Marcos Lages, BM; (2017) Functional profiling of the quiescence-to-proliferation dynamics in fission yeast. Doctoral thesis , UCL (University College London).

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Abstract

Cellular quiescence can be defined as a proliferation stand-by, whereby cells direct their focus towards survival and endurance until growth is favored.10,25 In fact, most cells spend most of their time in a quiescent state.25 Therefore, for microbial cells, a fast and steady transition from this non-dividing state into proliferation confers a fitness advantage and an increased likelihood of self-perpetuation.9,89,90 Unicellular organisms in the wild must thus exhibit a highly efficient regulation of this crucial step or otherwise risk gradual elimination. Moreover, the cellular machineries responsible for this transition appear to be in place, ready for quick activation upon requirement, although it is not yet clear how this is accomplished.102,105 Barcode-sequencing (bar-seq) is a state-of-the-art strategy that allows massive parallel profiling of genome-wide deletion libraries in a pooled environment.136,137 We implemented, established and optimized this high-throughput platform in our Lab, with the purpose of routinely employing this technique in future research. We successfully validated bar-seq to allow kinetic studies during a time-course experiment. Furthermore, we employed this technique to carry out a functional profiling study focusing on the dynamics of the quiescence-proliferation (q-p) transition. Together with the q-p transition profiles for over 2,000 mutants, we identified several genes whose deletion leads to altered q-p dynamics, together with others involved in biological processes important for this transition, namely autophagy and chromatin regulation. Furthermore, we uncovered several promising players exhibiting unusual q-p transition profiles, where the kinetics of this transition 7 was found to be clearly affected. This study thus constitutes a valuable framework for follow-up studies aiming to further elucidate the basis of the dynamic behavior associated with the quiescence-proliferation transition. In addition, we carried out a detailed characterization of the dynamics of this transition in fission yeast using mathematical models specifically developed for that purpose. Furthermore, we carried out growth fitness profiling of a genome-wide deletion library of non-essential genes in a pool environment, using bar-seq. We identified the mutants that were most affected with respect to growth kinetics during the q-p transition, and estimated their doubling times and respective growth rates. This project should prove to be a valuable resource to guide future work using this deletion library in general, and research on the q-p transition in particular.

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