Systematic comparison of genetically encoded F-actin probes for visualizing actomyosin dynamics in vivo using C. elegans

Genetically encoded F-actin probes enable visualization of actin organization and dynamics in living cells, but their performance and effects on cellular function can vary by probe, fluorescent protein, and biological context. Here, we systematically compared three F-actin probes - F-tractin, LifeAct, and Utrophin - fused to mScarlet-I, mNeonGreen, or mStayGold in vivo, using Caenorhabditis elegans. We used single-copy transgenic strains and evaluated fluorescence intensity, photostability, developmental and reproductive outcomes, cytokinetic ring closure, and probe localization across diverse cellular contexts. mStayGold provided the greatest brightness and photostability, making it well suited for long-term time-lapse imaging, whereas mNeonGreen offers an alternative for applications requiring photobleaching. Most of the strains exhibited minimal detectable effects on development and cellular function and outperformed previously generated strains tested. Probe performance varied across tissues and developmental stages. F-tractin generally exhibited lower fluorescence intensity than LifeAct and Utrophin, particularly in embryos. Although the probes largely exhibited subcellular localization patterns consistent with phalloidin staining, differences in their labeling of specific F-actin structures were observed. Together, these results establish a practical framework for selecting genetically encoded F-actin probes for C. elegans, they suggest best candidates for other in vivo systems, and they highlight the importance of considering both fluorescent protein and actin-binding probe when designing live-cell imaging experiments.