Budded baculoviruses as a receptor display system to quantify ligand binding with TIRF microscopy

Laasfeld, Tõnis; Ehrminger, Robin; Tahk, Maris‐Johanna; Veikšina, Santa; Kõlvart, Karl Rene; Min, Mart; Kopanchuk, Sergei; Rinken, Ago

doi:10.1039/d0nr06737g

Cited by 14 publications

(16 citation statements)

References 39 publications

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“…Conventional pharmacological studies require measurements of concentration dependences of numerous ligands, agonists, partial agonists, and antagonists to provide conclusions about the protein as a drug target. Here we present the proof of principle assay system, which has a real potential to study Sig1R intracellular dynamics and automatization of the imaging for increased throughput screening [ 52 ] will allow to evaluate the activity of novel compounds acting at Sig1R.…”

Section: Discussionmentioning

confidence: 99%

Intracellular dynamics of the Sigma-1 receptor observed with super-resolution imaging microscopy

et al. 2022

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Sigma-1 receptor (Sig1R) is an endoplasmic reticulum (ER)-related membrane protein, that forms heteromers with other cellular proteins. As the mechanism of action of this chaperone protein remains unclear, the aim of the present study was to detect and analyze the intracellular dynamics of Sig1R in live cells using super-resolution imaging microscopy. For that, the Sig1R-yellow fluorescent protein conjugate (Sig1R-YFP) together with fluorescent markers of cell organelles were transfected into human ovarian adenocarcinoma (SK-OV-3) cells with BacMam technology. Sig1R-YFP was found to be located mainly in the nuclear envelope and in both tubular and vesicular structures of the ER but was not detected in the plasma membrane, even after activation of Sig1R with agonists. The super-resolution radial fluctuations approach (SRRF) performed with a highly inclined and laminated optical sheet (HILO) fluorescence microscope indicated substantial overlap of Sig1R-YFP spots with KDEL-mRFP, slight overlap with pmKate2-mito and no overlap with the markers of endosomes, peroxisomes, lysosomes, or caveolae. Activation of Sig1R with (+)-pentazocine caused a time-dependent decrease in the overlap between Sig1R-YFP and KDEL-mRFP, indicating that the activation of Sig1R decreases its colocalization with the marker of vesicular ER and does not cause comprehensive translocations of Sig1R in cells.

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Section: Discussionmentioning

confidence: 99%

Intracellular dynamics of the Sigma-1 receptor observed with super-resolution imaging microscopy

et al. 2022

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Section: Methodsmentioning

confidence: 99%

“…Fluorescence anisotropy-based (FA-based) Y 1 R binding studies with fluorescent ligand 39 were performed at hY 1 R displaying budded baculovirus particles (termed BBVs below), which were prepared as described previously. 59 The obtained viruses were collected by centrifugation at 1600g for 10 min, and the virus titer was determined with an image-based cell-size estimation assay as described elsewhere. 60 The viruses were amplified using a multiplicity of infection (MOI) between 0.01 and 0.1 until a high-titer baculovirus stock (virus concentration of at least 1.0 × 10 8 infectious viral particles/mL) was acquired.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Structure-Based Design of High-Affinity Fluorescent Probes for the Neuropeptide Y Y₁ Receptor

et al. 2022

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The recent crystallization of the neuropeptide Y Y 1 receptor (Y 1 R) in complex with the argininamide-type Y 1 R selective antagonist UR-MK299 (2) opened up a new approach toward structure-based design of nonpeptidic Y 1 R ligands. We designed novel fluorescent probes showing excellent Y 1 R selectivity and, in contrast to previously described fluorescent Y 1 R ligands, considerably higher (∼100-fold) binding affinity. This was achieved through the attachment of different fluorescent dyes to the diphenylacetyl moiety in 2 via an amine-functionalized linker. The fluorescent ligands exhibited picomolar Y 1 R binding affinities (pK i values of 9.36−9.95) and proved to be Y 1 R antagonists, as validated in a Fura-2 calcium assay. The versatile applicability of the probes as tool compounds was demonstrated by flow cytometry-and fluorescence anisotropy-based Y 1 R binding studies (saturation and competition binding and association and dissociation kinetics) as well as by widefield and total internal reflection fluorescence (TIRF) microscopy of live tumor cells, revealing that fluorescence was mainly localized at the plasma membrane.

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“…The following treatment of cells with compounds 6 or 7 (5 μM or 2.5 μM final concentrations) was performed as described above. Imaging was carried out with the TIRF microscopy setup as described in [ 72 ]. Briefly, widefield epifluorescence and Highly Inclined and Laminated Optical sheet (HILO) imaging [ 73 ] was conducted using an inverted microscope built around a Till iMIC body (Till Photonics/FEI; Munich, Germany), equipped with TIRF APON 60× oil (NA 1.49) objective lenses (Olympus Corp., Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family

et al. 2021

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We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.

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Budded baculoviruses as a receptor display system to quantify ligand binding with TIRF microscopy

Abstract: Characterization of ligand binding properties to receptors and other membrane proteins in budded baculovirus nanoparticles using TIRF microscopy.

Cited by 14 publications

References 39 publications

Intracellular dynamics of the Sigma-1 receptor observed with super-resolution imaging microscopy

Intracellular dynamics of the Sigma-1 receptor observed with super-resolution imaging microscopy

Structure-Based Design of High-Affinity Fluorescent Probes for the Neuropeptide Y Y₁ Receptor

Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family

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Budded baculoviruses as a receptor display system to quantify ligand binding with TIRF microscopy

Abstract: Characterization of ligand binding properties to receptors and other membrane proteins in budded baculovirus nanoparticles using TIRF microscopy.

Cited by 14 publications

References 39 publications

Intracellular dynamics of the Sigma-1 receptor observed with super-resolution imaging microscopy

Intracellular dynamics of the Sigma-1 receptor observed with super-resolution imaging microscopy

Structure-Based Design of High-Affinity Fluorescent Probes for the Neuropeptide Y Y1 Receptor

Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family

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Product

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Structure-Based Design of High-Affinity Fluorescent Probes for the Neuropeptide Y Y₁ Receptor