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Final Report Summary - 3DCELLART (Cytoskeleton architecture in host cells during Listeria infection using cryo-electron tomography)


Summary overview of the results and conclusions

Listeria monocytogenes is a pathogenic bacterium causing Listeriosis, an infection that mainly affects pregnant women and their foetuses and may be fatal. The bacteria live in cells and can spread to neighbouring cells. Spreading involves remodelling the actin cytoskeleton of its host cells such that ‘comet tails’ are assembled powering its movement within cells and enabling the cell-to-cell spread. We used cryo-electron tomography (cryo-ET) to visualize the three-dimensional structure of the comet tails in situ at the level of individual filaments. We have performed a quantitative analysis of their supramolecular architecture revealing the existence of bundles of nearly parallel hexagonally packed filaments with spacings of 12 to 13 nm. Similar configurations were observed in stress fibers and filopodia, suggesting that nanoscopic bundles are a generic feature of actin filament assemblies involved in motility; presumably they provide the necessary stiffness. We proposed a mechanism for the initiation of comet tail assembly and two scenarios that occur either independently or in concert for the ensuing actin-based motility, both emphasizing the role of filament bundling. These findings constitute a breakthrough in the field of bacterial infectious diseases.

Impact of the project:

For an understanding of the molecular mechanism of actin-based motility, knowledge of the underlying molecular architectures is indispensible. We have used cryo-ET to study the supramolecular arrangements of actin filaments in unperturbed cellular environments. An in-depth quantitative analysis of comet tails, stress fibers and filopodia has revealed the existence of bundles of nearly parallel actin filaments, some of them hexagonally packed and with strikingly similar spacings between the filaments. This common feature of actin filament architecture has important implications for the mechanical properties of actin supramolecular assemblies and for the mechanism of force generation.

Contribution to European excellence and European competitiveness

At present, cryo-ET represents the most promising technique to visualize cellular components in life-like conditions. The project constitutes a breakthrough in the field of bacterial infectious diseases. It provides unprecedented cryo-EM data on eukaryotic cells infected by a pathogenic bacterium. This project created a unique research environment in Europe for cell biology of bacterial infectious diseases, by unifying the efforts of two pioneering laboratories in cryo-ET (Baumeister laboratory, MPIB, Martinsried, Germany) and microbiology (Cossart laboratory, Institut Pasteur, Paris, France). The work was published in PNAS (doi: 10.1073/pnas.1320155110) in December 2013.

Furthermore, the new tools developed have already been exploited for cell biological investigations of actin network architecture in Dictyostelium cells, thus demonstrating the broader impact of the work on Listeria comet tails. This work is in press (Heinrich D, Ecke M, Jasnin M, Engel U, Gerisch G Reversible membrane pearling in live cells upon destruction of the actin cortex, Accepted in Biophys J). It thus contributes to the expansion of European highly competitive, cutting-edge research on an international level.

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