Photoactivatable dyes enable single-molecule imaging and tracking in biology. Despite progress in the development of new fluorophores and labeling strategies, many intracellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid domains, e.g., membranes and droplets, remain difficult to image with nanometric resolution. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid domains, most notably droplets, with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment, and a competent nucleophile to produce a fluorescent product. The combination of these inputs results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolution beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipid trafficking between droplets and the endoplasmic reticulum.
Extracellular vesicles (EVs) are emerging as promising diagnostic and therapeutic tools for a variety of diseases. The characterization of EVs requires a series of orthogonal techniques that are overall time-and material-consuming. Here, a microfluidic device is presented that exploits the combination of diffusion sizing and multiwavelength fluorescence detection to simultaneously provide information on EV size, concentration, and composition. The latter is achieved with the nonspecific staining of lipids and proteins combined with the specific staining of EV markers such as EV-associated tetraspanins via antibodies. The device can be operated as a single-step immunoassay thanks to the integrated separation and quantification of free and EV-bound fluorophores. This microfluidic technique is capable of detecting and quantifying components associated to EV subtypes and impurities and thus to measure EV purity in a time scale of minutes, requiring less than 5 µL of sample and minimal sample handling before the analysis. Moreover, the analysis is performed directly in solution without immobilization steps. Therefore, this method can accelerate screening of EV samples and aid the evaluation of sample reproducibility, representing an important complementary tool to the current array of biophysical methods for EV characterization, particularly valuable for instance for bioprocess development.
Chromatin is spatially organized into functional states that are defined by both the presence of specific histone post-translational modifications (PTMs) and a defined set of chromatin-associated "reader" proteins. Different models for the underlying mechanism of such compartmentalization have been proposed, including liquid−liquid phase separation (LLPS) of chromatin-associated proteins to drive spatial organization. Heterochromatin, characterized by lysine 9 methylation on histone H3 (H3K9me3) and the presence of heterochromatin protein 1 (HP1) as a multivalent reader, represents a prime example of a spatially defined chromatin state. Heterochromatin foci exhibit features of protein condensates driven by LLPS; however, the exact nature of the physicochemical environment within heterochromatin in different cell types is not completely understood. Here we present tools to interrogate the environment of chromatin subcompartments in the form of modular, cell-permeable, multivalent, and fluorescent peptide probes. These probes can be tuned to target specific chromatin states by providing binding sites to reader proteins and can thereby integrate into the PTM-reader interaction network. Here we generate probes specific to HP1, directing them to heterochromatin at chromocenters in mouse fibroblasts. Moreover, we use a polarity-sensing photoactivatable probe that photoconverts to a fluorescent state in phase-separated protein droplets and thereby reports on the local microenvironment. Equipped with this dye, our probes indeed turn fluorescent in murine chromocenters. Image analysis and single-molecule tracking experiments reveal that the compartments are less dense and more dynamic than HP1 condensates obtained in vitro. Our results thus demonstrate that the local organization of heterochromatin in chromocenters is internally more complex than an HP1 condensate.
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