Figure 2.
Reversible vimentin repositioning by optogenetic recruitment of kinesin motors to vimentin. (A) Schematic overview of optogenetic vimentin constructs. The constructs are similar to those shown in Fig. 1 A, except that FRB is substituted for iLID and FKBP for SSBmicro. GFP and mVenus are used as fluorescent markers in ppKin14 and KIF5A constructs, respectively. (B and C) Schematics illustrating the action of the optogenetic vimentin constructs. (B) Upon blue light activation, the iLID module changes conformation, uncaging an SsrA peptide, which binds to SspB. By tagging vimentin with SspB and ppKin14 and KIF5A with iLID, vimentin can be inducibly pulled toward microtubule minus or plus ends, respectively. (C) Transfection of cells with Vim-mCh-SspB combined with one of the two kinesin constructs and blue light activation results in vimentin pulling to microtubule minus or plus ends, causing vimentin clustering either at the MTOC (ppKin14) or both the cell periphery and MTOC (KIF5A). (D) U2OS cells expressing the Vim-mCh-SspB construct. The overexpressed vimentin network is visualized by mCherry fluorescence, while total vimentin intensity is detected via anti-vimentin immunostaining. Images were captured using scanning confocal microscopy. (E–I) U2OS cells co-transfected with Vim-mCh-SspB and iLID-GFP-GCN4-ppKin14 were either fixed in a dark room (DARK) or exposed to 45 min of blue light (LIT) prior to fixation and staining for vimentin (total vimentin). (E) Representative images, with transfected cells outlined with a dashed line. (F) Quantification of the fraction of the cell area occupied by vimentin. (G) Mean iLID-GFP-GCN4-ppKin14 fluorescence intensity per cell, normalized to untransfected cells. (H) Mean cell Vim-mCh-SspB fluorescence intensity. (I) Mean total cell area. (F–I)n = 23–29 cells per treatment analyzed over three experiments; bars show mean ± SD. ns, not significant; ∗∗∗∗, P < 0.0001 by Mann–Whitney test (G and H) or Kruskal–Wallis with Dunn’s test (F and I). (J–L) U2OS cells co-transfected with Vim-mCh-SspB and iLID-GFP-GCN4-ppKin14 were locally illuminated with 488-nm light pulses for 40 min before fixing and staining for vimentin. (J) Scheme of the experiment. (K) Stills with the region illuminated with blue light are indicated with a blue dashed box. (L) Vimentin staining in the cell shown in K was fixed after local illumination. (M and N) U2OS cells co-transfected with Vim-mCh-SspB and iLID-GFP-GCN4-ppKin14 were exposed to whole-cell 488-nm light pulses for 30 min to cluster vimentin, and then 488-nm pulses were stopped to allow cells to recover over 4 h. (M) Representative cell directly before light pulses (−30 min), after 30 min of 488-nm light pulsing (0 min), and after 4 h of recovery without blue light activation (4 h). (N) Quantification of Vim-mCh-SspB mean intensity at an ROI at the cell periphery over time; plot shows a line for every individual cell, n = 9 cells analyzed over three independent experiments.

Reversible vimentin repositioning by optogenetic recruitment of kinesin motors to vimentin. (A) Schematic overview of optogenetic vimentin constructs. The constructs are similar to those shown in Fig. 1 A, except that FRB is substituted for iLID and FKBP for SSBmicro. GFP and mVenus are used as fluorescent markers in ppKin14 and KIF5A constructs, respectively. (B and C) Schematics illustrating the action of the optogenetic vimentin constructs. (B) Upon blue light activation, the iLID module changes conformation, uncaging an SsrA peptide, which binds to SspB. By tagging vimentin with SspB and ppKin14 and KIF5A with iLID, vimentin can be inducibly pulled toward microtubule minus or plus ends, respectively. (C) Transfection of cells with Vim-mCh-SspB combined with one of the two kinesin constructs and blue light activation results in vimentin pulling to microtubule minus or plus ends, causing vimentin clustering either at the MTOC (ppKin14) or both the cell periphery and MTOC (KIF5A). (D) U2OS cells expressing the Vim-mCh-SspB construct. The overexpressed vimentin network is visualized by mCherry fluorescence, while total vimentin intensity is detected via anti-vimentin immunostaining. Images were captured using scanning confocal microscopy. (E–I) U2OS cells co-transfected with Vim-mCh-SspB and iLID-GFP-GCN4-ppKin14 were either fixed in a dark room (DARK) or exposed to 45 min of blue light (LIT) prior to fixation and staining for vimentin (total vimentin). (E) Representative images, with transfected cells outlined with a dashed line. (F) Quantification of the fraction of the cell area occupied by vimentin. (G) Mean iLID-GFP-GCN4-ppKin14 fluorescence intensity per cell, normalized to untransfected cells. (H) Mean cell Vim-mCh-SspB fluorescence intensity. (I) Mean total cell area. (F–I)n = 23–29 cells per treatment analyzed over three experiments; bars show mean ± SD. ns, not significant; ∗∗∗∗, P < 0.0001 by Mann–Whitney test (G and H) or Kruskal–Wallis with Dunn’s test (F and I). (J–L) U2OS cells co-transfected with Vim-mCh-SspB and iLID-GFP-GCN4-ppKin14 were locally illuminated with 488-nm light pulses for 40 min before fixing and staining for vimentin. (J) Scheme of the experiment. (K) Stills with the region illuminated with blue light are indicated with a blue dashed box. (L) Vimentin staining in the cell shown in K was fixed after local illumination. (M and N) U2OS cells co-transfected with Vim-mCh-SspB and iLID-GFP-GCN4-ppKin14 were exposed to whole-cell 488-nm light pulses for 30 min to cluster vimentin, and then 488-nm pulses were stopped to allow cells to recover over 4 h. (M) Representative cell directly before light pulses (−30 min), after 30 min of 488-nm light pulsing (0 min), and after 4 h of recovery without blue light activation (4 h). (N) Quantification of Vim-mCh-SspB mean intensity at an ROI at the cell periphery over time; plot shows a line for every individual cell, n = 9 cells analyzed over three independent experiments.

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