A Study of the chaperonin TCP-1 in mammalian cells: Investigating GFP-tagged CCTε monomer interactions with the Cytoskeleton

Examensarbete för masterexamen
Master Thesis
Abdollahi Lakelayeh, Mona
The cytosolic chaperonin containing TCP-1, (CCT) is an essential hexadecameric protein consisting of two back-to-back rings each composed of 8-9 subunits. CCT is essential for correct folding of actin and tubulin in order for them to adopt native conformation. However, the role of CCT in cytoskeletal organization is to be further elucidated. The role of CCTε subunit in binding and folding of both actin and tubulin, while it is within the oligomer has been established. This study aimed to investigate CCT ε interactions -as a free monomer -with F-actin and microtubules. In this study, GFP tag, owing to its intrinsic fluorescent property was used for the purpose of rendering CCT ε incapable of being incorporated into the oligomer, thereby allowing its tracing as a free monomer within the cell. A GFP-tagged CCT ε construct was successfully transfected into murine BALB-3T3 and sucrose gradient fractionation confirmed that it was not incorporated into the CCT oligomer. Immunofluorescence staining of the cells for F-actin and microtubules did not reveal any co-localization of GFP tagged-CCTε to filamentous actin or microtubules. A previous study provided evidence that CCTε co-localizes to F-actin, therefore the absence of co-localization between GFP-tagged CCT ε may be due to the conformational alterations arising from GFP fusion to CCTε. The levels of CCT subunits α, β, γ, ε, η were compared between control cells and cells transfected with GFP-tagged CCTε using Image J analysis program in a single attempt. CCT β, γ, and η showed reduced levels in transfected cells. A previous study demonstrated that CCT is up-regulated during growth, therefore the lower levels of subunits in transfected cells may be due to the different cell cycle stages the cells were undergoing, with the control cells probably being in the growth phase.
Livsvetenskaper, Industriell bioteknik, Life Science, Industrial Biotechnology
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