Synthesis and vesicle formation from hybrid bolaphile/amphiphile ion-pairs. Evidence of membrane property modulation by molecular design

Four new hybrid (bolaphile/amphiphile) ion-pairs were synthesized. Electron microscopy indicated that each of these forms bilayer membranes upon dispersion in aqueous media. Membrane properties have also been examined by differential scanning calorimetry, microcalorimetry, temperature-dependent fluorescence anisotropy measurements, and UV-vis spectroscopy. The T-m values for the vesicular 1, 2, 3, 4, and 5 were 38, 12, 85, 31.3, and 41.6 degrees C, respectively. Interestingly the T-m values for 1 and 3 were found to depend on their concentration. The entrapment of small solute and the release capability have also been examined to demonstrate that these bilayers form enclosed vesicles. X-ray diffraction of the cast films has been performed to understand the nature and the thickness of these membrane organizations. The membrane widths ranged from 33 to 47 Angstrom. Finally, the above observations have been analyzed in light of the results obtained from molecular modeling studies. Thus we have demonstrated that membrane properties can be modulated by simple structural changes at the amphiphile level. It was shown that by judicious incorporation of central, isomeric, disubstituted aromatic units as structural anchors into different bolaphiles, one can modulate the properties of the resulting vesicles.

Vesicle formation from dimeric surfactants through ion-pairing. Adjustment of polar headgroup separation leads to control over vesicular thermotropic properties

Eight new vesicle-forming dimeric surfactants are synthesized: the polar headgroup separation in such dimeric amphiphiles strongly influences their vesicular thermotropic phase-transition behaviour.

Synthesis and vesicle formation from novel pseudoglyceryl dimeric lipids. Evidence of formation of widely different membrane organizations with exceptional thermotropic properties

Eight new bis-cationic dimeric lipids 2a-h have been synthesized; TEM of their aqueous dispersions confirmed the vesicle formation and from the thermal, spectroscopic, DLS and XRD studies it has been revealed that they form three different kinds of membranous aggregate depending on the m-value.

Synthesis and vesicle formation from dimeric pseudoglyceryl lipids with (CH2)(m) spacers: pronounced m-value dependence of thermal properties, vesicle fusion, and cholesterol complexation

Eight new dimeric lipids, in which the two Me2N+ ion headgroups are separated by a variable number of polymethylene units [-(CH2)(m)-], have been synthesized. The electron micrograph (TEM) and dynamic light scattering (DLS) of their aqueous dispersions confirmed the formation of vesicular-type aggregates. The vesicle sizes and morphologies were found to depend strongly on the m value, the method, and thermal history of the vesicle preparation. Information on the thermotropic properties of the resulting vesicles was obtained from microcalorimetry and temperature-dependent fluorescence anisotropy measurements. Interestingly, the T-m values for these vesicles revealed a nonlinear dependence on spacer chain length (m value). These vesicles were able to entrap riboflavin. The rates of permeation of the OH- ion under an imposed transmembrane pH gradient were also found to depend significantly on the m value. X-Ray diffraction of the cast films of the lipid dispersions elucidated the nature and the thickness of these membrane organizations, and it was revealed that these lipids organize in three different ways depending on the m value. The EPR spin-probe method with the doxylstearic acids 5NS, 12NS, and 16NS, spin-labeled at various positions of stearic acid, was used to establish, the chain-flexibility gradient and homogeneity of these bilayer assemblies. The apparent fusogenic propensities of these bipolar tetraether lipids were investigated in the presence of Na2SO4 with fluorescence-resonance energy-transfer fusion assay. Small unilamellar vesicles formed from 1 and three representative biscationic lipids were also studied with fluorescence anisotropy and H-1 NMR spectroscopic techniques in the absence and the presence of varying amounts of cholesterol.

Vesicle formation from dimeric ion-paired amphiphiles. Control over vesicular thermotropic and ion-transport properties as a function of intra-amphiphilic headgroup separation

A novel series of vesicle-forming ion-paired amphiphiles, bis(hexadecyldimethylammonium)alkane dipalmitate (1a-1h), containing four chains were synthesized with two isolated headgroups. In each of these amphiphiles, the two headgroup charges are separated by a flexible polymethylene spacer chain -[(CH2)(m)]- of varying lengths (m) such that the length and the conformation of the spacer chain determine the intra-"monomer" headgroup separation. Transmission electron microscopy indicated that each of these forms bilayer membranes upon dispersion in aqueous media. The vesicular properties of these aggregates have been examined by differential scanning calorimetry and temperature-dependent fluorescence anisotropy measurements. Interestingly, their T-m values decreased with the increase in the m value. Thus while the apparent T-m of the lipid with m = 2 (1a) is 74.1 degrees C, the corresponding value observed for the lipid with m = 12 (1h) is 38.9 degrees C. The fluorescence anisotropy values (r) for 1b-1g were quite high (r similar to 0.3) compared to that of 1h (r similar to 0.23) at 20-30 degrees C in their gel states. On the other hand, the r value for vesicular 1b beyond melting was higher (0.1) compared to any of those for 1c-1h (similar to 0.04-0.06). X-ray diffraction of the cast films was performed to understand the nature and the thickness of these membrane organizations. The membrane widths ranged from 30 to 51 A as the m values varied. The entrapment of a small water-soluble solute, riboflavin, by the individual vesicular aggregates, and their sustenance: under an imposed transmembrane pH gradient have also been examined. These results show that all lipid vesicles entrap riboflavin and that generally the resistance to OH- permeation decreases with the increase in m value. Finally,all the above observations were comparatively analyzed, and on the basis of the calculated structures of these lipids, it was possible to conclude that membrane propel-ties can be modulated by spacer chain length variation of the ion-paired amphiphiles.

ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation.

Examining how key components of coat protein I (COPI) transport participate in cargo sorting, we find that, instead of ADP ribosylation factor 1 (ARF1), its GTPase-activating protein (GAP) plays a direct role in promoting the binding of cargo proteins by coatomer (the core COPI complex). Activated ARF1 binds selectively to SNARE cargo proteins, with this binding likely to represent at least a mechanism by which activated ARF1 is stabilized on Golgi membrane to propagate its effector functions. We also find that the GAP catalytic activity plays a critical role in the formation of COPI vesicles from Golgi membrane, in contrast to the prevailing view that this activity antagonizes vesicle formation. Together, these findings indicate that GAP plays a central role in coupling cargo sorting and vesicle formation, with implications for simplifying models to describe how these two processes are coupled during COPI transport.

Simple models of complex aggregation: Vesicle formation by soft repulsive spheres with dipolelike interactions

Structural and thermodynamic properties of spherical particles carrying classical spins are investigated by Monte Carlo simulations. The potential energy is the sum of short range, purely repulsive pair contributions, and spin-spin interactions. These last are of the dipole-dipole form, with however, a crucial change of sign. At low density and high temperature the system is a homogeneous fluid of weakly interacting particles and short range spin correlations. With decreasing temperature particles condense into an equilibrium population of free floating vesicles. The comparison with the electrostatic case, giving rise to predominantly one-dimensional aggregates under similar conditions, is discussed. In both cases condensation is a continuous transformation, provided the isotropic part of the interatomic potential is purely repulsive. At low temperature the model allows us to investigate thermal and mechanical properties of membranes. At intermediate temperatures it provides a simple model to investigate equilibrium polymerization in a system giving rise to predominantly two-dimensional aggregates.

A Rab2 Mutant with Impaired GTPase Activity Stimulates Vesicle Formation from Pre-Golgi Intermediates

Rab2 immunolocalizes to pre-Golgi intermediates (vesicular-tubular clusters [VTCs]) that are the first site of segregation of anterograde- and retrograde-transported proteins and a major peripheral site for COPI recruitment. Our previous work showed that Rab2 Q65L (equivalent to Ras Q61L) inhibited endoplasmic reticulum (ER)-to-Golgi transport in vivo. In this study, the biochemical properties of Rab2 Q65L were analyzed. The mutant protein binds GDP and GTP and has a low GTP hydrolysis rate that suggests that Rab2 Q65L is predominantly in the GTP-bound–activated form. The purified protein arrests vesicular stomatitis virus glycoprotein transport from VTCs in an assay that reconstitutes ER-to-Golgi traffic. A quantitative binding assay was used to measure membrane binding of β-COP when incubated with the mutant. Unlike Rab2 that stimulates recruitment, Rab2 Q65L showed a dose-dependent decrease in membrane-associated β-COP when incubated with rapidly sedimenting membranes (ER, pre-Golgi, and Golgi). The mutant protein does not interfere with β-COP binding but stimulates the release of slowly sedimenting vesicles containing Rab2, β-COP, and p53/gp58 but lacking anterograde grade-directed cargo. To complement the biochemical results, we observed in a morphological assay that Rab2 Q65L caused vesiculation of VTCs that accumulated at 15°C. These data suggest that the Rab2 protein plays a role in the low-temperature–sensitive step that regulates membrane flow from VTCs to the Golgi complex and back to the ER.

Effect of Shear Flow on the Formation of Ring-Shaped ABA Amphiphilic Triblock Copolymer Micelles

We have investigated the effect of Shear flow on the formation of rill.-shaped ABA triblock copolymer (P4VP(43)-b-PS260-b-P4VP(43)) micelles. The results reveal that Shear flow Plays an important role in the formation of the rings Both ring size and its, distribution are found to be dependent sensitively on the stirring rate. Sizable rings are more likely to be formed at moderate stirring rate, Interestingly, the ring formation mechanism is also dependent oil the Shear flow. Copolymers are likely to form rings via end-to-end cylinder connection at low stirring rates, whereas they tend to form rings via the pathway of the rod-sphere-vesicle-ring it high stirring rates.

ARFGAP1 promotes the formation of COPI vesicles, suggesting function as a component of the coat.

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPgammaS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.

Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.

Expressions- und Funktionsanalysen von "Tetraspan Vesicle Membrane Proteins" in Caenorhabditis elegans

Tetraspan vesicle membrane proteins (TVPs) sind ubiquitäre Komponenten von Transportvesikeln. Bei den Säugetieren unterscheidet man drei Familien, die Physine, Gyrine und SCAMPs (secretory carrier-associated membrane proteins). Ihre Funktion ist weitgehend unbekannt, es wird jedoch vermutet, dass sie eine Rolle bei der Vesikelbildung und der Vesikelrezirkulierung spielen. In Caenorhabditis elegans existiert von jeder Familie jeweils nur ein einziges Polypeptid: für die Physine Synaptophysin (SPH-1), für die Gyrine Synaptogyrin (SNG-1) und für die SCAMPs SCAMP (SCM-1). Ziel der Arbeit war es die Verteilung der C. elegans TVPs zu untersuchen und ihre Funktion unter besonderer Berücksichtigung der vesikelvermittelten synaptischen Kopplung zu bestimmen. Wenn die C. elegans TVPs in humanen Epithelzellen synthetisiert werden, lokalisieren sie in zytoplasmatischen Vesikeln. In Kotransfektionsexperimenten wurde gezeigt, dass sie größtenteils in den gleichen Strukturen enthalten sind. In C. elegans synthetisierte TVP-Reporterkonstrukte können in unterschiedlichen Geweben nachgewiesen werden. Dabei ist SNG-1 fast ausschließlich in Neuronen zu finden. SPH-1 und SCM-1 hingegen weisen komplexe und teilweise überlappende Verteilungsmuster auf. Während für SPH-1 eine starke Fluoreszenz im Pharynx, auf der apikalen Seite der Darmzellen oberhalb des sog. terminal webs und in adluminalen Regionen von exkretorischen Geweben gefunden wurde, war SCM-1 stark in der Muskulatur und den Coelomozyten vertreten. Die Expression von SCM-1 in Pharynx und Darm war deutlich schwächer. Die C. elegans TVPs werden früh in der Entwicklung ab der Gastrulation (SPH-1 und SCM-1) bzw. ab der Neurulation im sog. Komma-Stadium (SNG-1) produziert. Um die Funktion der TVPs in C. elegans zu untersuchen, wurden TVP-Mutanten analysiert. Durch Kombination aller drei TVP-Gen-Mutanten wurden TVP-Dreifachmutanten generiert. Diese wiesen keinen offensichtlichen Defekt im Bewegungsmuster auf, entwickelten sich normal und bildeten ein normales Nervensystem aus. Auch auf unterschiedliche chemische und physikalische Reize in sensorischen Tests reagierten die TVP-Dreifachmutanten in gleicher Weise wie Wildtyptiere. Ebenso zeigen die TVP-Dreifachmutanten elektrophysiologisch unter normalen Bedingungen keine anormalen Reaktionsmuster. In ultrastrukturellen Untersuchungen wurde lediglich eine signifikant erhöhte Anzahl Clathrin-ummantelter Vesikel in cholinergen Synapsen gefunden. Erst unter Stressbedingungen, hervorgerufen durch den GABA-Antagonisten Pentylentetrazol (PTZ), wiesen sowohl die TVP-Dreifach- als auch die TVP-Einzelmutanten eine deutlich erhöhte Krampfbereitschaft auf. Zusammengenommen zeigen die Analysen, dass TVPs zwar für grundlegende neuronale Prozesse nicht notwendig sind, dass sie aber auf der anderen Seite vermutlich an alternativen redundanten Wegen der Neurotransmitterfreisetzung beteiligt sind.

VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation

The conversion of light to chemical energy by the process of photosynthesis is localized to the thylakoid membrane network in plant chloroplasts. Although several pathways have been described that target proteins into and across the thylakoids, little is known about the origin of this membrane system or how the lipid backbone of the thylakoids is transported and fused with the target membrane. Thylakoid biogenesis and maintenance seem to involve the flow of membrane elements via vesicular transport. Here we show by mutational analysis that deletion of a single gene called VIPP1 (vesicle-inducing protein in plastids 1) is deleterious to thylakoid membrane formation. Although VIPP1 is a hydrophilic protein it is found in both the inner envelope and the thylakoid membranes. In VIPP1 deletion mutants vesicle formation is abolished. We propose that VIPP1 is essential for the maintenance of thylakoids by a transport pathway not previously recognized.

APP controls the formation of PI(3,5)P2 vesicles through its binding of the PIKfyve complex

Phosphoinositides are signalling lipids that are crucial for major signalling events as well as established regulators of membrane trafficking. Control of endosomal sorting and endosomal homeostasis requires phosphatidylinositol-3-phosphate (PI(3)P) and phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2), the latter a lipid of low abundance but significant physiological relevance. PI(3,5)P2 is formed by phosphorylation of PI(3)P by the PIKfyve complex which is crucial for maintaining endosomal homeostasis. Interestingly, loss of PIKfyve function results in dramatic neurodegeneration. Despite the significance of PIKfyve, its regulation is still poorly understood. Here we show that the Amyloid Precursor Protein (APP), a central molecule in Alzheimer’s disease, associates with the PIKfyve complex (consisting of Vac14, PIKfyve and Fig4) and that the APP intracellular domain directly binds purified Vac14. We also show that the closely related APP paralogues, APLP1 and 2 associate with the PIKfyve complex. Whether APP family proteins can additionally form direct protein–protein interaction with PIKfyve or Fig4 remains to be explored. We show that APP binding to the PIKfyve complex drives formation of PI(3,5)P2 positive vesicles and that APP gene family members are required for supporting PIKfyve function. Interestingly, the PIKfyve complex is required for APP trafficking, suggesting a feedback loop in which APP, by binding to and stimulating PI(3,5)P2 vesicle formation may control its own trafficking. These data suggest that altered APP processing, as observed in Alzheimer’s disease, may disrupt PI(3,5)P2 metabolism, endosomal sorting and homeostasis with important implications for our understanding of the mechanism of neurodegeneration in Alzheimer’s disease.