerize to form stable filaments with lengths ranging from twenty to several hundred nanometers. These data suggest that LACTB filaments define a distinct microdomain in the intermembrane space. A possible role of LACTB filaments is proposed in the intramitochondrial membrane organization and microcompartmentation. The implications of these findings offer novel insight into the evolution of mitochondria. Further studies of the LACTB function might provide a tool to treat mitochondria-related metabolic diseases.

Mathematical models on the impact of noise and dyadic molecular structures on the properties of a cardiac myocyte

In cardiac myocytes (heart muscle cells), coupling of electric signal known as the action potential to contraction of the heart depends crucially on calcium-induced calcium release (CICR) in a microdomain known as the dyad. During CICR, the peak number of free calcium ions (Ca) present in the dyad is small, typically estimated to be within range 1-100. Since the free Ca ions mediate CICR, noise in Ca signaling due to the small number of free calcium ions influences Excitation-Contraction (EC) coupling gain. Noise in Ca signaling is only one noise type influencing cardiac myocytes, e.g., ion channels playing a central role in action potential propagation are stochastic machines, each of which gates more or less randomly, which produces gating noise present in membrane currents. How various noise sources influence macroscopic properties of a myocyte, how noise is attenuated and taken advantage of are largely open questions. In this thesis, the impact of noise on CICR, EC coupling and, more generally, macroscopic properties of a cardiac myocyte is investigated at multiple levels of detail using mathematical models. Complementarily to the investigation of the impact of noise on CICR, computationally-efficient yet spatially-detailed models of CICR are developed. The results of this thesis show that (1) gating noise due to the high-activity mode of L-type calcium channels playing a major role in CICR may induce early after-depolarizations associated with polymorphic tachycardia, which is a frequent precursor to sudden cardiac death in heart failure patients; (2) an increased level of voltage noise typically increases action potential duration and it skews distribution of action potential durations toward long durations in cardiac myocytes; and that (3) while a small number of Ca ions mediate CICR, Excitation-Contraction coupling is robust against this noise source, partly due to the shape of ryanodine receptor protein structures present in the cardiac dyad.

Optimization of rheological properties of photopolymerizable alumina suspensions for ceramic microstereolithography

Microstereolithography (MSL) is a rapid prototyping technique to fabricate complex three-dimensional (3D) structure in the microdomain involving different materials such as polymers and ceramics. The present effort is to fabricate microdimensional ceramics by the MSL system from a non-aqueous colloidal slurry of alumina. This slurry predominantly consists of two phases i.e. sub-micrometer solid alumina particles and non-aqueous reactive difunctional and trifunctional acrylates with inert diluent. The first part of the work involves the study of the stability and viscosity of the slurry using different concentrations of trioctyl phosphine oxide (TOPO) as a dispersant. Based on the optimization, the highest achievable solid loadings of alumina has been determined for this particular colloidal suspension. The second part of the study highlights the fabrication of several micro-dimensional alumina structures by the MSL system. (C) 2013 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

Functional evaluation of noncovalent interactions in neuroreceptors and progress toward the expansion of unnatural amino acid methodology

This dissertation primarily describes chemical-scale studies of G protein-coupled receptors and Cys-loop ligand-gated ion channels to better understand ligand binding interactions and the mechanism of channel activation using recently published crystal structures as a guide. These studies employ the use of unnatural amino acid mutagenesis and electrophysiology to measure subtle changes in receptor function.

In chapter 2, the role of a conserved aromatic microdomain predicted in the D3 dopamine receptor is probed in the closely related D2 and D4 dopamine receptors. This domain was found to act as a structural unit near the ligand binding site that is important for receptor function. The domain consists of several functionally important noncovalent interactions including hydrogen bond, aromatic-aromatic, and sulfur-π interactions that show strong couplings by mutant cycle analysis. We also assign an alternate interpretation for the linear fluorination plot observed at W6.48, a residue previously thought to participate in a cation-π interaction with dopamine.

Chapter 3 outlines attempts to incorporate chemically synthesized and in vitro acylated unnatural amino acids into mammalian cells. While our attempts were not successful, method optimizations and data for nonsense suppression with an in vivo acylated tRNA are included. This chapter is aimed to aid future researchers attempting unnatural amino acid mutagenesis in mammalian cells.

Chapter 4 identifies a cation-π interaction between glutamate and a tyrosine residue on loop C in the GluClβ receptor. Using the recently published crystal structure of the homologous GluClα receptor, other ligand-binding and protein-protein interactions are probed to determine the similarity between this invertebrate receptor and other more distantly related vertebrate Cys-loop receptors. We find that many of the interactions previously observed are conserved in the GluCl receptors, however care must be taken when extrapolating structural data.

Chapter 5 examines inherent properties of the GluClα receptor that are responsible for the observed glutamate insensitivity of the receptor. Chimera synthesis and mutagenesis reveal the C-terminal portion of the M4 helix and the C-terminus as contributing to formation of the decoupled state, where ligand binding is incapable of triggering channel gating. Receptor mutagenesis was unable to identify single residue mismatches or impaired protein-protein interactions within this domain. We conclude that M4 helix structure and/or membrane dynamics are likely the cause of ligand insensitivity in this receptor and that the M4 helix has an role important in the activation process.