Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604683
Title: Characterisation of a Salmonella actin-binding protein
Author: Hsu, C.-R.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2009
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Abstract:
Salmonella invasion protein C (SipC) and SipA bind actin directly. SipC inserts into the host cell plasma membrane from where it promotes effector delivery and is essential for Salmonella invasion. SipC-C (residues 200-409) directly nucleates actin polymerisation in vitro and induces cytoskeletal rearrangements at the leading edge when expressed in cells. By analysis a panel of SipC-C derivatives in transfected cells, a region spanning residues 377-409 was defined as necessary for actin reorganisation and leading-edge localisation. Residues 377-409 were sufficient to localise to zones of actin polymerisation in cells. SipC-N (residues 1-120) is sufficient to pair actin filaments in vitro and to induce elongated filopodia in cells. Two adjacent regions spanning residues 41-60 and 51-70 were identified as essential for inducing elongated filopodia. Corresponding proteins (SipC-NΔ41-60) and SipC-NΔ51-70) were expressed and purified. TEM of protein-F-actin mixtures revealed a deficiency in F-actin pairing. These data define the minimal N-terminal region required for filament pairing in vitro and corresponding filopodial elongation in cells. SipC derivatives containing equivalent deletions (SipC Δ41-60 and SipC Δ51-70) exhibited significantly reduced F-actin bundling activity. Derivatives with an extended deletion (SipC Δ31-70) exhibited an equivalent defect. TEM of a C-terminal truncate (SipC1-377) in complex with F-actin revealed an unanticipated defect in F-actin bundling, and a recombinant derivative containing a double deletion  (SipC1-377 Δ31-70) was unable to bundle or pair F-actin. These data demonstrate that SipC-induced F-actin bundling requires unexpected interplay between the N-terminal and C-terminal domains. SipC is proposed to bundle F-actin via the N-terminal region to pair filaments and the C-terminal actin-binding domain to efficiently cluster filaments for formation of highly dense actin bundles.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.604683  DOI: Not available
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