A robust split-luciferase-based cell fusion screening for discovering myogenesis-promoting molecules

Li, Qiaojing; Yoshimura, Hideyuki; Maki, Kazuhiro; Tajiri, Ken; Uesugi, Motonari; Hata, Yoshinobu; Ozawa, Toshio

doi:10.1039/c8an00285a

Cited by 7 publications

(4 citation statements)

References 27 publications

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“…As the model phenomenon requiring long-term observation, we selected cell fusion during myogenesis. Myogenesis is the successive process in which stem cells differentiate to myoblast, myocyte, myotubes, and finally matured myofibers. , For the in vitro study of myogenesis, mouse myoblast C2C12 cells are often used, and their fusion into nascent myotubes and subsequent fusion into elongated myotubes are observed over several days. , Previously, we developed a BL-based assay system to evaluate the progress of myogenesis using split firefly luciferase reconstitution technology and protein trans-splicing, but live-cell imaging has never been achieved. In this work, we constructed a NanoKAZ (NKAZ) version of the myogenesis assay system and conducted cellular imaging under microscopy.…”

Section: Results and Discussionmentioning

confidence: 99%

“…We previously developed a split firefly luciferase-based assay system to quantify the progress of myocyte fusion by counting the total BL intensity from the cell samples, but the state of fusing cells was not individually observed. 47 In this work, Ad-FMZ-OH was used in a split NKAZ system for long-term continuous visualization of the cell fusion progress over a day without further addition of the substrate during the observation. The results exhibited the potential of Ad-FMZ-OH for sensitive and prolonged BL imaging with single-cell resolution and expanded the application of the NLuc/NKAZ−FMZ system.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Cell fusion during myogenesis was selected as the model life phenomenon requiring observation over several days. We previously developed a split firefly luciferase-based assay system to quantify the progress of myocyte fusion by counting the total BL intensity from the cell samples, but the state of fusing cells was not individually observed . In this work, Ad-FMZ-OH was used in a split NKAZ system for long-term continuous visualization of the cell fusion progress over a day without further addition of the substrate during the observation.…”

Section: Introductionmentioning

confidence: 99%

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A Series of Furimazine Derivatives for Sustained Live-Cell Bioluminescence Imaging and Application to the Monitoring of Myogenesis at the Single-Cell Level

et al. 2022

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Bioluminescence (BL) imaging, which utilizes light emitted through the enzymatic reaction of luciferase oxidizing its substrate luciferin, enables sensitive and noninvasive monitoring of life phenomena. Herein, we developed a series of caged furimazine (FMZ) derivatives by introducing a protective group at the C-3 position and a hydroxy group at the C-6 phenyl ring to realize long-term live-cell BL imaging based on the NanoLuc (NLuc)/ NanoKAZ (NKAZ)−FMZ system. The membrane permeability and cytotoxicity of the substrates were evaluated and related to their hydrophobicity. Among the series, the derivative with the bulkiest protective group (adamantanecarbonyl group) and a hydroxy substituent (named Ad-FMZ-OH) showed significantly prolonged and constant BL signal in cells expressing NLuc compared to the native FMZ substrate. This derivative enabled continuous BL imaging at the single-cell level for 24 h. Furthermore, we applied Ad-FMZ-OH to BL imaging of myocyte fusion and succeeded in the consecutive and sensitive monitoring at a single-cell level over a day. In summary, NLuc/NKAZ-caged FMZ derivatives have the potential to be applied to live-cell BL imaging of various life phenomena that require long-term observation.

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Section: Results and Discussionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Series of Furimazine Derivatives for Sustained Live-Cell Bioluminescence Imaging and Application to the Monitoring of Myogenesis at the Single-Cell Level

et al. 2022

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“…This reaction is at the center of a variety of biochemical assays that have been used for decades to quantify biological processes, including transcription, translation, posttranscriptional regulation, and protein-protein interaction. FFLUC's dynamic range of many orders-of-magnitude coupled with a clear readout of activity has lent itself to high throughput screens to identify inhibitory compounds [48]. In the HIV field, FFLUC activity has been routinely used in viral pseudotyping assays with TZMbl target cells to measure the potency and breadth of anti-HIV broadly neutralizing antibodies, in addition to quantifying antibodydependent cellular cytotoxicity or ADCC [49].…”

Section: Introductionmentioning

confidence: 99%

Cell-based and cell-free firefly luciferase complementation assay to quantify Human Immunodeficiency Virus type 1 Rev-Rev interaction

Hansen

Baris

Zhao

et al. 2021

Preprint

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Rev is an essential regulatory protein of Human Immunodeficiency Virus type 1 (HIV) that is found in the nucleus of infected cells. Rev multimerizes on the Rev-response element (RRE) of HIV RNA to facilitate the export of intron-containing HIV mRNAs from the nucleus to the cytoplasm, and, as such, HIV cannot replicate in the absence of Rev. We have developed cell-intact and cell-free assays based upon a robust firefly split-luciferase complementation system, both of which quantify Rev-Rev interaction. Using the cell-based system we show that additional Crm1 did not impact the interaction whereas excess Rev reduced it. Furthermore, when a series of mutant Revs were tested, there was a strong correlation between the results of the cell-based assay and the results of a functional Rev trans-complementation infectivity assay. Of interest, a camelid nanobody (NB) that was known to inhibit Rev function enhanced Rev-Rev interaction in the cell-based system. We observed a similar increase in Rev-Rev interaction in a cell-free system, when cell lysates expressing NLUC-Rev or CLUC-Rev were simply mixed. In the cell-free system Rev-Rev interaction occurred within minutes and was inhibited by excess Rev. The levels of interaction between the mutant Revs tested varied by mutant type. Treatment of Rev lysates with RNAse minimally reduced the degree of interaction whereas addition of HIV RRE RNA enhanced the interaction. Purified GST-Rev protein inhibited the interaction. The Z-factor (Z') for the cell-free system was ~0.85 when tested in 96-well format, and anti-Rev NB enhanced the interaction in the cell-free system. Thus, we have developed both cell-intact and cell-free systems that can reliably, rapidly, and reproducibly quantify Rev-Rev interaction. These assays, particularly the cell-free one, may be useful in screening and identifying compounds that inhibit Rev function on a high throughput basis.

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A Split-Luciferase-Based Cell Fusion Assay for Evaluating the Myogenesis-Promoting Effects of Bioactive Molecules

Yoshimura

Ozawa

2021

Methods in Molecular Biology

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A robust split-luciferase-based cell fusion screening for discovering myogenesis-promoting molecules

Cited by 7 publications

References 27 publications

A Series of Furimazine Derivatives for Sustained Live-Cell Bioluminescence Imaging and Application to the Monitoring of Myogenesis at the Single-Cell Level

A Series of Furimazine Derivatives for Sustained Live-Cell Bioluminescence Imaging and Application to the Monitoring of Myogenesis at the Single-Cell Level

Cell-based and cell-free firefly luciferase complementation assay to quantify Human Immunodeficiency Virus type 1 Rev-Rev interaction

A Split-Luciferase-Based Cell Fusion Assay for Evaluating the Myogenesis-Promoting Effects of Bioactive Molecules

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