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osu1247691189.pdf (65.11 MB)
ETD Abstract Container
Abstract Header
Assembly Dynamics of Intermediate Filaments
Author Info
Colakoglu, Gulsen
Permalink:
http://rave.ohiolink.edu/etdc/view?acc_num=osu1247691189
Abstract Details
Year and Degree
2009, Doctor of Philosophy, Ohio State University, Molecular, Cellular, and Developmental Biology.
Abstract
The cytoskeleton of eukaryotic cells comprises three classes of protein polymers: actin filaments, microtubules and intermediate filaments. 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 SW13 vim- 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 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 fluorescent filaments 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 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.
Committee
Anthony Brown, PhD (Advisor)
Hay-Oak Park, PhD (Committee Member)
Dale D Vandre, PhD (Committee Member)
Jian-Qiu Wu, PhD (Committee Member)
Pages
227 p.
Subject Headings
Cellular Biology
;
Molecular Biology
Keywords
intermediate filament
;
neurofilament
;
vimentin
;
assembly
;
dynamics
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Citations
Colakoglu, G. (2009).
Assembly Dynamics of Intermediate Filaments
[Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1247691189
APA Style (7th edition)
Colakoglu, Gulsen.
Assembly Dynamics of Intermediate Filaments.
2009. Ohio State University, Doctoral dissertation.
OhioLINK Electronic Theses and Dissertations Center
, http://rave.ohiolink.edu/etdc/view?acc_num=osu1247691189.
MLA Style (8th edition)
Colakoglu, Gulsen. "Assembly Dynamics of Intermediate Filaments." Doctoral dissertation, Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1247691189
Chicago Manual of Style (17th edition)
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Document number:
osu1247691189
Download Count:
632
Copyright Info
© 2009, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.