Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P 2 ] is a low-abundance phosphoinositide presumed to be localized to endosomes and lysosomes, where it recruits cytoplasmic peripheral proteins and regulates endolysosome-localized membrane channel activity. Cells lacking PI(3,5)P 2 exhibit lysosomal trafficking defects, and human mutations in the PI(3,5)P 2 -metabolizing enzymes cause lysosome-related diseases. The spatial and temporal dynamics of PI (3,5)P 2 , however, remain unclear due to the lack of a reliable detection method. Of the seven known phosphoinositides, only PI (3,5)P 2 binds, in the low nanomolar range, to a cytoplasmic phosphoinositide-interacting domain (ML1N) to activate late endosome and lysosome (LEL)-localized transient receptor potential Mucolipin 1 (TRPML1) channels. Here, we report the generation and characterization of a PI(3,5)P 2 -specific probe, generated by the fusion of fluorescence tags to the tandem repeats of ML1N. The probe was mainly localized to the membranes of Lamp1-positive compartments, and the localization pattern was dynamically altered by either mutations in the probe, or by genetically or pharmacologically manipulating the cellular levels of PI(3,5)P 2 . Through the use of time-lapse live-cell imaging, we found that the localization of the PI(3,5)P 2 probe was regulated by serum withdrawal/addition, undergoing rapid changes immediately before membrane fusion of two LELs. Our development of a PI(3,5)P 2 -specific probe may facilitate studies of both intracellular signal transduction and membrane trafficking in the endosomes and lysosomes.TRP channel | PIKfyve | vesicle fusion P hosphorylated phosphoinositide lipids are produced on the cytosolic side of cellular lipid bilayer membranes (1, 2). There are seven different known phosphoinositides lipids, which localize to distinct membrane subdomains to regulate organellespecific membrane signaling pathways and membrane-trafficking events (1, 2). One such phosphoinositide lipid is phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], which is predominantly localized to the plasma membrane (PM). PI(4,5)P 2 serves as a permissive cofactor that is required for the activity of PM channels/transporters, as a precursor for the generation of second messengers, and as a PM recruiter for cytosolic peripheral proteins (3, 4). Likewise, PI(3,4)P 2 and PI(3,4,5)P 3 are transiently produced at the PM to regulate various signaling effectors, such as v-akt-murine thymoma viral oncogene homolog 1 (Akt) (1, 5). Conversely, PI(3)P is primarily found on early endosomes, phagosomes, and autophagosomes to regulate the maturation of these compartments (5, 6).Unlike the aforementioned phosphoinositides, the subcellular localization and functions of PI(3,5)P 2 are poorly understood. PI (3,5)P 2 is proposed to be mainly localized to late endosomes and lysosomes (LELs) (7), and also to early endosomes (8), based on the location of its synthesizing enzyme complex, which in mammalian cells consists of the phosphoinositide kinase, FYVE finger-containing (...

Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the field of cell biology and has become an indispensable tool in current biological studies. In order to minimize the disturbance to the biological system in live cell imaging, the probe concentration needs to be significantly lower than the analyte concentration. Because of this, any irreversible reaction-based GSH probe can only provide qualitative results within a short reaction time and will exhibit maximum response regardless of the GSH concentration if the reaction is completed. A reversible reaction-based probe with an appropriate equilibrium constant allows measurement of an analyte at much higher concentrations and, thus, is a prerequisite for GSH quantification inside cells. In this contribution, we report the first fluorescent probe—ThiolQuant Green (TQ Green)—for quantitative imaging of GSH in live cells. Due to the reversible nature of the reaction between the probe and GSH, we are able to quantify mM concentrations of GSH with TQ Green concentrations as low as 20 nM. Furthermore, the GSH concentrations measured using TQ Green in 3T3-L1, HeLa, HepG2, PANC-1, and PANC-28 cells are reproducible and well correlated with the values obtained from cell lysates. TQ Green imaging can also resolve the changes in GSH concentration in PANC-1 cells upon diethylmaleate (DEM) treatment. In addition, TQ Green can be conveniently applied in fluorescence activated cell sorting (FACS) to measure GSH level changes. Through this study, we not only demonstrate the importance of reaction reversibility in designing quantitative reaction-based fluorescent probes but also provide a practical tool to facilitate redox biology studies.

A fundamental question regarding autophagosome formation is how the shape of the double-membrane autophagosomal vesicle is generated. Here we show that in mammalian cells assembly of an actin scaffold inside the isolation membrane (the autophagosomal precursor) is essential for autophagosomal membrane shaping. Actin filaments are depolymerized shortly after starvation and actin is assembled into a network within the isolation membrane. When formation of actin puncta is disrupted by an actin polymerization inhibitor or by knocking down the actin-capping protein CapZβ, isolation membranes and omegasomes collapse into mixed-membrane bundles. Formation of actin puncta is PtdIns(3)P dependent, and inhibition of PtdIns(3)P formation by treating cells with the PI(3)K inhibitor 3-MA, or by knocking down Beclin-1, abolishes the formation of actin puncta. Binding of CapZ to PtdIns(3)P, which is enriched in omegasomes, stimulates actin polymerization. Our findings illuminate the mechanism underlying autophagosomal membrane shaping and provide key insights into how autophagosomes are formed.

Protein-protein interactions (PPIs) play a central role in most biological processes, and therefore represent an important class of targets for therapeutic development. However, disrupting PPIs using small-molecule inhibitors (SMIs) is challenging and often deemed as "undruggable." We developed a cell-based functional assay for highthroughput screening to identify SMIs for steroid receptor coactivator-3 (SRC-3 or AIB1), a large and mostly unstructured nuclear protein.Without any SRC-3 structural information, we identified SI-2 as a highly promising SMI for SRC-3. SI-2 meets all of the criteria of Lipinski's rule [Lipinski et al. (2001) Adv Drug Deliv Rev 46(1-3):3-26] for a drug-like molecule and has a half-life of 1 h in a pharmacokinetics study and a reasonable oral availability in mice. As a SRC-3 SMI, SI-2 can selectively reduce the transcriptional activities and the protein concentrations of SRC-3 in cells through direct physical interactions with SRC-3, and selectively induce breast cancer cell death with IC 50 values in the low nanomolar range (3-20 nM), but not affect normal cell viability. Furthermore, SI-2 can significantly inhibit primary tumor growth and reduce SRC-3 protein levels in a breast cancer mouse model. In a toxicology study, SI-2 caused minimal acute cardiotoxicity based on a hERG channel blocking assay and an unappreciable chronic toxicity to major organs based on histological analyses. We believe that this work could significantly improve breast cancer treatment through the development of "first-in-class" drugs that target oncogenic coactivators.steroid receptor coactivator | small-molecule inhibitor | breast cancer | drug development | protein-protein interactions P rotein-protein interactions (PPIs) play a central role in most biological processes, and therefore represent an important class of targets for therapeutic development (1). Biologics-based therapeutics, such as antibodies, exemplify success in PPI regulation (2). However, antibodies usually can only be applied to protein targets on cell surfaces because of their impermeability to plasma membranes (2). Although small-molecule drugs can readily cross membranes, applying small-molecule inhibitors (SMIs) to disrupt PPIs is a challenging task because ∼750-1,500 Å 2 of protein surface area is involved at the interface of PPIs (3), which is too large for SMIs to cover. In addition, these interacting protein surfaces do not have pocket-like small-molecule binding sites (2). Therefore, these PPI sites are deemed as "undruggable" targets for SMIs. The Holy Grail of drug development is to render small molecules the power of biologics to regulate PPIs.The current strategies for designing small-molecule PPI inhibitors primarily rely on the structural information of the protein targets (4). Clackson and Wells discovered that only a small set of residues at the PPI interface are critical for their interactions, known as "hot spots" (5). Therefore, current drug design for PPIs is mainly focused on small hot spots that can be covered by a dru...

Triple negative breast cancer (TNBC) has the poorest prognosis of all types of breast cancer and currently lacks efficient targeted therapy. Chemotherapy is the traditional standard-of-care for TNBC, but is frequently accompanied by severe side effects. Despite the fact that high expression of steroid receptor coactivator 3 (SRC–3) is correlated with poor survival in estrogen receptor positive breast cancer patients, its role in TNBC has not been extensively investigated. Here, we show that high expression of SRC–3 correlates with both poor overall survival and post progression survival in TNBC patients, suggesting that SRC–3 can serve as a prognostic marker for TNBC. Furthermore, we demonstrated that bufalin, a SRC–3 small molecule inhibitor, when introduced even at nM concentrations, can significantly reduce TNBC cell viability and motility. However, because bufalin has minimal water solubility, its in vivo application is limited. Therefore, we developed a water soluble prodrug, 3-phospho-bufalin, to facilitate its in vivo administration. In addition, we demonstrated that 3-phospho-bufalin can effectively inhibit tumor growth in an orthotopic TNBC mouse model, suggesting its potential application as a targeted therapy for TNBC treatment.

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Aims: Quantitative imaging of glutathione (GSH) with high spatial and temporal resolution is essential for studying the roles of GSH in redox biology. To study the long-standing question of compartmentalization of GSH, especially its distribution between the nucleus and cytosol, an organelle-targeted quantitative probe is needed. Results: We developed a reversible reaction-based ratiometric fluorescent probe-HaloRT-that can quantitatively measure GSH dynamics with subcellular resolution in real time. Using HaloRT, we quantitatively measured the GSH concentrations in the nucleus and cytosol of HeLa cells and primary hepatocytes under different treatment conditions and found no appreciable concentration gradients between these two organelles. Innovation and Conclusion: We developed the first reversible ratiometric GSH probe that can be universally targeted to any organelle of interest. Taking advantage of this new tool, we provided definitive evidence showing that GSH concentrations are not significantly different between the nucleus and cytosol, challenging the view of nuclear compartmentalization of GSH.

Imaging hydrogen sulfide (H2S) at the subcellular resolution will greatly improve the understanding of functions of this signaling molecule. Taking advantage of the protein labeling technologies, we report a general strategy for the development of organelle specific H2S probes, which enables sub-cellular H2S imaging essentially in any organelles of interest.