Time-lapse imaging of a dynamic phosphorylation-dependent protein-protein interaction in mammalian cells

Targeting the interaction of proteins with weak binding affinities or low solubility represents a particular challenge for drug screening. The NanoLucâ ® Binary Technology (NanoBiTâ ®) was originally developed to detect protein-protein interactions in live mammalian cells. Here we report the successful translation of the NanoBit cellular assay into a biochemical, cell-free format using mammalian cell lysates. We show that the assay is suitable for the detection of both strong and weak protein interactions such as those involving the binding of RAS oncoproteins to either RAF or phosphoinositide 3-kinase (PI3K) effectors respectively, and that it is also effective for the study of poorly soluble protein domains such as the RAS binding domain of PI3K. Furthermore, the RAS interaction assay is sensitive and responds to both strong and weak RAS inhibitors. Our data show that the assay is robust, reproducible, cost-effective, and can be adapted for small and large-scale screening approaches. The NanoBit Biochemical Assay offers an attractive tool for drug screening against challenging protein-protein interaction targets, including the interaction of RAS with PI3K.

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CaM kinase Iα–induced phosphorylation of Drp1 regulates mitochondrial morphology

The Journal of Cell Biology ◽

10.1083/jcb.200802164 ◽

2008 ◽

Vol 182 (3) ◽

pp. 573-585 ◽

Cited By ~ 284

Author(s):

Xiao-Jian Han ◽

Yun-Fei Lu ◽

Shun-Ai Li ◽

Taku Kaitsuka ◽

Yasufumi Sato ◽

...

Keyword(s):

Protein Kinase ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Time Lapse ◽

Cell Types ◽

Mitochondrial Morphology ◽

Voltage Dependent ◽

Dependent Protein ◽

Time Lapse Imaging

Mitochondria are dynamic organelles that frequently move, divide, and fuse with one another to maintain their architecture and functions. However, the signaling mechanisms involved in these processes are still not well characterized. In this study, we analyze mitochondrial dynamics and morphology in neurons. Using time-lapse imaging, we find that Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) causes a rapid halt in mitochondrial movement and induces mitochondrial fission. VDCC-associated Ca2+ signaling stimulates phosphorylation of dynamin-related protein 1 (Drp1) at serine 600 via activation of Ca2+/calmodulin-dependent protein kinase Iα (CaMKIα). In neurons and HeLa cells, phosphorylation of Drp1 at serine 600 is associated with an increase in Drp1 translocation to mitochondria, whereas in vitro, phosphorylation of Drp1 results in an increase in its affinity for Fis1. CaMKIα is a widely expressed protein kinase, suggesting that Ca2+ is likely to be functionally important in the control of mitochondrial dynamics through regulation of Drp1 phosphorylation in neurons and other cell types.

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Biochemical Characterization of RAR1 Cysteine- and Histidine-Rich Domains (CHORDs): A Novel Class of Zinc-Dependent Protein−Protein Interaction Modules†

Biochemistry ◽

10.1021/bi062174k ◽

2007 ◽

Vol 46 (6) ◽

pp. 1612-1623 ◽

Cited By ~ 14

Author(s):

Charles T. Heise ◽

Cécile S. Le Duff ◽

Marta Boter ◽

Catarina Casais ◽

Joanne E. Airey ◽

...

Keyword(s):

Protein Interaction ◽

Biochemical Characterization ◽

Protein Protein Interaction ◽

Dependent Protein

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Knock-in of labeled proteins into 5’UTR enables highly efficient generation of stable cell lines

10.1101/2020.08.03.234252 ◽

2020 ◽

Author(s):

Faryal Ijaz ◽

Koji Ikegami

Keyword(s):

Cell Lines ◽

Mammalian Cells ◽

Time Lapse ◽

Alpha Tubulin ◽

Endogenous Gene ◽

Stable Cell Lines ◽

Long Time ◽

Endogenous Proteins ◽

Endogenous Loci ◽

Time Lapse Imaging

AbstractStable cell lines and animal models expressing tagged proteins are important tools for studying behaviors of cells and molecules. Several molecular biological technologies have been applied with varying degrees of success and efficiencies to establish cell lines expressing tagged proteins. Here we applied CRISPR/Cas9 for the knock-in of tagged proteins into the 5’UTR of the endogenous gene loci. With this 5’UTR-targeting knock-in strategy, stable cell lines expressing Arl13b-Venus, Reep6-HA, and EGFP-alpha-tubulin were established with high knock-in efficiencies ranging from 50 to 80%. The localization of the knock-in proteins were identical to that of the endogenous proteins in wild-type cells and showed homogenous expression. Moreover, the expression of knock-in EGFP-alpha-tubulin from the endogenous promoter was stable over long-term culture. We further demonstrated that the fluorescent signals were enough for a long time time-lapse imaging. The fluorescent signals were distinctly visible during the whole duration of the time-lapse imaging and showed specific subcellular localizations. Altogether, our strategy demonstrates that 5’UTR is a ‘hotspot’ for targeted insertion of gene sequences and allows the stable expression of tagged proteins from endogenous loci in mammalian cells.

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Development of a cell-free split-luciferase biochemical assay as a tool for screening for inhibitors of challenging protein-protein interaction targets

Wellcome Open Research ◽

10.12688/wellcomeopenres.15675.2 ◽

2020 ◽

Vol 5 ◽

pp. 20

Author(s):

Rachel Cooley ◽

Neesha Kara ◽

Ning Sze Hui ◽

Jonathan Tart ◽

Chloë Roustan ◽

...

Keyword(s):

Protein Interaction ◽

Drug Screening ◽

Protein Interactions ◽

Mammalian Cells ◽

Large Scale ◽

Cost Effective ◽

Biochemical Assay ◽

Protein Protein Interactions ◽

Protein Protein Interaction ◽

Phosphoinositide 3 Kinase

Targeting the interaction of proteins with weak binding affinities or low solubility represents a particular challenge for drug screening. The NanoLuc ® Binary Technology (NanoBiT ®) was originally developed to detect protein-protein interactions in live mammalian cells. Here we report the successful translation of the NanoBit cellular assay into a biochemical, cell-free format using mammalian cell lysates. We show that the assay is suitable for the detection of both strong and weak protein interactions such as those involving the binding of RAS oncoproteins to either RAF or phosphoinositide 3-kinase (PI3K) effectors respectively, and that it is also effective for the study of poorly soluble protein domains such as the RAS binding domain of PI3K. Furthermore, the RAS interaction assay is sensitive and responds to both strong and weak RAS inhibitors. Our data show that the assay is robust, reproducible, cost-effective, and can be adapted for small and large-scale screening approaches. The NanoBit Biochemical Assay offers an attractive tool for drug screening against challenging protein-protein interaction targets, including the interaction of RAS with PI3K.

Download Full-text