Optogenetic minus end–directed motor recruitment and vimentin pulling by plus end–directed kinesin. (A) U2OS cells co-transfected with Vim-mCh-SspB and iLID-GFP-GCN4-ppKin14 were either fixed in a dark room (DARK) or exposed to 5 min of blue light (LIT) prior to fixation and staining for anti-GFP. Dashed box shows the region of the cell enlarged in the zoom panel. Images were captured using Airyscan confocal microscopy. (B) U2OS cells co-transfected with Vim-mCh-SspB and iLID-GFP-GCN4-ppKin14 and imaged live using spinning disc confocal microscopy. Cells were pulsed with 488 nm light for 30 min over the entire cell and then allowed to recover their vimentin distribution in the absence of blue light stimulation for 4 h. The graph shows quantification of construct expression levels based on fluorescence at the first frame, with background subtracted. Cells were categorized either as having no vimentin clustering (red square), vimentin pulling with recovery of vimentin spreading after 4 h (blue circle), or vimentin pulling without complete recovery of vimentin spreading after 4 h (green triangle). (C and D) Schematic overview of constructs for optogenetic plus end–directed vimentin pulling. (E) U2OS cells co-transfected with Vim-mCh-SspB and KIF5A-mVenus-iLID show vimentin relocalization to clusters in the cell periphery and at the cell center upon blue light activation. Live-cell imaging stills are shown before blue light activation (0 min) and 10 and 30 min after 488-nm pulsing.