Analyzing Mitochondrial Calcium Influx in Isolated Mitochondria.

Mitochondria play a crucial role in Ca2+ signaling and homeostasis and can contribute to shaping the cytosolic Ca2+ landscape as well as regulate a variety of pathways including energy production and cell death. Dysregulation of mitochondrial Ca2+ homeostasis promotes pathologies including neurodegenerative diseases, cardiovascular disorders, and metabolic syndromes. The significance of mitochondria to Ca2+ signaling and regulation underscores the value of methods to assess mitochondrial Ca2+ import. Here we present a plate reader-based method using the Ca2+-sensitive fluorescent probe calcium green-5 N to measure mitochondrial Ca2+ import in isolated cardiac mitochondria. This technique can be expanded to measure Ca2+ uptake in mitochondria isolated from other tissue types and from cultured cells.

Fluorescent Reporters, Imaging, and Artificial Intelligence Toolkits to Monitor and Quantify Autophagy, Heterophagy, and Lysosomal Trafficking Fluxes.

Lysosomal compartments control the clearance of cell-own material (autophagy) or of material that cells endocytose from the external environment (heterophagy) to warrant supply of nutrients, to eliminate macromolecules or parts of organelles present in excess, aged, or containing toxic material. Inherited or sporadic mutations in lysosomal proteins and enzymes may hamper their folding in the endoplasmic reticulum (ER) and their lysosomal transport via the Golgi compartment, resulting in lysosomal dysfunction and storage disorders. Defective cargo delivery to lysosomal compartments is harmful to cells and organs since it causes accumulation of toxic compounds and defective organellar homeostasis. Assessment of resident proteins and cargo fluxes to the lysosomal compartments is crucial for the mechanistic dissection of intracellular transport and catabolic events. It might be combined with high-throughput screenings to identify cellular, chemical, or pharmacological modulators of these events that may find therapeutic use for autophagy-related and lysosomal storage disorders. Here, discuss qualitative, quantitative and chronologic monitoring of autophagic, heterophagic and lysosomal protein trafficking in fixed and live cells, which relies on fluorescent single and tandem reporters used in combination with biochemical, flow cytometry, light and electron microscopy approaches implemented by artificial intelligence-based technology.

Fluorescent non-canonical amino acid provides insight into the human serotonin transporter.

The serotonin transporter (SERT), responsible for the reuptake of released serotonin, serves as a major target for antidepressants and psychostimulants. Nevertheless, refining the mechanistic models for SERT remains challenging. Here, we expand the molecular understanding of the binding of ions, substrates, and inhibitors to SERT by incorporating the fluorescent non-canonical amino acid Anap through genetic code expansion. We elucidate steady-state changes in conformational dynamics of purified SERT with Anap inserted at intracellular- or extracellular sites. This uncovers the competitive mechanisms underlying cation binding and assigns distinct binding- and allosteric coupling patterns for several inhibitors and substrates. Finally, we track in real-time conformational transitions in response to the interaction with Na+ or serotonin. In this work, we present a methodological platform reporting on SERT conformational dynamics, which together with other approaches will deepen our insights into the molecular mechanisms of SERT.

Determination of Initial Rates of Lipopolysaccharide Transport.

Nonvesicular lipid trafficking pathways are an important process in every domain of life. The mechanisms of these processes are poorly understood in part due to the difficulty in kinetic characterization. One important class of glycolipids, lipopolysaccharides (LPS), are the primary lipidic component of the outer membrane of Gram-negative bacteria. LPS are synthesized in the inner membrane and then trafficked to the cell surface by the lipopolysaccharide transport proteins, LptB2FGCADE. By characterizing the interaction of a fluorescent probe and LPS, we establish a quantitative assay to monitor the flux of LPS between proteoliposomes on the time scale of seconds. We then incorporate photocaged ATP into this system, which allows for light-based control of the initiation of LPS transport. This control allows us to measure the initial rate of LPS transport (3.0 min-1 per LptDE). We also find that the rate of LPS transport by the Lpt complex is independent of the structure of LPS. In contrast, we find the rate of LPS transport is dependent on the proper function of the LptDE complex. Mutants of the outer membrane Lpt components, LptDE, that cause defective LPS assembly in live cells display attenuated transport rates and slower ATP hydrolysis compared to wild type proteins. Analysis of these mutants reveals that the rates of ATP hydrolysis and LPS transport are correlated such that 1.2 ± 0.2 ATP are hydrolyzed for each LPS transported. This correlation suggests a model where the outer membrane components ensure the coupling of ATP hydrolysis and LPS transport by stabilizing a transport-active state of the Lpt bridge.

Endocytosis of Synaptic Vesicle in Motor Nerve Endings of FUS Transgenic Mice with a Model of Amyotrophic Lateral Sclerosis.

In experiments on the motor nerve endings of the diaphragm of transgenic FUS mice with a model of amyotrophic lateral sclerosis at the pre-symptomatic stage of the disease, the processes of transmitter release and endocytosis of synaptic vesicles were studied. In FUS mice, the intensity of transmitter release during high-frequency stimulation of the motor nerve (50 imp/sec) was lowered. At the same duration of stimulation, the loading of fluorescent dye FM1-43 was lower in FUS mice. However, at the time of stimulation, during which an equal number of quanta are released in wild-type and FUS mice, no differences in the intensity of dye loading were found. Thus, endocytosis is not the key factor in the mechanism of synaptic dysfunction in FUS mice at the pre-symptomatic stage.

Tracking induced pluripotent stem cell differentiation with a fluorescent genetically encoded epigenetic probe.

Epigenetic modifications (methylation, acetylation, etc.) of core histones play a key role in regulation of gene expression. Thus, the epigenome changes strongly during various biological processes such as cell differentiation and dedifferentiation. Classical methods of analysis of epigenetic modifications such as mass-spectrometry and chromatin immuno-precipitation, work with fixed cells only. Here we present a genetically encoded fluorescent probe, MPP8-Green, for detecting H3K9me3, a histone modification associated with inactive chromatin. This probe, based on the chromodomain of MPP8, allows for visualization of H3K9me3 epigenetic landscapes in single living cells. We used this probe to track changes in H3K9me3 landscapes during the differentiation of induced pluripotent stem cells (iPSCs) into induced neurons. Our findings revealed two major waves of global H3K9me3 reorganization during 4-day differentiation, namely on the first and third days, whereas nearly no changes occurred on the second and fourth days. The proposed method LiveMIEL (Live-cell Microscopic Imaging of Epigenetic Landscapes), which combines genetically encoded epigenetic probes and machine learning approaches, enables classification of multiparametric epigenetic signatures of single cells during stem cell differentiation and potentially in other biological models.

Emerging trends on the uptake of fluorescent probes based on glucose analogs by cancer cells: From basic studies to therapeutics.

The cancer cell metabolism, notably characterized by the Warburg effect, has been the focus of intense investigation regarding the mechanisms of the uptake of glucose analogs, opening up perspectives for diagnosis and treatment of cancer disease. In this review, we delve into the ever-evolving landscape of cancer research, centering on fluorescent probes based on glucose analogs. These analogs, resulting from modifications in the carbohydrate structure with functional groups, have stood out as versatile molecules in applications ranging from disease comprehension to therapeutic innovation, especially when combined with fluorescent compounds. Fluorescence-based assays have provided valuable contributions to the revelation of complex biological mechanisms in life sciences. This review presents selected studies from about the past six years up to 2024 related to the use of glucose-based fluorescent probes, for the investigation of their uptake profile as well as for therapeutic purposes. We believe that these investigations offer insights into the intricate interaction between glucose analogs and cancer cell metabolism, guiding future research and clinical applications in this field.

Measurement of Intracellular Ca2+ for Studying GPCR-Mediated Lipid Signaling.

Intracellular Ca2+ can be conveniently monitored by sensitive Ca2+ fluorescent dyes in live cells. The Gαq involved lipid signaling pathways and, thus, can be studied by intracellular Ca2+ imaging. Here we describe the protocols to measure intracellular Ca2+ for studying PEG2-EP1 activity in esophageal smooth muscle cells. The ratiometric Fura-2 imaging provides quantitative data, and the Fluo-4 confocal microscopic imaging has high-spatial resolution.

A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction.

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis upon metabolic demands. Detection of these contact sites at the nanometer scale over time in living cells is challenging. We developed a tool kit for detecting contact sites based on fluorogen-activated bimolecular complementation at CONtact sites, FABCON, using a reversible, low-affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic regulation.

Cell-penetrating activity of a short-chain ε-poly-l-α-lysine.

Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. We previously demonstrated that two representative bacterial polycationic isopeptides, ε-poly-l-α-lysine consisting of 25-35 l-α-lysine residues (ε-PαL25-35) and ε-poly-l-ß-lysine consisting of l-ß-lysine residues (ε-PßL4-13), were internalized into mammalian cells by both energy-independent direct penetration and energy-dependent endocytosis/macropinocytosis, and then diffused throughout the cytosol. In this study, we investigated the cell-penetrating activity of an ε-PαL short-chain derivative consisting of 5-14 l-α-lysine residues (ε-PαL5-14) to gain insight into the relationship between the isopeptide-chain length and the manner of cellular internalization. We prepared a conjugate of ε-PαL5-14 and a fluorescent dye (FAM) by click chemistry, and incubated the resulting polymer, ε-PαL5-14-FAM, with HeLa cells. Unlike ε-PαL25-35-FAM, ε-PαL5-14-FAM was internalized into cells only by energy-dependent endocytosis/macropinocytosis. Furthermore, a high concentration (>50 µM) was required for the internalization events. ε-PαL5-14 has a chain length almost equal to that of the membrane permeable ε-PßL4-13, which can enter cells at low concentrations. Considering that the basicity of the ß-amino group is higher than that of α-amino acid at physiological pH, ε-PßL is expected to have a greater cell-penetrating capacity than ε-PαL, provided their isopeptide-chain lengths are similar, suggesting that a more extended chain derivative of ε-PßL would be more advantageous for cellular internalization of cargo proteins than ε-PαL25-35.

Development of a selective novel fluorescent substrate for sodium-dependent transporters.

AIM: To synthesize, characterize, and validate 6FGA, a fluorescent glucose modified with a Cyanine5.5 at carbon-6 position, for probing the function of sodium-dependent glucose transporters, SGLT1 and SGLT2. MAIN METHODS: The synthesis of fluorescent glucose analogue was achieved through "click chemistry" of Cyanine5.5-alkyne and 6-azido-6-deoxy-d-glucose. Cell system studies were conducted to characterize the in vivo transport properties. KEY FINDINGS: Optical analyses revealed that 6FGA displayed similar spectral profiles to Cyanine5.5 in DMSO, allowing for concentration determination, thus supporting its utility in quantitative kinetic studies within biological assays. Uptake studies in cell system SGLT models, LLC-PK1 and HEK293 cells, exhibited concentration and time-dependent behavior, indicating saturation at specific concentrations and durations which are hallmarks of transported-mediated uptake. The results of cytotoxicity assays suggested cell viability at micromolar concentrations, enabling usage in assays for at least 1 h without significant toxicity. The dependence of 6FGA uptake on sodium, the co-transported cation, was demonstrated in LLC-PK1 and HEK293 cells. Fluorescence microscopy confirmed intracellular localization of 6FGA, particularly near the nucleus. Competition studies revealed that glucose tends to weakly reduce 6FGA uptake, although the effect did not achieve statistical significance. Assessments using standard SGLT and GLUT inhibitors highlighted 6FGA's sensitivity for probing SGLT-mediated transport. SIGNIFICANCE: 6FGA is a new fluorescent glucose analog offering advantages over existing probes due to its improved photophysical properties, greater sensitivity, enabling subcellular resolution and efficient tissue penetration in near-infrared imaging. 6FGA presents practicality and cost-effectiveness, making it a promising tool for nonradioactive, microplate-based assays at investigating SGLT-mediated glucose transport mechanisms.

Optimization of the fluorogen-activating protein tag for quantitative protein trafficking and colocalization studies in S. cerevisiae.

Spatial and temporal tracking of fluorescent proteins (FPs) in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active FPs fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae.

A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro.

In nature, bacteria usually exist as mixed-species biofilms, where they engage in a range of synergistic and antagonistic interactions that increase their resistance to environmental challenges. Biofilms are a major cause of persistent infections, and dispersal from initial foci can cause new infections at distal sites thus warranting further investigation. Studies of development and spatial interactions in mixed-species biofilms can be challenging due to difficulties in identifying the different bacterial species in situ. Here, we apply CellTrace dyes to studies of biofilm bacteria and present a novel application for multiplex labeling, allowing identification of different bacteria in mixed-species, in vitro biofilm models. Oral bacteria labeled with CellTrace dyes (far red, yellow, violet, and CFSE [green]) were used to create single- and mixed-species biofilms, which were analyzed with confocal spinning disk microscopy (CSDM). Biofilm supernatants were studied with flow cytometry (FC). Both Gram-positive and Gram-negative bacteria were well labeled and CSDM revealed biofilms with clear morphology and stable staining for up to 4 days. Analysis of CellTrace labeled cells in supernatants using FC showed differences in the biofilm dispersal between bacterial species. Multiplexing with different colored dyes allowed visualization of spatial relationships between bacteria in mixed-species biofilms and relative coverage by the different species was revealed through segmentation of the CSDM images. This novel application, thus, offers a powerful tool for studying structure and composition of mixed-species biofilms in vitro.IMPORTANCEAlthough most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofilm structure and dispersal. Critically, labeled bacteria can be multiplexed for identification of different species in mixed-species biofilms using confocal spinning disk microscopy, facilitating investigation of biofilm development and spatial interactions under different environmental conditions. The study is an important step in increasing the tools available for such complex and challenging studies.

Targeting of membrane proteins with fluoronanogold probes for high-resolution correlative microscopy.

Correlative light and electron microscopy (CLEM) can provide valuable information about a biological sample by giving information on the specific localization of a molecule of interest within an ultrastructural context. In this work, we describe a simple CLEM method to obtain high-resolution images of neurotransmitter receptor distribution in synapses by electron microscopy (EM). We use hippocampal organotypic slices from a previously reported mouse model expressing a modified AMPA receptor (AMPAR) subunit that binds biotin at the surface (Getz et al., 2022). This tag can be recognized by StreptAvidin-Fluoronanogold™ conjugates (SA-FNG), which reach receptors at synapses (synaptic cleft is 50-100nm thick). By using pre-embedding labeling, we found that SA-FNG reliably bind synaptic receptors and penetrate around 10-15µm in depth in live tissue. However, the silver enhancement was only reaching the surface of the slices. We show that permeabilization with triton is highly effective at increasing the in depth-gold amplification and that the membrane integrity is well preserved. Finally, we also apply high-resolution electron tomography, thus providing important information about the 3D organization of surface AMPA receptors in synapses at the nanoscale.

Unlocking Precision in Callose Staining: Unveiling the Role of Sirofluor.

Callose is a vital component in plant biology, contributing to essential processes like pollen maturation and defense against pathogens. However, misconceptions surrounding callose staining persist, particularly regarding the role of aniline blue. It is now known that commercial aniline blue contains sirofluor, and it is this fluorophore, rather than aniline blue itself, that is responsible for the observed fluorescence during callose detection. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Using Förster Resonance Energy Transfer (FRET) to Understand the Ubiquitination Landscape.

Förster resonance energy transfer (FRET) is a fluorescence technique that allows quantitative measurement of protein interactions, kinetics and dynamics. This review covers the use of FRET to study the structures and mechanisms of ubiquitination and related proteins. We survey FRET assays that have been developed where donor and acceptor fluorophores are placed on E1, E2 or E3 enzymes and ubiquitin (Ub) to monitor steady-state and real-time transfer of Ub through the ubiquitination cascade. Specialized FRET probes placed on Ub and Ub-like proteins have been developed to monitor Ub removal by deubiquitinating enzymes (DUBs) that result in a loss of a FRET signal upon cleavage of the FRET probes. FRET has also been used to understand conformational changes in large complexes such as multimeric E3 ligases and the proteasome, frequently using sophisticated single molecule methods. Overall, FRET is a powerful tool to help unravel the intricacies of the complex ubiquitination system.

A novel nanoparticle fluorescent probe based on a water-soluble conjugated polymer for real-time monitoring of ATP fluctuation and configuration of the Golgi apparatus during the inhibition of glycolysis.

BACKGROUND: Adenosine 5'-triphosphate (ATP) plays an important role in cell metabolism and has been regarded as an indicator of cell survival and damage. Golgi apparatus participates in the signal transduction processes of substance transport, ion homeostasis and stress when extracellular substances enter cells. Till now, there is no fluorescent probe for monitoring Golgi ATP level fluctuation and visualizing the configuration change of the Golgi apparatus during the inhibition of glycolysis. RESULTS: Herein, we report the synthesis of a novel water-soluble cationic polythiophene derivative (PEMTEA) that can be employed as a fluorescent sensor for measuring ATP in the Golgi apparatus. PEMTEA self-assembles into PT-NP nanoparticles in aqueous solution with a diameter of approximately 2 nm. PT-NP displays high sensitivity and superb selectivity towards ATP with a detection limit of 90 nM and a linear detection range from 0 to 3.0 µM. The nanoparticles show low toxicity to HepG2 cells and good photostability in the Golgi apparatus. With the stimulation of Ca2+, PT-NP was practically applied to real-time monitor of endogenous ATP levels in the Golgi apparatus through fluorescence microscopy. Finally, we studied the relationship between the concentration of ATP and configuration of the Golgi apparatus during the inhibition of glycolysis using PT-NP. SIGNIFICANCE: We have demonstrated that PT-NP can not only indicate the fluctuation and distribution of ATP in the Golgi apparatus, but also give the information of the configuration change of the Golgi apparatus at the single-cell level during the inhibition of glycolysis.

Miniaturized Implantable Fluorescence Probes Integrated with Metal-Organic Frameworks for Deep Brain Dopamine Sensing.

Continuously monitoring neurotransmitter dynamics can offer profound insights into neural mechanisms and the etiology of neurological diseases. Here, we present a miniaturized implantable fluorescence probe integrated with metal-organic frameworks (MOFs) for deep brain dopamine sensing. The probe is assembled from physically thinned light-emitting diodes (LEDs) and phototransistors, along with functional surface coatings, resulting in a total thickness of 120 µm. A fluorescent MOF that specifically binds dopamine is introduced, enabling a highly sensitive dopamine measurement with a detection limit of 79.9 nM. A compact wireless circuit weighing only 0.85 g is also developed and interfaced with the probe, which was later applied to continuously monitor real-time dopamine levels during deep brain stimulation in rats, providing critical information on neurotransmitter dynamics. Cytotoxicity tests and immunofluorescence analysis further suggest a favorable biocompatibility of the probe for implantable applications. This work presents fundamental principles and techniques for integrating fluorescent MOFs and flexible electronics for brain-computer interfaces and may provide more customized platforms for applications in neuroscience, disease tracing, and smart diagnostics.

A Quantitative Method to Distinguish Cytosolic from Endosome-Trapped Cell-Penetrating Peptides.

Cell-penetrating peptides are known to penetrate cells through endocytosis and translocation. The two pathways are hardly distinguished in current cell assays. We developed a reliable, simple and robust method to distinguish and quantify independently the two routes. The assay requires (DABCYL) 4-(dimethylaminoazo)benzene-4-carboxylic acid- and (CF) carboxyfluorescein-labeled peptides. When the labeled peptide is intact, the fluorescence signal is weak thanks to the dark quenching property of DABCYL. A 10-fold higher fluorescence signal is measured when the labeled peptide is degraded. By referring to a standard fluorescent curve according to the concentration of the hydrolyzed peptide, we have access to the internalized peptide quantity. Therefore, cell lysis after internalization permits to determine the total quantity of intracellular peptide. The molecular state of the internalized peptide (intact or degraded), depends on its location in cells (cytosol vs endo-lysosomes), and can be blocked by boiling cells. This boiling step results indeed in denaturation and inhibition of the cellular enzymes. The advantage of this method is the possibility to quantify translocation at 37 °C and to compare it to the 4 °C condition, where all endocytosis processes are inhibited. We found that ranking of the translocation efficacy is DABCYL-R6-(ϵCF)K≫DABCYL-R4-(ϵCF)K≥CF-R9.

Molecular Design of SERTlight: A Fluorescent Serotonin Probe for Neuronal Labeling in the Brain.

The serotonergic transmitter system plays fundamental roles in the nervous system in neurotransmission, synaptic plasticity, pathological processes, and therapeutic effects of antidepressants and psychedelics, as well as in the gastrointestinal and circulatory systems. We introduce a novel small molecule fluorescent agent, termed SERTlight, that specifically labels serotonergic neuronal cell bodies, dendrites, and axonal projections as a serotonin transporter (SERT) fluorescent substrate. SERTlight was developed by an iterative molecular design process, based on an aminoethyl-quinolone system, to integrate structural elements that impart SERT substrate activity, sufficient fluorescent brightness, and a broad absence of pharmacological activity, including at serotonin (5-hydroxytryptamine, 5HT) receptors, other G protein-coupled receptors (GPCRs), ion channels, and monoamine transporters. The high labeling selectivity is not achieved by high affinity binding to SERT itself but rather by a sufficient rate of SERT-mediated transport of SERTlight, resulting in accumulation of these molecules in 5HT neurons and yielding a robust and selective optical signal in the mammalian brain. SERTlight provides a stable signal, as it is not released via exocytosis nor by reverse SERT transport induced by 5HT releasers such as MDMA. SERTlight is optically, pharmacologically, and operationally orthogonal to a wide range of genetically encoded sensors, enabling multiplexed imaging. SERTlight enables labeling of distal 5HT axonal projections and simultaneous imaging of the release of endogenous 5HT using the GRAB5HT sensor, providing a new versatile molecular tool for the study of the serotonergic system.