Biology subjects

Ezoe, T.

Publications and source records attributed to Ezoe, T..

3 recordsLinked to original sources

bioRxiv2021

A small-molecule fluorescent probe for live-cell imaging of endocytosis

Endocytosis involves plasma membrane-derived vesicles for the recycling of intra- and extracellular components. Increasing evidence suggests that endocytosis is related to maintaining intracellular homeostasis and defense against disease. Consequently, investigation of the endocytic pathway attracts considerable scientific interest. This study reports live-cell imaging of endocytosis using the newly-developed fluorescent probe ECGreen. We demonstrate that ECGreen is not membrane permeable and its fluorescence signal increases in acidic conditions. Because of these characteristics, ECGreen remains on the plasma membrane, and then shows increased fluorescence when it is internalized into the acidic vesicles formed in the endocytic process. ECGreen allows direct observation of the internalized vesicle; it is a valuable new probe for endocytic imaging.

biochemistry↗

bioRxiv2020

Amphipathic Fluorescent Dyes for Sensitive and Long-Term Monitoring of Plasma Membranes

The plasma membrane (PM) plays a critical role in many cellular processes, and PM dysfunction is a key biomarker related to the cell status and several diseases. Imaging techniques using small fluorescent probes have become increasingly important tools for visualizing living cells, particularly their PMs. Among the commercially available PM-specific probes, PKH dyes are widely used; however, the utility of these dyes is limited by their short membrane retention times and high cytotoxicity. Herein, PlasMem Bright Green and Red are implemented as new PM-specific fluorescent probes, which employ a polycyclic aromatic fluorophore to improve their retention ability and a strong acid moiety to reduce their transmembrane diffusion and cytotoxicity. We demonstrate that the long retention and low cytotoxicity of the PlasMem Bright dyes enable them to be applied for observing neuronal PMs and monitoring PM dynamics involving the endocytic pathway. Furthermore, we successfully detected mitochondria in nerve axons over long periods using PlasMem Bright dyes. Finally, the combined use of exosome staining probes and PlasMem Bright dyes allowed clear visualization of the endocytic pathway. TOC graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2020

New Lipophilic Fluorescent Dyes for Exosome Labeling: Monitoring of Cellular Uptake of Exosomes

We have developed three types of exosomal membrane binding fluorescent probes, Mem Dye-Green, Mem Dye-Red and Mem Dye-Deep Red, to monitor exosome uptake into cells. The dyes contain a cyanine group as a fluorescent scaffold, which allows for highly sensitive fluorescence imaging of the exosome. These dyes can also be used to observe the dynamics of exosomes in live cells. The use of PKH dyes (Figure 1), which are currently the most widely-used fluorescent probes for exosome labeling, has some limitations. For example, PKH dyes tend to aggregate to form exosome-like nanoparticles, and these nanoparticles are uptaken by cells. Moreover, Mehdi suggested that the use of PKH dyes triggers an enlargement of the exosome size owing to membrane fusion or intercalation. To overcome the limitations of PKH dyes, we introduce amphiphilic moieties to the cyanine. To investigate the characteristics of the Mem Dyes as exosome labeling probes, we perform nanoparticle tracking analysis (NTA), zeta potential measurement and confocal microscopy. The Mem Dyes show excellent performance for exosome labeling (no aggregation and less size shift). O_FIG O_LINKSMALLFIG WIDTH=91 HEIGHT=200 SRC="FIGDIR/small/931295v1_fig1.gif" ALT="Figure 1"> View larger version (8K): org.highwire.dtl.DTLVardef@e978aeorg.highwire.dtl.DTLVardef@1f208cdorg.highwire.dtl.DTLVardef@11fdb1dorg.highwire.dtl.DTLVardef@6a7373_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Chemical structure of PKH26. C_FIG

biochemistry↗