Biology subjects

Rivera-Fuentes, P.

Publications and source records attributed to Rivera-Fuentes, P..

4 recordsLinked to original sources

bioRxiv2023

A Far-Red Fluorescent Probe to Visualize Staphylococcus aureus in Patient Samples

Staphylococcus aureus (S. aureus) is the leading bacterial cause of death in high-income countries and can cause invasive infections at various body sites. These infections are associated with prolonged hospital stays, a large economic burden, considerable treatment failure, and mortality rates. So far, there is only limited knowledge about the specific locations where S. aureus resides in the human body during various infections. Hence, the visualization of S. aureus holds significant importance in microbiological research. Herein, we report the development and validation of a far-red-fluorescent probe to detect S. aureus in human biopsies from deep-seated infections. This probe displays strong fluorescence and low background in human tissues, outperforming current tools for S. aureus detection. Several applications are demonstrated, including fixed- and live-cell imaging, flow cytometry, and super-resolution bacterial imaging. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/556223v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1bcf4e4org.highwire.dtl.DTLVardef@71bf45org.highwire.dtl.DTLVardef@1c468fdorg.highwire.dtl.DTLVardef@1c8baf3_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

A general strategy to develop fluorogenic polymethine dyes for bioimaging

Fluorescence imaging is an invaluable tool to study biological processes and further progress depends on the development of advanced probes. Fluorogenic dyes are crucial to reach intracellular targets and label them with high specificity. Excellent fluorogenic rhodamine dyes have been reported, but they often require a long and low-yielding synthesis and are spectrally limited to the visible range. Here, we present a general strategy to transform polymethine compounds into fluorogenic dyes using an intramolecular ring closure approach. We illustrate the generality of this method by creating both spontaneously blinking and no-wash, turn-on polymethine dyes with emissions across the visible and near-infrared spectrum. These probes are compatible with self-labeling proteins and small-molecule targeting ligands and can be combined with rhodamine-based dyes for multicolor and fluorescence lifetime multiplexing imaging. This strategy provides access to bright, fluorogenic dyes that emit at wavelengths that are significantly more red-shifted than those of existing rhodamine-based dyes.

biochemistry↗

bioRxiv2022

A Locally Activatable Sensor for Robust Quantification of Organellar Glutathione

Glutathione (GSH) is the main determinant of intracellular redox potential and participates in multiple cellular signaling pathways. Achieving a detailed understanding of intracellular GSH trafficking and regulation depends on the development of tools to map GSH compartmentalization and intra-organelle fluctuations. Herein, we present a new GSH sensing platform, TRaQ-G, for live-cell imaging. This small-molecule/protein hybrid sensor possesses a unique reactivity turn-on mechanism that ensures that the small molecule is only sensitive to GSH in the desired location. Furthermore, TRaQ-G can be fused to a fluorescent protein of choice to give a ratiometric response. Using TRaQ-G-mGold, we demonstrated that the nuclear and cytosolic GSH pools are independently regulated during cell proliferation. We also used this sensor, in combination with roGFP, to quantify redox potential and GSH concentration simultaneously in the endoplasmic reticulum. Finally, by exchanging the fluorescent protein, we created a near-infrared, targetable and quantitative GSH sensor.

cell biology↗

bioRxiv2021

Multivalent peptide ligands to probe the chromocenter microenvironment in living cells

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 sub-compartments 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. As a target, 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. However, 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.

genetics↗