Distinct actin microfilament localization during early cell plate formation through deep learning-based image restoration.

Using deep learning-based image restoration, we achieved high-resolution 4D imaging with minimal photodamage, revealing distinct localization and suggesting Lifeact-RFP-labeled actin microfilaments play a role in initiating cell plate formation. Phragmoplasts are plant-specific intracellular structures composed of microtubules, actin microfilaments (AFs), membranes, and associated proteins. Importantly, they are involved in the formation and the expansion of cell plates that partition daughter cells during cell division. While previous studies have revealed the important role of cytoskeletal dynamics in the proper functioning of the phragmoplast, the localization and the role of AFs in the initial phase of cell plate formation remain controversial. Here, we used deep learning-based image restoration to achieve high-resolution 4D imaging with minimal laser-induced damage, enabling us to investigate the dynamics of AFs during the initial phase of cell plate formation in transgenic tobacco BY-2 cells labeled with Lifeact-RFP or RFP-ABD2 (actin-binding domain 2). This computational approach overcame the limitation of conventional imaging, namely laser-induced photobleaching and phototoxicity. The restored images indicated that RFP-ABD2-labeled AFs were predominantly localized near the daughter nucleus, whereas Lifeact-RFP-labeled AFs were found not only near the daughter nucleus but also around the initial cell plate. These findings, validated by imaging with a long exposure time, highlight distinct localization patterns between the two AF probes and suggest that Lifeact-RFP-labeled AFs play a role in initiating cell plate formation.