Intermediate filaments incorporate subunits along their length. The cells were imaged immediately after photoactivation (0 h) or 8–20 h later (examples i and ii). The line scans show the green and red fluorescence intensity profiles along the filaments indicated by the white arrowheads. For clarity, we selected isolated filaments for most of the images. (a) Obtained by fusion of SW13 vim⁻ cells coexpressing PAGFP-NFM and NFL with SW13 vim⁻ cells coexpressing mCherry-NFM and NFL. (b) Obtained by fusion of SW13 vim⁻ cells coexpressing NFL-PAGFP and NFM with SW13 vim⁻ cells coexpressing NFL-mCherry and NFM. (c) Obtained by fusion of SW13 vim⁻ cells coexpressing PAGFP-vimentin and untagged vimentin with SW13 vim⁻ cells coexpressing mCherry-vimentin and untagged vimentin. (d) Obtained by photoactivation and bleaching of SW13 vim⁻ cells coexpressing PAGFP-NFM, mCherry-NFM, and NFL. (e) Obtained by fusion of SW13 vim⁺ cells stably expressing PAGFP-vimentin with SW13 vim⁺ cells stably expressing mCherry-vimentin. (f) Frequency distribution of the number of annealing events per filament at 2–4, 10–12, and 16–24 h after fusion for the same five cells analyzed in g and h (number of filaments counted = 850, 639, and 712, respectively). (g and h) Quantification of subunit exchange on green and red fluorescent filaments in cells obtained by the fusion of SW13 vim⁻ cells coexpressing NFL-PAGFP and NFM with SW13 vim⁻ cells coexpressing NFL-mCherry and NFM. Data are averaged from five different cells at 0, 8, and 20 h after PAGFP activation (at least 160 filaments per time point). The error bars represent mean ± standard deviation. NF, neurofilament. Bars, 2 µm.