Multi-Parameter Analysis of Spindle and Cell Cycle Dynamics in Asymmetric Cell Division

Abstract

Budding yeast is a unicellular organism which undergoes an asymmetric cell division. Successful completion of this process requires the assembly and translocation of the mitotic spindle from the mother cavity interior to the plane of cell division prior to segregation of the chromosomes. We use confocal fluorescence microscopy and automated image analysis algorithms to quantify the movement of the mitotic spindle, throughout the cell life cycle, at high spatial and temporal resolution. By selecting fluorescent proteins which specifically label the spindle poles and cell periphery, the spindle dynamics can be characterised within a coordinate system relevant to the cell division. Spindle length and orientation as well as the mother and bud cavity volumes have been extensively quantified in wild type cells. Our observations reveal fluctuations in spindle length and angle in the short pre-anaphase spindles that are distinct from longer anaphase spindles. Furthermore they indicate an apparent correlation between cell morphology and spindle dynamics. To elucidate the contributions of individual intercellular force generators to the fluctuations observed during spindle assembly and positioning, we apply the above methods to cells bearing deletions of the kinesins cin8, kip1 and kip3. It is expected that this work will ultimately enable a connection between observable single cell dynamics and biochemical signal pathways measurable by bulk assays.