Technical Reference #3109

3109.

Endocytosis of the Anthrax Toxin Is Mediated by Clathrin; Actin and Unconventional Adaptors Laurence Abrami; Mirko Bischofberger; Be´atrice Kunz; Romain Groux; F. Gisou van der Goot, Ecole Polytechnique Federale de Lausanne, PLoS Pathogens, 6(3109), (2010)
Link To Paper

Abstract:
The anthrax toxin is a tripartite toxin where the two enzymatic subunits require the third subunit the protective antigen (PA) to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors TEM8 and CMG2. Interestingly the toxin times and triggers its own endocytosis in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a b-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally we show that endocytosis of PA is strongly dependent on actin. Unexpectedly actin was also found to be essential for efficient heptamerization of PA but only when bound to one of its 2 receptors TEM8 due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitindependent clathrin-mediated endocytosis of the anthrax toxin.

Materials & Methods:
Fluorescence recovery after photobleaching (FRAP) HeLa cells were seeded and transfected with GFP-tagged receptors in 35 mm glass bottom dishes (MatTek). Samples were analyzed on a Leica SP2 confocal scanning microscope using a 63x oil immersion objective. After 10 scans of a chosen region a rectangle of 163 mm on the edge of a cell was irreversibly bleached using the full power of the 488 458 and 476 nm laser lines. The bleaching resulted in a 80% depletion of fluorescence. Recovery of fluorescence was monitored over 80 seconds with a 2 second interval by scanning the region with a low 488 nm laser power to minimize photobleaching during sampling. The fluorescence of the bleached area was normalized at each time point using a non-bleached control area. Recovery kinetics were fitted to an exponential function.

Microscopic Technique
FRAP microscopy, confocal scanning microscope

Cell Type(s)
HeLa cells