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. 2020 Nov 11;10:19525. doi: 10.1038/s41598-020-76076-4

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Vimentin protects differentiating stem cells from protein aggregation stress. (a) mESCs differentiated with retinoic acid (1 µg/ml) for 4 days. Protein folding stresses (for 2 h) such as (1) Heat (44 °C), (2) Puromycin (4 µg/ml) and (3) Arsenite (200 µM) were induced to the cells and fixed. Immuno-fluorescence for vimentin (green) and nucleus (white) is shown at each time point. During stress, vimentin retracts to the juxtanuclear region. Bottom panels illustrate vimentin (red) collapse in a counterstained cell that is also expressing synphilin (green). Both the vimentin and synphilin foci collapse while the rest of the cell remains intact. Statistics for the cells with vimentin cages compared between (1) no stress (2) stress. The number of cages during all the three stresses were combined and plotted as one value. 100 cells were counted for each repeat. Error bars indicate standard deviation. Statistics were performed using two tailed student t-test. (P < 0.001). (b) Differentiating mESCs (retinoic acid − 1 µg/ml), overexpressed with Synphilin-GFP and anti-vimentin chromobodies (RFP). Arsenite stress was induced (150 µM/2 h). The vimentin filament collapses along with synphilin aggregates with the cell size being intact. (c) Differentiating mESCs (retinoic acid − 1 µg/ml), over-expressed with Synphilin-GFP and VHL-GFP. Confocal images of Vimentin-RFP attaching and forming a cage along with the misfolded protein Synphilin-GFP (Top panel) and VHL-GFP (Bottom panel). Statistics in the right panel show the histogram for the Vimentin—aggregate colocalization. (d) Confocal time-lapse of Vimentin (green) recruiting Synphilin (red) to the vimentin cage. (e) Vimentin—ULF—GFP (green) interacting with Synphilin—RFP focis (red). (f) Confocal imaging of differentiating mESCs with Vimentin (red), Synphilin (green) and Nucleus (white). Top Panels—cells in M-phase with vimentin cage and aggregates. Bottom panels—cells during anaphase and telophase. (g) Confocal images of differentiating Vimentin knockout mESCs with Vimentin (red), Synphilin (green) and Nucleus (white). Top Panels—cells in M-phase with aggregates all over the cell. Bottom panels—cells during telophase. In Vimentin knockout cells, the Synphilin aggregates unable to collapse and form a JUNQ is symmetrically inherited during division. (h) Statistics for the Vimentin/aggregate asymmetry levels with respect to expression values between the two daughter cells after divisions (P < 0.01). Graph for ratio of expression values of the misfolded proteins between the daughter cells after division was plotted between Wildtype and Vimentin Knockout cells (P < 0.001). Error bars represent standard deviation. (i) Colonies of Vimentin Knockout (top panel) and vimentin complemented (Bottom panel) cells. Knockout cells have higher number of larger Synphilin aggregates (green) after 4 days of differentiation compared to wildtype or knockout complements.