Fig 2: Cav-1 was negatively regulated by vimentin filaments. (A) Representative immunofluorescence images showed the subcellular distribution of endogenous vimentin and Cav-1. The white dotted outline showed the perinuclear vimentin-rich region. Bars, 10 µm. (B) The correlation analysis indicated by Pearson’s coefficient of the fluorescence signals of endogenous vimentin and Cav-1. The correlation between exogenous expressed Cav-1-mCherry and endogenous antibody stained Cav-1 was used as a positive control. (C) Representative images from time-lapse videos (Supplementary Materials) of U2OS cells expressing vimentin-GFP and Cav-1-mCherry. Magnified panels showed representative images at different time points in the yellow box of the left panel, and white arrows indicated the positions of individual Cav-1 vesicle observed in the video (Supplementary Materials). Bars, 10 µm in the left panels and 2 µm in the right magnified panels, respectively. (D) The extracts of U2OS cells with wild-type and vimentin-knockout (VIM KO) were subjected to Western blot analysis. (E) The quantified relative levels of phosphorylated Cav-1 (P-Cav-1) protein normalized to the internal control GAPDH in wild-type and vimentin-knockout cells from Western blots. The data was from five independent experiments (n = 5). (F,G) The intensity of P-Cav-1 was increased in vimentin-knockout U2OS cells and was able to recover in vimentin full-length (FL) reintroducing cells. In (F), it was showed the representative images of wild-type (arrowheads in WT and VIM KO Mix), vimentin-knockout and vimentin-knockout/full-length (arrowheads in VIM KO and FL Mix) cells. In (G), it was showed the quantification of normalized relative fluorescence intensities in wild-type (n = 21), vimentin-knockout cells (n = 13) and full-length rescue cells (n = 22). Bars, 10 µm. All the quantified data was presented as mean ± s.e.m. ** p = 0.01, *** p = 0.001 (t-test).

Fig 3: Proper VIF dynamics are important for lamellipodia formation and cell migration. a Equal amounts of whole cell lysates from MCF7 cells stably expressing mCherry-VIM WT, Y117D, and Y117F or mCherry alone (control) were analyzed by immunoblotting with anti-mCherry or anti-actin. b MCF7 cells (5 × 103) were subjected to a trans-well cell migration assay in the presence (+) or absence (-) 200 ng/mL EGF for 24 h. The cells that migrated to the lower chamber were fixed, stained, and counted. Representative micrographs are shown, scale bars 100 µm. Data are expressed as a proportion of the control cell levels in the absence of EGF. Values (means ± SD) are from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. c The migration of MCF7 cells was analyzed by a wound-healing assay with an ibidi Culture-Insert. The cells (3 × 104) were grown as a monolayer and serum-starved for 24 h. A 500 ± 50 µm cell-free gap was generated and the cells were treated with (+) or without (-) 200 ng/mL EGF. The closure of the gap was visualized with time-lapse microscopy with micrographs taken every 5 min for 6 h. Representative micrographs at 0, 3, and 6 h are shown. Scale bars 100 µm. Data are expressed as a percentage of the gap closure at 6 h. Values (means ± SD) are from three independent experiments. *P < 0.05, ***P < 0.001. d MCF7 cells were serum-starved for 24 h and then treated with (+) or without (-) 200 ng/mL EGF for 1.5 h. The cells were fixed and stained for cortactin, which serves as a marker for lamellipodia. Images were acquired with a Zeiss ApoTome2 microscope imaging system. Arrows indicate lamellipodia. Scale bars 10 µm. The proportion of cells with lamellipodia relative to the total counted cells (n = 300) was determined. Values (means ± SD) are from three independent experiments. ***P < 0.001

Fig 4: Substitution of vimentin Tyr117 with Asp prevents its polymerization in vitro. a Purified His-vimentin proteins were stored in a buffer without sodium chloride, fractionated by SDS-PAGE, and visualized by Coomassie blue stain. b The in vitro polymerization of His-vimentin was performed by the addition of 150 mM sodium chloride (NaCl) and incubation at 30 °C for 30 min. The proteins were then centrifuged at 100,000 × g at 4 °C for 20 min. The supernatants were collected and the pellets were solubilized in vimentin extraction buffer containing 7 M urea. Equal proportions of His-vimentin in the supernatant (S) and pellet (P) fractions were fractionated by SDS-PAGE and visualized by Coomassie blue stain. The ratio of His-vimentin in the supernatant and pellet fractions was measured with ImageJ software. The in vitro polymerization of His-vimentin is expressed as the net percentage of His-vimentin in the pellet with and without NaCl. Values (means ± SD) are from three independent experiments. **P < 0.01. c His-vimentin proteins were polymerized in the presence or absence of 150 mM NaCl at 30 °C for 30 min and visualized by immunofluorescence staining with anti-vimentin or anti-lamin (control). Images were acquired using a Zeiss ApoTome2 microscope imaging system. Scale bars 50 µm. The fluorescence intensity of VIFs per 400 µm2 was measured with the microscope imaging system (n = 30). The VIF density was measured with Photoshop S6 (n = 30). Data are expressed as a percentage relative to the WT level. Values (means ± SD) are from three independent experiments. **P < 0.01, ***P < 0.001. d The polymerized His-vimentin proteins were visualized by cryo-electron microscopy. Representative images are shown. Arrow indicates the aggregates formed by the His-VIM Y117F mutant. Scale bars 200 nm

Fig 5: Src directly phosphorylates vimentin at Tyr117. a Mass spectrometry was used to analyze vimentin purified from v-Src-transformed MEFs. The mass spectrum indicates that vimentin Tyr117 is phosphorylated. b Tyr117 is located in the coil 1A region of vimentin, which is conserved in most, but not all, intermediate filaments. c Bacterially expressed His-tagged vimentin (His-VIM) and the Y117F mutant were purified and served as substrates for GFP-SrcY527F or the kinase-deficient (KD) mutant in vitro. The tyrosine phosphorylation of His-vimentin by GFP-SrcY527F was analyzed by immunoblotting with anti-PY. The amount of GFP-Src proteins used in the assay was analyzed by immunoblotting with anti-GFP. Purified His-vimentin proteins used in the assay were visualized by Coomassie blue stain. The tyrosine phosphorylation of His-vimentin was quantified and expressed as a percentage relative to the WT level. d mCherry-vimentin and the Y117F mutant were transiently co-expressed with GFP-cSrcY527F in HEK293 cells. mCherry-vimentin was immunoprecipitated with anti-mCherry and the immunocomplexes were analyzed by immunoblotting with anti-PY or anti-mCherry. The tyrosine phosphorylation of mCherry-vimentin was quantified and expressed as a percentage relative to the WT level. An equal amount of whole cell lysates was analyzed by immunoblotting with anti-GFP. e GFP-vimentin (GFP-VIM) and the Y117F mutant were immunoprecipitated by anti-GFP and the immunocomplexes were analyzed by immunoblotting with anti-GFP and anti-VIM pY117 in the presence of 5 µM Y117 phosphopeptide (pY117 peptide) or Y117 peptide (control). The tyrosine phosphorylation of GFP-vimentin at pY117 was quantified and expressed as a percentage relative to the WT level. f The Y117 phosphorylation of vimentin in MEFs and v-Src-transformed MEFs was analyzed. Vimentin was immunoprecipitated with anti-vimentin and the immunocomplexes were analyzed by immunoblotting with anti-VIM pY117. An equal amount of whole cell lysates was analyzed by immunoblotting with anti-Src and anti-actin