Figure 3.
mTORC1 is activated in FAs.(A) HeLa cells grown in full-nutrient medium were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS) in the absence or presence of the mTORC1 inhibitor rapamycin (100 nM) and immunostained for p-S6 and the FA protein paxillin. (B) Colocalization (Manders coefficient) between p-S6 and paxillin was analyzed. (C) Fluorescence intensity line profile plots corresponding to lines exemplified by an arrow in A. (D and E) Analysis of p-S6 in ROIs corresponding to FAs and adjacent (control) areas. Representative confocal image of paxillin (top) and p-S6 (bottom) in refed HeLa cells (D). The ROIs corresponding to FAs (top inset) and adjacent (control) areas (bottom inset) are indicated by white borders in zoom insets (D; right), and p-S6 IntDens in ROIs was quantified (E). (F) Diagram demonstrating the principle of the TORCAR biosensor (Zhou et al., 2015). (G) TORCAR biosensor analysis. Representative confocal image of GFP-paxillin (top) and GFP-paxillin intensity-scaled ratio image of TORCAR FRET-based biosensor (bottom) in serum-starved and refed HeLa cells. Note that to visualize the differences, the images were gamma adjusted, and white borders in GFP-paxillin zoom insets highlight the ROIs corresponding to FAs (top inset) and control areas (bottom inset) used for quantification. (H and I) mTORC1 activity was quantified in serum-starved versus refed conditions (H) and before and after stimulation with 25 mM membrane-permeable leucine methylester (I), presented as the normalized ratio of TORCAR biosensor in FAs and control areas. Each data point represents a coverslip including several cells. A/D, acceptor-to-donor ratio. (J) HeLa cells grown in full-nutrient medium were starved of amino acids and FCS (−aa −FCS) and then recovered in amino acid– and insulin-containing medium for 30 min (30 min aa +ins) in the presence of L-HPG with and without rapamycin. HPG incorporation was visualized via Click-IT reaction with Alexa Fluor 488 azide before cells were immunostained for p-S6 and paxillin. (K) Colocalization (Manders coefficient) between p-S6 and HPG was analyzed. (L) Fluorescence intensity line profile plots corresponding to lines as exemplified by an arrow in J. (M) Quantification of HPG IntDens in ROIs corresponding to FAs and adjacent (control) areas. Scale bars, 20 µm (insets 10 µm). Nuclei were visualized with DAPI. Error bars represent SEM; n = 3 independent experiments. For B, E, K and M, *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-sided Student’s t test. For H and I, ****, P < 0.0001; two-way ANOVA with Sidak's multiple comparisons test.

mTORC1 is activated in FAs. (A) HeLa cells grown in full-nutrient medium were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS) in the absence or presence of the mTORC1 inhibitor rapamycin (100 nM) and immunostained for p-S6 and the FA protein paxillin. (B) Colocalization (Manders coefficient) between p-S6 and paxillin was analyzed. (C) Fluorescence intensity line profile plots corresponding to lines exemplified by an arrow in A. (D and E) Analysis of p-S6 in ROIs corresponding to FAs and adjacent (control) areas. Representative confocal image of paxillin (top) and p-S6 (bottom) in refed HeLa cells (D). The ROIs corresponding to FAs (top inset) and adjacent (control) areas (bottom inset) are indicated by white borders in zoom insets (D; right), and p-S6 IntDens in ROIs was quantified (E). (F) Diagram demonstrating the principle of the TORCAR biosensor (Zhou et al., 2015). (G) TORCAR biosensor analysis. Representative confocal image of GFP-paxillin (top) and GFP-paxillin intensity-scaled ratio image of TORCAR FRET-based biosensor (bottom) in serum-starved and refed HeLa cells. Note that to visualize the differences, the images were gamma adjusted, and white borders in GFP-paxillin zoom insets highlight the ROIs corresponding to FAs (top inset) and control areas (bottom inset) used for quantification. (H and I) mTORC1 activity was quantified in serum-starved versus refed conditions (H) and before and after stimulation with 25 mM membrane-permeable leucine methylester (I), presented as the normalized ratio of TORCAR biosensor in FAs and control areas. Each data point represents a coverslip including several cells. A/D, acceptor-to-donor ratio. (J) HeLa cells grown in full-nutrient medium were starved of amino acids and FCS (−aa −FCS) and then recovered in amino acid– and insulin-containing medium for 30 min (30 min aa +ins) in the presence of L-HPG with and without rapamycin. HPG incorporation was visualized via Click-IT reaction with Alexa Fluor 488 azide before cells were immunostained for p-S6 and paxillin. (K) Colocalization (Manders coefficient) between p-S6 and HPG was analyzed. (L) Fluorescence intensity line profile plots corresponding to lines as exemplified by an arrow in J. (M) Quantification of HPG IntDens in ROIs corresponding to FAs and adjacent (control) areas. Scale bars, 20 µm (insets 10 µm). Nuclei were visualized with DAPI. Error bars represent SEM; n = 3 independent experiments. For B, E, K and M, *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-sided Student’s t test. For H and I, ****, P < 0.0001; two-way ANOVA with Sidak's multiple comparisons test.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal