scholarly journals Genetically encoded live cell sensor for tyrosinated microtubules

2020 ◽  
Author(s):  
Shubham Kesarwani ◽  
Prakash Lama ◽  
Anchal Chandra ◽  
P. Purushotam Reddy ◽  
AS Jijumon ◽  
...  
Keyword(s):  
Living Cells ◽  
Live Cell ◽  
Live Cells ◽  
Yeast Display ◽  
Post Translational Modification ◽  
Alpha Tubulin ◽  
Microtubule Stability ◽  
Specific Manner ◽  
Potential Applications ◽  
Stability Dynamics

AbstractMicrotubule cytoskeleton exists in various biochemical forms in different cells due to tubulin post-translational modification (PTMs). These PTMs are known to affect microtubule stability, dynamics and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present there exist no tool that can specifically mark tubulin PTMs in live cells, thus severely limiting our understanding of tubulin PTMs. Using yeast display library, we identified a binder against terminal tyrosine of alpha tubulin, a unique PTM site. Extensive characterization validates the robustness and non-perturbing nature of our binder as tyrosination sensor, a live cell tubulin nanobody specific towards tyrosinated or unmodified microtubules. Using which, in real time we followed nocodazole, colchicine and vincristine induced depolymerization events of unmodified microtubules, and found each distinctly perturb microtubule polymer. Together, our work describes the tyrosination sensor and potential applications to study microtubule and PTM processes in living cells.

scholarly journals Genetically encoded live-cell sensor for tyrosinated microtubules

2020 ◽  
Vol 219 (10) ◽  
Author(s):  
Shubham Kesarwani ◽  
Prakash Lama ◽  
Anchal Chandra ◽  
P. Purushotam Reddy ◽  
A.S. Jijumon ◽  
...  
Keyword(s):  
Real Time ◽  
Posttranslational Modifications ◽  
Living Cells ◽  
Live Cell ◽  
Yeast Display ◽  
Cellular Functions ◽  
Microtubule Stability ◽  
Specific Manner ◽  
Potential Applications ◽  
Stability Dynamics

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin posttranslational modifications (PTMs). Tubulin PTMs are known to affect microtubule stability, dynamics, and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present, there exists no tool that can specifically mark tubulin PTMs in living cells, thus severely limiting our understanding of their dynamics and cellular functions. Using a yeast display library, we identified a binder against terminal tyrosine of α-tubulin, a unique PTM site. Extensive characterization validates the robustness and nonperturbing nature of our binder as tyrosination sensor, a live-cell tubulin nanobody specific towards tyrosinated microtubules. Using this sensor, we followed nocodazole-, colchicine-, and vincristine-induced depolymerization events of tyrosinated microtubules in real time and found each distinctly perturbs the microtubule polymer. Together, our work describes a novel tyrosination sensor and its potential applications to study the dynamics of microtubule and their PTM processes in living cells.

scholarly journals The changing face of live-cell imaging: From phase contrast to single photon

2004 ◽  
Vol 26 (3) ◽  
pp. 30-34
Author(s):  
Mark Jepson ◽  
Darran Clements
Keyword(s):  
Live Cell Imaging ◽  
Phase Contrast ◽  
Cell Imaging ◽  
Cell Function ◽  
Molecular Level ◽  
Single Photon ◽  
Living Cells ◽  
Live Cell ◽  
Live Cells ◽  
The Past

The imaging of live cells using light microscopy has come a long way from the early days of phase contrast. There have been many exciting developments in technology that now deliver live-cell images that previously would not have been thought possible. The study of dynamic processes right down to the molecular level as they happen in living cells is now common practice in the drive to understand cell function. So what has happened over the past 50 years to make live-cell imaging so much more accessible today?

Increased microtubule stability and alpha tubulin acetylation in cells transfected with microtubule-associated proteins MAP1B, MAP2 or tau

1992 ◽  
Vol 103 (4) ◽  
pp. 953-964 ◽  
Author(s):  
R. Takemura ◽  
S. Okabe ◽  
T. Umeyama ◽  
Y. Kanai ◽  
N.J. Cowan ◽  
...  
Keyword(s):  
Post Translational Modification ◽  
3T3 Cells ◽  
Microtubule Organization ◽  
Alpha Tubulin ◽  
Transfected Cells ◽  
Microtubule Associated Proteins ◽  
Cos Cells ◽  
Tubulin Acetylation ◽  
Microtubule Stability ◽  
Associated Proteins

We previously transfected MAP2, tau and MAP1B cDNA into fibroblasts and have studied the effect of expression of these microtubule-associated proteins on microtubule organization. In this study, we examined some additional characteristics of microtubule bundles and arrays formed in fibroblasts transfected with these microtubule-associated proteins. It was found that microtubule bundles formed in MAP2c- or tau-transfected cells were stabilized against microtubule depolymerizing reagents and were enriched in acetylated alpha tubulin. When mouse MAP1B cDNA was expressed following transfection into COS cells, MAP1B was localized along microtubule arrays, but no extensive reorganization of microtubules such as bundle formation was observed, in agreement with our previous finding using HeLa and 3T3 cells. However, stabilization of microtubules was indicated: (a) microtubules in MAP1B-transfected cells were stabilized against a microtubule depolymerizing reagent, although stabilization was less efficient than that seen in MAP2c- or tau-transfected cells, and (b) microtubules in MAP1B-transfected cells were enriched in acetylated alpha tubulin. These results suggest that neuronal microtubule-associated proteins introduced into fibroblasts by cDNA transfection stabilize microtubules and affect the state of post-translational modification of tubulin.

4-dimensional, high-resolution imaging of living cells

1992 ◽  
Vol 50 (1) ◽  
pp. 416-417
Author(s):  
Shinya Inoué
Keyword(s):  
Light Microscope ◽  
Living Cells ◽  
Live Cells ◽  
Video Tape ◽  
Active Living ◽  
High Resolution Imaging ◽  
3 Dimensional ◽  
Dynamic Architecture ◽  
Resolution Imaging ◽  
Disk Memory

This paper reports progress of our effort to rapidly capture, and display in time-lapsed mode, the 3-dimensional dynamic architecture of active living cells and developing embryos at the highest resolution of the light microscope. Our approach entails: (A) real-time video tape recording of through-focal, ultrathin optical sections of live cells at the highest resolution of the light microscope; (B) repeat of A at time-lapsed intervals; (C) once each time-lapsed interval, an image at home focus is recorded onto Optical Disk Memory Recorder (OMDR); (D) periods of interest are selected using the OMDR and video tape records; (E) selected stacks of optical sections are converted into plane projections representing different view angles (±4 degrees for stereo view, additional angles when revolving stereos are desired); (F) analysis using A - D.

α-Methylene-β-Lactone Probe for Measuring Live-Cell Reactions of Small Molecules

2020 ◽  
Author(s):  
Lei Wang ◽  
Louis Riel ◽  
Bekim Bajrami ◽  
Bin Deng ◽  
Amy Howell ◽  
...  
Keyword(s):  
Mass Spectra ◽  
Live Cell ◽  
Live Cells ◽  
The Novel ◽  
Proteomics Analysis ◽  
Chemical Proteomics ◽  
Tandem Mass Spectra ◽  
Modified Peptides ◽  
Covalent Probes ◽  
Ht 29

The novel use of the α-methylene-β-lactone (MeLac) moiety as a warhead of multiple electrophilic sites is reported. In this study, we demonstrate that a MeLac-alkyne is a competent covalent probe and reacts with diverse proteins in live cells. Proteomics analysis of affinity-enriched samples identifies probe-reacted proteins, resolves their modified peptides/residues, and thus characterizes probe-protein reactions. Unique methods are developed to evaluate confidence in the identification of the reacted proteins and modified peptides. Tandem mass spectra of the peptides reveal that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael addition or acyl addition. A peptide-centric proteomics platform, using MeLac-alkyne as the measurement probe, successfully analyzes the Orlistat selectivity in live HT-29 cells. MeLac is a versatile warhead demonstrating enormous potential to expedite the development of covalent probes and inhibitors in interrogating protein (re)activity. MeLac-empowered platforms in chemical proteomics are widely adaptable for measuring the live-cell action of reactive molecules.

Potential Pharmaceutical and Food Applications of Postbiotics: A Review

2020 ◽  
Vol 21 (15) ◽  
pp. 1576-1587 ◽  
Author(s):  
Aziz H. Rad ◽  
Amin Abbasi ◽  
Hossein S. Kafil ◽  
Khudaverdi Ganbarov
Keyword(s):  
Functional Foods ◽  
Animal Health ◽  
Pharmaceutical Formulations ◽  
Live Cells ◽  
Beneficial Effects ◽  
Safe Product ◽  
Potential Applications ◽  
Food Applications ◽  
Potential Alternative ◽  
Bioactive Agents

In recent decades, functional foods with ingredients comprising probiotics, prebiotics and postbiotics have been gaining a lot of attention from scientists. Probiotics and postbiotics are usually applied in pharmaceutical formulations and/or commercial food-based products. These bioactive agents can be associated with host eukaryotic cells and have a key role in maintaining and restoring host health. The review describes the concept of postbiotics, their quality control and potential applications in pharmaceutical formulations and commercial food-based products for health promotion, prevention of disease and complementary treatment. Despite the effectiveness of probiotic products, researchers have introduced the concept of postbiotic to optimize their beneficial effects as well as to meet the needs of consumers to provide a safe product. The finding of recent studies suggests that postbiotics might be appropriate alternative agents for live probiotic cells and can be applied in medical, veterinary and food practice to prevent and to treat some diseases, promote animal health status and develop functional foods. Presently scientific literature confirms that postbiotics, as potential alternative agents, may have superiority in terms of safety relative to their parent live cells, and due to their unique characteristics in terms of clinical, technological and economical aspects, can be applied as promising tools in the drug and food industry for developing health benefits, and therapeutic aims.

scholarly journals 3D super-resolved imaging in live cells using sub-diffractive plasmonic localization of hybrid nanopillar arrays

Nanophotonics ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2847-2859
Author(s):  
Soojung Kim ◽  
Hyerin Song ◽  
Heesang Ahn ◽  
Seung Won Jun ◽  
Seungchul Kim ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Imaging Techniques ◽  
Optical Loss ◽  
Super Resolution ◽  
Living Cells ◽  
Live Cells ◽  
Complex Biological System ◽  
Observation Volume ◽  
Resolution Imaging ◽  
Nanopillar Arrays

AbstractAnalysing dynamics of a single biomolecule using high-resolution imaging techniques has been had significant attentions to understand complex biological system. Among the many approaches, vertical nanopillar arrays in contact with the inside of cells have been reported as a one of useful imaging applications since an observation volume can be confined down to few-tens nanometre theoretically. However, the nanopillars experimentally are not able to obtain super-resolution imaging because their evanescent waves generate a high optical loss and a low signal-to-noise ratio. Also, conventional nanopillars have a limitation to yield 3D information because they do not concern field localization in z-axis. Here, we developed novel hybrid nanopillar arrays (HNPs) that consist of SiO2 nanopillars terminated with gold nanodisks, allowing extreme light localization. The electromagnetic field profiles of HNPs are obtained through simulations and imaging resolution of cell membrane and biomolecules in living cells are tested using one-photon and 3D multiphoton fluorescence microscopy, respectively. Consequently, HNPs present approximately 25 times enhanced intensity compared to controls and obtained an axial and lateral resolution of 110 and 210 nm of the intensities of fluorophores conjugated with biomolecules transported in living cells. These structures can be a great platform to analyse complex intracellular environment.

scholarly journals Fluorescent Probes for Live Cell Thiol Detection

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3575
Author(s):  
Shenggang Wang ◽  
Yue Huang ◽  
Xiangming Guan
Keyword(s):  
Fluorescent Probes ◽  
Biological System ◽  
High Sensitivity ◽  
Intracellular Distribution ◽  
Live Cell ◽  
Live Cells ◽  
High Demand ◽  
Non Invasive

Thiols play vital and irreplaceable roles in the biological system. Abnormality of thiol levels has been linked with various diseases and biological disorders. Thiols are known to distribute unevenly and change dynamically in the biological system. Methods that can determine thiols’ concentration and distribution in live cells are in high demand. In the last two decades, fluorescent probes have emerged as a powerful tool for achieving that goal for the simplicity, high sensitivity, and capability of visualizing the analytes in live cells in a non-invasive way. They also enable the determination of intracellular distribution and dynamitic movement of thiols in the intact native environments. This review focuses on some of the major strategies/mechanisms being used for detecting GSH, Cys/Hcy, and other thiols in live cells via fluorescent probes, and how they are applied at the cellular and subcellular levels. The sensing mechanisms (for GSH and Cys/Hcy) and bio-applications of the probes are illustrated followed by a summary of probes for selectively detecting cellular and subcellular thiols.

scholarly journals A rapid “on–off–on” mitochondria-targeted phosphorescent probe for selective and consecutive detection of Cu2+ and cysteine in live cells and zebrafish

RSC Advances ◽  
2021 ◽  
Vol 11 (13) ◽  
pp. 7610-7620 ◽  
Author(s):  
Peipei Deng ◽  
Yongyan Pei ◽  
Mengling Liu ◽  
Wenzhu Song ◽  
Mengru Wang ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
Living Cells ◽  
Live Cells ◽  
Mitochondria Targeting

An iridium(iii) complex-based mitochondria targeting phosphorescent probe for selectively detecting Cu2+ and Cys in aqueous solution, living cells and zebrafish has been developed.

scholarly journals Ligand-Mediated Assembly and Real-Time Cellular Dynamics of Estrogen Receptor α-Coactivator Complexes in Living Cells

2001 ◽  
Vol 21 (13) ◽  
pp. 4404-4412 ◽  
Author(s):  
David L. Stenoien ◽  
Anne C. Nye ◽  
Maureen G. Mancini ◽  
Kavita Patel ◽  
Martin Dutertre ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Real Time ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Steroid Receptor ◽  
Estrogen Receptor Α ◽  
Living Cells ◽  
Live Cells ◽  
Creb Binding Protein ◽  
High Accumulation

ABSTRACT Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor α (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-taggedlac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integratedlac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP–SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP–SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP–SRC-1, while antagonist additions diminish YFP–SRC-1–CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER–SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP–SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.

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