ArticleThe Journal of biological chemistry2009
Determination of in vivo dissociation constant, KD, of Cdc42-effector complexes in live mammalian cells using single wavelength fluorescence cross-correlation spectroscopy.
Article in The Journal of biological chemistry, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 32 papers.
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Who cites it
32 citing papers in PubMed, 63 citations in OpenAlex.
- Effective in vivo binding energy landscape illustrates kinetic stability of RBPJ-DNA binding.Nature communications · 2025Article
- Cytoplasmic fluidization contributes to breaking spore dormancy in fission yeast.Proceedings of the National Academy of Sciences of the United States of America · 2024Article
- Computational Approaches to Predict Protein-Protein Interactions in Crowded Cellular Environments.Chemical reviews · 2024Review
- Generation of extracellular morphogen gradients: the case for diffusion.Nature reviews. Genetics · 2021Review
- The intracellular environment affects protein-protein interactions.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
- Article
- Influence of FRET and fluorescent protein maturation on the quantification of binding affinity with dual-channel fluorescence cross-correlation spectroscopy.Biomedical optics express · 2020Article
- Single-cell quantification of the concentrations and dissociation constants of endogenous proteins.The Journal of biological chemistry · 2019Article
- AUC measurements of diffusion coefficients of monoclonal antibodies in the presence of human serum proteins.European biophysics journal : EBJ · 2018Article
- Anosmin1 Shuttles Fgf to Facilitate Its Diffusion, Increase Its Local Concentration, and Induce Sensory Organs.Developmental cell · 2018Article
- Relationship Between Homodimeric Glucocorticoid Receptor and Transcriptional Regulation Assessed via an In Vitro Fluorescence Correlation Spectroscopy-Microwell System.Scientific reports · 2018Article
- Quantifying membrane protein oligomerization with fluorescence cross-correlation spectroscopy.Methods (San Diego, Calif.) · 2018Article
- Toward an understanding of biochemical equilibria within living cells.Biophysical reviews · 2018Review
- A Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell.Scientific reports · 2017Article
- Macromolecular Crowding In Vitro, In Vivo, and In Between.Trends in biochemical sciences · 2016Review
- Article
- Quantitative in vivo fluorescence cross-correlation analyses highlight the importance of competitive effects in the regulation of protein-protein interactions.Molecular and cellular biology · 2014Article
- The IQGAP-related protein DGAP1 mediates signaling to the actin cytoskeleton as an effector and a sequestrator of Rac1 GTPases.Cellular and molecular life sciences : CMLS · 2014Article
- Time-resolved fluorescence spectroscopy measures clustering and mobility of a G protein-coupled receptor opsin in live cell membranes.Journal of the American Chemical Society · 2014Article
- Quantifying intracellular protein binding thermodynamics during mechanotransduction based on FRET spectroscopy.Methods (San Diego, Calif.) · 2014Article
Corrections and comments
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Authors and funding
7 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The RhoGTPase Cdc42 coordinates cell morphogenesis, cell cycle, and cell polarity decisions downstream of membrane-bound receptors through distinct effector pathways. Cdc42-effector protein interactions represent important elements of cell signaling pathways that regulate cell biology in systems as diverse as yeast and humans. To derive mechanistic insights into cell signaling pathways, it is vital that we generate quantitative data from in vivo systems. We need to be able to measure parameters such as protein concentrations, rates of diffusion, and dissociation constants (K(D)) of protein-protein interactions in vivo. Here we show how single wavelength fluorescence cross-correlation spectroscopy in combination with Förster resonance energy transfer analysis can be used to determine K(D) of Cdc42-effector interactions in live mammalian cells. Constructs encoding green fluorescent protein or monomeric red fluorescent protein fusion proteins of Cdc42, an effector domain (CRIB), and two effectors, neural Wiskott-Aldrich syndrome protein (N-WASP) and insulin receptor substrate protein (IRSp53), were expressed as pairs in Chinese hamster ovary cells, and concentrations of free protein as well as complexed protein were determined. The measured K(D) for Cdc42V12-N-WASP, Cdc42V12-CRIB, and Cdc42V12-IRSp53 was 27, 250, and 391 nm, respectively. The determination of K(D) for Cdc42-effector interactions opens the way to describe cell signaling pathways quantitatively in vivo in mammalian cells.
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