Fig 2: 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

Fig 3: mTORC1-induced megalin S4577 phosphorylation induces binding to ARH and increases proteolytic processing of megalin C-terminus. (A and B) Representative western blots of pulldown assay using GST-coupled autosomal recessive hypercholesterolemia (ARH) or disabled homolog 2 (Dab2) and megalin C-termini S4577 wildtype, S4577A and S4577D mutants (A). Graph of respective densitometric evaluation (B). Values are mean ± SEM. n = 5 independent experiments were evaluated. Kruskal-Wallis test followed by Dunns post test, * P < 0.05. (C - E) Quantitative colocalization analysis of super-resolution STED images using Pearson correlation coefficient (PCC) between megalin MMR2 mutants and Rab35 obtained from MDCK-II cells upon starvation condition (C) or 5 min albumin uptake (D) double stained with anti-megalin to identify megalin mutants in green and with Rab35 in red (E). Values were normalized to values obtained from S4577 megalin wildtype construct. Scale bar = 5 μm and in higher magnification STED image scale bar = 1 μm. (F and G) Representative western blots of megalin C-terminus fragments in megalin MMR2 mutants transfected HEK293 cells (F) and respective graph of densitometrical evaluation (G). fl. MMR2 is full length megalin mini-construct, MCTF is megalin cytoplasmic tail fragment and MICD is soluble megalin intracellular domain. Values are mean ± SEM. n = 8 independent experiments were evaluated. Mann-Whitney-U test, * P < 0.05

Fig 4: Megalin supports cell proliferation and assists for redistribution of ARH and recycling endosomes to intercellular bridges. (A and B) Representative images of Ki67- or BrdU-stained BN16 mixed wildtype and megalin-deficient cells (A). Ki67 or BrdU in red, megalin in green and nuclei in blue. Scale bar = 10 μm. Arrow heads point to megalin wildtype cells and asterisks mark the megalin-deficient cells. Graph of respective evaluations (B). Values are mean ± SEM. n = 4–5 independent experiments were evaluated. Mann-Whitney-U test, * P < 0.05, ** P < 0.01. (C) Graph of results obtained from the evaluation of the nuclear size. Values are mean ± SEM. n = 5 independent experiments were evaluated. Mann-Whitney-U test, * P < 0.05. (D) Graph of number of megalin-positive vesicles at the pole of the intercellular bridge (ICB) in BN16 wildtype cells. Individual ICBs are presented in black dots, means of independent experiments (n = 6) in green. (E) Graph of number of ARH signals at the ICB pole in BN16 wildtype and megalin-deficient cells. Individual ICBs are presented in black dots, means of independent experiments (n = 3) in green for megalin wildtype cells and in red for megalin-deficient cells. Mann-Whitney-U test, ** P < 0.01. (F) Representative confocal and super-resolution STED image (below) of quadruple-stained mixture of BN16 wildtype and megalin-deficient cells of megalin in green, ARH in red, α-tubulin in blue and nuclei in magenta. Scale bar = 10 μm and in higher magnification STED image scale bar = 1 μm. Section character marks a megalin-expression neighboring cell and the hash marks midbodies. (G) Graph of number of Rab11-positive vesicles at the ICB pole in BN16 wildtype and megalin-deficient cells. Individual ICBs are presented in black dots, means of independent experiments (n = 3) in green for megalin wildtype cells and in red for megalin-deficient cells. Mann-Whitney-U test, ** P < 0.01. (H) Representative confocal and super-resolution image (below) of quadruple-stained mixture of BN16 wildtype and megalin-deficient cells of megalin in green, Rab11 in red, α-tubulin in blue and nuclei in magenta. The hash marks midbodies. Scale bar = 10 μm and in higher magnification STED image scale bar = 1 μm. (I) Graph of midbody length in BN16 wildtype and megalin-deficient cells during cytokinesis. Individual midbodies are presented in black dots, means of independent experiments (n = 4) in green for megalin wildtype cells and in red for megalin-deficient cells. (J) Representative images of Ki67-stained BN16 mixed wildtype and ARH-deficient cells. Ki67 in red, ARH in green and nuclei in blue. Scale bar = 10 μm. Arrow heads point to ARH wildtype cells and asterisks mark the ARH-deficient cells. (K) Evaluation of Ki67-positive cells in BN16 wildtype and ARH-deficient cells. Values are mean ± SEM. n = 4 independent experiments were evaluated. Mann-Whitney-U test, *P < 0.05. (L) Representative confocal and super-resolution STED image (below) of quadruple-stained mixture of BN16 wildtype and ARH-deficient cells of megalin in green, ARH in red, α-tubulin in blue and nuclei in magenta. Scale bar = 10 μm and in higher magnification STED image scale bar = 1 μm. (M) Graph of number of megalin-positive vesicles at the ICB pole in BN16 wildtype and ARH-deficient cells. Individual ICBs are presented in black dots, means of independent experiments (n = 4) in green for ARH wildtype cells and in red for ARH-deficient cells. Mann-Whitney-U test, ** P < 0.01