Improved tools for live imaging of F-actin structures in yeast.

Abstract

For over 20 years, the most effective probe for live imaging of yeast actin cables has been Abp140-GFP. Here, we report that endogenously-tagged Abp140-GFP poorly decorates actin patches and cables in the bud compartment of yeast cells, while robustly decorating these structures in the mother cell. Using mutagenesis, we found that asymmetric decoration by Abp140 requires F-actin binding. By expressing integrated Bni1-Bnr1 and Bnr1-Bni1 chimeras, we demonstrate that asymmetric cable decoration by Abp140 also does not depend on which formin assembles the cables in each compartment. In contrast, the short actin-binding fragment of Abp140 (known as "Lifeact"), fused to 1x or 3xmNeonGreen and expressed from the endogenous ABP140 promoter, uniformly decorates patches and cables in both compartments. Further, this probe dramatically improves live imaging detection of cables (and patches) without altering their in vivo dynamics or cell growth. Improved detection allows us to visualize cables growing inward from the cell cortex and dynamically interacting with the vacuole. This probe also robustly decorates the cytokinetic actomyosin ring. Because Lifeact-3xmNeon expressed at relatively low levels provides intense labeling of cellular F-actin structures, this tool may improve live imaging in other organisms where higher levels of Lifeact expression are detrimental.