Investigating the potential role of mechanistic target of rapamycin complex 1 (mTORC1) in regulating cell fate decisions in Dictyostelium discoideum

Understanding the underlying mechanisms of cell fate decisions in response to genetic and environmental factors will help to develop advanced stem cell therapies such as tissue regeneration or replacement of damaged or diseased cells. The social amoebae Dictyostelium discoideum is used as a lineage model which has a growth-to-development transition triggered by starvation. When vegetatively growing cells are starved, they aggregate and generate a multicellular organism, which ultimately develops into a fruiting body comprising two major cell types, spores and stalk cells. In mixed aggregates, heterogeneities in genetic background or environmental conditions during growth influence cell fate decisions. Environmental factors such as nutrient history, intracellular calcium (Ca2+) levels and acidic vesicle pH are well-documented to bias cell fate, however, the underlying mechanism remains unclear. Two assays were used to study cell fates in this thesis: chimeric mixing experiments assessing spore formation in multicellular development and monolayer stalk induction assays monitoring the sensitivity to a stalk inducer differentiation inducing factor-1 (DIF-1) in the absence of cell contact.
One potential link between the factors biasing cell fate decisions is the activity of a protein kinase complex known as mechanistic target of rapamycin complex 1 (mTORC1). In mammalian cells, mTORC1 is an important activator of cell growth and proliferation whose activity is dependent on nutrient and energy status and is present on endo-lysosomal acidic compartments when activated. Also, mTORC1 interacts with and regulates Ca2+ release from lysosomes through a Ca2+ channel protein two-pore channel 2 (TPC2). In Dictyostelium, tpc2- strains have altered Ca2+ signalling, increased acidic vesicle pH and increased mTORC1 activity. Consistent with a role for mTORC1, tpc2- cells prefer a stalk cell fate. To investigate whether altering mTORC1 activity can underlie cell fate bias, mTORC1 pathway activity was analysed in cells with different cell fate preferences and high activity correlated with stalk cell fate bias. To determine if this was causal, the consequences of both pharmacological inhibition of mTORC1 and genetic modulation of genes encoding mTORC1 regulators, such as AMP-activated protein kinase (AMPK) and Ras homolog enriched in brain (Rheb), on cell fate were determined. Use of inhibitors suggested that altering mTORC1 pathway activity has a different effect during growth than during development, indicating distinct roles at different stages in cell fate determination.
One mechanism to regulate mTORC1 activity may be through changes in gene expression. The ratio of expression during growth of two genes (rrgA and rigA) was validated as a marker to predict the cell fate bias prior to development in a range of mutant strains and in different conditions. The expression ratio of these genes is dependent on the presence of specific histone modifications, methylation of histone H3 on lysine 4 and subsequent acetylation, and high rrgA/rigA ratio correlates with high mTORC1 pathway activity and a stalk cell fate bias.
Overall, this thesis reveals a potential role of mTORC1 pathway activity in regulating cell fate decisions in Dictyostelium with wide-spread implications for manipulating this pathway to direct pluripotent stem cells towards defined fates.

Keywords: mTOR signalling pathway
genetic regulation
genetics
developmental biology
cell fate decision
cell biology
Advisor: Pears, C
Galione, A