Regulation of the Activity of Integrins During the Cell Cycle

Castell, Salvador Soriano (1993) Regulation of the Activity of Integrins During the Cell Cycle. PhD thesis, University of Glasgow.

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I compared the expression and activity of integrins in HeLa cells between interphase and mitosis. Cultures enriched in mitotic cells (>90%) were obtained by thymidine and nocodazole blocks. Early synchronisation experiments suggested that serum improved the rate of exit from metaphase in nocodazole-blocked cells. This was confirmed by quantification of postmetaphase cells at different times after release of the nocodazole block. Immunoprecipitation and immunoblotting showed that HeLa cells express at least three integrin subunits, probably alpha5, alphav and beta1. However, I could not detect beta3 subunits. Flow cytometry showed that integrins in HeLa cells were not expressed differently in mitosis and interphase. However, I found that, in spreading assays, cells arrested in metaphase with the microtubule inhibitor nocodazole were unable to spread on fibronectin, whereas control interphase cells spread normally in the presence of nocodazole. I could just detect spreading of mitotic cells on fibronectin if 1mM Mn2+ was present in the medium, or on glass surfaces in growth medium if the metaphase block was continued for 3-4 hours. Immunofluorescence showed that integrins were redistributed in this limited spreading, as well as in the process of respreading of non blocked, mitotic cells. Analysis of fibronectin receptor activity using affinity chromatography of detergent extracts on the cell binding fragment of fibronectin showed that the mitotic receptor partially loses its activity. No phosphorylation of either mitotic or interphase receptors could be detected by immunoprecipitation from 32P-labelled cells. I further investigated integrin phosphorylation and activity in cellular rounding caused by the phosphatase inhibitor calyculin A (CL-A), as it offered a possible model for cellular rounding at mitosis. Although fully spread HeLa cells did not respond to CL-A at concentreitions reported to specifically stimulate CDK activity (i.e. 1-2nM), cellular rounding was achieved at ~50nM CL-A. Fibronectin receptor activity was lost from CL-A-treated cells, to a much larger extent than from mitotic cells, but again no phosphorylation was detected. Interestingly, low concentrations of CL-A (0.5-2nM) caused small but significant improvement of cell spreading, both on haemoglobin and on fibronectin, at the early stages (i.e. less than 30 min.). However, this effect was reversed on the long term. Analysis of the biosynthesis of integrins during the cell cycle was also carried out using immunoprecipitation from [35]S-labelled cells. Asynchronous cultures were compared with cell populations enriched in Gl+S phases. The evidence suggests that, although there is a basal level of synthesis throughout the cell cycle, there may be a peak of synthesis in G2, especially of alpha subunits. The results also show the presence of a large cytoplasmic pool of beta subunit.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: John Edwards
Keywords: Cellular biology
Date of Award: 1993
Depositing User: Enlighten Team
Unique ID: glathesis:1993-75637
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 19:02
Last Modified: 19 Nov 2019 19:02

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