Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Influence of fluorophore and linker length on the localization and trafficking of fluorescent sterol probes

ABSTRACT: Fluorescent sterol probes, comprising a fluorophore connected to a sterol backbone by means of a linker, are promising tools for enabling high-resolution imaging of intracellular cholesterol. In this study, we evaluated how the size of the linker, site of its attachment and nature of the fluorophore, affect the localization and trafficking properties of fluorescent sterol probes. Varying lengths of linker using the same fluorophore affected cell penetration and retention in specific cell compartments. A C-4 linker was confirmed as optimal. Derivatives of heterocyclic sterol precursors attached with identical C-4 linker to different fluorophores at diverse positions also showed significant differences in their binding properties to various intracellular compartments and kinetics of trafficking. Two novel red-emitting probes with good cell permeability, fast intracellular labelling and slightly different distribution displayed very promising characteristics for sterol probes. These probes also strongly labelled endo/lysosomal compartment in cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, that overlapped with filipin staining of cholesterol. Overall, the present study demonstrates that the physicochemical properties of the fluorophore/linker pairing determine the kinetics of uptake and distribution and subsequently influence the applicability of final probes.

SUBMITTER: Kralova J

PROVIDER: S-EPMC7745015 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Heterocyclic sterol probes for live monitoring of sterol trafficking and lysosomal storage disorders.

Project description:The monitoring of intracellular cholesterol homeostasis and trafficking is of great importance because their imbalance leads to many pathologies. Reliable tools for cholesterol detection are in demand. This study presents the design and synthesis of fluorescent probes for cholesterol recognition and demonstrates their selectivity by a variety of methods. The construction of dedicated library of 14 probes was based on heterocyclic (pyridine)-sterol derivatives with various attached fluorophores. The most promising probe, a P1-BODIPY conjugate FP-5, was analysed in detail and showed an intensive labelling of cellular membranes followed by intracellular redistribution into various cholesterol rich organelles and vesicles. FP-5 displayed a stronger signal, with faster kinetics, than the commercial TF-Chol probe. In addition, cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, exhibited strong and fast FP-5 signal in the endo/lysosomal compartment, co-localizing with filipin staining of cholesterol. Hence, FP-5 has high potential as a new probe for monitoring cholesterol trafficking and its disorders.

| S-EPMC6158244 | biostudies-literature

Fluorescent Probes for Subcellular Localization during Osteclast Formation.

Project description:Labeling of a small bioactive molecule with fluorescent probe has been becoming an essential tool in cell biology to reveal the subcellular distribution and the location of a molecular target. QOA-8a is a novel molecule with potent antiosteoporotic effect in vivo. To investigate the molecular mechanism of QOA-8a, novel fluorescence-tagged chemical probes as bioactive as their parent molecule were designed and synthesized. The fluorescent compound 12 showed a more potent inhibitory activity on RANKL-induced osteoclastogenesis at 2 ?M compared with that of QOA-8a. Microscopy experiments revealed that almost all of probe 12 accumulated in the fusing region, with little in the osteoclast precursors or the mature osteoclasts during osteoclast formation. The result suggests the location of the binding target of QOA-8a, which might greatly narrow down the search field of the target protein(s).

| S-EPMC4137367 | biostudies-literature

Use of fluorescent probes in the study of phospholipid--sterol bilayers.

Project description:1. The transfer of excitation energy between the fluorescent probes 1,6-diphenylhexa-1,3,5-triene and 12-(9-anthroyl)stearic acid and the cholesterol analogue cholesta-4,6-dien-3-one in phosphatidylcholine liposomes has been investigated. 2. The results indicate that probes and steroid are randomly distributed in the bilayer at steroid concentrations up to 35 mol%. 3. The degree of polarization of diphenylhexatriene fluorescence increases with increasing cholesterol content. Other sterols, differing in structure in the region of the polar group or in the side chain at position-17, produce similar but not identical effects. 4. the results are consistent with the proposal that diphenylhexatriene gives a general picture of the state of the bilayer and that there is no segregation of sterols in liquid-crystalline phosphatidylcholine bilayers.

| S-EPMC1162278 | biostudies-other

Linker length in fluorophore-cholesterol conjugates directs phase selectivity and cellular localisation in GUVs and live cells.

Project description:Lipid membrane fluorescent probes that are both domain-selective and compatible with demanding microscopy methods are crucial to elucidate the presence and function of rafts and domains in cells and biophysical models. Whereas targeting fluorescent probes to liquid-disordered (Ld) domains is relatively facile, it is far more difficult to direct probes with high selectivity to liquid-ordered (Lo) domains. Here, a simple, one-pot approach to probe-cholesterol conjugation is described using Steglich esterification to synthesise two identical BODIPY derivatives that differ only in the length of the aliphatic chain between the dye and cholesterol. In the first, BODIPY-Ar-Chol, the probe and cholesterol were directly ester linked and in the second BODIPY-Ahx-Chol, a hexyl linker separated probe from cholesterol. Uptake and distribution of each probe was compared in ternary, phase separated giant unilamellar vesicles (GUVs) using a commercial Ld marker as a reference. BODIPY-Ar-Chol targets almost exclusively the Ld domains with selectivity of >90% whereas by contrast introducing the C6 linker between the probe and cholesterol drove the probe to Lo with excellent selectivity (>80%). The profound impact of the linker length extended also to uptake and distribution in live mammalian cells. BODIPY-Ahx-Chol associates strongly with the plasma membrane where it partitioned preferably into opposing micron dimensioned do-mains to a commercial Ld marker and its concentration at the membrane was reduced by cyclodextrin treatment of the cells. By contrast the BODIPY-Ahx-Chol permeated the membrane and localised strongly to lipid droplets within the cell. The data demonstrates the profound influence of linker length in cholesterol bioconjugates in directing the probe.

| S-EPMC9067298 | biostudies-literature

Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates.

Project description:A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. We have synthesized over 100 polyamide-fluorophore conjugates and assayed their nuclear uptake profiles in 13 mammalian cell lines. The compiled dataset, comprising 1300 entries, establishes a benchmark for the nuclear localization of polyamide-dye conjugates. Compounds in this series were chosen to provide systematic variation in several structural variables, including dye composition and placement, molecular weight, charge, ordering of the aromatic and aliphatic amino-acid building blocks and overall shape. Nuclear uptake does not appear to be correlated with polyamide molecular weight or with the number of imidazole residues, although the positions of imidazole residues affect nuclear access properties significantly. Generally negative determinants for nuclear access include the presence of a beta-Ala-tail residue and the lack of a cationic alkyl amine moiety, whereas the presence of an acetylated 2,4-diaminobutyric acid-turn is a positive factor for nuclear localization. We discuss implications of these data on the design of polyamide-dye conjugates for use in biological systems.

| S-EPMC419610 | biostudies-literature

Fluorescent probes of the apoptolidins and their utility in cellular localization studies.

Project description:Apoptolidin A has been described among the top 0.1% most-cell-selective cytotoxic agents to be evaluated in the NCI 60 cell line panel. The molecular structure of apoptolidin A consists of a 20-membered macrolide with mono- and disaccharide moieties. In contrast to apoptolidin A, the aglycone (apoptolidinone) shows no cytotoxicity (>10 μM) when evaluated against several tumor cell lines. Apoptolidin H, the C27 deglycosylated analogue of apoptolidin A, displayed sub-micromolar activity against H292 lung carcinoma cells. Selective esterification of apoptolidins A and H with 5-azidopentanoic acid afforded azido-functionalized derivatives of potency equal to that of the parent macrolide. They also underwent strain-promoted alkyne-azido cycloaddition reactions to provide access to fluorescent and biotin-functionalized probes. Microscopy studies demonstrate apoptolidins A and H localize in the mitochondria of H292 human lung carcinoma cells.

| S-EPMC4293314 | biostudies-literature

Influence of linker length in shape recognition of B* DNA by dimeric aminoglycosides.

Project description:DNA-protein recognition has shown us the importance of DNA shapes in the recognition process. Specific high-affinity targeting of DNA shapes by small molecules is desirable for many biological applications that involve regulation of DNA based processes. Here, the effect of linker length and rigidity on the affinity of a conjugated neomycin dimer for a specific DNA shape (B* form) AT-rich DNA was explored. Binding constants approximating 10(8)M(-1) for optimal linker lengths of 18-19 atoms are reported herein.

| S-EPMC4837466 | biostudies-literature

Synthesis, Biological Evaluation of Fluorescent 23-Hydroxybetulinic Acid Probes, and Their Cellular Localization Studies.

Project description:23-Hydroxybetulinic acid (23-HBA) is a complex lupane triterpenoid, which has attracted increasing attention as an anticancer agent. However, its detailed mechanism of anticancer action remains elusive so far. To reveal its anticancer mode of action, a series of fluorescent 23-HBA derivatives conjugated with coumarin dyes were designed, synthesized, and evaluated for their antiproliferative activities. Subcellular localization and uptake profile studies of representative fluorescent 23-HBA probe 26c were performed in B16F10 cells, and the results suggested that probe 26c was rapidly taken up into B10F10 cells in a dose-dependent manner and mitochondrion was the main site of its accumulation. Further mode of action studies implied that the mitochondrial pathway was involved in 23-HBA-mediated apoptosis. Together, our results provided new clues for revealing the molecular mechanism of natural product 23-HBA for its further development into an antitumor agent.

| S-EPMC6187402 | biostudies-literature

Linker length and fusion site composition improve the optical signal of genetically encoded fluorescent voltage sensors.

Project description:Several genetically encoded fluorescent sensors of voltage were created by systematically truncating the length of the linker sequence between the voltage-sensing domain and the position of the fluorescent protein, Super Ecliptic A227D. In addition to varying the length, the amino acid composition at the fusion site for the fluorescent protein was modified. Both linker length and amino acid composition affected the size and voltage sensitivity of the optical signal. The truncation mutants revealed a potential structural periodicity with a maximum signal three amino acids from the voltage-sensing domain and another maximum 11 amino acids from the voltage-sensing domain. These results confirm that the linker length and composition can fine tune the size and voltage range of the sensor. The potential periodicity suggests that the orientation of the fluorescent protein could be important for improving the signal size implicating dimerization of the fluorescent protein.

| S-EPMC4478964 | biostudies-literature

Single molecule localization microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes.

Project description:Several approaches have been described to fluorescently label and image DNA and chromatin in situ on the single-molecule level. These superresolution microscopy techniques are based on detecting optically isolated, fluorescently tagged anti-histone antibodies, fluorescently labeled DNA precursor analogs, or fluorescent dyes bound to DNA. Presently they suffer from various drawbacks such as low labeling efficiency or interference with DNA structure. In this report, we demonstrate that DNA minor groove binding dyes, such as Hoechst 33258, Hoechst 33342, and DAPI, can be effectively employed in single molecule localization microscopy (SMLM) with high optical and structural resolution. Upon illumination with low intensity 405 nm light, a small subpopulation of these molecules stochastically undergoes photoconversion from the original blue-emitting form to a green-emitting form. Using a 491 nm laser excitation, fluorescence of these green-emitting, optically isolated molecules was registered until "bleached". This procedure facilitated substantially the optical isolation and localization of large numbers of individual dye molecules bound to DNA in situ, in nuclei of fixed mammalian cells, or in mitotic chromosomes, and enabled the reconstruction of high-quality DNA density maps. We anticipate that this approach will provide new insights into DNA replication, DNA repair, gene transcription, and other nuclear processes.

| S-EPMC4152347 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data