The G1/S regulator Whi5 shapes the proliferative structure of clonal cell cultures

Abstract Clonal cultures exhibit phenotypic variation in spite of being composed of genetically identical cells under equal environmental conditions. Proliferation rate is one of the characters that have been described to show this heterogeneity, but the mechanisms underlying this phenomenon are still poorly understood. Cell cycle regulation controls proliferative capacity and previous transcriptomic studies revealed that budding yeast microcolonies with low proliferation rates display high levels of the G1-S transition inhibitor Whi5. This regulator, which, in budding yeast, plays a similar role to Retinoblastoma protein in mammalian cells, has been also linked to replicative aging in very late steps of mother cells lifespan. In this work, we combined single cell microencapsulation with confocal microscopy to study heterogeneity in clonal cultures. Using this new method, we found that a significant proportion of slow-growing microcolonies are founded by mother cells with a very short number of cell cycles. We also found that the reduction in the proliferation capacity of microcolonies founded by young mother cells is related to the expression levels of Whi5, which increases with the number of mother cell replication rounds since early stages. Our results establish that the proliferative structure of a cell population is cumulatively shaped in every mitotic cycle, when a mother cell has the opportunity to increase Whi5 expression levels and develop a slow proliferating lineage. Significance Statement Proliferative heterogeneity has been widely described in clonal cultures, but the mechanisms underlying this phenomenon are still poorly understood. In this work we have developed a new technology combining single cell microencapsulation with confocal microscopy to dissect cell lineages. We found that a proportion of microcolonies, predominantly founded by mother cells with a short number of divisions, present reduced proliferation rates and increased levels of the cell cycle regulator Whi5. Our results indicate that stably reduced proliferation is not exclusively linked to aged cells and open a new perspective to understand heterogeneity of clonal cell populations.