Interconnected Membrane Research Articles

Fabrication of Dense La<SUB>0.7</SUB>Ca<SUB>0.3</SUB>Cr<SUB>0.97</SUB>O<SUB>3−<I>δ</I></SUB> Inter-connect Membrane on Novel SOFC Composite Support by Co-firing

Fabrication of Dense La<SUB>0.7</SUB>Ca<SUB>0.3</SUB>Cr<SUB>0.97</SUB>O<SUB>3−<I>δ</I></SUB> Inter-connect Membrane on Novel SOFC Composite Support by Co-firing

In situ fabrication mechanism of a dense Sr and Ca doped lanthanum chromite interconnect on Ni-YSZ anode of a solid oxide fuel cell during co-sintering

In this study fabrication mechanism of a dense Sr and Ca doped lanthanum chromite interconnect on Ni-YSZ anode of a solid oxide fuel cell after co-sintering is investigated. Based on the understanding of sintering mechanism, a dense La1−x−ySrxCayCrO3−δ interconnect membrane was successfully prepared on the anode support of NiO-YSZ by a novel method. In this method a base layer of La0.8Sr0.2CrO3−δ was screen printed on NiO-YSZ substrate and a top layer of CaCrO4 was then coated and co-sintered at 1400°C. CaCrO4 not only melts and fills the open pores in the base layer, but it also dissolves in it forming La1−x−y SrxCayCrO3−δ as a single layer. The mechanism of CaCrO4 dissolution in the base layer, densification characteristics and microstructure of interconnect are discussed.

Designing and simulating a nitinol-based micro ejector

This paper describes pico-droplet ejector design and simulation. The actuation system was based on two interconnected nitinol membranes' shape memory effect. Ejected volume was 12pL and it operated at 30°C to 64°C. Ejecting excitation voltage was 12V and the ejecting energy required by actuator operation was 26µJ per drop. These pico-liter ejectors could have applications in making, lubricating and cooling integrated circuits.

Self-assembly of amphiphilic patchy particles with different cross-linking densities

The influence of deformability of two-patch particles on their self-assembly behavior is studied via computer simulations. The softness and the deformability of the patchy particles can be controlled by varying the cross-linking densities in different parts of the particles. The patchy particles in a solvent that is bad for patches but good for the matrix form linear thread-like structures when cross-linking densities are low, whereas they form three-dimensional network structures at relatively high cross-linking densities. For patchy particles in a solvent that is good for patches but bad for the matrix, inter-connected membrane structures are obtained at relatively low cross-linking densities, and some cluster structures emerge when cross-linking densities are high. Bicontinuous membranes with better morphologies can be observed by tuning the cross-linking densities of different parts of the patchy particles.

A novel approach to co-sintering of doped lanthanum chromite interconnect on Ni–YSZ anode substrate for SOFC applications

A cost-effective method was developed to fabricate dense doped LaCrO3 bases interconnect membrane on anode substrate for solid oxide fuel cell (SOFC) applications. As conventional lanthanum chromite interconnect is difficult to be co-sintered with green anode, a simple and cost effective screen printing/co-sintering process was employed, and dense La1−x−ySrxCayCrO3−δ interconnect membrane was successfully prepared on the anode support of NiO–YSZ. In this method a base layer of doped lanthanum chromite (La1−xSrxCrO3−δ) was applied on NiO–YSZ substrate and a top layer of CaCrO4 was then coated and co-sintered to melt the top layer. By means of this method, not only CaCrO4 melts and fills the open pores in the base layer, but it also dissolves in base layer forming La1−x−ySrxCayCrO3−δ as a single layer. The sintering characteristics, microstructure and electrical conductivity of interconnect were investigated. In addition, the effects of composition on sintering and electrical properties were studied.

Architecture and Regulation of the HIV-1 Assembly and Holding Compartment in Macrophages

Productive infection of macrophages is central to HIV-1 pathogenesis. Newly formed virions bud into a tubular membranous compartment that is contiguous with the plasma membrane. However, little is known about the structure of this compartment and its potential regulation by infection. Here we characterized this compartment in macrophages using electron tomography and electron microscopy with stereology. We found an intricate, interconnected membrane network that constitutes a preexisting physiologic structure in macrophages but which expands in size upon HIV-1 infection. Membranes required for this expansion were apparently derived from preexisting pools of plasma membrane. Physical connections between this compartment and the extracellular milieu were frequently made by tube-like structures of insufficient diameter for virion passage. We conclude that HIV-1 induces the expansion of a complex membranous labyrinth in macrophages in which the virus buds and can be retained, with potential consequences for transmission and immune evasion.

Preparation, structure and supercapacitance of bonded carbon nanofiber electrode materials

Interconnected carbon nanofibrous membranes were prepared by conventional electrospinning and bicomponent electrospinning to produce polyvinylpyrrolidone (PVP)/polyacrylonitrile (PAN) blend nanofibers and PVP/PAN side-by-side bicomponent nanofibers, followed by a direct pyrolysis treatment. The inter-fiber connection was highly affected by the PVP/PAN ratio and electrospinning method. The carbon nanofibers prepared from the side-by-side PVP/PAN nanofibers were found to have higher electrochemical capacitance than those from the PVP/PAN blend nanofibers.

Use of nanoporous columnar thin film in the wafer-level packaging of MEMS devices

This paper presents a new packaging technology that uses a nanoporous columnar thin film to seal microelectromechanical system (MEMS) devices at the wafer level. In the proposed packaging process, the processing temperature is 350 °C. The process is relatively inexpensive compared to wafer level packaging processes, because the wafer-bonding step is eliminated and the die size is shrunk. In the suggested approach, a sputtered columnar thin film at room temperature forms vertical nanopores as etch holes, and an air cavity is formed by the removal of a sacrificial layer through the nanopores in the columnar membrane. Subsequent hermetic vacuum packaging of the cavity is achieved by depositing thin films over the membrane under low pressure. The hermeticity of the packaging was verified by using an optical surface morphology microscope to measure the deflection change of the sealing membrane before and after breaking of the vacuum through an interconnected membrane. The long-term hermeticity was monitored by measuring the maximum central deflection of the PECVD sealing layer over a period of 170 days. The precise pressure (0.7 Torr) and short-term (30 days) pressure change inside the cavity were measured by encapsulated Ni Pirani gauges, representing packaged freestanding MEMS devices.

Membrane Biogenesis: Networking at the ER with Atlastin

The peripheral endoplasmic reticulum forms a dynamic network of interconnected membrane tubules. Although some determinants of this striking architecture are known, the mechanism underlying fusion of individual tubules has remained elusive. Two studies now identify atlastin proteins as key mediators of homotypic fusion of endoplasmic reticulum membranes.

Fabrication of dense LaCrO 3-based interconnect thin membrane on anode substrates by co-firing

In order to develop cost-effective method to prepare ceramic interconnect membrane for solid oxide fell cell stacks, the triple-phase composites of NiO/Sm 0.2Ce 0.8O 2− δ /La 0.7Ca 0.3Cr 0.97O 3− δ with the weight ratios of 6:4: x ( x = 0, 2, 3, 4, 5) were, respectively prepared, and then examined as novel anode supports. The sintering character, microstructure, electrical conductivity, fracture strength, and thermal expansion coefficient of the anodes were, respectively investigated in detail as a function of La 0.7Ca 0.3Cr 0.97O 3− δ content. Results indicate the above-mentioned performances of NiO/Sm 0.2Ce 0.8O 2− δ /La 0.7Ca 0.3Cr 0.97O 3−δ anode at x ≤ 3 have no significant reduction, and are still acceptable for the practical requirements for solid oxide fell cells. Furthermore, by using a simple and cost-effective drop-coating/co-firing process, dense La 0.7Ca 0.3Cr 0.97O 3− δ interconnect membrane was successfully prepared on the anode support of NiO/Sm 0.2Ce 0.8O 2− δ /La 0.7Ca 0.3Cr 0.97O 3−δ at x = 3. This work presents a simple technical route for developing dense interconnect membranes for Sm 0.2Ce 0.8O 2− δ -based fuel cell stacks.

The regulation of proplatelet production

Hematologists have long been fascinated by thrombopoiesis. While it has long been accepted that platelets are derived from megakaryocytes,1 the process by which this occurs and its regulation are still incompletely understood. As they mature, megakaryocytes acquire competency for platelet formation through up-modulation of a vast array of cytoskeletal, membrane and granule regulatory proteins,2,3 differentiating into cells that are large, polyploid, and have a cytoplasm filled with a complex system of interconnected cytoplasmic membranes (dermarcation membrane system or DMS), as well as stores of ribosomes, alpha granules and dense granules.4,5 Although the growth and differentiation of megakaryocytes requires thrombopoietin, the role of this cytokine in the subsequent steps of platelet formation is less well established.6

Theoretical analysis of the effects of asymmetric membrane structure on fouling during microfiltration

AbstractThere is a growing interest in the use of both asymmetric and composite membranes for microfiltration and ultrafiltration processes. This includes particle removal applications in the semiconductor industry and virus clearance in biopharmaceutical applications. Filter fouling plays an important role in these processes. Although flux decline models have been developed for homogeneous membranes, the effects of asymmetric membrane structure on flux decline behavior remain poorly understood on a fundamental level. Here, we develop a theoretical model to describe the effects of asymmetric membrane structure on flux decline. The asymmetric structure was described by the spatial variation in Darcy permeability in the directions normal to and parallel to the membrane surface. The velocity profile and flux decline because of pore blockage were described using Darcy's law and a pore blockage and cake filtration model. Flux decline data were obtained using pseudocomposite membranes with highly interconnected polyvinylidene fluoride membranes (PVDF) and straight through pore polycarbonate track‐etched membranes (PCTE). Model composite membranes were formed by layering PCTE or PVDF membranes with different pore sizes on top of each other. Flux decline data for the composite membrane were in good agreement with model calculations. The results provide important insights into the effects of asymmetric membrane pore structures on flux decline. © 2009 American Institute of Chemical Engineers AIChE J, 2009

In Vitro Reconstitution of Dynamic, ER-like, Nanotubular Networks, and of Small, Tubulo-Vesicular Transport Entities by Interactions of Cytoplasmic Dynein and Spectrin with Liposomes

Cells contain intricate networks of membrane tubes of nanoscale dimensions, such as the endoplasmic reticulum (ER). Much smaller tubular entities, derived by extraction from donor compartments (e.g., those emerging from recycling endosomes or the trans-Golgi network) or generated by vesicle fusion (e.g., the ER-to-Golgi transport units), function in intracellular transport. Different mechanisms are thought to underlie the morphogenesis of the complex, tubular ER network, and the formation of the small tubular transport entities that travel along microtubules. Here, we show that the molecular machinery that powers retrograde vesicle motility in neurons can interact with membranes to generate these different types of tubes. We reconstituted in vitro elaborate networks of interconnected membrane tubes (with ER-characteristic ring closures and three-way junctions), as well as freely moving, stable tubes and tubulo-vesicular clusters, from mixtures of the minus-end motor, cytoplasmic dynein, its regulatory complex, dynactin, the anchoring protein, spectrin, and liposomes containing acidic phospholipids, in the presence of microtubules and ATP. The tubulo-vesicular clusters contained trains of spherical liposomes attached to a small tube via elastic linkers, likely maintained together through a supravesicular spectrin meshwork that encompasses both the tube and the associated vesicles. Recruitment of dynein-dynactin and spectrin from the cytosol to liposomes was stimulated by phospholipase D-induced conversion of neutral phospholipids to acidic forms, and by activation of small GTPases. We conclude that similar mechanisms underlie the generation of ER-like tube networks and small tubular transport entities. Both may be generated and maintained by the action of soluble microtubule motor complexes and anchoring proteins, which bind to phospholipids, and do not require membrane proteins. Supported by March of Dimes grant 1-FY04-240 and NIH grant R01GM068596.

A role for Rab5 in structuring the endoplasmic reticulum

The endoplasmic reticulum (ER) is a contiguous network of interconnected membrane sheets and tubules. The ER is differentiated into distinct domains, including the peripheral ER and nuclear envelope. Inhibition of two ER proteins, Rtn4a and DP1/NogoA, was previously shown to inhibit the formation of ER tubules in vitro. We show that the formation of ER tubules in vitro also requires a Rab family GTPase. Characterization of the 29 Caenorhabditis elegans Rab GTPases reveals that depletion of RAB-5 phenocopies the defects in peripheral ER structure that result from depletion of RET-1 and YOP-1, the C. elegans homologues of Rtn4a and DP1/NogoA. Perturbation of endocytosis by other means did not affect ER structure; the role of RAB-5 in ER morphology is thus independent of its well-studied requirement for endocytosis. RAB-5 and YOP-1/RET-1 also control the kinetics of nuclear envelope disassembly, which suggests an important role for the morphology of the peripheral ER in this process.

In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53

In macrophages, HIV-1 has been shown to bud into intracellular structures that contain the late endosome marker CD63. We show that these organelles are not endosomes, but an internally sequestered plasma membrane domain. Using immunofluorescence microscopy and immunoelectron microscopy, we find that HIV-1 buds into a compartment that contains the tetraspanins CD81, CD9, and CD53. On uninfected macrophages, these proteins are seen at the cell surface and in intracellular vacuole-like structures with a complex content of vesicles and interconnected membranes that lack endosome markers, including CD63. Significantly, these structures are accessible to small tracers (horseradish peroxidase or ruthenium red) applied to cells at 4°C, indicating that they are connected to the cell surface. HIV assembles on, and accumulates within, these intracellular compartments. Furthermore, CD63 is recruited to the virus-containing structures and incorporated into virions. These results indicate that, in macrophages, HIV-1 exploits a previously undescribed intracellular plasma membrane domain to assemble infectious particles.

Morphogenesis of the Endoplasmic Reticulum: Beyond Active Membrane Expansion

The endoplasmic reticulum (ER) of higher eukaryotic cells is a dynamic network of interconnected membrane tubules that pervades almost the entire cytoplasm. On the basis of the morphological changes induced by the disruption of the cytoskeleton or molecular motor proteins, the commonly accepted model has emerged that microtubules and conventional kinesin (kinesin-1) are essential determinants in establishing and maintaining the structure of the ER by active membrane expansion. Surprisingly, very similar ER phenotypes have now been observed when the cytoskeleton-linking ER membrane protein of 63 kDa (CLIMP-63) is mutated, revealing stable attachment of ER membranes to the microtubular cytoskeleton as a novel requirement for ER maintenance. Additional recent findings suggest that ER maintenance also requires ongoing homotypic membrane fusion, possibly controlled by the p97/p47/VICP135 protein complex. Work on other proteins proposed to regulate ER structure, including huntingtin, the EF-hand Ca(2+)-binding protein p22, the vesicle-associated membrane protein-associated protein B and kinectin isoforms further contribute to the new emerging concept that ER shape is not only determined by motor driven processes but by a variety of different mechanisms.

SGEF, a RhoG guanine nucleotide exchange factor that stimulates macropinocytosis.

SGEF (SH3-containing Guanine Nucleotide Exchange Factor) is a RhoGEF of unknown function. We found the SGEF protein to be expressed in many established cell lines and highly expressed in human liver tissue. SGEF stimulated the formation of large interconnected membrane ruffles across dorsal surfaces when expressed in fibroblasts. SGEF required its proline-rich amino-terminus to generate dorsal, but not lateral, membrane ruffles and a functional SH3 domain to colocalize with filamentous actin at sites of membrane protrusion. Full-length SGEF activated RhoG, but not Rac, when expressed in fibroblasts. Further, recombinant SGEF DH/PH protein exchanged nucleotide on RhoG, but not on Rac1 or Rac3, in vitro. Scanning electron microscopy of fibroblasts demonstrated that SGEF induced dorsal ruffles that were morphologically similar to those generated by constitutively active RhoG, but not constitutively active Rac1. Transient expression of SGEF stimulated fibroblast uptake of 10-kDa dextran, a marker of macropinocytosis. This required the full-length protein and a catalytically active DH domain. Finally, activated RhoG was found to be more effective than activated Rac, and comparable to SGEF, in its ability to trigger dextran uptake. Together, this work establishes SGEF as a RhoG exchange factor and provides evidence that both SGEF and RhoG regulate membrane dynamics in promotion of macropinocytosis.

Brefeldin A-dependent membrane tubule formation reconstituted in vitro is driven by a cell cycle-regulated microtubule motor.

Treatment of cultured cells with brefeldin A (BFA) induces the formation of extensive membrane tubules from the Golgi apparatus, trans-Golgi network, and early endosomes in a microtubule-dependent manner. We have reconstituted this transport process in vitro using Xenopus egg cytosol and a rat liver Golgi-enriched membrane fraction. The presence of BFA results in the formation of an intricate, interconnected tubular membrane network, a process that, as in vivo, is inhibited by nocodazole, the H1 anti-kinesin monoclonal antibody, and by membrane pretreatment with guanosine 5'-O-(3-thiotriphosphate). Surprisingly, membrane tubule formation is not due to the action of conventional kinesin or any of the other motors implicated in Golgi membrane dynamics. Two candidate motors of approximately 100 and approximately 130 kDa have been identified using the H1 antibody, both of which exhibit motor properties in a biochemical assay. Finally, BFA-induced membrane tubule formation does not occur in metaphase cytosol, and because membrane binding of both candidate motors is not altered after incubation in metaphase compared with interphase cytosol, these results suggest that either the ATPase or microtubule-binding activity of the relevant motor is cell cycle regulated.

ARF is required for maintenance of yeast Golgi and endosome structure and function.

ADP ribosylation factor (ARF) is thought to play a critical role in recruiting coatomer (COPI) to Golgi membranes to drive transport vesicle budding. Yeast strains harboring mutant COPI proteins exhibit defects in retrograde Golgi to endoplasmic reticulum protein transport and striking cargo-selective defects in anterograde endoplasmic reticulum to Golgi protein transport. To determine whether arf mutants exhibit similar phenotypes, the anterograde transport kinetics of multiple cargo proteins were examined in arf mutant cells, and, surprisingly, both COPI-dependent and COPI-independent cargo proteins exhibited comparable defects. Retrograde dilysine-mediated transport also appeared to be inefficient in the arf mutants, and coatomer mutants with no detectable anterograde transport defect exhibited a synthetic growth defect when combined with arf1Delta, supporting a role for ARF in retrograde transport. Remarkably, we found that early and medial Golgi glycosyltransferases localized to abnormally large ring-shaped structures. The endocytic marker FM4-64 also stained similar, but generally larger ring-shaped structures en route from the plasma membrane to the vacuole in arf mutants. Brefeldin A similarly perturbed endosome morphology and also inhibited transport of FM4-64 from endosomal structures to the vacuole. Electron microscopy of arf mutant cells revealed the presence of what appear to be hollow spheres of interconnected membrane tubules which likely correspond to the fluorescent ring structures. Together, these observations indicate that organelle morphology is significantly more affected than transport in the arf mutants, suggesting a fundamental role for ARF in regulating membrane dynamics. Possible mechanisms for producing this dramatic morphological change in intracellular organelles and its relation to the function of ARF in coat assembly are discussed.

Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis.

The mechanisms of localization and retention of membrane proteins in the inner nuclear membrane and the fate of this membrane system during mitosis were studied in living cells using the inner nuclear membrane protein, lamin B receptor, fused to green fluorescent protein (LBR-GFP). Photobleaching techniques revealed the majority of LBR-GFP to be completely immobilized in the nuclear envelope (NE) of interphase cells, suggesting a tight binding to heterochromatin and/or lamins. A subpopulation of LBR-GFP within ER membranes, by contrast, was entirely mobile and diffused rapidly and freely (D = 0. 41 +/- 0.1 microm2/s). High resolution confocal time-lapse imaging in mitotic cells revealed LBR-GFP redistributing into the interconnected ER membrane system in prometaphase, exhibiting the same high mobility and diffusion constant as observed in interphase ER membranes. LBR-GFP rapidly diffused across the cell within the membrane network defined by the ER, suggesting the integrity of the ER was maintained in mitosis, with little or no fragmentation and vesiculation. At the end of mitosis, nuclear membrane reformation coincided with immobilization of LBR-GFP in ER elements at contact sites with chromatin. LBR-GFP-containing ER membranes then wrapped around chromatin over the course of 2-3 min, quickly and efficiently compartmentalizing nuclear material. Expansion of the NE followed over the course of 30-80 min. Thus, selective changes in lateral mobility of LBR-GFP within the ER/NE membrane system form the basis for its localization to the inner nuclear membrane during interphase. Such changes, rather than vesiculation mechanisms, also underlie the redistribution of this molecule during NE disassembly and reformation in mitosis.