Poster abstracts

Poster number 17 submitted by Gulsen Colakoglu

Intermediate Filaments Exchange Subunits along Their Length and Elongate by End-to-End Annealing

Gulsen Colakoglu (Department of Neuroscience, MCDB), Anthony Brown (Department of Neuroscience, Center for Molecular Neurobiology)

Abstract:
Actin filaments and microtubules are known to lengthen and shorten by addition and loss of subunits at their ends. In contrast, several studies suggest that intermediate filaments may lengthen by end-to-end annealing of pre-existing filaments and that addition and loss of subunits is not confined to the filament ends. To address these hypotheses, we expressed mCherry-tagged, GFP-tagged or photoactivatable GFP-tagged neurofilament or vimentin proteins in cultured SW13vim- cells, which lack endogenous cytoplasmic intermediate filaments. To test for annealing, we used polyethylene glycol-induced cell fusion to create cells containing a mixture of red and green filaments. Within five hours, we observed many examples of single intermediate filaments comprised of alternating red and green segments, indicating that end-to-end annealing is a common event. To determine the site of subunit incorporation, we used cell fusion to create cells containing a mixture of red and photoactivatable green fluorescent filaments and then used whole-cell photoactivation to mark those filaments that lacked red fluorescence. Alternatively, we co-expressed red and photoactivatable green fluorescent intermediate filament proteins in the same cells to make filaments containing both red and green fluorescence, then photobleached the red fluorescence and used partial-cell photoactivation to mark a sub-population of the bleached filaments. In both cases, we observed the incorporation of newly synthesized or pre-existing red fluorescent intermediate filament subunits along the length of the pre-existing photoactivated green fluorescent filaments over a period of eight to twenty hours, and no preferential addition of subunits to the filament ends. These data provide the first direct evidence that intermediate filaments in cells can elongate by end-to-end annealing and that filaments can incorporate subunits along their length, a process that we term intercalary subunit exchange.

Keywords: intermediate filament, neurofilament, vimentin