Elastometry of Complex Fluid Pendant Capsules.

Oil/water interfaces are ubiquitous in nature. Opposing polarities at these interfaces attract surface-active molecules, which can seed complex viscoelastic or even solid interfacial structure. Biorelevant proteins such as hydrophobin, polymers such as PNIPAM, and the asphaltenes in crude oil (CRO) are examples of some systems where such layers can occur. When a pendant drop of CRO is aged in brine, it can form an interfacial elastic membrane of asphaltenes so stiff that it wrinkles and crumples upon retraction. Most of the work studying CRO/brine interfaces focuses on the viscoelastic liquid regime, leaving a wide range of fully solidified, elastic interfaces largely unexplored. In this work, we quantitatively measure elasticity in all phases of drop retraction. In early retraction, the interface shows a fluid viscoelasticity measurable using a Gibbs isotherm or dilatational rheology. Further retraction causes a phase transition to a 2D elastic solid with nonisotropic, nonhomogeneous surface stresses. In this regime, we use new techniques in the elastic membrane theory to fit for the elasticities of these solid capsules. These elastic measurements can help us develop a deeper understanding not only of CRO interfaces but also of the myriad fluid systems with solid interfacial layers.

Membrane Binding of Parkinson's Protein α-Synuclein: Effect of Phosphorylation at Positions 87 and 129 by the S to D Mutation Approach.

Human α-synuclein, a protein relevant in the brain with so-far unknown function, plays an important role in Parkinson's disease. The phosphorylation state of αS was related to the disease, prompting interest in this process. The presumed physiological function and the disease action of αS involves membrane interaction. Here, we study the effect of phosphorylation at positions 87 and 129, mimicked by the mutations S87A, S129A (nonphosphorylated) and S87D, S129D (phosphorylated) on membrane binding. Local binding is detected by spin-label continuous-wave electron paramagnetic resonance. For S87A/D, six positions (27, 56, 63, 69, 76, and 90) are probed; and for S129A/D, three (27, 56, and 69). Binding to large unilamellar vesicles of 100 nm diameter of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in a 1 : 1 composition is not affected by the phosphorylation state of S129. For phosphorylation at S87, local unbinding of αS from the membrane is observed. We speculate that modulating the local membrane affinity by phosphorylation could tune the way αS interacts with different membranes; for example, tuning its membrane fusion activity.

Membrane-Bound Alpha Synuclein Clusters Induce Impaired Lipid Diffusion and Increased Lipid Packing.

The aggregation of membrane-bound α-synuclein (αS) into oligomers and/or amyloid fibrils has been suggested to cause membrane damage in in vitro model phospholipid membrane systems and in vivo. In this study, we investigate how αS interactions that precede the formation of well-defined aggregates influence physical membrane properties. Using three truncated variants of αS with different aggregation propensities and comparable phospholipid membrane binding affinities we show, using fluorescence recovery after photobleaching (FRAP) and fluorescence anisotropy measurements, that formation of αS clusters on supported lipid bilayers (SLBs) impairs lateral lipid diffusion and increases lipid packing beneath the αS clusters. Formation of protein clusters starts immediately after monomer addition. The magnitudes of the changes in effective lipid diffusion and lipid order increase with the protein cluster size. Our results show that the combination of inter-αS and αS-membrane interactions can drive the formation of more ordered lipid domains. Considering the functional involvement of membrane micro-domains in biological membranes, αS-induced domain formation may be relevant for alternative disease mechanisms.

The Impact of N-terminal Acetylation of α-Synuclein on Phospholipid Membrane Binding and Fibril Structure.

Human α-synuclein (αS) has been shown to be N terminally acetylated in its physiological state. This modification is proposed to modulate the function and aggregation of αS into amyloid fibrils. Using bacterially expressed acetylated-αS (NTAc-αS) and endogenous αS (Endo-αS) from human erythrocytes, we show that N-terminal acetylation has little impact on αS binding to anionic membranes and thus likely not relevant for regulating membrane affinity. N-terminal acetylation does have an effect on αS aggregation, resulting in a narrower distribution of the aggregation lag times and rates. 2D-IR spectra show that acetylation changes the secondary structure of αS in fibrils. This difference may arise from the slightly higher helical propensity of acetylated-αS in solution leading to a more homogenous fibril population with different fibril structure than non-acetylated αS. We speculate that N-terminal acetylation imposes conformational restraints on N-terminal residues in αS, thus predisposing αS toward specific interactions with other binding partners or alternatively decrease nonspecific interactions.

Fluorescence Methods for Unraveling Oligomeric Amyloid Intermediates.

Amyloid oligomers are considered to be the relevant toxic species in many amyloid diseases and much research effort has been devoted to fully characterize these oligomers. Despite their importance, oligomers have proven to be difficult to characterize structurally. Information on their aggregation number is scarce, largely because standard techniques struggle to provide reliable results. In this chapter, we present two different methods that reproducibly yield fluorescently labeled α-Synuclein oligomers. We then discuss a new approach, combining single-molecule photobleaching and sub-stoichiometric fluorescent labeling, that we have developed to determine the aggregation number of supramolecular protein assemblies.

Parkinson's Protein α-Synuclein Binds Efficiently and with a Novel Conformation to Two Natural Membrane Mimics.

Binding of human α-Synuclein, a protein associated with Parkinson's disease, to natural membranes is thought to be crucial in relation to its pathological and physiological function. Here the binding of αS to small unilamellar vesicles mimicking the inner mitochondrial and the neuronal plasma membrane is studied in situ by continuous wave and pulsed electron paramagnetic resonance. Local binding information of αS spin labeled by MTSL at positions 56 and 69 respectively shows that also helix 2 (residues 50-100) binds firmly to both membranes. By double electron-electron resonance (DEER) on the mutant spin labeled at positions 27 and 56 (αS 27/56) a new conformation on the membrane is found with a distance of 3.6 nm/ 3.7 nm between residues 27 and 56. In view of the low negative charge density of these membranes, the strong interaction is surprising, emphasizing that function and pathology of αS could involve synaptic vesicles and mitochondria.

Expression and purification of the C-terminal fragments of TRPV5/6 channels.

The transient receptor potential vanniloid 5 and 6 (TRPV5 and TRPV6) Ca(2+)-ion channels are crucial for the regulation of minute-to-minute whole body calcium homeostasis. They act as the gatekeepers of active Ca(2+) reabsorption in kidney and intestine, respectively. In spite of the great progress in the TRP channels characterization, very little is known at the atomic level about their structure and interactions with other proteins. To the major extent it is caused by difficulties in obtaining suitable samples. Here, we report expression and purification of 36 intracellular C-terminal fragments of TRPV5 and TRPV6 channels, for which no structural information is reported thus far. We demonstrate that these proteins contain intrinsically disordered regions and identify fragments suitable for biophysical characterization. By combining bioinformatic predictions and experimental results, we propose several criteria that may aid in designing a scheme for large-scale production of difficult proteins.

Molecular mechanisms of calmodulin action on TRPV5 and modulation by parathyroid hormone.

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry gate for active Ca(2+) reabsorption in the kidney. Ca(2+) influx through TRPV5 induces rapid channel inactivation, preventing excessive Ca(2+) influx. This inactivation is mediated by the last ∼30 residues of the carboxy (C) terminus of the channel. Since the Ca(2+)-sensing protein calmodulin has been implicated in Ca(2+)-dependent regulation of several TRP channels, the potential role of calmodulin in TRPV5 function was investigated. High-resolution nuclear magnetic resonance (NMR) spectroscopy revealed a Ca(2+)-dependent interaction between calmodulin and a C-terminal fragment of TRPV5 (residues 696 to 729) in which one calmodulin binds two TRPV5 C termini. The TRPV5 residues involved in calmodulin binding were mutated to study the functional consequence of releasing calmodulin from the C terminus. The point mutants TRPV5-W702A and TRPV5-R706E, lacking calmodulin binding, displayed a strongly diminished Ca(2+)-dependent inactivation compared to wild-type TRPV5, as demonstrated by patch clamp analysis. Finally, parathyroid hormone (PTH) induced protein kinase A (PKA)-dependent phosphorylation of residue T709, which diminished calmodulin binding to TRPV5 and thereby enhanced channel open probability. The TRPV5-W702A mutant exhibited a significantly increased channel open probability and was not further stimulated by PTH. Thus, calmodulin negatively modulates TRPV5 activity, which is reversed by PTH-mediated channel phosphorylation.

MEIS homeobox genes in neuroblastoma.

The common pediatric tumor neuroblastoma originates from primitive neural crest-derived precursor cells of the peripheral nervous system. Neuroblastoma especially affects very young children, and can already be present at birth. Its early onset and cellular origin predict the involvement of developmental control genes in neuroblastoma etiology. These genes are indispensable for the tight regulation of normal embryonic development but as a consequence cause cancer and congenital diseases upon mutation or aberrant expression. To date however, the connotation of these genes in neuroblastoma pathogenesis is scant. This review recapitulates data on the MEIS homeobox control genes in cancer and focuses on neuroblastoma.

The role of the MEIS homeobox genes in neuroblastoma.

We recently found amplification of the TALE homeobox gene MEIS1 in the IMR32 neuroblastoma cell line. We now demonstrate high-level expression of the MEIS1 and MEIS2 genes, as well as efficient expression of most other TALE family member genes in a panel of neuroblastoma cell lines. Stable transfection of MEIS1-expressing cell lines with cDNA encoding a naturally occurring dominant-negative splice variant of MEIS1 (MEIS1E) yielded clones with impaired cell proliferation, gain of differentiated phenotype, and increased contact inhibition and cell death. This indicated a relevance of MEIS expression for neuroblastoma cell growth and proliferation. We therefore determined the gene expression profiles of several MEIS1E transfectants using serial analysis of gene expression (SAGE). A large number of genes showed differential expression as a result of MEIS1E expression. These include genes involved in developmental signalling pathways, chromatin binding, cell cycle control, proliferation, and apoptosis. The results presented provide important clues for the oncogenic function of MEIS1 in neuroblastoma.