About the Project
We squared this circle, providing direct evidence that TPC2 carries lysosomal calcium flux, using a recombinant human TPC2 mutant fused at its cytoplasmic N terminus to the calcium indicator GCaMP 5 stably expressed in HEK293 cells, and cells acutely isolated from Tpcn2 knockout mice. Perhaps of greater importance, we demonstrated that that calcium flux through TPC2 is controlled by mTORC1, a complex formed by mechanistic target of Rapamycin and Raptor. mTORC1 inhibits autophagy and promotes cell growth and proliferation, and coordinates necessary adjustments to cellular metabolism, in part through translation control. However, we do not understand the precise, cell-specific mechanisms by which metabolic signals are received and processed by lysosomes. You will determine: (1) How TPC2 interacts with and is regulated by mTORC1 in different cell types; (2) The upstream pathways (e.g. AMP-activated protein kinase) that modulate mTORC1 TPC2 signalling; (3) How TPC2 contributes to autophagy during cell specification and proliferation.
To achieve these goals you will use and receive training in confocal imaging, immunocytochemistry and a variety of molecular techniques, such as qRT-PCR and RNAseq.
kematsu, N., Dallas, M. L., Ross, F.A., Lewis, R.W., Rafferty, J.N., David, J.A., Suman, R., Peers, C., Hardie, D.G., Evans, A.M. (2011). Phosphorylation of the voltage-gated potassium channel Kv2.1 by AMP-activated protein kinase regulates membrane excitability. PNAS USA, 108, 18132-18137.
Ogunbayo O.A., Zhu Y., Rossi D., Sorrentino V., Ma J., Zhu M.X., Evans A.M. (2011). Cyclic adenosine diphosphate ribose activates ryanodine receptors, whereas NAADP activates two-pore domain channels. J. Biol. Chem., 286, 9136-9140.
Ogunbayo, O.A., Zhu, Y., Shen, B., Agbani, E., Li, J., Ma, J., Zhu, M.X., Evans, A.M. (2015). Organelle-specific Subunit Interactions of the Vertebrate Two-pore Channel Family. J. Biol. Chem., 290, 1086-1095.
Ogunbayo OA, Duan J, Xiong J, Wang Q, Feng X, Ma J, Zhu MX and Evans AM. mTORC1 controls lysosomal Ca2+ release through the two-pore channel TPC2. Science Signaling. 2018;11. doi: 10.1126/scisignal.aao5775.
Duan J., Navarro-Dorado J., Clark J.H., Kinnear N.P., Meinke P., Schirmer E.C., Evans A.M. (2019). The cell wide web coordinates cellular processes by directing site-specific Ca2+ flux through cytoplasmic nanocourses. Nat Comm 10, 2299, doi:10.1038/s41467-019-10055-w.
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