Strategies to test for end-to-end annealing of intermediate filaments. (a) Schematic of the cell fusion strategy. (b) A cell obtained by fusion of SW13 vim⁻ cells coexpressing NFM-mCherry and NFL with SW13 vim⁻ cells coexpressing NFM-GFP and NFL and imaged 3.5 h after fusion. (c) Schematic of the photoactivation and bleaching strategy. (d) An SW13 vim⁻ cell coexpressing mCherry-NFM, PAGFP-NFM, and NFL imaged before bleaching and photoactivation (i), and immediately after bleaching and photoactivation before the filaments had mixed (ii). Bars, 10 µm.

Strategies to test for subunit incorporation along intermediate filaments. (a) Schematic of the cell fusion and photoactivation strategy. (b) A cell obtained by fusion of SW13 vim⁻ cells coexpressing NFL-PAGFP and NFM with SW13 vim⁻ cells coexpressing NFL-mCherry and NFM and imaged before photoactivation (4.5 h after fusion; i) and immediately after photoactivation (ii). (c) Schematic of the photoactivation and bleaching strategy. (d) An SW13 vim⁻ cell coexpressing mCherry-NFM, PAGFP-NFM, and NFL imaged before bleaching and photoactivation (i) and immediately after bleaching and photoactivation (ii). Bars, 10 µm.

Models for intermediate filament end-to-end annealing and intercalary subunit exchange. The filaments are depicted as being composed of eight tetramers per cross section, each composed of two staggered coiled-coil dimers (shown as cylinders). (a) End-to-end annealing. For diagrammatic purposes, we show the end of a red filament annealing with the end of a green filament. The annealing mechanism may involve interdigitation of the protruding dimer overhangs at the filament ends. (b) Intercalary subunit exchange. For diagrammatic purposes, we show red tetramer subunits incorporating along the length of a green filament. Both annealing and intercalary subunit exchange may occur spontaneously in cells, although it is likely that there are also accessory factors that facilitate or regulate these processes.

Intermediate filaments undergo end-to-end annealing. Sites of end-to-end annealing (red/green junctions) are marked with white arrowheads. Two examples (i and ii) are shown for each experiment. For clarity, we selected isolated filaments for most of the images. (a) Obtained by fusion of SW13 vim⁻ cells coexpressing mCherry-NFM and NFL with SW13 vim⁻ cells coexpressing GFP-NFM and NFL. (b) Obtained by fusion of SW13 vim⁻ cells coexpressing NFM-mCherry and NFL with SW13 vim⁻ cells coexpressing NFM-GFP and NFL. (c) Obtained by fusion of SW13 vim⁻ cells coexpressing mCherry-vimentin and untagged vimentin with SW13 vim⁻ cells coexpressing GFP-vimentin and untagged vimentin. (d) Obtained by photoactivation and bleaching of SW13 vim⁻ cells coexpressing mCherry-NFM, PAGFP-NFM, and NFL. (e) Excerpts from a video of a neurofilament in a cell formed by the fusion of an SW13 vim⁻ cell coexpressing NFM-GFP and NFL with an SW13 vim⁻ cell coexpressing NFM-mCherry and NFL (Video 1). (f) Obtained by fusion of SW13 vim⁻ cells coexpressing NFM and NFL with SW13 vim⁻ cells coexpressing NFH and NFL. 5 h after fusion, the cells were fixed and immunostained for NFH and NFM. (g) Obtained by fusion of SW13 vim⁺ cells stably expressing mCherry-vimentin with SW13 vim⁺ cells stably expressing GFP-vimentin. (h) The edge of a cell obtained by fusion of SW13 vim⁺ cells stably expressing mCherry-vimentin with SW13 vim⁺ cells stably expressing GFP-vimentin. (i) Frequency distribution of the number of annealing events per filament 2–5 h after fusion of SW13 vim⁻ cells coexpressing NFM-GFP and NFL with SW13 vim⁻ cells coexpressing NFM-mCherry and NFL (991 filaments counted in five cells). NF, neurofilament. Bars, 2 µm.

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.