scholarly journals A dual-labeling probe to track functional mitochondria–lysosome interactions in live cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Qixin Chen ◽  
Hongbao Fang ◽  
Xintian Shao ◽  
Zhiqi Tian ◽  
Shanshan Geng ◽  
...  
Keyword(s):  
Fluorescence Intensity ◽  
Fluorescent Probes ◽  
Living Cells ◽  
Molecular Probe ◽  
Live Cells ◽  
Blue Fluorescence ◽  
Red Fluorescence ◽  
Dynamic Tracking ◽  
A Cell

AbstractMitochondria–lysosome interactions are essential for maintaining intracellular homeostasis. Although various fluorescent probes have been developed to visualize such interactions, they remain unable to label mitochondria and lysosomes simultaneously and dynamically track their interaction. Here, we introduce a cell-permeable, biocompatible, viscosity-responsive, small organic molecular probe, Coupa, to monitor the interaction of mitochondria and lysosomes in living cells. Through a functional fluorescence conversion, Coupa can simultaneously label mitochondria with blue fluorescence and lysosomes with red fluorescence, and the correlation between the red–blue fluorescence intensity indicates the progress of mitochondria–lysosome interplay during mitophagy. Moreover, because its fluorescence is sensitive to viscosity, Coupa allowed us to precisely localize sites of mitochondria–lysosome contact and reveal increases in local viscosity on mitochondria associated with mitochondria–lysosome contact. Thus, our probe represents an attractive tool for the localization and dynamic tracking of functional mitochondria–lysosome interactions in living cells.

Non-centrosomal microtubule formation and measurement of minus end microtubule dynamics in A498 cells

1997 ◽  
Vol 110 (19) ◽  
pp. 2391-2401 ◽  
Author(s):  
A.M. Yvon ◽  
P. Wadsworth
Keyword(s):  
Cell Line ◽  
De Novo ◽  
Human Kidney ◽  
Light Level ◽  
Pause Duration ◽  
Living Cells ◽  
Live Cells ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
A Cell

Experiments performed on a cell line (A498) derived from a human kidney carcinoma revealed non-centrosomal microtubules in the peripheral lamella of many cells. These short microtubules were observed in glutaraldehyde-fixed cells by indirect immunofluorescence, and in live cells injected with rhodamine-labeled tubulin. The non-centrosomal microtubules were observed to form de novo in living cells, and their complete disassembly was also observed. Low-light-level fluorescence microscopy, coupled to imaging software, was utilized to record and measure the dynamic behavior of both ends of the non-centrosomal microtubules in these cells. For each, the plus end was differentiated from the minus end using the ratio of their transition frequencies and by measuring total assembly at each end. For comparative purposes, dynamics of the plus ends of centrosomally nucleated microtubules were also analyzed in this cell line. Our data reveal several striking differences between the plus and minus ends. The average pause duration was nearly 4-fold higher at the minus ends; the percentage of time spent in pause was 92% at the minus ends, compared to 55% at plus ends. Dynamicity was decreased 4-fold at the minus ends, and the average number of events per minute was reduced from 7.0 at the plus end to 1.5 at the minus ends. The minus ends also showed a 6-fold decrease in frequency of catastrophe over the plus ends. These data demonstrate that in living cells, microtubules can form at sites distant from the perinuclear microtubule organizing center, and once formed, non-centrosomal microtubules can persist for relatively long periods.

scholarly journals Graphene oxide-enhanced cytoskeleton imaging and mitosis tracking

2017 ◽  
Vol 53 (23) ◽  
pp. 3373-3376 ◽  
Author(s):  
Qian-Ru Li ◽  
Jin-Biao Jiao ◽  
Li-Li Li ◽  
Xiao-Peng He ◽  
Yi Zang ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Live Cells ◽  
Peptide Probe ◽  
Dynamic Tracking ◽  
A Cell

Graphene oxide enhances the imaging ability of a cell-impermeable peptide probe that targets microtubules, enabling the dynamic tracking of mitosis in live cells.

scholarly journals Permeant Fluorescent Probes Visualize the Activation of SARM1 and Uncover an Anti-neurodegenerative Drug Candidate

2021 ◽  
Author(s):  
Wan Hua Li ◽  
Ke Huang ◽  
Yang Cai ◽  
Qian Wen Wang ◽  
Wen Jie Zhu ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Neurodegenerative Disorders ◽  
Drug Target ◽  
Axonal Degeneration ◽  
Live Cells ◽  
Drug Candidate ◽  
Library Screening ◽  
Inactive Conformation ◽  
A Cell ◽  
Covalent Inhibitor

AbstractSARM1 regulates axonal degeneration through its NAD-metabolizing activity and is a drug target for neurodegenerative disorders. We designed and synthesized fluorescent conjugates of styryl derivative with pyridine to serve as substrates of SARM1, which exhibited large red-shifts after conversion. With the conjugates, SARM1 activation was visualized in live cells following elevation of endogenous NMN or treatment with a cell-permeant NMN-analog. In neurons, imaging documented SARM1 activation preceded vincristine-induced axonal degeneration by hours. Library screening identified a derivative of nisoldipine as a covalent inhibitor of SARM1 that reacted with Cys311 in its Armadillo-domain and blocked its NMN-activation, protecting axons from degeneration. CryoEM showed that SARM1 was locked into an inactive conformation by the inhibitor, uncovering an unsuspected neuroprotective mechanism of dihydropyridines.

scholarly journals Permeant fluorescent probes visualize the activation of SARM1 and uncover an anti-neurodegenerative drug candidate

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Wan Hua Li ◽  
Ke Huang ◽  
Yang Cai ◽  
Qian Wen Wang ◽  
Wen Jie Zhu ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Neurodegenerative Disorders ◽  
Drug Target ◽  
Axonal Degeneration ◽  
Live Cells ◽  
Drug Candidate ◽  
Library Screening ◽  
Inactive Conformation ◽  
A Cell ◽  
Covalent Inhibitor

SARM1 regulates axonal degeneration through its NAD-metabolizing activity and is a drug target for neurodegenerative disorders. We designed and synthesized fluorescent conjugates of styryl derivative with pyridine to serve as substrates of SARM1, which exhibited large red-shifts after conversion. With the conjugates, SARM1 activation was visualized in live cells following elevation of endogenous NMN or treatment with a cell-permeant NMN-analog. In neurons, imaging documented mouse SARM1 activation preceded vincristine-induced axonal degeneration by hours. Library screening identified a derivative of nisoldipine as a covalent inhibitor of SARM1 that reacted with the cysteines, especially Cys311 in its ARM domain and blocked its NMN-activation, protecting axons from degeneration. The Cryo-EM structure showed that SARM1 was locked into an inactive conformation by the inhibitor, uncovering a potential neuroprotective mechanism of dihydropyridines.

scholarly journals Discovery of catalytic-site-fluorescent probes for tracing phosphodiesterase 5 in living cells

RSC Advances ◽  
2021 ◽  
Vol 11 (51) ◽  
pp. 31967-31971
Author(s):  
Meiying Qiu ◽  
Deyan Wu ◽  
Yi-You Huang ◽  
Yue Huang ◽  
Qian Zhou ◽  
...  
Keyword(s):  
Drug Screening ◽  
Fluorescent Probes ◽  
Drug Target ◽  
Living Cells ◽  
Catalytic Site ◽  
Live Cells ◽  
Tissue Slices ◽  
Phosphodiesterase 5

To enhance the understanding of PDE5 as the drug target. Herein, we designed catalytic-site-fluorescent probes that can be applied to PDE5 visualization in live cells and tissue slices, implying the potential in diagnosis and drug screening.

scholarly journals Translocation or just location? Pseudopodia affect fluorescent signals

2009 ◽  
Vol 184 (2) ◽  
pp. 197-203 ◽  
Author(s):  
Sharon Dewitt ◽  
Richard L. Darley ◽  
Maurice B. Hallett
Keyword(s):  
Optical Properties ◽  
Fluorescent Probes ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Probe Signal ◽  
Fluorescent Signal ◽  
Geometrical Properties ◽  
Dynamic Events ◽  
A Cell ◽  
Protein Constructs

The use of fluorescent probes is one of the most powerful techniques for gaining spatial and temporal knowledge of dynamic events within living cells. Localized increases in the signal from cytosolic fluorescent protein constructs, for example, are frequently used as evidence for translocation of proteins to specific sites within the cell. However, differences in optical and geometrical properties of cytoplasm can influence the recorded intensity of the probe signal. Pseudopodia are especially problematic because their cytoplasmic properties can cause abrupt increases in fluorescent signal of both GFP and fluorescein. Investigators should therefore be cautious when interpreting fluorescence changes within a cell, as these can result from either translocation of the probe or changes in the optical properties of the milieu surrounding the probe.

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.

scholarly journals Structure modulation on fluorescent probes for biothiols and the reversible imaging of glutathione in living cells

RSC Advances ◽  
2021 ◽  
Vol 11 (34) ◽  
pp. 21116-21126
Author(s):  
Yu Li ◽  
Li Chen ◽  
Yan Zhu ◽  
Liming Chen ◽  
Xianglin Yu ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Fluorescent Probes ◽  
Living Cells ◽  
Structure Modulation

A reversible fluorescent probe for GSH was obtained through structure modulation, by which the intracellular GSH fluctuation was imaged.

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.

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