Effects of vimentin clustering on the distribution of mitochondria and lysosomes. (A and D) U2OS cell, co-transfected with Vim-mCh-SspB, iLID-GFP-GCN4-ppKin14, and Halo-MitoTag to label mitochondria (A) or LAMP1-Halo to label lysosomes (D), locally illuminated with 488-nm pulses inside an ROI (blue dashed box) for 10 min (A) or with global illumination for 20 min (D). The cell is shown before application of blue light (0 min) and at the indicated time points after blue light pulsing. (B and E) Imaging of U2OS cells transfected with Vim-mCh-FKBP and FRB-BFP-GCN4-ppKin14 constructs. After 1 h of treatment with or without rapalog, cells were subjected to immunostaining for total vimentin and cytochrome C to label mitochondria (B) or LAMTOR4 to label lysosomes (E). Transfected cells are indicated by a dotted outline. Images were acquired with Airyscan mode. (C and F) The distribution of mitochondria (C) or lysosomes (F) relative to the MTOC. Organelles were detected using the ComDet plugin, and their distance from the MTOC was calculated using a radius plugin. The graph shows the percentage of mitochondria or lysosomes located in the peripheral region, which was defined as an area beyond a 13.81-μm radius from the MTOC, with each dot representing data from an individual cell. Measurements were collected from n = 20–29 cells in C and from n = 20–22 cells in F across three independent experiments. The plots display the mean ± SD, with individual cell measurements represented as dots. ns, not significant; ∗∗, P < 0.01 by Mann–Whitney test.