Figure 7.
MYCT1 restricts endothelial endocytosis and IFITM2/3-dependent mTORC1 activation. (A) Experimental workflow for in vivo Atto647–labeled plasma protein uptake experiment. (B) WAT ECs take up higher amounts of labeled plasma proteins compared with colon ECs. Representative flow cytometry histograms showing Atto647-MFI in ECs from s.c. (sc. fat), visceral (vis. fat) WAT, and colon of two wild-type mice, comparing an injected one (colored) and a non-injected control (gray). (C)Myct1 ablation increases labeled plasma protein uptake in WAT ECs. Atto647-MFI of ECs from visceral WAT of control mice or Myct1ecKO mice, plotted as a function of corresponding plasma Atto647-fluorescence levels. Each dot corresponds to an individual mouse. Linear regression showing the 95% confidence bands of the best-fit line and the goodness of fit (R2). a.u., arbitrary units, MFI, mean fluorescence intensity. Similar results were obtained for s.c. fat (data not shown). (D)MYCT1 knockdown promotes lysosomal degradation activity. Control and MYCT1KD cells were treated for 1 h with DQ-BSA. DQ-BSA fluorescence (gray/black) and staining for β-catenin (magenta) and DNA (blue). Lower panels show DQ-BSA. Scale bar, 20 µm. (E) Quantification of the percentage of DQ-BSA signal per cell. n = 4 independent experiments; 60–100 cells were analyzed per condition for each experiment; mean ± SD; unpaired t test, P = 0.0059 (*). (F)IFITM2/3 knockdown rescues RAB5+ endosome enlargement in MYCT1-deficient ECs. Staining of ECs for RAB5 (gray/black), VE-cadherin (magenta), and DAPI (blue). Lower panels show RAB5. Scale bar, 10 μm. (G) Quantification of RAB5+ vesicle volume in control, MYCT1KD, IFITM2/3KD, and MYCT1–IFITM2/3KD cells. n = 3 independent experiments; 1,000–3,000 vesicles were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Tukey’s multiple comparisons test, P = 0.028 (*) for MYCT1 knockdown effect, P = 0.0060 (*) for comparison between MYCT1 and IFITM2/3 knockdowns, and P = 0.0070 (*) for rescue effect by IFITM2/3 simultaneous knockdown. (H)IFITM2/3 knockdown rescues mTORC1 hyperactivation in MYCT1-deficient ECs. Staining for p-S6 (gray), β-catenin (magenta), and DAPI (blue). Scale bar, 20 μm. (I) Quantification of mTORC1 activation in control, MYCT1KD, IFITM2/3KD, and MYCT1–IFITM2/3KD cells. The percentage of p-S6+ cells was quantified in the indicated conditions. n = 4 independent experiments; 1,500–8,000 cells were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Tukey’s multiple comparisons test, P = 0.0195 (*) for MYCT1 knockdown effect and P = 0.0118 (*) for rescue effect by IFITM2/3 simultaneous knockdown. (J) MYCT1 ablation leads to accumulation of IFITM2/3, which drives enlargement of early endosomes, increases cargo uptake, and enhances lysosomal degradation. This process results in greater amino acid delivery, thereby activating mTORC1. These phenotypes are reversed upon simultaneous knockdown of IFITM2/3 and MYCT1. Icons used in A and J were created with BioRender.com and modified in Affinity. See also Fig. S5, H–Q. Refer to the image caption for details. Panel A shows the experimental workflow for plasma protein uptake. Panel B presents flow cytometry histograms comparing Atto 647-M F I in endothelial cells from subcutaneous and visceral white adipose tissue and colon of wildtype mice, with injected and non-injected controls. Panel C displays a line graph plotting Atto 647-M F I of endothelial cells from visceral white adipose tissue of control and M y c t 1 e c K O mice against plasma Atto 647-fluorescence levels, with linear regression and confidence bands. Panel D shows fluorescence images of control and M Y C T 1 knockdown cells treated with D Q-B S A, stained for beta-catenin and D N A, with lower panels highlighting D Q-B S A fluorescence. Panel E quantifies the percentage of D Q-B S A signal per cell. Panel F includes images of endothelial cells stained for R A B 5, V E-cadherin, and DAPI, with lower panels showing R A B 5 staining. Panel G quantifies R A B 5 plus vesicle volume in different cell conditions. Panel H presents images of cells stained for p-S 6, beta-catenin, and DAPI, with a focus on p-S 6 staining. Panel I quantifies m T O R C 1 activation in various cell conditions. Panel J provides a schematic illustration summarizing the findings, showing the impact of M Y C T 1 ablation on I F I T M 2 slash 3 accumulation, endosome enlargement, cargo uptake, lysosomal degradation, and m T O R C 1 activation.

MYCT1 restricts endothelial endocytosis and IFITM2/3-dependent mTORC1 activation. (A) Experimental workflow for in vivo Atto647–labeled plasma protein uptake experiment. (B) WAT ECs take up higher amounts of labeled plasma proteins compared with colon ECs. Representative flow cytometry histograms showing Atto647-MFI in ECs from s.c. (sc. fat), visceral (vis. fat) WAT, and colon of two wild-type mice, comparing an injected one (colored) and a non-injected control (gray). (C)Myct1 ablation increases labeled plasma protein uptake in WAT ECs. Atto647-MFI of ECs from visceral WAT of control mice or Myct1ecKO mice, plotted as a function of corresponding plasma Atto647-fluorescence levels. Each dot corresponds to an individual mouse. Linear regression showing the 95% confidence bands of the best-fit line and the goodness of fit (R2). a.u., arbitrary units, MFI, mean fluorescence intensity. Similar results were obtained for s.c. fat (data not shown). (D)MYCT1 knockdown promotes lysosomal degradation activity. Control and MYCT1KD cells were treated for 1 h with DQ-BSA. DQ-BSA fluorescence (gray/black) and staining for β-catenin (magenta) and DNA (blue). Lower panels show DQ-BSA. Scale bar, 20 µm. (E) Quantification of the percentage of DQ-BSA signal per cell. n = 4 independent experiments; 60–100 cells were analyzed per condition for each experiment; mean ± SD; unpaired t test, P = 0.0059 (*). (F)IFITM2/3 knockdown rescues RAB5+ endosome enlargement in MYCT1-deficient ECs. Staining of ECs for RAB5 (gray/black), VE-cadherin (magenta), and DAPI (blue). Lower panels show RAB5. Scale bar, 10 μm. (G) Quantification of RAB5+ vesicle volume in control, MYCT1KD, IFITM2/3KD, and MYCT1–IFITM2/3KD cells. n = 3 independent experiments; 1,000–3,000 vesicles were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Tukey’s multiple comparisons test, P = 0.028 (*) for MYCT1 knockdown effect, P = 0.0060 (*) for comparison between MYCT1 and IFITM2/3 knockdowns, and P = 0.0070 (*) for rescue effect by IFITM2/3 simultaneous knockdown. (H)IFITM2/3 knockdown rescues mTORC1 hyperactivation in MYCT1-deficient ECs. Staining for p-S6 (gray), β-catenin (magenta), and DAPI (blue). Scale bar, 20 μm. (I) Quantification of mTORC1 activation in control, MYCT1KD, IFITM2/3KD, and MYCT1–IFITM2/3KD cells. The percentage of p-S6+ cells was quantified in the indicated conditions. n = 4 independent experiments; 1,500–8,000 cells were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Tukey’s multiple comparisons test, P = 0.0195 (*) for MYCT1 knockdown effect and P = 0.0118 (*) for rescue effect by IFITM2/3 simultaneous knockdown. (J) MYCT1 ablation leads to accumulation of IFITM2/3, which drives enlargement of early endosomes, increases cargo uptake, and enhances lysosomal degradation. This process results in greater amino acid delivery, thereby activating mTORC1. These phenotypes are reversed upon simultaneous knockdown of IFITM2/3 and MYCT1. Icons used in A and J were created with BioRender.com and modified in Affinity. See also Fig. S5, H–Q.

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