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. Author manuscript; available in PMC: 2022 Sep 24.
Published in final edited form as: Cell Rep. 2022 Aug 30;40(9):111278. doi: 10.1016/j.celrep.2022.111278

Figure 3. In neurons, DEPDC5 is essential for leucine, arginine, and glutamine signaling through mTORC1, whereas TSC2 is essential for growth factor signaling through mTORC1.

Figure 3.

(A) Immunoblots from scramble control (CTL) and DEPDC5 knockdown (KD) primary cortical neurons after 90-min amino acid (AA) and/or growth factor (GF) withdrawal.

(B) Reduced mTORC1 sensitivity of DEPDC5 KD neurons to AA withdrawal as measured by the ratio of quantified pS6 from AA withdrawal versus AA replete conditions from (A).

(C) Immunoblots demonstrate that mTORC1 (pS240/244-S6), but not mTORC2 (pS473-AKT), is insensitive to withdrawal of leucine (L), arginine (R), and/or glutamine (Q) in DEPDC5 KD primary cortical neurons.

(D) Representative images of mTOR lysosomal localization in primary neurons transduced with scramble or shDepdc5 lentivirus stained with mTOR (red) and the lysosomal marker LAMP1 (gray) after incubation with or without AA and/or GF for 90 min. Scale bars = 20 μm, 2× inset. (E) mTOR colocalization with LAMP1 after 90-min AA or leucine withdrawal is reduced by all AA withdrawal but insensitive to leucine alone withdrawal in DEPDC5 KD neurons by Mander’s colocalization index from at least 50 cells quantified across three independent experiments.

(F) Immunoblots from scramble control (CTL) and TSC2 knockdown (KD) primary cortical neurons after 90-min amino acid (AA) and/or growth factor (GF) withdrawal.

(G) Ratio of quantified pS6 from GF withdrawal versus GF replete conditions in (F) demonstrates that TSC2 KD neurons are insensitive to GF withdrawal independent of AAs.

(H) Representative images of mTOR lysosomal localization in primary neurons transduced with scramble or TSC2 knockdown lentivirus stained with mTOR (red) and the lysosomal marker LAMP1 (gray) after incubation with or without AA and/or GF for 90 min. Scale bars, 20 μm, 2× inset.

(I) mTOR colocalization with LAMP1 is reduced by 90-min AA or leucine withdrawal in both TSC2 KD and control neurons by Mander’s colocalization index from at least 50 cells quantified across three independent experiments.

(J) Immunoblots from scramble control (CTL) and double-knockdown of DEPDC5 and TSC2 (DTKD) primary cortical neurons after 90-min AA and/or GF withdrawal. For all experiments, graphs of mean ± SD, with symbols representing individual replicates from at least three independent experiments, two-way ANOVA with Tukey’s post hoc analysis. *p < 0.05, **p < 0.01, ****p < 0.001, ****p < 0.0001.