A semisynthetic fluorescent protein assembly-based FRET probe for real-time profiling of cell membrane protease functions in situ

2019 ◽  
Vol 55 (15) ◽  
pp. 2218-2221 ◽  
Author(s):  
Sujuan Sun ◽  
Yanan Liu ◽  
Julan Xia ◽  
Miao Wang ◽  
Rui Tang ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Real Time ◽  
Fluorescent Protein ◽  
Protein Assembly

A semisynthetic fluorescent protein assembly-based FRET probe (sFPAP) was proposed for cell membrane protease function assay.

Detection of Mitochondrial Caspase Activity in Real Time In Situ in Live Cells

2004 ◽  
Vol 10 (4) ◽  
pp. 442-448 ◽  
Author(s):  
Yingpei Zhang ◽  
Catherine Haskins ◽  
Marisa Lopez-Cruzan ◽  
Jianhua Zhang ◽  
Victoria E. Centonze ◽  
...  
Keyword(s):  
Real Time ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Subcellular Location ◽  
Live Cells ◽  
Excitation Peak ◽  
Caspase Substrate

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP–caspase 3 substrate–YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.

Real-time effects of Cd(ii) on the cellular membrane permeability

The Analyst ◽  
2021 ◽  
Vol 146 (19) ◽  
pp. 5973-5979
Author(s):  
Biao Zhang ◽  
Na Pan ◽  
Xiaoyin Fan ◽  
Liping Lu ◽  
Xiayan Wang
Keyword(s):  
Cell Membrane ◽  
Membrane Permeability ◽  
Real Time ◽  
Cellular Membrane ◽  
Cell Membrane Permeability ◽  
Time Effects

Using SECM to determine the cell membrane permeability has the advantages of being real-time, in situ and sensitive. Compared with x-scan study, DPV technology shows a higher performance in dectecting changes in the membrane permeability.

In Situ and Real-Time SFG Measurements Revealing Organization and Transport of Cholesterol Analogue 6-Ketocholestanol in a Cell Membrane

2014 ◽  
Vol 5 (3) ◽  
pp. 419-424 ◽  
Author(s):  
Sulan Ma ◽  
Hongchun Li ◽  
Kangzhen Tian ◽  
Shuji Ye ◽  
Yi Luo
Keyword(s):  
Cell Membrane ◽  
Real Time ◽  
A Cell

Amide I SFG Spectral Line Width Probes the Lipid–Peptide and Peptide–Peptide Interactions at Cell Membrane In Situ and in Real Time

Langmuir ◽  
2018 ◽  
Vol 34 (25) ◽  
pp. 7554-7560 ◽  
Author(s):  
Baixiong Zhang ◽  
Junjun Tan ◽  
Chuanzhao Li ◽  
Jiahui Zhang ◽  
Shuji Ye
Keyword(s):  
Cell Membrane ◽  
Spectral Line ◽  
Real Time ◽  
Line Width ◽  
Spectral Line Width ◽  
Peptide Interactions

Monitoring of tumor burden in vivo by optical imaging in a xenograft SCID mouse model: evaluation of two fluorescent proteins of the GFP-superfamily

2018 ◽  
Vol 60 (3) ◽  
pp. 315-326 ◽  
Author(s):  
Ingrid Böhm ◽  
Stephan Gehrke ◽  
Beate Kleb ◽  
Martin Hungerbühler ◽  
Rolf Müller ◽  
...  
Keyword(s):  
Mouse Model ◽  
Real Time ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Severe Combined Immunodeficiency ◽  
Liver Parenchyma ◽  
Human Malignant Melanoma ◽  
Signal Intensities

Background Mouse models of human-malignant-melanoma (MM) are important tools to study tumor dynamics. The enhanced green fluorescent protein (EGFP) is widely used in molecular imaging approaches, together with optical scanners, and fluorescence imaging. Purpose Currently, there are no data available as to whether other fluorescent proteins are more suitable. The goal of this preclinical study was to analyze two fluorescent proteins of the GFP superfamily under real-time in vivo conditions using fluorescence reflectance imaging (FRI). Material and Methods The human melanoma cell line MeWo was stable transfected with one plasmid: pEGFP-C1 or pDsRed1-N1. We investigated two severe combined immunodeficiency (SCID)-mice groups: A (solid xenografts) and B (xenografts as metastases). After three weeks, the animals were weekly imaged by FRI. Afterwards the mice were euthanized and metastases were imaged in situ: to quantify the cutis-dependent reduction of emitted light, we compared signal intensities obtained by metastases in vivo with signal intensities obtained by in situ liver parenchyma preparations. Results More than 90% of cells were stable transfected. EGFP-/DsRed-xenograft tumors had identical growth kinetics. In vivo the emitted light by DsRed tumors/metastases was much brighter than by EGFP. DsRed metastases were earlier (3 vs. 5 weeks) and much more sensitive detectable than EGFP metastases. Cutis-dependent reduction of emitted light was greater in EGFP than in DsRed mice (tenfold). Autofluorescence of DsRed was lower than of EGFP. Conclusion We established an in vivo xenograft mouse model (DsRed-MeWo) that is reliable, reproducible, and superior to the EGFP model as a preclinical tool to study innovative therapies by FRI under real-time in vivo conditions.

Live imaging of cell membrane-localized MT1-MMP activity on a microfluidic chip

2018 ◽  
Vol 54 (81) ◽  
pp. 11435-11438 ◽  
Author(s):  
Nan Li ◽  
Weifei Zhang ◽  
Ling Lin ◽  
Ziyi He ◽  
Mashooq Khan ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Real Time ◽  
Microfluidic Chip ◽  
Live Imaging ◽  
Activatable Probe

We designed an enzyme-activatable probe for real time in situ tracking of MT1-MMP activity.

Probing the Dynamic Self-Assembly Behaviour of Photoswitchable Wormlike Micelles in Real Time

2018 ◽  
Author(s):  
Elaine A. Kelly ◽  
Judith E. Houston ◽  
Rachel Evans
Keyword(s):  
Real Time ◽  
Absorption Spectroscopy ◽  
Self Assembly ◽  
Uv Light ◽  
Wormlike Micelles ◽  
Tetraethylene Glycol ◽  
Light Illumination ◽  
First Time ◽  
Dependent Processes

Understanding the dynamic self-assembly behaviour of azobenzene photosurfactants (AzoPS) is crucial to advance their use in controlled release applications such as<i></i>drug delivery and micellar catalysis. Currently, their behaviour in the equilibrium <i>cis-</i>and <i>trans</i>-photostationary states is more widely understood than during the photoisomerisation process itself. Here, we investigate the time-dependent self-assembly of the different photoisomers of a model neutral AzoPS, <a>tetraethylene glycol mono(4′,4-octyloxy,octyl-azobenzene) </a>(C<sub>8</sub>AzoOC<sub>8</sub>E<sub>4</sub>) using small-angle neutron scattering (SANS). We show that the incorporation of <i>in-situ</i>UV-Vis absorption spectroscopy with SANS allows the scattering profile, and hence micelle shape, to be correlated with the extent of photoisomerisation in real-time. It was observed that C<sub>8</sub>AzoOC<sub>8</sub>E<sub>4</sub>could switch between wormlike micelles (<i>trans</i>native state) and fractal aggregates (under UV light), with changes in the self-assembled structure arising concurrently with changes in the absorption spectrum. Wormlike micelles could be recovered within 60 seconds of blue light illumination. To the best of our knowledge, this is the first time the degree of AzoPS photoisomerisation has been tracked <i>in</i><i>-situ</i>through combined UV-Vis absorption spectroscopy-SANS measurements. This technique could be widely used to gain mechanistic and kinetic insights into light-dependent processes that are reliant on self-assembly.

The role of (bio)surfactant sorption in promoting the bioavailability of nutrients localized at the solid-water interface

1999 ◽  
Vol 39 (7) ◽  
pp. 91-98 ◽  
Author(s):  
Ryan N. Jordan ◽  
Eric P. Nichols ◽  
Alfred B. Cunningham
Keyword(s):  
Mass Transfer ◽  
Cell Membrane ◽  
Substrate Concentration ◽  
Water Interface ◽  
Industrial Systems ◽  
Solid Liquid Interface ◽  
Solid Liquid ◽  
Surfactant Sorption

Bioavailability is herein defined as the accessibility of a substrate by a microorganism. Further, bioavailability is governed by (1) the substrate concentration that the cell membrane “sees,” (i.e., the “directly bioavailable” pool) as well as (2) the rate of mass transfer from potentially bioavailable (e.g., nonaqueous) phases to the directly bioavailable (e.g., aqueous) phase. Mechanisms by which sorbed (bio)surfactants influence these two processes are discussed. We propose the hypothesis that the sorption of (bio)surfactants at the solid-liquid interface is partially responsible for the increased bioavailability of surface-bound nutrients, and offer this as a basis for suggesting the development of engineered in-situ bioremediation technologies that take advantage of low (bio)surfactant concentrations. In addition, other industrial systems where bioavailability phenomena should be considered are addressed.

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