Stanford University, Stanford, USA.
Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
Sci Rep. 2024 Oct 5;14(1):23159. doi: 10.1038/s41598-024-73887-7.
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.
细胞内蛋白质的区室化和差异分布维持着细胞的功能和活力。CapG 是一种与凝胶蛋白相关的肌动蛋白结合蛋白，在稳态下弥散分布于细胞质和细胞核中。为了在单细胞水平上检测 CapG 核质穿梭在对外界刺激的反应中的变化，我们建立了重复 FRAP 实验。通过这种实验设置，我们发现 ATP 耗竭可使 CapG 向细胞核的穿梭可逆性减少。表皮生长因子（EGF）的加入使 CapG 的核穿梭在数分钟内增加。血清饥饿使乳腺癌细胞的数量增加了一倍，从 40%增加到 80%，对 EGF 的 CapG 穿梭增加。对五个不同的潜在 CapG 磷酸化位点进行测试，我们发现丝氨酸 70 介导 EGF 触发的 CapG 核穿梭增加。因此，细胞核中 CapG 的重复 FRAP 可以作为同一单个活乳腺癌细胞中信号级联的功能读出。
