Development of small fluorescent probes for the analysis of autophagy kinetics.

Autophagy is a dynamic process that degrades subcellular constituents, and its activity is measured by autophagic flux. The tandem proteins RFP-GFP-LC3 and GFP-LC3-RFP-LC3ΔG, which enable the visualization of autophagic vacuoles of different stages by differences in their fluorescent color, are useful tools to monitor autophagic flux, but they require plasmid transfection. In this study, we hence aimed to develop a new method to monitor autophagic flux using small cell-permeable fluorescent probes. We previously developed two green-fluorescent probes, DALGreen and DAPGreen, which detect autolysosomes and multistep autophagic vacuoles, respectively. We here developed a red-fluorescent autophagic probe, named DAPRed, which recognizes various autophagic vacuoles. By the combinatorial use of these green- and red-fluorescent probes, we were able to readily detect autophagic flux. Furthermore, these probes were useful not only for the visualization of canonical autophagy but also for alternative autophagy. DAPRed was also applicable for the detection of autophagy in living organisms.

A tandem activity-based sensing and labeling strategy enables imaging of transcellular hydrogen peroxide signaling.

Reactive oxygen species (ROS) like hydrogen peroxide (H2O2) are transient species that have broad actions in signaling and stress, but spatioanatomical understanding of their biology remains insufficient. Here, we report a tandem activity-based sensing and labeling strategy for H2O2 imaging that enables capture and permanent recording of localized H2O2 fluxes. Peroxy Green-1 Fluoromethyl (PG1-FM) is a diffusible small-molecule probe that senses H2O2 by a boronate oxidation reaction to trigger dual release and covalent labeling of a fluorescent product, thus preserving spatial information on local H2O2 changes. This unique reagent enables visualization of transcellular redox signaling in a microglia-neuron coculture cell model, where selective activation of microglia for ROS production increases H2O2 in nearby neurons. In addition to identifying ROS-mediated cell-to-cell communication, this work provides a starting point for the design of chemical probes that can achieve high spatial fidelity by combining activity-based sensing and labeling strategies.

Small fluorescent molecules for monitoring autophagic flux.

We have developed two types of fluorescent probes, DALGreen and DAPGreen, for monitoring autophagy, that exhibit fluorescence upon being incorporated into autophagosomes. DALGreen enhances its fluorescence at acidic pH, which is favorable for monitoring late-phase autophagy, whereas DAPGreen remains fluorescent with almost constant brightness during the autophagic process. With these probes that stain autophagosomes as they are being formed, the real-time change of autophagic phenomena of live cells may be traced, which is an advantage over conventional approaches with small molecules that stain mature autophagosomes. The use of both dyes allows monitoring of the membrane dynamics of autophagy in any type of cell without the need for genetic engineering, and therefore, will be useful as a tool to study autophagic phenomena.

Live Cell Imaging of Mitochondrial Autophagy with a Novel Fluorescent Small Molecule.

There has been a growing interest in mitophagy, mitochondria-selective autophagy, which plays an essential role in maintaining intracellular homeostasis. We have developed a small-molecule fluorescent probe, Mtphagy Dye, for visualizing mitophagy, which was readily synthesized from a known perylene derivative, perylene-3,4-dicarboxylic anhydride. Mtphagy Dye has suitable fluorescent properties for detecting mitochondrial acidification during mitophagy in the long-wavelength region that does not damage mitochondria. Using Mtphagy Dye, we were able to visualize mitophagy with both cases of Parkin-dependent and -independent HeLa cells.

Secreted cerberus1 as a marker for quantification of definitive endoderm differentiation of the pluripotent stem cells.

To date, CXCR4 and E-cadherin double-positive cells detected by flow cytometry have been used to identify the differentiation of embryonic stem (ES) cells or induced pluripotent stem (iPS) cells into definitive endoderm (DE) lineages. Quantification of DE differentiation from ES/iPS cells by using flow cytometry is a multi-step procedure including dissociation of the cells, antibody reaction, and flow cytometry analysis. To establish a quick assay method for quantification of ES/iPS cell differentiation into the DE without dissociating the cells, we examined whether secreted Cerberus1 (Cer1) protein could be used as a marker. Cer1 is a secreted protein expressed first in the anterior visceral endoderm and then in the DE. The amount of Cer1 secreted correlated with the proportion of CXCR4+/E-Cadherin+ cells that differentiated from mouse ES cells. In addition, we found that human iPS cell-derived DE also expressed the secreted CER1 and that the expression level correlated with the proportion of SOX17+/FOXA2+ cells present. Taken together, these results show that Cer1 (or CER1) serves as a good marker for quantification of DE differentiation of mouse and human ES/iPS cells.