Poster abstracts

Poster number 29 submitted by Gulsen Colakoglu

Assembly dynamics of intermediate filaments

Gulsen Colakoglu (MCDB), Anthony Brown (Departmant of Neuroscience)

Abstract:
Actin filaments and microtubules lengthen and shorten by addition and loss of subunits at their ends. In contrast, several studies suggest that intermediate filaments (IFs) 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 IFs. To test for annealing, we used PEG-induced cell fusion to create cells containing a mixture of red and green filaments. Within five hours, we observed many examples of single IFs 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 filaments and then used whole-cell photoactivation to mark those filaments that lacked red fluorescence. Alternatively, we co-expressed red and photoactivatable green neurofilament 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 IFs over a period of eight to twenty hours, and no preferential addition of subunits to the filament ends. We confirmed our results in SW13 vim+ cells with endogenous vimentin networks and in MFT-16 fibroblasts derived from vimentin knockout mice. These data provide the first direct evidence that IFs 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 filaments, assembly, dynamics