scholarly journals Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takanobu A. Katoh ◽  
Takashi Daiho ◽  
Kazuo Yamasaki ◽  
Stefania Danko ◽  
Shoko Fujimura ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Fluorescence Microscopy ◽  
Total Internal Reflection ◽  
High Speed ◽  
Atp Hydrolysis ◽  
Orientation Imaging ◽  
Angle Change ◽  
Fixed Orientation ◽  
Set Up ◽  
Domain Motions

AbstractThe sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) transports Ca2+ ions across the membrane coupled with ATP hydrolysis. Crystal structures of ligand-stabilized molecules indicate that the movement of actuator (A) domain plays a crucial role in Ca2+ translocation. However, the actual structural movements during the transitions between intermediates remain uncertain, in particular, the structure of E2PCa2 has not been solved. Here, the angle of the A-domain was measured by defocused orientation imaging using isotropic total internal reflection fluorescence microscopy. A single SERCA1a molecule, labeled with fluorophore ReAsH on the A-domain in fixed orientation, was embedded in a nanodisc, and stabilized on Ni–NTA glass. Activation with ATP and Ca2+ caused angle changes of the fluorophore and therefore the A-domain, motions lost by inhibitor, thapsigargin. Our high-speed set-up captured the motion during EP isomerization, and suggests that the A-domain rapidly rotates back and forth from an E1PCa2 position to a position close to the E2P state. This is the first report of the detection in the movement of the A-domain as an angle change. Our method provides a powerful tool to investigate the conformational change of a membrane protein in real-time.

scholarly journals Imaging Constitutive Exocytosis with Total Internal Reflection Fluorescence Microscopy

2000 ◽  
Vol 149 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Jan Schmoranzer ◽  
Mark Goulian ◽  
Dan Axelrod ◽  
Sanford M. Simon
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Fluorescence Microscopy ◽  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Green Fluorescent ◽  
Kinetics Of ◽  
Flattening Process

Total internal reflection fluorescence microscopy has been applied to image the final stage of constitutive exocytosis, which is the fusion of single post-Golgi carriers with the plasma membrane. The use of a membrane protein tagged with green fluorescent protein allowed the kinetics of fusion to be followed with a time resolution of 30 frames/s. Quantitative analysis allowed carriers undergoing fusion to be easily distinguished from carriers moving perpendicularly to the plasma membrane. The flattening of the carriers into the plasma membrane is seen as a simultaneous rise in the total, peak, and width of the fluorescence intensity. The duration of this flattening process depends on the size of the carriers, distinguishing small spherical from large tubular carriers. The spread of the membrane protein into the plasma membrane upon fusion is diffusive. Mapping many fusion sites of a single cell reveals that there are no preferred sites for constitutive exocytosis in this system.

Scanning x-ray fluorescence microscopy and principal component analysis

1992 ◽  
Vol 50 (2) ◽  
pp. 1752-1753
Author(s):  
Brian Cross
Keyword(s):  
Principal Component Analysis ◽  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
High Speed ◽  
Image Acquisition ◽  
Principal Component ◽  
Fluorescence Microscope ◽  
Acquisition Rate ◽  
X Ray ◽  
Speed Up

A relatively new entry, in the field of microscopy, is the Scanning X-Ray Fluorescence Microscope (SXRFM). Using this type of instrument (e.g. Kevex Omicron X-ray Microprobe), one can obtain multiple elemental x-ray images, from the analysis of materials which show heterogeneity. The SXRFM obtains images by collimating an x-ray beam (e.g. 100 μm diameter), and then scanning the sample with a high-speed x-y stage. To speed up the image acquisition, data is acquired "on-the-fly" by slew-scanning the stage along the x-axis, like a TV or SEM scan. To reduce the overhead from "fly-back," the images can be acquired by bi-directional scanning of the x-axis. This results in very little overhead with the re-positioning of the sample stage. The image acquisition rate is dominated by the x-ray acquisition rate. Therefore, the total x-ray image acquisition rate, using the SXRFM, is very comparable to an SEM. Although the x-ray spatial resolution of the SXRFM is worse than an SEM (say 100 vs. 2 μm), there are several other advantages.

scholarly journals High-speed label-free two-photon fluorescence microscopy of metabolic transients during neuronal activity

2021 ◽  
Vol 118 (8) ◽  
pp. 081104
Author(s):  
Andrew J. Bower ◽  
Carlos Renteria ◽  
Joanne Li ◽  
Marina Marjanovic ◽  
Ronit Barkalifa ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Neuronal Activity ◽  
High Speed ◽  
Label Free ◽  
Two Photon ◽  
Metabolic Transients ◽  
Two Photon Fluorescence ◽  
Photon Fluorescence

scholarly journals A sensitive immunoassay for rat fatty acid translocase (CD36) using phage antibodies selected on cell transfectants: abundant presence of fatty acid translocase/CD36 in cardiac and red skeletal muscle and up-regulation in diabetes

1999 ◽  
Vol 337 (3) ◽  
pp. 407-414 ◽  
Author(s):  
Maurice M. A. L. PELSERS ◽  
Jan T. LUTGERINK ◽  
Frans A. van NIEUWENHOVEN ◽  
Narendra N. TANDON ◽  
Ger J. van der VUSSE ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Membrane Protein ◽  
Diabetic Rats ◽  
Direct Selection ◽  
Antibody Library ◽  
Fatty Acid Binding ◽  
Fatty Acid Translocase ◽  
Phage Antibody ◽  
Phage Antibodies ◽  
Set Up

The rat membrane protein fatty acid translocase (FAT), which shows sequence similarity to human CD36 (a membrane protein supposedly involved in a variety of membrane processes), is implicated in the transport of long-chain fatty acids across cellular membranes. To set up an immunoassay for quantification of FAT in different tissues, we isolated a series of anti-FAT antibodies by panning a large naive phage antibody library on FAT-transfected H9c2 cells. All seven different phage antibody fragments isolated reacted specifically with FAT, and most likely recognize the same or closely located immunodominant sites on FAT, as a competitive monoclonal antibody (mAb) (CLB-IV7) completely blocked the binding of all these phage antibodies to cells. A sandwich ELISA was set up using mAb 131.4 (directed against purified CD36 from human platelets) as capture antibody and phage antibodies and anti-phage sera as detector. With this ELISA (sensitivity 0.05 µg/ml), the FAT content in isolated cardiomyocytes was found to be comparable with that of total heart (≈ 3 mg/g of protein), while liver tissue and endothelial cells were below the detection limit (< 0.1 mg of FAT/g of protein). During rat heart development, protein levels of FAT rose from 1.7±0.7 mg/g of protein on the day before birth to 3.6±0.4 mg/g of protein on day 70. Comparing control with streptozotocin-induced diabetic rats, a statistically significant (P< 0.05) 2–4-fold increase of FAT was seen in heart (from 4.2±2.3 to 11.0±5.7 mg/g of protein), soleus (from 0.6±0.1 to 1.4±0.5 mg/g of protein) and extensor digitorum longus (EDL) muscle (from 0.3±0.1 to 1.2±0.8 mg/g of protein). In addition, the FAT contents of each of these muscles were found to be of similar magnitude to the contents of cytoplasmic heart-type fatty-acid-binding protein in both diabetic rats and controls, supporting the suggested roles of these two proteins in cellular fatty acid metabolism. This is the first time phage display technology has been succesfully applied for direct selection, from a large naive antibody library, of antibodies that recognize selected membrane proteins in their natural context.

Study on Magnetic Barrel Machine Equipped with Three-Dimensional Arrangement of Magnets

2007 ◽  
Vol 329 ◽  
pp. 761-766 ◽  
Author(s):  
Y. Zhang ◽  
Masato Yoshioka ◽  
Shin-Ichiro Hira
Keyword(s):  
High Speed ◽  
Permanent Magnets ◽  
Three Dimensional ◽  
Video Camera ◽  
High Speed Video ◽  
High Speed Video Camera ◽  
The Media ◽  
Set Up

At present, a commercially available magnetic barrel machine equipped with permanent magnets has some faults arising from constructional reason. That is, grinding or finishing ability is different from place to place in the machining region, resulting in the limitation on the region we can use in the container of workpieces. Therefore, in this research, authors made the new magnetic barrel machine equipped with three dimensional (3D) magnet arrangement to overcome these faults. The grinding ability of the new 3D magnetic barrel machine converted was experimentally examined, and compared with that of the traditional magnetic barrel machine. As a result, it was shown that we can use much broader region in the new 3D machine. It was also shown that the grinding ability became higher. The distribution of barrel media in action was recorded by means of a high speed video camera. It was clarified that the media rose up higher and were distributed more uniformly in the container by the effect of the magnet block newly set up. It was supposed that this must be the reason for the above-mentioned improvement of grinding ability.

Effect of Nanosized Filler on Matrix Dominated Properties of Fiber Reinforced Epoxy

2011 ◽  
Author(s):  
Yuanxin Zhou ◽  
Shaik Jeelani
Keyword(s):  
Compression Strength ◽  
High Speed ◽  
High Vacuum ◽  
Trapped Air ◽  
Nano Fiber ◽  
Open Hole ◽  
Set Up ◽  
Mechanical Agitator ◽  
Laminar Shear ◽  
Nanosized Filler

In this study, a high-intensity ultrasonic liquid processor was used to obtain a homogeneous molecular mixture of epoxy resin and carbon nano fiber. The carbon nano fibers were infused into the part A of SC-15 (diglycidylether of Bisphenol A) through sonic cavitations and then mixed with part B of SC-15 (cycloaliphatic amine hardener) using a high-speed mechanical agitator. The trapped air and reaction volatiles were removed from the mixture using high vacuum. Nanophased epoxy with 2 wt.% CNF was then utilized in a vacuum assisted resin transfer molding (VARTM) set up with carbon fabric to fabricate laminated composites. The effectiveness of CNF addition on matrix dominated properties of composites has been evaluated by compression, open hole compression and inter-laminar shear. The compression strength, open hole compression strength and ILS were improved by 21%, 23% and 15%, respectively as compared to the neat composite.

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