Fig 2: BFC localizes to vacuoles in response to amino acid starvation and augments TORC1 activation in response to amino acid stimulationRepresentative fluorescence microscopy images depicting the co-localization of (A) Bhd1-GFP and (B) Fnp1-GFP with vacuoles (FM4-64-Red) for cells grown continuously in minimal media supplemented with amino acids (+aa) or starved for amino acids for 90 min (-aa). Graphs depicting the fluorescent intensity signal of Bhd1-GFP (C) and Fnp1-GFP (D) along with FM4-64 after drawing a line from one end of the cell to another. Representative graphs used to calculate the Pearson correlation coefficient (R) of overlap between Bhd1-GFP (E) and Fnp1-GFP (F) with FM4-64. DIC - differential interference contrast; au.–arbitrary units; GFP – green fluorescent protein. 15 cells per replicate were analyzed for calculating R using ImageJ software. Three biological replicates were performed. See also Figures S2 and S3. Scale bar – 10µm.(G) A representative Western blot depicting the kinetics of Rps6 phosphorylation (P-Rps6) after amino acid supplementation. Log phase wild-type (WT), bhd1? and fnp1? cells were starved for amino acids (-aa) for 140 min (red text), and then incubated in pre-warmed EMM supplemented with amino acids (+aa) for the indicated times (in hours). Because we were unable to find a commercial antibody for detecting total Rps6 levels, similar to previous studies (Chica et al., 2016; Ma et al., 2013; Martin et al., 2017; Matsuda et al., 2020; Nakashima et al., 2010; Nakashima and Tamanoi, 2010; Rodland et al., 2014; Valbuena et al., 2012a, 2012b), tubulin or actin were used as loading controls for Western blot analyses. Three biological replicates were performed. PS – pre-starvation.See also Figure S4 for two other replicates.

Fig 3: HfrTMS leads to selective localization of rpS6 immunostaining in activated sensorimotor cortex. (A) Distribution of rpS6 immunostaining neuronal cells as reveled by immunocytochemistry in a sham stimulation-treated control animal. (B) Distribution of rpS6 immunostaining neuronal cells as reveled by immunocytochemistry in an animal following 60 hfrTMS (400 Hz, 10 s intervals) trains. (C) Graph illustrating the average optical density (OD) of labeling across the pyramidal cell layer, i.e., layer V of the motor cortex in the case illustrated in control (A) and in real stimulated animal (B). Bars indicate the standard deviation of the five measurements at each level. CON, control; hfrTMS, high frequency repetitive transcranial stimulation.

Fig 4: Quantitative time course and stimulus intensity analyses of the increases in rpS6 levels after 400 Hz-hfrTMS as revealed by immunocytochemistry. Quantitative analyses of the increases in rpS6 levels after 400 Hz high frequency magnetic stimulation as revealed by immunocytochemistry. (A) The graph illustrates the average optical density in the layer V of the motor cortex 0, 15, 30, 60, 120, 180, and 360 min after 60 bursts (solid triangles) and 180 bursts (blank squares) of high frequency magnetic stimulation (treatment effect: P = 0.0003). The values represent the mean and standard deviation of the measurements. There were no significant differences between 60 bursts (10 min duration) and 180 bursts (30 min duration). (B) The graph illustrates the average optical density in the layer V of the motor cortex 30 min after high frequency magnetic stimulation at 0, 18.75, 25, 50, 75, and 100% of machine output (con. vs. 50%; P < 0.05; con. vs. 75%; P < 0.05; control vs. 100%; P < 0.008). The values represent the mean and standard deviation of the measurements. There is a sharp change in the extent of rpS6 phosphorylation between 25 and 50% stimulus intensity. A * denotes p < 0.05 [repeated measures two way ANOVA].

Fig 5: 400 Hz-hfrTMS leads to increases the co-activated rpS6-positive and Arc, c-fos protein-positive neurons in the motor cortex. (A–C) Pattern of immunostaining for rpS6 in sham control animal (A). Pattern of immunostaining for Arc (B) and c-fos (C) in sham control animal. (D–F) Pattern of immunostaining for rpS6 (D), Arc (E) and c-fos (F) 30 min, after 400 Hz hfrTMS. Note that many neurons including some pyramidal neurons in layer V were strongly positive for rpS6 and Arc protein, the larger pyramidal neurons in layer V were less intensely stained for Arc and that the distribution of c-fos did not align with the distribution of Arc-positive pyramidal neurons within the layer V.