Format

Send to

Choose Destination
Cell Microbiol. 2017 Jan;19(1). doi: 10.1111/cmi.12636. Epub 2016 Jul 15.

Actin ADP-ribosylation at Threonine148 by Photorhabdus luminescens toxin TccC3 induces aggregation of intracellular F-actin.

Author information

1
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.
2
Abteilung für Anatomie und Molekulare Embryologie, Ruhr-Universität Bochum, Bochum, Germany.
3
ETH Zürich, Institute for Biomechanics, University of Zürich, Balgrist Campus, Zürich, Switzerland.
4
Department of Cardiovascular Physiology, Ruhr-University Bochum, Bochum, Germany.
5
Focal Area Structural Biology and Biophysics, Biozentrum, University of Basel, Switzerland.
6
Centre for Biological Signalling Studies (BIOSS), University of Freiburg, Freiburg, Germany.
7
Freiburg Institute of Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany.

Abstract

Intoxication of eukaryotic cells by Photorhabdus luminescens toxin TccC3 induces cell rounding and detachment from the substratum within a few hours and compromises a number of cell functions like phagocytosis. Here, we used morphological and biochemical procedures to analyse the mechanism of TccC3 intoxication. Life imaging of TccC3-intoxicated HeLa cells transfected with AcGFP-actin shows condensation of F-actin into large aggregates. Life cell total internal reflection fluorescence (TIRF) microscopy of identically treated HeLa cells confirmed the formation of actin aggregates but also disassembly of F-actin stress fibres. Recombinant TccC3 toxin ADP-ribosylates purified skeletal and non-muscle actin at threonine148 leading to a strong propensity to polymerize and F-actin bundle formation as shown by TIRF and electron microscopy. Native gel electrophoresis shows strongly reduced binding of Thr148-ADP-ribosylated actin to the severing proteins gelsolin and its fragments G1 and G1-3, and to ADF/cofilin. Complexation of actin with these proteins inhibits its ADP-ribosylation. TIRF microscopy demonstrates rapid polymerization of Thr148-ADP-ribosylated actin to curled F-actin bundles even in the presence of thymosin β4, gelsolin or G1-3. Thr148-ADP-ribosylated F-actin cannot be depolymerized by gelsolin or G1-3 as verified by TIRF, co-sedimentation and electron microscopy and shows reduced treadmilling as indicated by a lack of stimulation of its ATPase activity after addition of cofilin-1.

PMID:
27341322
DOI:
10.1111/cmi.12636
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Wiley
Loading ...
Support Center