bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

2016 ◽  
Vol 212 (2) ◽  
pp. 2122OIA1
Author(s):  
Hui Zheng ◽  
Sungsoo Lee ◽  
Marc C. Llaguno ◽  
Qiu-Xing Jiang
Keyword(s):  
Membrane Proteins ◽  
Membrane System ◽  
Giant Vesicles

scholarly journals bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

2015 ◽  
Vol 147 (1) ◽  
pp. 77-93 ◽  
Author(s):  
Hui Zheng ◽  
Sungsoo Lee ◽  
Marc C. Llaguno ◽  
Qiu-Xing Jiang
Keyword(s):  
Ion Channels ◽  
Membrane Proteins ◽  
Lipid Bilayers ◽  
Lipid Composition ◽  
Targeted Delivery ◽  
Membrane System ◽  
Membrane Systems ◽  
Giant Vesicles ◽  
Planar Electrode ◽  
New System

Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo–electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2–20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2–0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid–protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.

scholarly journals Biogenesis of peroxisomes: immunocytochemical investigation of peroxisomal membrane proteins in proliferating rat liver peroxisomes and in catalase-negative membrane loops.

1989 ◽  
Vol 108 (6) ◽  
pp. 2221-2231 ◽  
Author(s):  
E Baumgart ◽  
A Völkl ◽  
T Hashimoto ◽  
H D Fahimi
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Normal Liver ◽  
Membrane System ◽  
Peroxisomal Membrane ◽  
Lowicryl K4m ◽  
Membrane Reservoir ◽  
Liver Peroxisomes

Treatment of rats with a new hypocholesterolemic drug BM 15766 induces proliferation of peroxisomes in pericentral regions of the liver lobule with distinct alterations of the peroxisomal membrane (Baumgart, E., K. Stegmeier, F. H. Schmidt, and H. D. Fahimi. 1987. Lab. Invest. 56:554-564). We have used ultrastructural cytochemistry in conjunction with immunoblotting and immunoelectron microscopy to investigate the effects of this drug on peroxisomal membranes. Highly purified peroxisomal fractions were obtained by Metrizamide gradient centrifugation from control and treated rats. Immunoblots prepared from such peroxisomal fractions incubated with antibodies to 22-, 26-, and 70-kD peroxisomal membrane proteins revealed that the treatment with BM 15766 induced only the 70-kD protein. In sections of normal liver embedded in Lowicryl K4M, all three membrane proteins of peroxisomes could be localized by the postembedding technique. The strongest labeling was obtained with the 22-kD antibody followed by the 70-kD and 26-kD antibodies. In treated animals, double-membraned loops with negative catalase reaction in their lumen, resembling smooth endoplasmic reticulum segments as well as myelin-like figures, were noted in the proximity of some peroxisomes. Serial sectioning revealed that the loops seen at some distance from peroxisomes in the cytoplasm were always continuous with the peroxisomal membranes. The double-membraned loops were consistently negative for glucose-6-phosphatase, a marker for endoplasmic reticulum, but were distinctly labeled with antibodies to peroxisomal membrane proteins. Our observations indicate that these membranous structures are part of the peroxisomal membrane system. They could provide a membrane reservoir for the proliferation of peroxisomes and the expansion of this intracellular compartment.

scholarly journals Pentanol and Benzyl Alcohol Attack Bacterial Surface Structures Differently

2015 ◽  
Vol 82 (1) ◽  
pp. 402-408 ◽  
Author(s):  
Takehisa Yano ◽  
Yoshiko Miyahara ◽  
Noriyuki Morii ◽  
Tetsuya Okano ◽  
Hiromi Kubota
Keyword(s):  
Membrane Proteins ◽  
Benzyl Alcohol ◽  
Efflux Pump ◽  
Public Health Issue ◽  
Membrane Structures ◽  
Giant Vesicles ◽  
Content Type ◽  
Cellular Accumulation ◽  
Bacterial Surface ◽  
Membrane Fluidization

ABSTRACTThe genusMethylobacteriumtolerates hygiene agents like benzalkonium chloride (BAC), and infection with this organism is an important public health issue. Here, we found that the combination of BAC with particular alcohols at nonlethal concentrations in terms of their solitary uses significantly reduced bacterial viability after only 5 min of exposure. Among the alcohols, Raman spectroscopic analyses showed that pentanol (pentyl alcohol [PeA]) and benzyl alcohol (BzA) accelerated the cellular accumulation of BAC. Fluorescence spectroscopic assays and morphological assays with giant vesicles indicated that PeA rarely attacked membrane structures, while BzA increased the membrane fluidity and destabilized the structures. Other fluorescent spectroscopic assays indicated that PeA and BzA inactivate bacterial membrane proteins, including an efflux pump for BAC transportation. These findings suggested that the inactivation of membrane proteins by PeA and BzA led to the cellular accumulation but that only BzA also enhanced BAC penetration by membrane fluidization at nonlethal concentrations.

Surface membrane regeneration in deciliated Tetrahymena

1983 ◽  
Vol 62 (1) ◽  
pp. 407-417
Author(s):  
N.E. Williams
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Surface Membrane ◽  
Membrane System ◽  
Ciliated Protozoa ◽  
Membrane Flow ◽  
Transmission Electron ◽  
Surface Marking

The induced synthesis of identified surface membrane proteins has been demonstrated in deciliated Tetrahymena. Cells in the process of regenerating cilia were also studied using transmission electron microscopy in order to obtain information on the deployment of new membrane at the cell surface. The results obtained suggest a pattern of membrane flow that includes the ‘pellicular alveoli’, a subsurface membrane system characteristically present in ciliated protozoa. The results of 125I surface-marking experiments were consistent with the notion that new membrane is added initially in non-ciliated regions, then subsequently flows laterally to cover regenerating cilia.

scholarly journals Expression of autofluorescent proteins reveals a novel protein permeable pathway between cells in the lens core

2000 ◽  
Vol 113 (11) ◽  
pp. 1913-1921 ◽  
Author(s):  
V.I. Shestopalov ◽  
S. Bassnett
Keyword(s):  
Membrane Proteins ◽  
Cell Communication ◽  
Membrane System ◽  
Mediated Communication ◽  
Large Molecules ◽  
Fiber Cells ◽  
Quiescent Cells ◽  
Fluorescent Markers ◽  
Cytoplasmic Proteins ◽  
Protein Components

The lens of the eye is composed of concentric layers of tightly packed fiber cells. The oldest fibers, those in the lens core, lose their nuclei and other organelles during terminal differentiation. This is thought to ensure the clarity of the lens. The anucleated core fibers are sustained by gap junction-mediated communication with metabolically active cells near the lens surface. In this study, we expressed autofluorescent proteins and microinjected fluorescent markers to probe cell-to-cell communication in different regions of the developing lens. Our data indicate that a novel cell-cell diffusion pathway becomes patent in the lens core during development. This pathway is remarkable in that it is permeable to proteins and other large molecules and is thus distinct from gap junctions. Diffusion of large molecules probably occurs through regions of membrane fusion observed between neighboring cells in the lens core. Further direct evidence for a continuous plasma membrane system was provided by the observation that exogenous membrane proteins expressed in one core fiber cell were able to diffuse laterally into the membranes of adjacent fibers. Thus, the lens core appears to represent a true syncytium within which both membrane proteins and cytoplasmic proteins freely diffuse. Significantly, the outermost edge of the core syncytium encompasses a shell of nucleated, transcriptionally-competent, fiber cells. This arrangement could facilitate the delivery of newly synthesized protein components to the aged and metabolically quiescent cells in the center of the lens.

A Freeze-Etch Study of Acinetobacter SP. Grown on a Hydrocarbon Substrate

1974 ◽  
Vol 32 ◽  
pp. 174-175
Author(s):  
C. L. Scott ◽  
W. R. Finnerty
Keyword(s):  
Membrane System ◽  
Structural Distortion ◽  
Intracytoplasmic Membrane ◽  
Gram Negative ◽  
Sectioned Material ◽  
Acinetobacter Sp

Acinetobacter sp. HO-1-N, a gram-negative hydrocarbon oxidizing bacterium previously designated Micrococcus cerificans, has been shown to sequester the hydrocarbon into intracytoplasmic pools as a result of growth on this substrate. In hydrocarbon grown cells, an intracytoplasmic membrane system was also observed along with a doubling of cellular phospholipids (Z). However, using conventional dehydration and embedding procedures in preparing thin sectioned material, the hydrocarbon is extracted from the cells. This may lead to structural distortion, consequently, the freeze-etch technique was applied to preserve the integrity of the cell.

Changes Occur in Plasma Membrane Turnover when K562 Leukemia Cells are Induced to Differentiate

1982 ◽  
Vol 40 ◽  
pp. 286-287
Author(s):  
L. M. Marshall
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Intracellular Transport ◽  
Parent Cell ◽  
Membrane Turnover ◽  
Coated Pits ◽  
Surface Distribution ◽  
Specific Proteins ◽  
Erythroleukemic Cell ◽  
Specific Membrane

A human erythroleukemic cell line, metabolically blocked in a late stage of erythropoiesis, becomes capable of differentiation along the normal pathway when grown in the presence of hemin. This process is characterized by hemoglobin synthesis followed by rearrangement of the plasma membrane proteins and culminates in asymmetrical cytokinesis in the absence of nuclear division. A reticulocyte-like cell buds from the nucleus-containing parent cell after erythrocyte specific membrane proteins have been sequestered into its membrane. In this process the parent cell faces two obstacles. First, to organize its erythrocyte specific proteins at one pole of the cell for inclusion in the reticulocyte; second, to reduce or abolish membrane protein turnover since hemoglobin is virtually the only protein being synthesized at this stage. A means of achieving redistribution and cessation of turnover could involve movement of membrane proteins by a directional lipid flow. Generation of a lipid flow towards one pole and accumulation of erythrocyte-specific membrane proteins could be achieved by clathrin coated pits which are implicated in membrane endocytosis, intracellular transport and turnover. In non-differentiating cells, membrane proteins are turned over and are random in surface distribution. If, however, the erythrocyte specific proteins in differentiating cells were excluded from endocytosing coated pits, not only would their turnover cease, but they would also tend to drift towards and collect at the site of endocytosis. This hypothesis requires that different protein species are endocytosed by the coated vesicles in non-differentiating than by differentiating cells.

Image Analysis of Intramembrane Particles in Freeze-Fractured Biomembranes Using Computer Simulation Techniques

1975 ◽  
Vol 33 ◽  
pp. 214-215
Author(s):  
D.J. Benefiel ◽  
R.S. Weinstein
Keyword(s):  
Membrane Proteins ◽  
Freeze Fracture ◽  
Integral Membrane Proteins ◽  
Systematic Evaluation ◽  
Intramembrane Particles ◽  
Modeling Methods ◽  
Simulation Techniques ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron ◽  
Computer Simulation Techniques

Intramembrane particles (IMP or MAP) are components of most biomembranes. They are visualized by freeze-fracture electron microscopy, and they probably represent replicas of integral membrane proteins. The presence of MAP in biomembranes has been extensively investigated but their detailed ultrastructure has been largely ignored. In this study, we have attempted to lay groundwork for a systematic evaluation of MAP ultrastructure. Using mathematical modeling methods, we have simulated the electron optical appearances of idealized globular proteins as they might be expected to appear in replicas under defined conditions. By comparing these images with the apearances of MAPs in replicas, we have attempted to evaluate dimensional and shape distortions that may be introduced by the freeze-fracture technique and further to deduce the actual shapes of integral membrane proteins from their freezefracture images.

A Novel Reconstitution Method for Inducing the Formation of Regular 2-Dimensional Arrays of Membrane Proteins and Lipids

1988 ◽  
Vol 46 ◽  
pp. 152-153 ◽  
Author(s):  
A. Engel ◽  
A. Holzenburg ◽  
K. Stauffer ◽  
J. Rosenbusch ◽  
U. Aebi
Keyword(s):  
Membrane Proteins ◽  
Flow Rate ◽  
Lipid Membranes ◽  
Functional Characterization ◽  
Dimensional Structure ◽  
Electron Crystallography ◽  
Peltier Element ◽  
Protein Ratio ◽  
Precise Control ◽  
3 Dimensional

Reconstitution of solubilized and purified membrane proteins in the presence of phospholipids into vesicles allows their functions to be studied by simple bulk measurements (e.g. diffusion of differently sized solutes) or by conductance measurements after transformation into planar membranes. On the other hand, reconstitution into regular protein-lipid arrays, usually forming at a specific lipid-to-protein ratio, provides the basis for determining the 3-dimensional structure of membrane proteins employing the tools of electron crystallography.To refine reconstitution conditions for reproducibly inducing formation of large and highly ordered protein-lipid membranes that are suitable for both electron crystallography and patch clamping experiments aimed at their functional characterization, we built a flow-dialysis device that allows precise control of temperature and flow-rate (Fig. 1). The flow rate is generated by a peristaltic pump and can be adjusted from 1 to 500 ml/h. The dialysis buffer is brought to a preselected temperature during its travel through a meandering path before it enters the dialysis reservoir. A Z-80 based computer controls a Peltier element allowing the temperature profile to be programmed as function of time.

The structure of membranes and membrane proteins embedded in amorphous ice

1992 ◽  
Vol 50 (1) ◽  
pp. 432-433
Author(s):  
Uwe Lücken ◽  
Joachim Jäger
Keyword(s):  
Phase Transition ◽  
Transition Temperature ◽  
Membrane Proteins ◽  
Phase Transition Temperature ◽  
Lipid Vesicles ◽  
Freezing Stress ◽  
Amorphous Ice ◽  
Different Temperatures ◽  
Band Pass ◽  
Lipid Polymorphism

TEM imaging of frozen-hydrated lipid vesicles has been done by several groups Thermotrophic and lyotrophic polymorphism has been reported. By using image processing, computer simulation and tilt experiments, we tried to learn about the influence of freezing-stress and defocus artifacts on the lipid polymorphism and fine structure of the bilayer profile. We show integrated membrane proteins do modulate the bilayer structure and the morphology of the vesicles.Phase transitions of DMPC vesicles were visualized after freezing under equilibrium conditions at different temperatures in a controlled-environment vitrification system. Below the main phase transition temperature of 24°C (Fig. 1), vesicles show a facetted appearance due to the quasicrystalline areas. A gradual increase in temperature leads to melting processes with different morphology in the bilayer profile. Far above the phase transition temperature the bilayer profile is still present. In the band-pass-filtered images (Fig. 2) no significant change in the width of the bilayer profile is visible.

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