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Intermediate Filaments Exchange Subunits Along Their Length and Elongate by End-To-End Annealing

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Intermediate Filaments Exchange Subunits Along Their Length and Elongate by End-To-End Annealing Published May 25, 2009 JCB: REPORT Intermediate filaments exchange subunits along their length and elongate by end-to-end annealing Gülsen Çolakoğlu1,2 and Anthony Brown1,2 1Center for Molecular Neurobiology and 2Department of Neuroscience, The Ohio State University, Columbus, OH 43210 ctin filaments and microtubules lengthen and using cell fusion, photobleaching, and photoactivation shorten by addition and loss of subunits at their strategies in combination with conventional and photoacti- A ends, but it is not known whether this is also true vatable fluorescent fusion proteins. We show that neuro- for intermediate filaments. In fact, several studies suggest filaments and vimentin filaments lengthen by end-to-end that in vivo, intermediate filaments may lengthen by end-to- annealing of assembled filaments. We also show that end annealing and that addition and loss of subunits is not neurofilaments and vimentin filaments incorporate subunits Downloaded from confined to the filament ends. To test these hypotheses, we along their length by intercalation into the filament wall investigated the assembly dynamics of neurofilament and with no preferential addition of subunits to the filament ends, vimentin intermediate filament proteins in cultured cells a process which we term intercalary subunit exchange. jcb.rupress.org Introduction Actin filaments and microtubules exist in dynamic equilibrium mers, whereas neurofilaments are obligate heteropolymers that with a soluble subunit pool, and it is well established that these are minimally comprised of the low molecular neurofilament polymers lengthen and shorten by addition and loss of subunits protein L (NFL) plus the medium and/or high molecular weight on October 9, 2012 at their ends. Intermediate filaments also exchange subunits with proteins neurofilament protein M (NFM) and neurofilament a soluble subunit pool (Albers and Fuchs, 1987; Angelides et al., protein H (NFH; Lee et al., 1993). 1989), but, remarkably, the site of subunit exchange is not clear (Alberts et al., 2002; Kim and Coulombe, 2007; Godsel et al., Results and discussion 2008; Lodish et al., 2008). In fact, several studies have suggested that subunit exchange is not confined to the ends of intermediate To test the hypothesis that intermediate filaments can anneal filaments and that these polymers can exchange subunits along end-to-end, we have taken advantage of the SW13 vim human their length (Ngai et al., 1990; Coleman and Lazarides, 1992; adrenal carcinoma cell line, which lacks endogenous cytoplas- THE JOURNAL OF CELL BIOLOGY Vikstrom et al., 1992). In addition, observations on vimentin and mic intermediate filaments because of spontaneous silencing of keratin filaments in living cells suggest that they may elongate vimentin expression (Sarria et al., 1990; Yamamichi-Nishina by end-to-end annealing in vivo (Prahlad et al., 1998; Woll et al., et al., 2003). We cotransfected SW13 vim cells with either NFL 2005), and this is supported by studies using electron micros- plus EGFP-tagged NFM to form green fluorescent neurofila- copy and mathematical modeling in vitro (Herrmann et al., 1999; ments or with NFL plus mCherry-tagged NFM to form red fluor- Wickert et al., 2005; Kirmse et al., 2007). escent neurofilaments. We then mixed the cells and fused them In this study, we describe experiments that directly test the with polyethylene glycol (PEG) to create cells containing dis- mechanisms of lengthening and subunit exchange of cytoplasmic tinct populations of red and green filaments (Fig. 1, a and b). intermediate filaments in cultured cells. We have focused on Alternatively, to confirm our data with a strategy that does not vimentin and neurofilament proteins, which are members of the involve cell fusion, we cotransfected cells with NFL plus photo- type III and IV families of intermediate filament proteins, respec- activatable GFP (PAGFP)–tagged NFM and mCherry-tagged tively (Coulombe and Wong, 2004). Vimentin forms homopoly- NFM to form a single population of neurofilaments containing Correspondence to Anthony Brown: [email protected] © 2009 Çolakoğlu and Brown This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publica- Abbreviations used in this paper: NFH, neurofilament protein H; NFL, neurofila- tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a ment protein L; NFM, neurofilament protein M; PAGFP, photoactivatable GFP; Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, PEG, polyethylene glycol; ULF, unit length filament. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). Supplemental Material can be found at: http://jcb.rupress.org/content/suppl/2009/05/25/jcb.200809166.DC1.html The Rockefeller University Press $30.00 J. Cell Biol. Vol. 185 No. 5 769–777 www.jcb.org/cgi/doi/10.1083/jcb.200809166 JCB 769 Published May 25, 2009 Downloaded from jcb.rupress.org on October 9, 2012 Figure 1. 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) Sche- matic 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. both red and photoactivatable green fluorescence along their filament, we routinely acquired several consecutive images over a entire length. To create distinct populations of green and red fil- period of 10–15 min. In each case, the red and green segments aments in these cells, we then bleached the red fluorescence in remained contiguous at all times, confirming that they were part of part of the cell and activated the green fluorescence in the same the same filament (Fig. 2 e and Videos 1 and 2). In rare instances, region (Fig. 1, c and d). With both of these strategies, we looked we even observed circular filaments, which is indicative of anneal- for the formation of chimeric filaments composed of contiguous ing between two ends of the same filament (Video 3). green and red segments. We also observed end-to-end annealing for N-terminally Both of these experimental strategies resulted in the appear- tagged NFM (coexpressed with untagged NFL) and N-terminally ance of filaments containing alternating contiguous red and green tagged vimentin (coexpressed with untagged vimentin) in MFT-16 segments within several hours, which is indicative of end-to-end cells, which are an immortalized fibroblast cell line derived annealing. We obtained the same result with N- or C-terminally from vimentin knockout mice (Fig. S1; Holwell et al., 1997), and tagged NFM (each coexpressed with untagged NFL; Fig. 2, a, b, in SW13 vim+ cells stably transfected with N-terminally tagged and d) and with N- or C-terminally tagged NFL (each coexpressed vimentin (Fig. 2, g and h). Thus, end-to-end annealing is not an with untagged NFM; not depicted) as well as with N-terminally artifact of fusion proteins or transient transfection, is not unique tagged vimentin (coexpressed with untagged vimentin; Fig. 2 c) to SW13 cells, and also occurs in the context of an endogenous and untagged neurofilament proteins (Fig. 2 f). To establish that intermediate filament network. the contiguous red and green segments were indeed part of the To quantify the annealing frequency, we measured the length same filament, we took advantage of the fact that the filaments and number of chimeric and nonchimeric filaments in SW13 vim undergo constant Brownian-like motion, presumably caused by the cells processed using the cell fusion strategy. For neurofila- thermal motion of their environment as well as jostling by other ments, 14% showed at least one annealing event (defined as a actively moving organelles in the cytoplasm. For each chimeric red/green junction) 2–5 h after fusion (n = 991 filaments from 770 JCB • VOLUME 185 • NUMBER 5 • 2009 Published May 25, 2009 Downloaded from jcb.rupress.org on October 9, 2012 Figure 2. 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).
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