Project description:Spinning disc confocal microscopical research was conducted on living mating hyphae of the tetrapolar basidiomycete Schizophyllum commune. Haploid strains with either the same or different A and B mating-type genes and expressing differently labelled histone 2B were confronted. In the haploid hyphae histone 2B mCherry and histone 2B EGFP were visualized as red and green nuclei, respectively. In hyphae with the same A but different B genes, the red and green nuclei were observed next to each other. This indicated that nuclear migration between strains, regulated by the B mating type, had taken place. The compatible mating with different A and B genes produced a high number of mixed EFGP/mCherry, yellow nuclei. The mixed nuclei resulted from nearby divisions of nuclei encoding different histones and mating-type genes. During this process, the histones with the different labels were incorporated in the same nuclei, along with the heterodimerized transcription factors encoded by the different A mating-type genes and present around the nuclei. This led to the activation of the A-regulated pathway and indicated that different A genes are important to the cell cycle activation of a compatible mating. Consequently, a yellow nuclear pair stuck together, divided synchronously and proceeded in the migration hyphae towards the colony periphery, where the dikaryotization was promoted by branch formation from the migration hyphae.
Project description:Characterizing the long-term nanometer-scale interactions between lysosomes and mitochondria in live cells is essential for understanding their functions but remains challenging due to limitations of the existing fluorescent probes. Here, we develop cell-permeable organic fluorescent probes for lysosomes with excellent specificity and high photostability. We also use an existing Atto 647N dye with high brightness and excellent photostability to achieve specific labeling of mitochondria in live cells. Using these probes, we obtain dual-color structured illumination microscopy (SIM) images of dynamic physical lysosome-mitochondrion interactions in live cells at an ~90-nm resolution over a long time course of ~13 min. We successfully record the consecutive dynamic processes of lysosomal fusion and fission, as well as four types of physical lysosome-mitochondrion interactions by super-resolution imaging. Our probes provide an avenue for understanding the functions and the dynamic interplay of lysosomes and mitochondria in live cells.
Project description:Mitochondria, crucial intracellular organelles, are central to energy metabolism, signal transduction, apoptosis, calcium homeostasis, and a myriad of other biological processes, making them a focal point in diverse research fields. The capacity to fluorescently label and visually track mitochondria is crucial for understanding their biological roles. We present mulberrin-Cy3, a novel small molecule fluorescent probe that selectively labels mitochondria in animal cells, including cancer cells, with relative ease. This protocol details the synthesis of mulberrin-Cy3 and its use for visualizing mitochondria in living cells. The synthesis is straightforward and time-efficient, and the labeling method is more accessible than traditional approaches, providing a cost-effective option for mitochondrial visualization at room temperature. The labeling is rapid, with effective labeling achieved within 5 min of incubation. The fluorescent signal is stable and brighter, offering a significant advantage over existing methods. Mulberrin-Cy3 represents a promising mitochondrial labeling compound, providing researchers with a novel experimental tool to explore the complex biological functions of mitochondria. This innovation has the potential to significantly advance our comprehension of mitochondrial dynamics and their role in cellular health and disease.
Project description:Single-molecule localization microscopy (SMLM) achieves super-resolution imaging beyond the diffraction limit but critically relies on the use of photo-modulatable fluorescent probes. Here we report a general strategy for constructing cell-permeable photo-modulatable organic fluorescent probes for live-cell SMLM by exploiting the remarkable cytosolic delivery ability of a cell-penetrating peptide (rR)3R2. We develop photo-modulatable organic fluorescent probes consisting of a (rR)3R2 peptide coupled to a cell-impermeable organic fluorophore and a recognition unit. Our results indicate that these organic probes are not only cell permeable but can also specifically and directly label endogenous targeted proteins. Using the probes, we obtain super-resolution images of lysosomes and endogenous F-actin under physiological conditions. We resolve the dynamics of F-actin with 10 s temporal resolution in live cells and discern fine F-actin structures with diameters of ~80 nm. These results open up new avenues in the design of fluorescent probes for live-cell super-resolution imaging.
Project description:Lipids regulate a wide range of biological activities. Since their local concentrations are tightly controlled in a spatiotemporally specific manner, the simultaneous quantification of multiple lipids is essential for elucidation of the complex mechanisms of biological regulation. Here, we report a new method for the simultaneous in situ quantification of two lipid pools in mammalian cells using orthogonal fluorescent sensors. The sensors were prepared by incorporating two environmentally sensitive fluorophores with minimal spectral overlap separately into engineered lipid-binding proteins. Dual ratiometric analysis of imaging data allowed accurate, spatiotemporally resolved quantification of two different lipids on the same leaflet of the plasma membrane or a single lipid on two opposite leaflets of the plasma membrane of live mammalian cells. This new imaging technology should serve as a powerful tool for systems-level investigation of lipid-mediated cell signaling and regulation.
Project description:Targeting live cell organelles is essential for imaging, understanding, and controlling specific biochemical processes. Typically, fluorescent probes with distinct structural scaffolds are used to target specific cell organelles. Here, we have designed a modular one-step synthetic strategy using a common reaction intermediate to develop new lysosomal, mitochondrial, and nucleus-targeting pH-activable fluorescent probes that are all based on a single boron dipyrromethane scaffold. The divergent cell organelle targeting was achieved by synthesizing probes with specific functional group changes to the central scaffold resulting in differential fluorescence and pKa . Specifically, we show that the functional group transformation of the same scaffold influences cellular localization and specificity of pH-activable fluorescent probes in live primary microglial cells with pKa values ranging from ∼3.2-6.0. We introduce a structure-organelle-relationship (SOR) framework to target nuclei (NucShine), lysosomes (LysoShine), and mitochondria (MitoShine) in live microglia. This work will result in future applications of SOR beyond imaging to target and control organelle-specific biochemical processes in disease-specific models.
Project description:Guanosine tetra- and pentaphosphate, (p)ppGpp, are important alarmone nucleotides that regulate bacterial survival in stressful environment. A direct detection of (p)ppGpp in living cells is critical for our understanding of the mechanism of bacterial stringent response. However, it is still challenging to image cellular (p)ppGpp. Here, we report RNA-based fluorescent sensors for the live-cell imaging of (p)ppGpp. Our sensors are engineered by conjugating a recently identified (p)ppGpp-specific riboswitch with a fluorogenic RNA aptamer, Broccoli. These sensors can be genetically encoded and enable direct monitoring of cellular (p)ppGpp accumulation. Unprecedented information on cell-to-cell variation and cellular dynamics of (p)ppGpp levels is now obtained under different nutritional conditions. These RNA-based sensors can be broadly adapted to study bacterial stringent response.
Project description:Bcl-2 family proteins have important roles in tumor initiation, progression and resistance to therapy. Pro-survival Bcl-2 proteins are regulated by their interactions with pro-death BH3-only proteins making these protein-protein interactions attractive therapeutic targets. Although these interactions have been extensively characterized biochemically, there is a paucity of tools to assess these interactions in cells. Here, we address this limitation by developing quantitative, high throughput microscopy assays to characterize Bcl-2 and BH3-only protein interactions in live cells. We use fluorescent proteins to label the interacting proteins of interest, enabling visualization and quantification of their mitochondria-localized interactions. Using tool compounds, we demonstrate the suitability of our assays to characterize the cellular activity of putative therapeutic molecules that target the interaction between pro-survival Bcl-2 and pro-death BH3-only proteins. In addition to the relevance of our assays for drug discovery, we anticipate that our work will contribute to an improved understanding of the mechanisms that regulate these important protein-protein interactions within the cell.
Project description:Ammonium serves as key nitrogen source and metabolic intermediate, yet excess causes toxicity. Ammonium uptake is mediated by ammonium transporters, whose regulation is poorly understood. While transport can easily be characterized in heterologous systems, measuring transporter activity in vivo remains challenging. Here we developed a simple assay for monitoring activity in vivo by inserting circularly-permutated GFP into conformation-sensitive positions of two plant and one yeast ammonium transceptors ('AmTrac' and 'MepTrac'). Addition of ammonium to yeast cells expressing the sensors triggered concentration-dependent fluorescence intensity (FI) changes that strictly correlated with the activity of the transporter. Fluorescence-based activity sensors present a novel technology for monitoring the interaction of the transporters with their substrates, the activity of transporters and their regulation in vivo, which is particularly valuable in the context of analytes for which no radiotracers exist, as well as for cell-specific and subcellular transport processes that are otherwise difficult to track. DOI:http://dx.doi.org/10.7554/eLife.00800.001.
Project description:Copper is an essential metal nutrient for life that often relies on redox cycling between Cu(I) and Cu(II) oxidation states to fulfill its physiological roles, but alterations in cellular redox status can lead to imbalances in copper homeostasis that contribute to cancer and other metalloplasias with metal-dependent disease vulnerabilities. Copper-responsive fluorescent probes offer powerful tools to study labile copper pools, but most of these reagents target Cu(I), with limited methods for monitoring Cu(II) owing to its potent fluorescence quenching properties. Here, we report an activity-based sensing strategy for turn-on, oxidation state-specific detection of Cu(II) through metal-directed acyl imidazole chemistry. Cu(II) binding to a metal and oxidation state-specific receptor that accommodates the harder Lewis acidity of Cu(II) relative to Cu(I) activates the pendant dye for reaction with proximal biological nucleophiles and concomitant metal ion release, thus avoiding fluorescence quenching. Copper-directed acyl imidazole 649 for Cu(II) (CD649.2) provides foundational information on the existence and regulation of labile Cu(II) pools, including identifying divalent metal transporter 1 (DMT1) as a Cu(II) importer, labile Cu(II) increases in response to oxidative stress induced by depleting total glutathione levels, and reciprocal increases in labile Cu(II) accompanied by decreases in labile Cu(I) induced by oncogenic mutations that promote oxidative stress.