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Figure 4
G3BP1 tethers the TSC complex to lysosomes
(A) PLA of TSC2-LAMP2 in insulin/aa-stimulated siG3BP1 cells (15 min, 1 μM insulin). PLA puncta, white dots; nuclei, blue (DAPI). Scale bar, 100 μm. n = 4.
(B) Quantitation of data in (A). Shown are data points and mean ± SEM. Control (0 min) normalized to 1. n = 12 technical replicates.
(C) IF of LAMP1-TSC2 co-localization in G3BP1 KO cells transfected with siRHEB; insulin/aa stimulation (1 μM insulin). Overlay: white, LAMP1-TSC2 co-localization; green, TSC2; magenta, LAMP1; insert, magnification of the yellow square. Scale bar, 10 μm. n = 3.
(D) Quantitation of data in (C). Shown are data points and mean ± SEM.
(E) TSC2 KO cells in full medium. n = 3.
(F) Quantitation of TSC2 in (E). Shown are data points and mean ± SEM.
(G) Quantitation of RPS6KB1-pT389 in (E). Data are shown as in (F).
(H) Size of G3BP1 KO cells. Mean ± SEM. *p < 0.05. n = 3.
(I) Quantitation of data in (J). Shown are data points and mean ± SEM.
(J) IF of MTOR-LAMP2 co-localization in G3BP1 KO cells. Overlay: white, MTOR-LAMP2 co-localization; green, MTOR; magenta, LAMP2; insert: magnification of the yellow square. Scale bar, 10 μm. n = 3.
(K) Insulin-stimulated TSC2 KO cells transfected with siG3BP1. n = 4.
(L) Quantitation of TSC2 in (K). Shown are data points and mean ± SEM. TSC2 was compared between control and TSC2 KO cells.
(M) Quantitation of G3BP1 in (K). Shown are data points and mean ± SEM. G3BP1 was compared between siControl and siG3BP1 in control or TSC2 KO cells.
(N) Quantitation of RPS6KB1-pT389 in (K). Data are shown as in (M).
See also
Reprinted from Cell, 184(3), Prentzell, M.T., Rehbein, U., Cadena Sandoval, M., De Meulemeester, A.S., Baumeister, R., Brohée, L., Berdel, B., Bockwoldt, M., Carroll, B., Chowdhury, S.R., von Deimling, A., Demetriades, C., Figlia, G., Genomics England Research Consortium, de Araujo, M.E.G., Heberle, A.M., Heiland, I., Holzwarth, B., Huber, L.A., Jaworski, J., Kedra, M., Kern, K., Kopach, A., Korolchuk, V.I., van 't Land-Kuper, I., Macias, M., Nellist, M., Palm, W., Pusch, S., Ramos Pittol, J.M., Reil, M., Reintjes, A., Reuter, F., Sampson, J.R., Scheldeman, C., Siekierska, A., Stefan, E., Teleman, A.A., Thomas, L.E., Torres-Quesada, O., Trump, S., West, H.D., de Witte, P., Woltering, S., Yordanov, T.E., Zmorzynska, J., Opitz, C.A., Thedieck, K., G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling, 655-674.e27, Copyright (2021) with permission from Elsevier. Full text @ Cell