Repeated FRAP of the actin-binding protein CapG in the cell nucleus—a functional assay for EGF signaling in the single live breast cancer cell

Compartmentalization and differential distribution of proteins within a cell maintain cellular function and viability. CapG is a gelsolin-related actin-binding protein that distributes in steady state diffusively throughout cytoplasm and cell nucleus. To detect changes in CapG’s nucleocytoplasmic sh...

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Bibliographic Details
Published in:Scientific reports 2024-10, Vol.14 (1), p.23159-8, Article 23159
Main Authors: Fernandez, M. K., Sinha, M., Kühnemuth, R., Renz, M.
Format: Article
Language:English
Subjects:
631/67
631/80
631/80/389
Actin
Breast cancer
Breast Neoplasms - metabolism
Breast Neoplasms - pathology
Cell Line, Tumor
Cell Nucleus - metabolism
Cytoplasm
Differential distribution
Epidermal growth factor
Epidermal Growth Factor - metabolism
Epidermal Growth Factor - pharmacology
External stimuli
Female
Fluorescence Recovery After Photobleaching - methods
Gelsolin
Humanities and Social Sciences
Humans
Microfilament Proteins - metabolism
multidisciplinary
Nuclear Proteins
Phosphorylation
Science
Science (multidisciplinary)
Signal Transduction
Single-Cell Analysis - methods
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Summary:Compartmentalization and differential distribution of proteins within a cell maintain cellular function and viability. CapG is a gelsolin-related actin-binding protein that distributes in steady state diffusively throughout cytoplasm and cell nucleus. To detect changes in CapG’s nucleocytoplasmic shuttling in response to external stimuli on the single cell level, we established repeated FRAP experiments of one and the same breast cancer cell. With this experimental set up, we found that ATP-depletion reversibly decreased CapG’s shuttling into the cell nucleus. The addition of epidermal growth factor (EGF) increased CapG’s nuclear shuttling within minutes. Serum-starvation doubled the number of breast cancer cells from 40 to 80% displaying increased CapG shuttling in response to EGF. Testing five different potential CapG phosphorylation sites, we found that serine 70 mediates the increase in CapG’s nuclear shuttling triggered by EGF. Thus, repeated FRAP of CapG in the cell nucleus can be used as functional readout of signaling cascades in the same single live breast cancer cell.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-024-73887-7