Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: mTORC1-signaling switches megalin function from endocytosis to cell cycle progression

doi: 10.1007/s00018-026-06247-5

Figure Lengend Snippet: mTORC1-induced megalin S4577 phosphorylation increases cell proliferation and megalin colocalizes with ARH at the spindle pole. ( A ) Graph of analysis determining the number of Ki67-positive cells in transfected megalin MMR2 mutant MDCK-II cells showing a significant higher number of Ki67-positive cells in megalin MMR2 S4577D mutants compared to MMR2 S4577A mutants. Values are mean ± SEM. n = 7 independent experiments were evaluated. Mann-Whitney-U test, * P < 0.05. ( B ) Graph of densitometrical evaluation of PCNA expression in transfected megalin MMR2 mutant MDCK-II cells showing a significant higher PCNA expression in megalin MMR2 S4577D mutants compared to MMR2 S4577A mutants. Values are mean ± SEM. n = 5 independent experiments were evaluated. Mann-Whitney-U test, * P < 0.05. ( C ) Representative super-resolution images of transfected megalin MMR2 mutants MDCK-II cells double stained with anti-megalin to identify mutants in green and anti-α-tubulin in red to mark mitotic spindles in cells undergoing mitosis. Arrow heads pointing to megalin protein clusters. Scale bar = 10 μm. ( D ) Coomassie stained SDS-Page of immunoprecipitated megalin at 600 kDa using BN16 cells synchronized to G1 phase, metaphase or telophase/cytokinesis. ( E ) Respective graphs of quantitative mass spectrometry analysis of total protein, megalin protein and S4577 phosphorylated megalin using BN16 cells synchronized to G1 phase, metaphase or telophase/cytokinesis. Values are mean ± SEM. n = 3 independent experiments were evaluated. Unpaired t-test, * P < 0.05. ( F ) Representative super-resolution image of a proximal tubule profile from vhl (von-Hippel-Lindau)-depleted cells triple staining for megalin (green), α-tubulin for mitotic spindles (red) and nuclei with DAPI (blue). Higher magnification images are depicted on the right. Arrow heads point to megalin protein clusters. Scale bar = 5 μm. ( G ) Representative super-resolution image of quadruple staining for ARH (magenta), megalin (yellow), α-tubulin (cyan) for mitotic spindles and nuclei with DAPI (grey) of megalin MMR2 S4577 transiently transfected MDCK-II cells undergoing mitosis. Scale bar = 10 μm. Pearson correlation coefficient (PCC) between megalin and ARH. ( H ) Representative super-resolution image of a proximal tubule profile from vhl -depleted cells quadruple staining for ARH (magenta), megalin (yellow), α-tubulin (cyan) for mitotic spindles and nuclei with DAPI (grey). Higher magnification images are depicted on the right. Arrow heads point to megalin protein clusters. Scale bar = 10 μm. ( I ) Representative super-resolution image of quadruple staining for clathrin (magenta), megalin (yellow), α-tubulin (cyan) for mitotic spindles and nuclei with DAPI (grey) of megalin MMR2 S4577 transiently transfected MDCK-II cells undergoing mitosis. Scale bar = 10 μm. Graph of Pearson correlation coefficient (PCC) between megalin and clathrin. ( J and K ) Representative images ( J ) and quantification ( K ) of Ki67-positive OKC treated with media containing amino acids (+ AA) for mTORC1 activation; with pepstatin A (PepA) and E-64 for lysosomal mTORC1 inhibition or media without amino acids (-AA) for inhibition of non-lysosomal mTORC1 and stained with Ki67 (green), actin filaments (phalloidin, red) and nuclei with DAPI (blue). Scale bar = 100 μm. Values are mean ± SEM. n = 5 independent experiments were evaluated. Kruskal-Wallis test followed by Dunns post test, * P < 0.05, ** P < 0.01

Article Snippet: Analogous to Fernandes et al. [ ], we used [ ] growth media containing amino acids (DMEM/Ham’s F12) [ ], growth media containing amino acids and supplemented with pepstatin A (50 μM, #2936, Roth) and E-64 (25 μM, HY-15282, MedChem Express, Sollentuna, Sweden) to block lysosomal mTORC1 [ ], growth media lacking amino acids (D9807-11, US Biological, Salem, Massachusetts, USA) to block non-lysosomal mTORC1, and [ ] growth media lacking amino acids supplemented with pepstatin A and E-64 to block all mTORC1.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, MANN-WHITNEY, Expressing, Staining, SDS Page, Immunoprecipitation, Mass Spectrometry, Activation Assay, Inhibition