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A study of intermediate filament formation using retrovirus-mediated gene transfer

Lu, Xin; (1991) A study of intermediate filament formation using retrovirus-mediated gene transfer. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Keratin filament formation is known to require the simultaneous presence of both type I and type II keratin proteins; type I and type II keratins are coexpressed in vivo as specific pairs, whose functions are unclear. The role of intermediate filament domains in protein stabilization and filament formation has therefore been studied using retrovirus vectors to express simple epithelial keratins as intact or deleted proteins in mesenchymal cells (fibroblasts) which do not normally express keratins. The distinct size of keratin-containing retroviral mRNAs (larger than endogenous intermediate filament mRNAs) allowed fibroblast lines to be established which contained single keratins; these were observed to be rapidly degraded, although mRNA was clearly present. The secondary introduction of a complementary type of keratin however led to immunofluorescence-detectable keratin filaments in the cytoplasm, indistinguishable from those of normal simple epithelial cells. A large number of combinations of keratins with deletions in the helix, head and tail domains were then analysed. Coexpression of two keratins of the same type, or two complementary type but expression-mismatched keratins, revealed that the protein stabilization is type-specific but not pair-specific. It was seen that (most of) the α-helical domain, but not the non-helical termini, is essential for the heterotypic type l-type II interactions required for protein stabilization. The terminal domains are both needed for effective filament formation, although only one of the two participating keratin species need be intact. This implies that a head with tail interaction of like (homotypic) keratins plays a role in filament formation. Two kinds of binding sites (helix, and head/tail) are thus involved in filament network formation, operating in different planes with respect to the main axis of the filaments. This has important implications for the morphology of the three-dimensional keratin filament network, and thus for its physical properties in different cell types.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: A study of intermediate filament formation using retrovirus-mediated gene transfer
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Pure sciences; Keratin filaments
URI: https://discovery.ucl.ac.uk/id/eprint/10120116
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