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Development of an integrin beta1A-green fluorescent protein chimera: A tool for studying real time integrin dynamics in vitro
Başlık:
Development of an integrin beta1A-green fluorescent protein chimera: A tool for studying real time integrin dynamics in vitro
Yazar:
Morrisroe, Lee, author.
ISBN:
9780438043435
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (267 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Özet:
Cell adhesion is a highly dynamic process in which regulated receptor-ligand interactions can be transient or stable attachment to substrata. Integrins form a major class of adhesion receptors that mediate cell-cell and cell-ECM interactions as well as cell motility. It is known that integrins are present in the endocytic system and are recycled, yet little is known about the molecular basis of the vesicular trafficking of integrins and it is not known whether this contributes to cell motility. In order to investigate the mechanisms of integrin trafficking, the aim of this thesis was to construct and express a C-terminal beta1A integrin-green fluorescent protein (GFP) chimera. Initially GFP was mutated at two positions (V163A and S175G) to improve thermostability and folding. The chimera was then cloned into pcDNA3 for initial transfection into MDCK cells. Results showed the GFP chimera to be retained in the ER of these cells, which may be an indication that the biosynthetic machinery of MDCK cells could not process the overexpressed protein efficiently. To test this possibility the GFP-chimera was then transfected into GD25 cells that have no endogenous beta1 integrin present. The expression pattern of the GFP chimera was improved, with some of the GFP chimera being found in focal adhesions, but this expression was at a low level and was not as efficient as that seen when wild-type beta1A integrin was restored in these cells. N-terminal chimeras were also constructed, however, these fusion proteins were held up in the ER much more than the C-terminal GFP constructs. It was hypothesised that the CMV (cytomegalovirus) promoter may not be optimal for the expression of the integrin-GFP chimera in these cells. An expression vector with an SV40 (simian virus 40) promoter, pZeoSV2(+) was therefore tested, and both wild-type and chimeric constructs were cloned into this vector. The integrin-GFP chimera expression in GD25 cells was greatly improved using pZeoSV2(+) and the GFP-chimera was localised to focal adhesions. Unfortunately generation of a stable cell line resulted in cells becoming resistant to ZeocinTM but not retaining the gene of interest. A further expression system using the SV40 vector, pECE, was tested. Wild-type 131A 131A-GFP constructs were cloned into pECE and transfected into GD25 cells. Initial transfection with this vector were not successful, however retransfection into GD25 cells yielded two functional cell lines, containing wild-type 131A and 131A-GFP integrins respectively. However, 131A-GFP cells when viewed under a microscope did fluoresce in approximately 10% of cells, but not very brightly and not at all in some cells. This is an encouraging result and this number of cells should be easy to clone to give a higher percentage of expressing cells where GFP fluorescence is visible in all cells. The results presented described for the first time the construction, expression and characterisation of a fully functional integrin-GFP chimera for use in the study of integrin trafficking in cells. However this study also suggests that care needs to be taken in the use of the integrin-GFP chimera.
Notlar:
School code: 1543
Tüzel Kişi Ek Girişi:
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(684280.1) | 684280-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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