Figure 4. Ragulator inhibits... | Rockefeller University Press

Figure 4.

$Figure 4. Ragulator inhibits BORC-mediated lysosome dispersal. (A) WT, lyspersin-KO, myrlysin-KO, diaskedin-KO, or MEF2BNB-KO HeLa cells cultured in regular medium were analyzed by SDS-PAGE and immunoblotting (IB) for phosphorylation (p) of the mTORC1 substrates S6K, 4E-BP1, and ULK1. (B) WT HeLa cells were transfected with nontargeting (control) and LAMTOR1 siRNAs and disrupted without detergent. Cytosolic and membrane fractions were separated by centrifugation of PNS for 1 h at 100,000 g and analyzed by SDS-PAGE and IB for the proteins indicated in the figure. In A and B, the positions of molecular mass markers (in kilodaltons) are indicated on the left. (C–H) shRNA- or siRNA-mediated KD was performed in WT HeLa or lyspersin-KO cells, as indicated in the figure. Nontargeting shRNA or siRNA were used as controls. The distribution of lysosomes and the expression and localization of Ragulator were visualized by immunostaining with antibodies to LAMP1 and LAMTOR4, respectively. Myc-lyspersin was detected by immunostaining for the myc epitope. Bar, 10 µm. (I) Lysosome distribution was quantified in the cells indicated in the figure (n = 13 cells from three independent experiments) using Imaris software. The distance between lysosomes and the MTOC was normalized to the longest distance between the cell periphery and the MTOC. Values are the mean ± SD. LAMTOR1 KD versus control KD, *, P < 0.05; **, P < 0.01; ***, P < 0.001; lyspersin KO or LAMTOR1 KD versus lyspersin KO, ###, P < 0.001 (Student’s t test).$

Ragulator inhibits BORC-mediated lysosome dispersal. (A) WT, lyspersin-KO, myrlysin-KO, diaskedin-KO, or MEF2BNB-KO HeLa cells cultured in regular medium were analyzed by SDS-PAGE and immunoblotting (IB) for phosphorylation (p) of the mTORC1 substrates S6K, 4E-BP1, and ULK1. (B) WT HeLa cells were transfected with nontargeting (control) and LAMTOR1 siRNAs and disrupted without detergent. Cytosolic and membrane fractions were separated by centrifugation of PNS for 1 h at 100,000 g and analyzed by SDS-PAGE and IB for the proteins indicated in the figure. In A and B, the positions of molecular mass markers (in kilodaltons) are indicated on the left. (C–H) shRNA- or siRNA-mediated KD was performed in WT HeLa or lyspersin-KO cells, as indicated in the figure. Nontargeting shRNA or siRNA were used as controls. The distribution of lysosomes and the expression and localization of Ragulator were visualized by immunostaining with antibodies to LAMP1 and LAMTOR4, respectively. Myc-lyspersin was detected by immunostaining for the myc epitope. Bar, 10 µm. (I) Lysosome distribution was quantified in the cells indicated in the figure (n = 13 cells from three independent experiments) using Imaris software. The distance between lysosomes and the MTOC was normalized to the longest distance between the cell periphery and the MTOC. Values are the mean ± SD. LAMTOR1 KD versus control KD, *, P < 0.05; **, P < 0.01; ***, P < 0.001; lyspersin KO or LAMTOR1 KD versus lyspersin KO, ^###, P < 0.001 (Student’s t test).

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