Fucci has proven effective in numerous applications, but Miyawaki’s team ran into complications while studying the differentiation of neuronal progenitors. “The nuclear targeting of the fluorescence signal did not permit us to identify cell types and differentiation states by their characteristic morphologies,” explains Miyawaki. To address this, his team developed a Fucci variant that illuminates the cytoplasm, enabling simultaneous monitoring of transitions in cell shape and cell cycle state**.
Normally, Geminin is strictly localized to the nucleus; however, by trimming a segment from the end of the protein, Miyawaki’s team was able to engineer Fucci-ready variants that linger in the cytoplasm. Upon testing their new Fucci markers in neural progenitors in the developing mouse brain, they found that they could now classify different types of neuroepithelial progenitors based on changes in growth and morphology as well as their cycling behavior.
For Fucci to maintain an accurate readout, the cell must degrade labeled Geminin at the end of each cycle, and Miyawaki’s team found that this requires the protein to be in the nucleus. Fortunately, the modified Geminin is only transiently located in the cytoplasm before being shuttled back to the nucleus, minimizing the overall impact on Fucci’s effectiveness.
Miyawaki’s future goals include expansion of the Fucci palette to incorporate additional color tags and spotlight different, specific segments of the cell cycle, but he sees immediate value in this latest variant. “This type of indicator, which traces the silhouette of individual cells with fluorescence in a cell cycle-dependent manner, will be useful for imaging mitotic cells with interesting shapes, including neural progenitor cells,” he says.
*Sakaue-Sawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H., Osawa, H. Kashiwagi, S., Fukami, K., Miyata, T., Miyosi, H. et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132, 487–498 (2008).
**Sakaue-Sawano, A., Ohtawa, K., Hama, H., Kawano, M., Ogawa, M. & Miyawaki, A. Tracing the silhouette of individual cells in S/G2/M phases with fluorescence. Chemistry & Biology 15, 1243–1248 (2008).