The mechanistic target of rapamycin (mTOR) signalling plays crucial roles in controlling mammalian cell behaviour and function, and thereby maintains tissue homeostasis and organism health. This signalling functions through two distinct multi-protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Both complexes are essential in embryo development as deficiency of either one in mouse leads to embryonic lethality. Considerable knowledge has been obtained regarding to mTORC1 while much less is known about mTORC2, including its function in human embryonic development. In the current study, I utilise CRISPR/Cas9 technology to generate mTORC2-deficient HEK 293T cells and human embryonic stem cells (hESCs) by disrupting one of the mTORC2 key components, RICTOR gene. With these RICTOR-knockout cell model systems, I explored the relationship between the two key subunits (RICTOR and mSIN1) of mTORC2 and their functions in mTORC2 assembly/activity. In particularly, I investigated the effect of mTORC2-deficiency on human pluripotent stem cell properties.

Using RICTOR-KO 293T cells, I have demonstrated that the protein expression of mSIN1 is substantially diminished upon RICTOR-deficiency and so does the mTORC2 activity. Further analyses demonstrate that RICTOR/mTORC2 is crucial for mSIN1 protein stability, which may be partly attributed to the mTORC2-associated phosphorylation on mSIN1-Ser260/270 residues. More importantly, this phosphorylation may also affect substrate recognition of mTORC2, suggesting that mTORC2 may have an auto-regulatory function which affects its substrate selection. In addition to the 293T cells, my data in hESCs reveal that mTORC2 signalling may be dispensable for the hESC self-renewal but may have a function in their cell fate determination, particularly in the differentiation of the mesendoderm lineages. Collectively, my findings advance our understanding in both regulation and function of RICTOR/mTORC2 and knockout cells generated from this work are valuable cell models for the further studies on this important signalling pathway, which will provide potential insights for both new therapies development and regenerative applications.