Aim 1: Determine the amino acid sensing specificity of folliculin. Since folliculin’s has novel molecular function in amino acid sensing I plan to further understand whether FLCN specifically senses a certain amino acid and if this level of detection is enough to elicit an mTORC1 response.
1.1. Determine if folliculin’s novel molecular function in amino acid sensing is triggered by glutamine. mTORC1 activation by amino acids requires FLCN, whereby its lysosomal residence during starvation interacts with inactive Rag complex and exerts GAP activity upon RagC/D during amino acid re-stimulation. This converts RagC/D to its active state initiating the binding between raptor and the Rag complex, thus, promoting mTORC1 lysosomal translocation and …show more content…
Lst7-Lst4 complex is recruited to and released from the vacuolar surface, equivalent to mammalian lysosome, upon amino acid starvation and refeeding (Peli-guilli et al., 2016). Furthermore, Lst7-Lst4 complex functions as a Gtr2, ortholog to mammalian RagC/D, in parallel with mammalian FLCN-FNIP function (Peli-guilli et al., 2016). Yeast research revealed the Lst7-Lst4 complex vacuolar dissociation and Gtr2 GAP function to regulate TORC1 activation is stimulated by glutamine (Peli-guilli et al., 2016). Since FLCN-FNIP complex is highly conserved, this glutamine phenomenon might shine insight on whether glutamine addicted mTORC1-dependent cancers may be mediated by the FLCN-FNIP complex (Peli-guilli et al., 2016). Given glutamine sensitivity in yeast orthologs, I propose mammalian FLCN-FNIP …show more content…
Immunofluorescence methods are restricted to capture a snap shot of live-cells; therefore, I plan to image FLCN lysosomal dissociation in cells that were designed by crispr methods to fluorescently tag FLCN with GFP and LAMP2 with RFP. Then with live time-lapse imaging using spinning-disk confocal to track this FLCN dissociation in HEK293T cells seeded on fibronectin-coated glass bottom dishes (Tsun). If mammalian FLCN-FNIP follows suit to its’ yeast orthologs, then glutamine addition to starved cells will trigger the dissociation of FLCN-FNIP from the lysosome, thus, promoting FLCN-FNIP has an amino acid specificity, glutamine. Next, I will observe if the FLCN interaction with the Rag complex during starvation is abrogated upon stimulation with glutamine. This will be biochemically analyzed through co-immunoprecipitation of HEK293T cells stably expressing FLAG-RagB who are starved from 50mins and then re-stimulated with glutamine for 10mins and immunoblot for endogenous FLCN levels. If FLCN is glutamine sensitive, you should see a transition from protein interaction between FLCN and RagB seen under
1.1. Determine if folliculin’s novel molecular function in amino acid sensing is triggered by glutamine. mTORC1 activation by amino acids requires FLCN, whereby its lysosomal residence during starvation interacts with inactive Rag complex and exerts GAP activity upon RagC/D during amino acid re-stimulation. This converts RagC/D to its active state initiating the binding between raptor and the Rag complex, thus, promoting mTORC1 lysosomal translocation and …show more content…
Lst7-Lst4 complex is recruited to and released from the vacuolar surface, equivalent to mammalian lysosome, upon amino acid starvation and refeeding (Peli-guilli et al., 2016). Furthermore, Lst7-Lst4 complex functions as a Gtr2, ortholog to mammalian RagC/D, in parallel with mammalian FLCN-FNIP function (Peli-guilli et al., 2016). Yeast research revealed the Lst7-Lst4 complex vacuolar dissociation and Gtr2 GAP function to regulate TORC1 activation is stimulated by glutamine (Peli-guilli et al., 2016). Since FLCN-FNIP complex is highly conserved, this glutamine phenomenon might shine insight on whether glutamine addicted mTORC1-dependent cancers may be mediated by the FLCN-FNIP complex (Peli-guilli et al., 2016). Given glutamine sensitivity in yeast orthologs, I propose mammalian FLCN-FNIP …show more content…
Immunofluorescence methods are restricted to capture a snap shot of live-cells; therefore, I plan to image FLCN lysosomal dissociation in cells that were designed by crispr methods to fluorescently tag FLCN with GFP and LAMP2 with RFP. Then with live time-lapse imaging using spinning-disk confocal to track this FLCN dissociation in HEK293T cells seeded on fibronectin-coated glass bottom dishes (Tsun). If mammalian FLCN-FNIP follows suit to its’ yeast orthologs, then glutamine addition to starved cells will trigger the dissociation of FLCN-FNIP from the lysosome, thus, promoting FLCN-FNIP has an amino acid specificity, glutamine. Next, I will observe if the FLCN interaction with the Rag complex during starvation is abrogated upon stimulation with glutamine. This will be biochemically analyzed through co-immunoprecipitation of HEK293T cells stably expressing FLAG-RagB who are starved from 50mins and then re-stimulated with glutamine for 10mins and immunoblot for endogenous FLCN levels. If FLCN is glutamine sensitive, you should see a transition from protein interaction between FLCN and RagB seen under