peroxisome-staining-mammalian-cells-using-peroxisome-specific

Peroxisome Staining in Mammalian Cells Using Peroxisome-Specific Probes

Peroxisomes are ubiquitous organelles with essential roles in human health, and their dysfunction leads to a spectrum of genetic disorders. Visualization ...

Ecole Polytechnique Fédérale de Lausanne (EPFL)

We report an optimized synthesis of all canonical 2'-O-TOM protected ribonucleoside phosphoramidites and solid supports containing [13C5]-labeled ribose moieties, their sequence-specific introduction into very short RNA sequences and their use for the structure determination of two protein-RNA complexes. These specifically labeled sequences facilitate RNA resonance assignments and are essential to assign a high number of sugar-sugar and intermolecular NOEs, which ultimately improve the precision and accuracy of the resulting structures. This labeling strategy is particularly useful for the study of protein-RNA complexes with single-stranded RNA in solution, which is rapidly an increasingly relevant research area in biology.

Short, synthetic and selectively 13C-labeled RNA sequences for the NMR structure determination of protein-RNA complexes.

While γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter, suitable tools to measure its concentration in living cells with high spatiotemporal resolution are missing. Herein, we describe the first ratiometric fluorescent sensor for GABA, dubbed GABA-Snifit, which senses GABA with high specificity and spatiotemporal resolution on the surface of living mammalian cells. GABA-Snifit is a semisynthetic fusion protein containing the GABA(B) receptor, SNAP- and CLIP-tag, a synthetic fluorophore and a fluorescent GABA(B) receptor antagonist. When assembled on cell surfaces, GABA-Snifit displays a GABA-dependent fluorescence emission spectrum in the range of 500-700 nm that permits sensing micromolar to millimolar GABA concentrations. The ratiometric change of the sensor on living cells is 1.8. Furthermore, GABA-Snifit can be utilized to quantify the relative binding affinities of GABA(B) receptor agonists, antagonists and the effect of allosteric modulators. These properties make GABA-Snifit a valuable tool to investigate the role of GABA and GABA(B) in biological systems.

A fluorescent sensor for GABA and synthetic GABA(B) receptor ligands.

The centrosome functions as the main microtubule-organizing center of animal cells and is crucial for several fundamental cellular processes. Abnormalities in centrosome number and composition correlate with tumor progression and other diseases. Although proteomic studies have identified many centrosomal proteins, their interactions are incompletely characterized. The lack of information on the precise localization and interaction partners for many centrosomal proteins precludes comprehensive understanding of centrosome biology. Here, we utilize a combination of selective chemical crosslinking and superresolution microscopy to reveal novel functional interactions among a set of 31 centrosomal proteins. We reveal that Cep57, Cep63, and Cep152 are parts of a ring-like complex localizing around the proximal end of centrioles. Furthermore, we identify that STIL, together with HsSAS-6, resides at the proximal end of the procentriole, where the cartwheel is located. Our studies also reveal that the known interactors Cep152 and Plk4 reside in two separable structures, suggesting that the kinase Plk4 contacts its substrate Cep152 only transiently, at the centrosome or within the cytoplasm. Our findings provide novel insights into protein interactions critical for centrosome biology and establish a toolbox for future studies of centrosomal proteins.

Selective chemical crosslinking reveals a Cep57-Cep63-Cep152 centrosomal complex.

A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.

A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

Cell-permeable rhodamine dyes are reductively quenched by NaBH4 into a non-fluorescent leuco-rhodamine form. Quenching is reversible, and their fluorescence is recovered when the dyes are oxidized. In living cells, oxidation occurs spontaneously, and can result in up to ten-fold higher densities of single molecule localizations, and more photons per localization as compared with unmodified dyes. These two parameters directly impact the achievable resolution, and we see a significant improvement in the quality of live-cell point-localization super-resolution images taken with reduced dyes. These improvements carry over to increase the density of trajectories for single-molecule tracking experiments.

Reduced dyes enhance single-molecule localization density for live superresolution imaging.

One of the most prominent self-labeling tags is SNAP-tag. It is an in vitro evolution product of the human DNA repair protein O (6)-alkylguanine-DNA alkyltransferase (hAGT) that reacts specifically with benzylguanine (BG) and benzylchloropyrimidine (CP) derivatives, leading to covalent labeling of SNAP-tag with a synthetic probe (Gronemeyer et al., Protein Eng Des Sel 19:309-316, 2006; Curr Opin Biotechnol 16:453-458, 2005; Keppler et al., Nat Biotechnol 21:86-89, 2003; Proc Natl Acad Sci U S A 101:9955-9959, 2004). SNAP-tag is well suited for the analysis and quantification of fused target protein using fluorescence microscopy techniques. It provides a simple, robust, and versatile approach to the imaging of fusion proteins under a wide range of experimental conditions.

Fluorescent labeling of SNAP-tagged proteins in cells.

Cell-permeable DNA stains are popular markers in live-cell imaging. Currently used DNA stains for live-cell imaging are either toxic, require illumination with blue light or are not compatible with super-resolution microscopy, thereby limiting their utility. Here we describe a far-red DNA stain, SiR-Hoechst, which displays minimal toxicity, is applicable in different cell types and tissues, and is compatible with super-resolution microscopy. The combination of these properties makes this probe a powerful tool for live-cell imaging.

SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy.

Here we present a far-red, silicon-rhodamine-based fluorophore (SiR700) for live-cell multicolor imaging. SiR700 has excitation and emission maxima at 690 and 715 nm, respectively. SiR700-based probes for F-actin, microtubules, lysosomes, and SNAP-tag are fluorogenic, cell-permeable, and compatible with superresolution microscopy. In conjunction with probes based on the previously introduced carboxy-SiR650, SiR700-based probes permit multicolor live-cell superresolution microscopy in the far-red, thus significantly expanding our capacity for imaging living cells.

Fluorogenic Probes for Multicolor Imaging in Living Cells.

We introduce luciferases whose emission maxima can be tuned to different wavelengths by chemical labeling. The luciferases are chimeras of NanoLuc with either SNAP-tag or HaloTag7. Labeling of the self-labeling tag with a fluorophore shifts the emission maximum of NanoLuc to that of the fluorophore. Luciferases with tunable colors have applications as reporter genes, for the construction of biosensors and in bioimaging.

Luciferases with Tunable Emission Wavelengths.

Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore's outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

Photoactivation of silicon rhodamines via a light-induced protonation.

Gene delivery using vector or viral-based methods is often limited by technical and safety barriers. A promising alternative that circumvents these shortcomings is the direct delivery of proteins into cells. Here we introduce a non-viral, ligand-mediated protein delivery system capable of selectively targeting primary skin cells in-vivo. Using orthologous self-labelling tags and chemical cross-linkers, we conjugate large proteins to ligands that bind their natural receptors on the surface of keratinocytes. Targeted CRE-mediated recombination was achieved by delivery of ligand cross-linked CRE protein to the skin of transgenic reporter mice, but was absent in mice lacking the ligand's cell surface receptor. We further show that ligands mediate the intracellular delivery of Cas9 allowing for CRISPR-mediated gene editing in the skin more efficiently than adeno-associated viral gene delivery. Thus, a ligand-based system enables the effective and receptor-specific delivery of large proteins and may be applied to the treatment of skin-related genetic diseases.

A ligand-based system for receptor-specific delivery of proteins.

This article describes four fluorescent membrane tension probes that have been designed, synthesized, evaluated, commercialized and applied to current biology challenges in the context of the NCCR Chemical Biology. Their names are Flipper-TR, ER Flipper-TR, Lyso Flipper-TR, and Mito Flipper-TR. They are available from Spirochrome.

Flipper Probes for the Community.

Kallikrein-related peptidases 5 (KLK5) and 7 (KLK7) are serine proteases with homeostatic functions in the epidermis that play a critical role in Netherton syndrome (NS), a rare yet life-threatening genetic disorder that currently lacks specific treatment. Previous research suggests that controlling KLKs could lead to the development of NS therapies, but existing synthetic inhibitors have limitations. Herein, we used phage display to screen libraries comprising more than 100 billion different cyclic peptides and found selective, high-affinity inhibitors of KLK5 ( = 2.2 ± 0.1 nM) and KLK7 ( = 16 ± 4 nM). By eliminating protease-prone sites and conjugating the inhibitors to an albumin-binding peptide, we enhanced the inhibitor stability and prolonged the elimination half-life to around 5 h in mice. In tissue sections taken from mice, a fluorescently labeled peptide was detected in the epidermis, suggesting that the inhibitors can reach the KLKs upon systemic delivery and should be suited to control deregulated protease activity in NS.

Phage Display Selected Cyclic Peptide Inhibitors of Kallikrein-Related Peptidases 5 and 7 and Their  Delivery to the Skin.

Microtubules, a critical component of the cytoskeleton, carry post-translational modifications (PTMs) that are important for the regulation of key cellular processes. Long-lived microtubules, in neurons particularly, exhibit both detyrosination of α-tubulin and polyglutamylation. Dysregulation of these PTMs can result in developmental defects and neurodegeneration. Owing to a lack of tools to study the regulation and function of these PTMs, the mechanisms that govern such PTM patterns are not well understood. Here we produce fully functional tubulin carrying precisely defined PTMs within its C-terminal tail. We ligate synthetic α-tubulin tails-which are site-specifically glutamylated-to recombinant human tubulin heterodimers by applying a sortase- and intein-mediated tandem transamidation strategy. Using microtubules reconstituted with these designer tubulins, we find that α-tubulin polyglutamylation promotes its detyrosination by enhancing the activity of the tubulin tyrosine carboxypeptidase vasohibin/small vasohibin-binding protein in a manner dependent on the length of polyglutamyl chains. We also find that modulating polyglutamylation levels in cells results in corresponding changes in detyrosination, corroborating the link between the detyrosination cycle to polyglutamylation.

Tubulin engineering by semi-synthesis reveals that polyglutamylation directs detyrosination.

Polo-like kinase (Plk4) is a serine/threonine-protein kinase that is essential for biogenesis of the centriole organelle and is enriched at centrioles. Herein, we introduce Cen-TCO, a chemical probe based on the Plk4 inhibitor centrinone, to image Plk4 and centrioles in live or fixed cultured human cells. Specifically, we established a bio-orthogonal two-step labeling system that enables the Cen-TCO-mediated imaging of Plk4 by STED super-resolution microscopy. Such direct labeling of Plk4 results in an increased resolution in STED imaging compared with using anti-Plk4 antibodies, underlining the importance of direct labeling strategies for super-resolution microscopy. We anticipate that Cen-TCO will become an important tool for investigating the biology of Plk4 and of centrioles.

Chemical Probe for Imaging of Polo-like Kinase 4 and Centrioles.

Peroxisomes are eukaryotic organelles that are essential for multiple metabolic pathways, including fatty acid oxidation, degradation of amino acids, and biosynthesis of ether lipids. Consequently, peroxisome dysfunction leads to pediatric-onset neurodegenerative conditions, including Peroxisome Biogenesis Disorders (PBD). Due to the dynamic, tissue-specific, and context-dependent nature of their biogenesis and function, live cell imaging of peroxisomes is essential for studying peroxisome regulation, as well as for the diagnosis of PBD-linked abnormalities. However, the peroxisomal imaging toolkit is lacking in many respects, with no reporters for substrate import, nor cell-permeable probes that could stain dysfunctional peroxisomes. Here we report that the BODIPY-C12 fluorescent fatty acid probe stains functional and dysfunctional peroxisomes in live mammalian cells. We then go on to improve BODIPY-C12, generating peroxisome-specific reagents, PeroxiSPY650 and PeroxiSPY555. These probes combine high peroxisome specificity, bright fluorescence in the red and far-red spectrum, and fast non-cytotoxic staining, making them ideal tools for live cell, whole organism, or tissue imaging of peroxisomes. Finally, we demonstrate that PeroxiSPY enables diagnosis of peroxisome abnormalities in the PBD CRISPR/Cas9 cell models and patient-derived cell lines.

Fluorescent fatty acid conjugates for live cell imaging of peroxisomes.

Rhodamines have been continuously optimized in brightness, biocompatibility, and color to fulfill the demands of modern bioimaging. However, the problem of phototoxicity caused by the excited fluorophore under long-term illumination has been largely neglected, hampering their use in time-lapse imaging. Here we introduce cyclooctatetraene (COT) conjugated rhodamines that span the visible spectrum and exhibit significantly reduced phototoxicity. We identified a general strategy for the generation of Gentle Rhodamines, which preserved their outstanding spectroscopic properties and cell permeability while showing an efficient reduction of singlet-oxygen formation and diminished cellular photodamage. Paradoxically, their photobleaching kinetics do not go hand in hand with reduced phototoxicity. By combining COT-conjugated spirocyclization motifs with targeting moieties, these Gentle Rhodamines compose a toolkit for time-lapse imaging of mitochondria, DNA, and actin, and synergize with covalent and exchangeable HaloTag labeling of cellular proteins with less photodamage than their commonly used precursors. Taken together, the Gentle Rhodamines generally offer alleviated phototoxicity and allow advanced video recording applications, including voltage imaging.

Gentle Rhodamines for Live-Cell Fluorescence Microscopy.

Molecular recognition events between proteins drive biological processes in living systems. However, higher levels of mechanistic regulation have emerged, in which protein-protein interactions are conditioned to small molecules. Despite recent advances, computational tools for the design of new chemically induced protein interactions have remained a challenging task for the field. Here we present a computational strategy for the design of proteins that target neosurfaces, that is, surfaces arising from protein-ligand complexes. To develop this strategy, we leveraged a geometric deep learning approach based on learned molecular surface representations and experimentally validated binders against three drug-bound protein complexes: Bcl2-venetoclax, DB3-progesterone and PDF1-actinonin. All binders demonstrated high affinities and accurate specificities, as assessed by mutational and structural characterization. Remarkably, surface fingerprints previously trained only on proteins could be applied to neosurfaces induced by interactions with small molecules, providing a powerful demonstration of generalizability that is uncommon in other deep learning approaches. We anticipate that such designed chemically induced protein interactions will have the potential to expand the sensing repertoire and the assembly of new synthetic pathways in engineered cells for innovative drug-controlled cell-based therapies.

Targeting protein-ligand neosurfaces with a generalizable deep learning tool.

Mitochondria are central to cellular metabolism. Various fluorescence tools have been developed for imaging the mitochondrial environment. Yet, new reporters and imaging methods for directly reading the mitochondrial status are needed for high spatial-temporal resolution imaging. Here, we introduce PK Mito Deep Red (PKMDR), a low-phototoxicity mitochondrial probe for time-lapse imaging, whose fluorescence lifetime serves as a sensitive indicator of mitochondrial membrane potential (Δψ). The positively charged PKMDR accumulates within mitochondria under a higher Δψ, leading to concentration-induced quenching and a measurable decrease in fluorescence lifetime. Since mitochondrial respiration primarily regulates Δψ, PKMDR's fluorescence lifetime effectively reports on the status of oxidative phosphorylation. Using PKMDR with fluorescence lifetime imaging microscopy (FLIM), we visualize heterogeneous Δψ across individual cells, organoids, and tissues over time. This method reliably reveals the heterogeneity between metabolically active peripheral mitochondria and relatively inactive perinuclear mitochondria in various cell types. Overall, PKMDR-FLIM is a robust tool for directly visualizing Δψ with high spatiotemporal resolution.

Article	Year
Short, synthetic and selectively 13C-labeled RNA sequences for the NMR structure determination of protein-RNA complexes. Nucleic acids research\| PubMed ID: 16807315	2006
A fluorescent sensor for GABA and synthetic GABA(B) receptor ligands. Journal of the American Chemical Society\| PubMed ID: 23095089	2012
Selective chemical crosslinking reveals a Cep57-Cep63-Cep152 centrosomal complex. Current biology : CB\| PubMed ID: 23333316	2013
A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red. ACS chemical biology\| PubMed ID: 24471525	2014
Reduced dyes enhance single-molecule localization density for live superresolution imaging. Chemphyschem : a European journal of chemical physics and physical chemistry\| PubMed ID: 24554553	2014
Fluorescent labeling of SNAP-tagged proteins in cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 25560070	2015
SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy. Nature communications\| PubMed ID: 26423723	2015
Fluorogenic Probes for Multicolor Imaging in Living Cells. Journal of the American Chemical Society\| PubMed ID: 27420907	2016
Luciferases with Tunable Emission Wavelengths. Angewandte Chemie (International ed. in English)\| PubMed ID: 28941028	2017
Photoactivation of silicon rhodamines via a light-induced protonation. Nature communications\| PubMed ID: 31594948	2019
A ligand-based system for receptor-specific delivery of proteins. Scientific reports\| PubMed ID: 31844114	2019
Flipper Probes for the Community. Chimia\| PubMed ID: 34920768	2021
Phage Display Selected Cyclic Peptide Inhibitors of Kallikrein-Related Peptidases 5 and 7 and Their Delivery to the Skin. Journal of medicinal chemistry\| PubMed ID: 35653695	2022
Tubulin engineering by semi-synthesis reveals that polyglutamylation directs detyrosination. Nature chemistry\| PubMed ID: 37386282	2023
Chemical Probe for Imaging of Polo-like Kinase 4 and Centrioles. JACS Au\| PubMed ID: 37654580	2023
Fluorescent fatty acid conjugates for live cell imaging of peroxisomes. Nature communications\| PubMed ID: 38773129	2024
Gentle Rhodamines for Live-Cell Fluorescence Microscopy. ACS central science\| PubMed ID: 39463828	2024
Targeting protein-ligand neosurfaces with a generalizable deep learning tool. Nature\| PubMed ID: 39814890	2025
Imaging mitochondrial membrane potential via concentration-dependent fluorescence lifetime changes. Nature communications\| PubMed ID: 41387403	2025

Luc Reymond

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