We were the first to demonstrate that two pore segment channels (TPC1-3) represent a family of calcium release channels that are preferentially targeted to endolysosomes. Only the genes encoding TPC1 (TPCN1) and TPC2 (TPCN2) are expressed in humans, rats and mice. Of these TPC2 is specifically targeted to lysosomes, indicating a role in autophagy which has been confirmed by our previous investigations on skeletal muscle. TPCs represent an evolutionary intermediate between some Calcium permeable transient receptor potential (TRP) channels and voltage-gated calcium channels. Nevertheless, our proposal that TPC2 supports a calcium conductance proved controversial, because others suggested that TPC2 is in fact a sodium-selective channel. Controversy also surrounded the mechanism(s) by which TPC2 is activated. We first proposed that TPC2 and thus lysosomal Ca2+ release is activated by nicotinic acid adenine dinucleotide phosphate (NAADP). Countering this, others proposed that phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), which is critical to endolysosomal operations, gates a TPC2-dependent Na+ conductance that is insensitive to NAADP. In turn, the latter proposal was countered on the grounds that cells derived from Tpcn1 and Tpcn2 knockout mice retained PI(3,5)P2-gated cation currents, but not NAADP-gated calcium currents, which were rescued by overexpression of wild type TPC2.
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.
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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.