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Characterization of the mechanisms underlying the asymmetric segregation of cell fate determinants in budding yeast Saccharomyces cerevisiae

Final Report Summary - ESCA-Y (Characterization of the mechanisms underlying the asymmetric segregation of cell fate determinants in budding yeast Saccharomyces cerevisiae)

A modern challenge in cell biology is to fully characterize the mechanisms underlying cell lineage decisions, which take place during the development and the lifetime of multicellular organisms. To do so, adult stem cells exit quiescence and divide asymmetrically to produce two daughter cells with different properties and fate, one being able to self-renew, the other to differentiate. How this is achieved is still unclear, mainly due to the scarcity and difficulty to manipulate stem cells in their environment. Of interest, cell fate determinants (i.e. DNA strands, histones and at least one associated epigenetic mark) have recently been shown to segregate in a biased manner in human or Drosophila adult stem cells. However the mechanisms responsible for the asymmetric segregation of chromatin components are unknown and will not be gathered easily in these biological systems where stem cells dividing asymmetrically are rare, dependent on cellular context (niche) and not amenable to genetic dissection.
As it has been the case already for many important and conserved biological processes, light may come from studying simpler model organisms. Important in this regard is a study on germinating yeast spores exiting quiescence, which revealed the existence of a single cell lineage in yeast defined by the asymmetric segregation of kinetochore components. In addition, one striking feature, which has gained little attention so far, is that all cells displaying asymmetric divisions to establish a specific cell lineage seem generally to exit from quiescence. This observation suggests that the first cell cycle following exit from quiescence is somehow different compared to that of cycling cells. Unfortunately, little is known about the specific feature of this first cell cycle, mostly for historical and technical reasons.
Within this project we aimed at (i) characterizing the first cell cycle following exit from quiescence and (ii) identifying cell fate determinants that segregate asymmetrically to unravel the mechanisms underlying cell lineage establishment using the budding yeast Saccharomyces cerevisiae as a working model. Several important results were obtained:
- A novel method was developed to improve cell cycle analysis in yeast using the thymidine analog EdU (5’-ethynyl-2’-deoxyuridine). This protocol is now used in routine to determine (i) whether some mutants have replication defects that may have escaped detection using the tools previously available, (ii) the duration of G1, S and G2+M cell cycle phases, and (iii) whether the fraction of cells in each phase of the cell cycle varies depending on the environment.
- We also devised methods to isolate large amounts of quiescent yeast cells that, upon release in rich medium, synchronously initiate and progress through the cell cycle. Surprisingly we found that the B-type cyclin Clb3, which normally promotes the G2/M transition in vegetative cells, is already expressed in G1 after G0 exit. This is the first indication that this cell cycle is different from the one traditionally studied.
- We found that the evolutionary conserved Greatwall/Rim15 pathway promotes cell cycle entry and cell size homeostasis during metabolic rewiring of cells as they shift from fermentation to respiration before quiescence entry.
- Strikingly we found that germinating yeast spores tend to co-segregate chromatids containing the older DNA strand, similar to what was observed in mouse adult stem cells exiting quiescence and establishing a new cell lineage.
- We compared the nuclear composition of cells arrested in late G1 either after G0 exit or after continuous proliferation, establishing a list of proteins (including histones and enzymes involved in dNTP synthesis) that are enriched after G0 exit. These are candidates to unravel the mechanisms underlying the asymmetric segregation of sister chromatids.

The results obtained within this project are at the intersection of cell cycle, metabolism and stem cell biology. They open promising new avenues to unravel the mechanism involved in the asymmetric segregation of cell fate determinants. Such studies are highly relevant for stem cell biology and therapeutic purposes. Indeed (i) the imbalance between stem cell self-renewal and differentiation may underlie several disorder (tumorigenesis, tissue ageing or degeneration), (ii) the discovery of induced pluripotent stem (iPS) cells has broadened the promises of regenerative medicine (iii) a hypothesis about cancer stem cells proposes that they would be necessary and sufficient to initiate and maintain the disease. Therefore, using stem cells or developing therapies without fully mastering the mechanisms leading to self-renewal and differentiation may lead to treatment failure.