Copyright © 2009 Çolakoğlu and Brown
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 43210Corresponding author.
Correspondence to Anthony Brown: Email: email@example.com
Received September 23, 2008; Accepted May 1, 2009.
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Actin filaments and microtubules lengthen and shorten by addition and loss of subunits at their ends, but it is not known whether this is also true for intermediate filaments.In fact, several studies suggest that in vivo, intermediate filaments may lengthen by end-to-end annealing and that addition and loss of subunits is not confined to the filament ends. To test these hypotheses, we investigated the assembly dynamics of neurofilament and vimentin intermediate filament proteins in cultured cells using cell fusion, photobleaching, and photoactivation strategies incombination with conventional and photoactivatable fluorescent fusion proteins. We show that neurofilaments and vimentin filaments lengthen by end-to-end annealing of assembled filaments. We also show that neurofilaments and vimentin filaments incorporate subunits along their length by intercalation into the filament wall with no preferential addition of subunits to the filament ends, a process whichwe term intercalary subunit exchange.
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Actin filaments and microtubules exist in dynamic equilibrium with a soluble subunit pool, and it is well established that these polymers lengthen and shorten by addition and loss of subunits at their ends. Intermediate filaments also exchange subunits with a soluble subunit pool (Albers and Fuchs, 1987; Angelides et al.,1989), but, remarkably, the site of subunit exchange is not clear (Alberts et al., 2002; Kim and Coulombe, 2007; Godsel et al., 2008; Lodish et al., 2008). In fact, several studies have suggested that subunit exchange is not confined to the ends of intermediate filaments and that these polymers can exchange subunits along their length (Ngai et al., 1990; Coleman and Lazarides, 1992; Vikstrom etal., 1992). In addition, observations on vimentin and keratin filaments in living cells suggest that they may elongate by end-to-end annealing in vivo (Prahlad et al., 1998; Woll et al., 2005), and this is supported by studies using electron microscopy and mathematical modeling in vitro (Herrmann et al., 1999; Wickert et al., 2005; Kirmse et al., 2007).
In this study, we describe experiments thatdirectly test the mechanisms of lengthening and subunit exchange of cytoplasmic intermediate filaments in cultured cells. We have focused on vimentin and neurofilament proteins, which are members of the type III and IV families of intermediate filament proteins, respectively (Coulombe and Wong, 2004). Vimentin forms homopolymers, whereas neurofilaments are obligate heteropolymers that are minimallycomprised of the low molecular neurofilament protein L (NFL) plus the medium and/or high molecular weight proteins neurofilament protein M (NFM) and neurofilament protein H (NFH; Lee et al., 1993).
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Results and discussion
To test the hypothesis that intermediate filaments can anneal end-to-end, we have taken advantage of the SW13 vim− human adrenal carcinoma cell line,...