Automated spectrophotometric assay for cell division regulation in yeast

Abstract

A spectrophotometric assay is presented for monitoring the regulation of cell division by the polypeptide alpha-factor in cultures of living cells of Saccharomyces cerevisiae yeast. This assay is simple, automated, and may have wider application in the study of other eucaryotic cells that do not require anchorage for cell growth. The kinetics of absorbance change were monitored continuously over time in yeast cell cultures that were mixed and aerated in cuvettes fitted with top-loading propeller stirrers. The absorbance doubling time. TD(Abs), was identical to the cell number doubling time in the absence of cell division arrest by alpha-factor. alpha-Factor lengthened the TD(Abs) during division arrest. At pH 5.8, 10(5) 381G cells/ml, the Khalf-maximal was 250 +/- 50 nM alpha-factor for the TD(Abs) increase during arrest, with a maximum increase of five-fold. After a period of time the TD(Abs) abruptly shortened. This is defined as the spectrophotometric recovery time (RTspec) and was compared to the time of recovery that is due to the reinitiation of cell division monitored by bud emergence (RTBE). RTBE occurred 40 +/- 5 min prior to RTspec when recovery was spontaneous or was artificially induced by the removal of alpha-factor (pH 5.8, 381G). The difference between RTBE and RTspec was independent of alpha-factor concentration between 0.05 and 1 microM and cell concentration between 1 and greater than or equal to 25 x 10(5) cells/ml (pH 5.8, 381G) but was both pH and cell strain dependent. At pH 5.8 and 2.7 the recovery from arrest occurred by inactivation of alpha-factor. The TD(Abs) increase during arrest appears to be due to an alpha-factor-induced inhibition of net cell mass increase, an effect that has not been reported previously. Evidence is presented that this process is also correlated with the formation of cell projections.