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Identification and characterization of an oncogenically modified actin associated protein

Shapland, Claire Elizabeth; (1994) Identification and characterization of an oncogenically modified actin associated protein. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The actin cytoskeleton is crucial for a variety of cellular events such as cell locomotion, phagocytosis, cytokinesis and cell surface receptor movement. The control mechanisms for these actin based cellular events are provided by a large number of actin-associated proteins which, acting in concert, regulate the polymerization status, interactions and geometry of actin. I have characterized a new actin associated 21 kDa polypeptide doublet, protein C4. In normal mesenchymal cells, protein C4 is associated with and uniformly distributed on actin stress fibres, while in cells and tissues that lack stress fibres, C41 is the only protein C4 isoform present. Protein C4 is present in all cells and tissues so far examined apart from neurones, erythrocytes and skeletal muscle. Protein C4 is evolutionarily conserved as it has been found in yeast. A number of actin associated proteins are down regulated in transformed cells in parallel with the reorganization of the actin cytoskeleton that often accompanies transformation. I have shown that the higher Mr protein C4 isoform, transgelin, is absent in oncogenically transformed cells where actin stress fibres are reduced in number or absent, while in contrast, the lower Mr protein C4 isoform, C41, is always present. Expression of transgelin can also be blocked by culturing normal, non-transformed mesenchymal cells in suspension. Re-expression of transgelin occurs 24 hours after these cells are returned to normal adherent culture conditions, but can be blocked by either actinomycin D or cycloheximide, suggesting that the expression of transgelin is regulated at the transcriptional level. I have purified transgelin and shown that it binds directly to actin filaments at a ratio of 1:6 actin monomers, with a binding constant (Ka) of 7.5 x 105M-1 and that it induces actin filament gelation within 2 minutes, without affecting actin polymerization. Although both actin binding and gelation activities of transgelin are controlled by ionic strength, and may be mediated by positively charged amino acid residues, the molecule remains as a monomer irrespective of ionic conditions. Electron microscopy reveals that transgelin converts actin filaments from a loose random distribution into tightly tangled aggregates. An 'add-back' permeabilization system shows that transgelin specifically rebinds to actin filaments in cells from which it has previously been removed by detergent extraction.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Identification and characterization of an oncogenically modified actin associated protein
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Actin cytoskeleton
URI: https://discovery.ucl.ac.uk/id/eprint/10108371
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