Selecting optogenetic modules to reposition organelles. (A) Assay and constructs. Recycling endosomes (red) were tagged with fluorescently labeled RAB11 fused to iLID. Blue light illumination induced conformational changes to expose Jα helix for transient binding to KIF1A(1–383)-GFP-SSPB, promoting transport to the cellular periphery. (B–D) Fixed-cell imaging (B) and quantification (C and D) of iLID-mCherry-RAB11 in COS-7 cells expressing KIF1A(1–383) fused to GFP-SSPB(milli), GFP-SSPB(micro), or GFP-SSPB(nano) in the dark or after 10 min of illumination with 20 µW cm⁻². Quantification in C shows the normalized iLID-mCherry-RAB11 intensity in the cellular periphery, defined as the 25% of cellular area that is most distal from the perinuclear region (gray in A). Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean and the mean ± two times the SD of control cells. Numbers show the percentage of responsive cells for each dataset (peripheral enrichment greater than mean + two times the SD of control). Data from at least three experiments. (D) Graph showing normalized cellular expression levels of KIF1A(1–383)-GFP-SSPB(micro) and iLID-mCherry-RAB11 per cell in the dark, color coded for peripheral RAB11 intensity to indicate cells that were not activated (gray) or showed dark-state activation (yellow, magenta, and blue). Scale bars are 10 µm.