Mitotic checkpoint gene expression is tuned by codon usage bias.

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Correct functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about the regulation of SAC gene expression. Here, we show in the fission yeast Schizosaccharomyces pombe that a combination of short mRNA half-lives and long protein half-lives supports stable SAC protein levels. For the SAC genes mad2+ and mad3+ , their short mRNA half-lives are caused, in part, by a high frequency of nonoptimal codons. In contrast, mad1+ mRNA has a short half-life despite a higher frequency of optimal codons, and despite the lack of known RNA-destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co-translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine-tuned to ensure proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.

Pomegranate: 2D segmentation and 3D reconstruction for fission yeast and other radially symmetric cells.

Three-dimensional (3D) segmentation of cells in microscopy images is crucial to accurately capture signals that extend across optical sections. Using brightfield images for segmentation has the advantage of being minimally phototoxic and leaving all other channels available for signals of interest. However, brightfield images only readily provide information for two-dimensional (2D) segmentation. In radially symmetric cells, such as fission yeast and many bacteria, this 2D segmentation can be computationally extruded into the third dimension. However, current methods typically make the simplifying assumption that cells are straight rods. Here, we report Pomegranate, a pipeline that performs the extrusion into 3D using spheres placed along the topological skeletons of the 2D-segmented regions. The diameter of these spheres adapts to the cell diameter at each position. Thus, Pomegranate accurately represents radially symmetric cells in 3D even if cell diameter varies and regardless of whether a cell is straight, bent or curved. We have tested Pomegranate on fission yeast and demonstrate its ability to 3D segment wild-type cells as well as classical size and shape mutants. The pipeline is available as a macro for the open-source image analysis software Fiji/ImageJ. 2D segmentations created within or outside Pomegranate can serve as input, thus making this a valuable extension to the image analysis portfolio already available for fission yeast and other radially symmetric cell types.